# Patent

## Title
Common and rare genetic variations associated with common variable immunodeficiency (CVID) and methods of use thereof for the treatment and diagnosis of the same

## Abstract

Compositions and methods useful for the diagnosis and treatment of common variable immunodeficiency are disclosed.

## Background
This application is a continuation in part application of PCT/US2011/051385 filed Sep. 13, 2011 which in turn claims priority to U.S. Provisional Application 61/382,231 filed Sep. 13, 2010, which is incorporated herein by reference as though set forth in full.
This invention relates to the fields of genetics and the diagnosis of common variable immunodeficiency (CVID). More specifically, the invention provides compositions and methods useful for the diagnosis and treatment of CVID.


BACKGROUND OF THE INVENTION
Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Common variable immunodeficiency (CVID) disorders are manifested by insufficient quantity and quality of immunoglobulin leading to susceptibility to bacterial infections1. CVID is considered to be a primary immunodeficiency disease (PIDD) as it is believed to result from intrinsic deficits affecting immunological functions. CVID is heterogeneous in presentation, presenting either early or later in life and associated with a group of known comorbidities2. Efforts to subcategorize CVID in order to predict outcomes and comorbid conditions both clinically and by immunologic phenotypes are ongoing3. BAFFR4, TACI5,6,7, and certain HLA haplotypes8,9 have been identified as potential gene candidates for susceptibility to CVID. ICOS10,11, CD8112, CD1913,14, CD2015, harbor disease causing mutations that presently explain only a small percentage of cases. The heterogeneous presentations of patients with CVID make management of these conditions difficult and searches for novel genetic predictors of disease causation or susceptibility have been of limited success.
SUMMARY OF THE INVENTION
In accordance with the present invention, methods are provided for the diagnosis and treatment of CVID. An exemplary method entails detecting the presence of at least one CVID associated CNV in a target polynucleotide wherein if said CNV(s) is/are present, said patient has an increased risk for developing CVID.
In one aspect of the present invention, a method for detecting a propensity for developing common variable immunodeficiency disorder (CVID) in a patient in need thereof is provided. An exemplary method entails detecting the presence of at least one SNP containing nucleic acid in a target polynucleotide, said SNP being informative of a the presence of a CVID associated copy number variation (CNV), wherein if said SNP is present, said patient has an increased risk for developing CVID, wherein said SNP containing nucleic acid is provided in Tables 1 to 5. In one embodiment, at least 1, 2, 3, 4, 5, 10, 15, 20 or all of the SNPs provided herein are detected.
In another embodiment of the invention a method for identifying agents which alter immune cell function or signaling is provided. Such a method comprises providing cells expressing at least one nucleic acid comprising the CVID associated CNVs of the invention, (step a); providing cells which express the cognate wild type sequences which lack the CNV (step b); contacting the cells from each sample with a test agent and analyzing whether said agent alters immune signaling or function of cells of step a) relative to those of step b), thereby identifying agents which alter immune cell signaling or function. Methods of treating CVID patients via administration of test agents identified using the methods described herein are also encompassed by the present invention. The invention also provides at least one isolated CVID related SNP-containing nucleic acid selected from the group listed in Tables 1 to 5. In one embodiment, a multiplex SNP panel containing all of the informative SNPs from the tables provided herein is disclosed. Such SNP containing nucleic acids which indicate the presence of CVID associated CNV(s) may optionally be contained in a suitable expression vector for expression in immune cells. Alternatively, they may be immobilized on a solid support. In yet another alternative embodiment, the panel may be provided in silico.
According to yet another aspect of the present invention, there is provided a method of treating CVID in a patient determined to have at least one prescribed single nucleotide polymorphism indicative of the presence of a CVID-associated copy number variation, as described hereinbelow, by administering to the patient a therapeutically effective amount of an agent useful for modulating immune function. This method provides a test and treat paradigm, whereby a patient's genetic profile is used to personalize treatment with therapeutics targeted towards specific gastrointestinal defects found in individuals exhibiting CVID. Such a test and treat model may benefit up to 50% of patients with CVID with greater efficacy and fewer side effects than non-personalized treatment.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. SNP based case:control significance is shown in negative log base 10 genome wide for A) Discovery and B) Replication inclusive CVID cohorts. Subsequently, CVID cases with specific disease subphenotypes were compared to CVID cases without the subphenotype. The single SNP tests are shown as single points. Multiple neighboring SNPs of similar significance boost confidence in the association as shown in the strong peak on 6p22.1-p21.32 of the Discovery case:control cohort. Conversely the median significance P value is kept low by minimizing population stratification which minimizes the genomic inflation factor.
FIG. 2. Frequency of CNV in cases and controls. CNVs are called on a single sample basis with deletions and duplications in specific genomic regions based on the genotype and intensity signal of contiguous SNPs. These single sample CNV profiles are plotted as a SNP-based statistic to allow for SNP-based association testing. Lastly, neighboring SNPs with similar significance define a CNVR. Red indicates case deletion; Blue indicates case duplication; Black indicates control deletion; Purple indicates control duplication.
FIG. 3. Duplication of ORC4L Found Exclusive to 15 CVID Cases. Vertical blue lines indicate the SNP probe coverage. Green rectangles delineate regions of copy number variation in individual cases. Three exons of ORC4L are shown duplicated in 15 cases and 0 controls. It is likely that the duplications could extend to a larger region of ORC4L based on coverage.
FIGS. 4A, 4B, and 4C. CNV Validation with Illumina Intensity Data Review and Independent Array Technology, Affymetrix 2.7M. FIG. 4A provides a visual validation of all reported CNV loci using Illumina Quad 610 Intensity Data. Gray rectangle represents normal diploid two copy state. FIG. 4B provides representative experimental validation of two of most significant deletions and two duplications, using Affymetrix 2.7M intensity data at a high resolution. Intensity values are plotted for each region listed based on samples with copy number variation calls. FIG. 4C provides experimental validation of all Table 2 loci using Affymetrix 2.7M intensity data. Intensity values are plotted for each region listed based on samples with copy number variation calls. Table 2 loci are labeled with corresponding intensity values for contiguous probes in the genomic region.
FIG. 5. The core diagnosis of CVID has many clinical progressions with varying frequency. The height and size of subphenotypes signify the frequency of the specific progression. Given significant SNP genotype associations for each CVID subphenotype progression vs. CVID patients without the progression, prediction can be made which may improve clinical outcome.
FIG. 6. Schematic Representation of Observed Data with Classification Divided by a Hyperplane. Binary SVM selects a hyperplane (bold line) that maximizes the width of the ‘gap’ (margin) between the two classes. The hyperplane is specified by ‘boundary’ training instances, called support vectors shown with circles. New cases are classified based on which side of the hyperplane they are mapped.
FIG. 7. SVM Model for CVID Case vs. individual not affected by CVID.
FIG. 8. SVM Model for CVID case with Bronchiectasis versus CVID case without Bronchiectasis.
FIG. 9. SVM Model for CVID case with OSAI (organ specific autoimmunity) versus CVID case without OSAI.
FIG. 10. Pedigree of a family with autosomal dominant conmon variable immunodeficiency FIG. 11. SVM Algorithm Hyperplane.
FIG. 12. S535N mutation in IRF2BP2. The WT nucleotide sequence is SEQ ID NO: 1; the WT amino acid sequence is SEQ ID NO: 3; the S535N nucleotide sequence is SEQ ID NO: 2; and the S535N amino acid sequence is SEQ ID NO: 4.
FIG. 13. RT-PCR or IRF2BP2.



DETAILED DESCRIPTION OF THE INVENTION
Common variable immunodeficiency disorders (CVID) are a group of uncommon heterogeneous immune defects characterized by hypogammaglobulinemia, failure of antibody production, susceptibility to bacterial infections and an array of serious comorbidities. To address the underlying immunopathogeneses of CVID, we conducted the first genome-wide association study of patients with CVID. 363 patients were genotyped with 610,000 SNPs. Due to the relative rarity of this group of disorders, the study cohort was recruited at four sites. Patients were randomly divided into a discovery cohort of 179 cases in comparison with 1,917 disease-free controls and a replication cohort of 109 cases in comparison with 1,114 controls, controlled for population stratification. Our analyses detected strong association with the MHC region and uncovered a novel association with a cluster of ADAM genes that was replicated in the independent case cohort. Analysis of the same cases for copy number variation (CNV) revealed 16 disease-associated deletions and duplications, as well as numerous unique rare intraexonic deletions and duplications suggesting multiple novel genetic etiologies for individual CVID cases. Analysis of CVID comorbidities identified significantly associated SNP genotypes with the major CVID clinical phenotypes. Taken together, our integrative genome-wide analysis of SNP genotypes and CNVs has uncovered multiple novel susceptibility loci for CVID, both common and rare, consistent with the highly heterogeneous nature of CVID. These results may allow for improved diagnosis of CVID, prediction of the CVID clinical phenotypes, and mechanistic insights into immune diseases based upon these unique genetic variations.
I. DEFINITIONS
For purposes of the present invention, “a” or “an” entity refers to one or more of that entity; for example, “a cDNA” refers to one or more cDNA or at least one cDNA. As such, the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein. It is also noted that the terms “comprising,” “including,” and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.
The term “genetic alteration” as used herein refers to a change from the wild-type or reference sequence of one or more nucleic acid molecules. Genetic alterations include without limitation, base pair substitutions, additions and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.
A “single nucleotide polymorphism (SNP)” refers to a change in which a single base in the DNA differs from the usual base at that position. These single base changes are called SNPs or “snips.” Millions of SNP's have been cataloged in the human genome. Some SNPs such as that which causes sickle cell are responsible for disease. Other SNPs are normal variations in the genome. Frequently SNPs are used to “mark” particular genes and genetic regions, particularly those containing CNVs for example.
A “copy number variation (CNV)” refers to the number of copies of a particular gene or segment thereof in the genome of an individual. CNVs represent a major genetic component of human phenotypic diversity. Susceptibility to genetic disorders is known to be associated not only with single nucleotide polymorphisms (SNP), but also with structural and other genetic variations, including CNVs. A CNV represents a copy number change involving a DNA fragment that is  ̃1 kilobases (kb) or larger. CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g.,  ̃6-kb KpnI repeats) to minimize the complexity of future CNV analyses. The term CNV therefore encompasses previously introduced terms such as large-scale copy number variants (LCVs), copy number polymorphisms (CNPs), and intermediate-sized variants (ISVs), but not retroposon insertions. The terminology “duplication-containing CNV” is also used herein below consistent with the CNV definition provided.
“CVID-associated SNP” or “CVID-associated specific marker” is a SNP or marker which is associated with an increased or decreased risk of developing CVID not found normal patients who do not have this disease. Such markers may include but are not limited to nucleic acids, proteins encoded thereby, or other small molecules. Thus, the phrase “CVID-associated SNP containing nucleic acid” is encompassed by the above description.
The term “solid matrix” as used herein refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter. The material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.
The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.
The phrase “partial informative CNV” is used herein to refer to a nucleic acid that hybridizes to sequences comprising a duplication on a chromosome however, the partial informative CNV may not be identical to the duplication, rather, the CNV may correspond to only a portion of the duplication, but yet is still informative for the presence of the same.
“Target nucleic acid” as used herein refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation which may or may not be associated with CVID. The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer. With regard to nucleic acids used in the invention, the term “isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An “isolated nucleic acid molecule” may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.
With respect to RNA molecules, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form.
By the use of the term “enriched” in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10−6-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Thus the term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.
The term “complementary” describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a “complement” of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
With respect to single stranded nucleic acids, particularly oligonucleotides, the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. For example, specific hybridization can refer to a sequence which hybridizes to any CVID specific marker gene or nucleic acid, but does not hybridize to other nucleotides. Also polynucleotide which “specifically hybridizes” may hybridize only to a single specific marker, such as an CVID-specific marker shown in the Tables contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated Tm of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the Tm of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.
The term “oligonucleotide,” as used herein is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.
The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.
The term “vector” relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of the expression construct into a prokaryotic or eukaryotic organism. The terms “transformation”, “transfection”, and “transduction” refer to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like.
The term “promoter element” describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. In one embodiment, the promoter element of the present invention precedes the 5′ end of the CVID specific marker nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than the promoter element and the CVID specific marker nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.
A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.
An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.
The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
The term “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell.
The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
The terms “recombinant organism” or “transgenic organism” refer to organisms which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The term “organism” relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, the phrase “a recombinant organism” encompasses a recombinant cell, as well as eukaryotic and prokaryotic organism.
The term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.
A “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
“Sample” or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably a CVID specific marker molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, gastric lavage, saliva, tears, pleural fluid and the like.
The terms “agent” and “compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the CNV or SNP-containing nucleic acids described herein or their encoded proteins. Agents and compounds may also be referred to as “test agents” or “test compounds” which are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.
The term “modulate” as used herein refers to increasing/promoting or decreasing/inhibiting a particular cellular, biological or signaling function associated with the normal activities of the genetic alteration containing molecules described herein or the proteins encoded thereby. For example, the term modulate refers to the ability of a test compound or test agent to interfere with signaling or activity of a gene or protein of the present invention. Alternatively, the term may refer to augmentation of the activity of such a protein.
II. METHODS OF USING CVID-ASSOCIATED CNVS AND/OR SNPS FOR DIAGNOSING A PROPENSITY FOR THE DEVELOPMENT OF CVID
The present invention provides methods of diagnosing CVID in a patient or methods for identifying a patient having an increased risk of developing CVID. Diagnosis, as used herein, includes not only the initial identification of CVID-associated with the genetic alterations described herein in a patient but confirmatory testing, or screening in patients who have previously been identified as having or likely to have CVID. The methods include the steps of providing a biological sample from the patient, measuring the amount of particular sets (e.g., those with the highest statistical significance), 1, 2, 3, 4, 5, 10, 10, 20 or all of the CVID associated markers (Tables herein) present in the biological sample, preferably a tissue and/or blood plasma sample, and determining if the patient has a greater likelihood of having or developing CVID based on the amount and/or type of CVID marker expression level determined relative to those expression levels identified in patient cohorts of known outcome. A patient has a greater likelihood of having CVID when the sample has a CNV marker expression profile associated with patients previously diagnosed with CVID. The compositions and methods of the invention are useful for the prognosis and diagnosis and management of CVID.
In another aspect, the patient sample may have been previously genotyped and thus the genetic expression profile in the sample may be available to the clinician. Accordingly, the method may entail storing reference CVID associated marker sequence information in a database, i.e., those CNVs statistically associated with a more favorable or less favorable prognosis as described in the tables herein, and performance of comparative genetic analysis on the computer, thereby identifying those patients having increased risk of CVID.
CVID-related CNV or SNP-containing nucleic acids, including but not limited to those listed below may be used for a variety of purposes in accordance with the present invention. CVID-associated CNV or SNP-containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of CVID specific markers. Methods in which CVID specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
Further, assays for detecting CVID-associated CNVs or SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage, cerebral spinal fluid), any type of cell (such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation) or body tissue.
Clearly, CVID-associated CNV or SNP-containing nucleic acids, vectors expressing the same, CVID CNV or SNP-containing marker proteins and anti-CVID specific marker antibodies of the invention can be used to detect CVID associated CNVs or SNPs in body tissue, cells, or fluid, and alter CVID CNV or SNP-containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of CVID.
In most embodiments for screening for CVID-associated CNVs or SNPs, the CVID-associated CNV or SNP-containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are important in the art.
Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as lug of total RNA. Using Resonance Light Scattering (RLS) technology, as opposed to traditional fluorescence techniques, multiple reads can detect low quantities of mRNAs using biotin labeled hybridized targets and anti-biotin antibodies. Another alternative to PCR amplification involves planar wave guide technology (PWG) to increase signal-to-noise ratios and reduce background interference. Both techniques are commercially available from Qiagen Inc. (USA).
Any of the aforementioned techniques may be used to detect or quantify CVID-associated CNV or SNP marker expression and accordingly, diagnose an increased risk for developing the same.
III. KITS AND ARTICLES OF MANUFACTURE
Any of the aforementioned products can be incorporated into a kit which may contain a CVID-associated CNV or SNP specific marker polynucleotide or one or more such markers immobilized on a solid support or Gene Chip, provided in silico in a database, one or more oligonucleotides, a polypeptide, a peptide, an antibody, a label, marker, reporter, a pharmaceutically acceptable carrier, reagents suitable for performance of PCR with positive and negative control nucleic acids, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.
IV. METHODS OF USING CVID-ASSOCIATED CNVS AND/OR SNPS FOR THE DEVELOPMENT OF THERAPEUTIC AGENTS
Since the CNVs and SNPs identified herein have been associated with the etiology of CVID, methods for identifying agents that modulate the activity of the genes and their encoded products containing such CNVs and/or SNPs should result in the generation of efficacious therapeutic agents for the treatment of this disorder.
Several regions of the human genome provide suitable targets for the rational design of therapeutic agents. Small nucleic acid molecules or peptide molecules corresponding to these regions may be used to advantage in the design of therapeutic agents that effectively modulate the activity of the encoded proteins.
Molecular modeling should facilitate the identification of specific organic molecules with capacity to bind to the active site of the proteins encoded by the CNV or SNP-containing nucleic acids based on conformation or key amino acid residues required for function. A combinatorial chemistry approach will be used to identify molecules with greatest activity and then iterations of these molecules will be developed for further cycles of screening. Molecules available for testing in this screening assay, include without limitation, Apilimod Mesylate (STA-5326), an oral small-molecule compound that selectively inhibits the production of the IL-12 family of proteins. Inhibition of IL-production may improve the gastrointestinal manifestations of CVID. Apilimod mesylate selectively inhibits this pathway and reduces over-production of IL-12 and IL-23. Other agents that may have therapeutic utility for treating CVID symptoms include the ACVR2A antagonist Sotaercept and the ACVR antibody PF3446962 as ACVR2A exhibits a significant CNV duplication associated with CVID. PEG-interleukin-2 and B-lymphocyte stimulators may also be screened for therapeutic utility in the treatment of CVID. The polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may determine, for example, formation of complexes between the polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between the polypeptide or fragment and a known substrate is interfered with by the agent being tested.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity for the encoded polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds, such as those described above, are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is then detected by methods well known in the art.
A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional or altered CVID associated gene. These host cell lines or cells are defective at the polypeptide level. The host cell lines or cells are grown in the presence of drug compound. Altered immune signaling or function of the host cells is measured to determine if the compound is capable of regulating this function in the defective cells. Host cells contemplated for use in the present invention include but are not limited to bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. However, mammalian cells, particularly immune cells are preferred. The CVID-associated CNV or SNP encoding DNA molecules may be introduced singly into such host cells or in combination to assess the phenotype of cells conferred by such expression. Methods for introducing DNA molecules are also well known to those of ordinary skill in the art. Such methods are set forth in Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y. 1995, the disclosure of which is incorporated by reference herein.
A wide variety of expression vectors are available that can be modified to express the novel DNA sequences of this invention. The specific vectors exemplified herein are merely illustrative, and are not intended to limit the scope of the invention. Expression methods are described by Sambrook et al. Molecular Cloning: A Laboratory Manual or Current Protocols in Molecular Biology 16.3-17.44 (1989). Expression methods in Saccharomyces are also described in Current Protocols in Molecular Biology (1989).
Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854). Examples of eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif. 92121); pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors such as YRP17, YIPS, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as pHIL-D1 (Phillips Petroleum Co., Bartlesville, Okla. 74004); retroviral vectors such as PLNCX and pLPCX (Clontech); and adenoviral and adeno-associated viral vectors.
Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof. Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters, baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter, as well as neuronal-specific platelet-derived growth factor promoter (PDGF), the Thy-1 promoter, the hamster and mouse Prion promoter (MoPrP), and the Glial fibrillar acidic protein (GFAP) for the expression of transgenes in glial cells.
In addition, a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell. Such markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.
Host cells expressing the CVID-associated CNVs and/or SNPs of the present invention or functional fragments thereof provide a system in which to screen potential compounds or agents for the ability to modulate the development of CVID. Thus, in one embodiment, the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents which modulate aspects of cellular metabolism associated with CVID and aberrant glutaminergic function. Also provided herein are methods to screen for compounds capable of modulating the function of proteins encoded by CNV and SNP-containing nucleic acids.
Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the CNV or SNP-containing nucleic acids on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the expressed peptide and the components of the chemical library may be detected. U.S. Pat. Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.
The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21. In one approach, discussed above, the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex, is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533). In addition, peptides may be analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based.
One can bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.
Thus, one may design drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity. By virtue of the availability of CNV or SNP-containing nucleic acid sequences described herein, sufficient amounts of the encoded polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
In another embodiment, the availability of CVID-associated CNV or SNP-containing nucleic acids enables the production of strains of laboratory mice carrying the CVID-associated SNPs or CNVs of the invention. Transgenic mice expressing the CVID-associated CNV or SNP of the invention provide a model system in which to examine the role of the protein encoded by the CNV or SNP-containing nucleic acid in the development and progression towards CVID. Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. the incorporation of genetically manipulated embryonic stem cells into an early embryo. Production of the transgenic mice described above will facilitate the molecular elucidation of the role that a target protein plays in various cellular metabolic processes, including: aberrant antibody production and function, altered immunoreceptor ligand signaling and aberrant responses to bacterial and viral infections. Such mice provide an in vivo screening tool to study putative therapeutic drugs in a whole animal model and are encompassed by the present invention.
The term “animal” is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A “transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term “transgenic animal” is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term “germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.
The alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information would encompass the introduction of CVID-associated CNV or SNP-containing nucleotide sequences.
The DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
A preferred type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated CVID-associated CNV or SNP genes as insertional cassettes to selectively inactivate a wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).
Techniques are available to inactivate or alter any genetic region to a mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. However, in comparison with homologous extrachromosomal recombination, which occurs at a frequency approaching 100%, homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10−6 and 10−3. Nonhomologous plasmid-chromosome interactions are more frequent occurring at levels 105-fold to 102 fold greater than comparable homologous insertion.
To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening of individual clones. Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly. One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists. The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter. Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil, (FIAU). By this counter selection, the number of homologous recombinants in the surviving transformants can be increased. Utilizing CVID-associated CNV or SNP-containing nucleic acid as a targeted insertional cassette provides means to detect a successful insertion as visualized, for example, by acquisition of immunoreactivity to an antibody immunologically specific for the polypeptide encoded by CVID-associated CNV or SNP nucleic acid and, therefore, facilitates screening/selection of ES cells with the desired genotype.
As used herein, a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human CVID-associated CNV or informative fragment thereof or SNP-containing gene of the invention. Such knock-in animals provide an ideal model system for studying the development of CVID.
As used herein, the expression of a CVID-associated CNV or SNP-containing nucleic acid, partial informative CNV fragment thereof, or an CVID-associated fusion protein in which the CNV or SNP is encoded can be targeted in a “tissue specific manner” or “cell type specific manner” using a vector in which nucleic acid sequences encoding all or a portion of an CVID-associated CNV or SNP are operably linked to regulatory sequences (e.g., promoters and/or enhancers) that direct expression of the encoded protein in a particular tissue or cell type. Such regulatory elements may be used to advantage for both in vitro and in vivo applications. Promoters for directing tissue specific proteins are well known in the art and described herein.
Methods of use for the transgenic mice of the invention are also provided herein. Transgenic mice into which a nucleic acid containing the CVID-associated CNV or SNP or its encoded protein have been introduced are useful, for example, to develop screening methods to screen therapeutic agents to identify those capable of modulating the development of CVID.
V. PHARMACEUTICAL AND PEPTIDE THERAPIES
The elucidation of the role played by the CVID associated CNVs and SNPs described herein in immunoreceptor ligand signaling and function facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of CVID. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
The following materials and methods are provided to facilitate the practice of the present invention.
Patients
The diagnosis of CVID was established in concordance with existing diagnostic criteria16,38. All patients were enrolled in institutionally approved research protocols to enable genetic analysis and collection of clinical data. Subsets of the patients reported here have been previously included in published studies2,22,39.
Illumina Infinium Assay for SNP Genotyping and CNV Discovery
We performed high-throughput, genome-wide SNP genotyping, using the InfiniumII HumanHap550 BeadChip technology (Illumina San Diego Calif.), at the Center for Applied Genomics at CHOP. The genotype data content together with the intensity data provided by the genotyping array provides high confidence for CNV calls. Importantly, the simultaneous analysis of intensity data and genotype data in the same experimental setting establishes a highly accurate definition for normal diploid states and any deviation from the norm. To call CNVs, we used the PennCNV algorithm12, which combines multiple sources of information, including Log R Ratio (LRR) and B Allele Frequency (BAF) at each SNP marker, along with SNP spacing and population frequency of the B allele to generate CNV calls. Rare recurrent CNVs were the focus of our study.
CNV Quality Control
We calculated Quality Control (QC) measures on our HumanHap550 GWAS data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of attempted SNPs which were successfully genotyped. Only samples with call rate >98% were included. The genome wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (LRR) <0.35 were included. All samples must have Caucasian ethnicity based on principle components analysis and all other samples were excluded. Furthermore, case and control matching was insured by calculating a genomic inflation factor between groups. Wave artifacts roughly correlating with GC content resulting from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations. Only samples where the GC corrected wave factor of LRR −0.02<x<0.02 were accepted. If the count of CNV calls made by PennCNV exceeds 100, it is suggestive of poor DNA quality, and those samples were excluded. Thus, only samples with CNV call count <100 were included. Any duplicate samples (such as monozygotic twins or repeats on the same patient) were identified and as a result one sample was excluded.
Statistical Analysis of CNVs
CNV frequency between cases and controls was evaluated at each SNP using Fisher's exact test. We only considered loci that were nominally significant between cases and controls (p<0.05) for which patients had the same variation, that were observed in multiple cohorts or were not observed in any of the control subjects, and were validated with an independent method. We report statistical local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other. Resulting nominally significant CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling; iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs. Three lines of evidence establish statistical significance: independent replication p<0.05, permutation of observations, and no loci observed with control enriched significance. We used DAVID (Database for Annotation, Visualization, and Integrated Discovery)42 to assess the significance of functional annotation clustering of independently associated results into InterPro categories.
The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.
Example 1
Genome wide single nucleotide polymorphism (SNP) arrays have enabled high throughput typing of genomic DNA with tagging of the whole genome based on linkage disequilibrium. Given both intensity and genotype content, copy number variations (CNVs) can also be detected using the same GWAS platforms. Both SNPs and CNVs have a significant difference in frequency between cases and controls in a variety of diseases. We genotyped 363 CVID cases and 3,031 healthy controls on the Illumina Infinium HumanHap550K BeadChip. We sought to associate SNPs and CNVs with CVID16, as well as with the characteristic clinical and immunologic phenotypes of this syndrome, to address disease heterogeneity. We additionally compared the CVID results with those obtained from Inflammatory Bowel Disease (IBD), another disease with an immunologic etiology but without susceptibility to infection, to serve as a “disease control” cohort and define associations unique to CVID subjects. Included in the cohort were 29 subjects with known mutations in TACI including 10 C104R, 8 A181E, 6 V220A and 1 occurrence of each of C104R/S144X, C104R/S194X, A181E/L171R, R72H, and S194X heterozygous.
Our CVID case cohort was composed of 223 patients from Mount Sinai School of Medicine, 76 patients from University of Oxford, 37 patients from The Children's Hospital of Philadelphia, and 27 patients from University of South Florida. The diagnosis in each case was validated against the ESID/PAGID diagnostic criteria16 and only patients confirmed by these criteria were included.
We first evaluated quality and suitability of the data for a case control study. The primary parameters were sample based call rates and population based stratification. Seven samples had call rates lower than 98% and were excluded. Based on principle components analysis by Eigenstrat17, the cases were stratified into three clusters: 179 cases were of confirmed European ancestry and another 109 Caucasian cases clustered separately, but very close to the other Caucasian cluster, whereas 30 samples were significant outliers from Caucasian and were omitted from this analysis. We also observed comparable clustering of the 179 cases to the 1,917 Caucasian controls. However, there were relatively few Caucasian controls that clustered well with the second Caucasian case cluster. To perform a more comprehensive search for additional control individuals that clustered with the latter case cohort, we ran 18,000 controls in 19 batches together with our cases through Eigenstrat. This identified over 1,000 control samples clustering similarly to the second Caucasian case cohort. We then reran those identified samples together with cases to yield a robustly stratified cluster with a total of 1,114 controls. This yielded a discovery cohort of 179 cases and 1,917 controls along with a replication cohort of 109 cases and 1,114 controls. We also excluded individuals with observed cryptic relatedness of genotypes as well as three known and two newly discovered 22q11 deletions. For CNV calling, samples with high noise (standard deviation of Log R Ratio >0.35) in intensity signal were additionally removed.
First, a genome wide association analysis (GWAS) was performed using the SNP genotype data. Genotype frequencies were compared between cases versus controls and a chi-square test statistic applied in Plink18 for SNPs with at least 90% call rate and 1% minor allele frequency. The discovery cohort of 179 cases and 1,917 controls yielded a low genomic inflation factor of 1.02783 (FIG. 1A). The 1,000 most significant SNPs were evaluated for enrichment in genomic regions to boost confidence in significance based on neighboring SNPs also with significance in the top 1,000. We found 87 such regions with multiple significant SNPs composed of 344 SNPs in total, the remainder of which we excluded as spurious signal (Table 1). The most significant association was the major histocompatibility complex (MHC) with 110 neighboring SNPs showing significance in the top 1,000, the most significant SNP rs3117426 having P=8.62×10−10. A total of 62 regions were supported by two SNPs, and 25 regions were supported by 3 or more SNPs, with 47 regions directly impacting genes while 40 did not.







TABLE 1







Genomic Regions with Multiple SNPs in the 1000 most Significant Associations



















Count






Region


Distance
SNPs


Genomic


From
Top

Best


Span
Gene
Distance
Exon
1000
Best P
SNP
F_A
F_U


















chr6: 27236785-32521295
MHC, Many
0
0
110
8.62E−10
rs3117426
0.3268
0.1907


chr8: 23746576-24681608
ADAM28, ADAM7, ADAMDEC1,
0
0
4
6.24E−6
rs4872262
0.03911
0.01069



STC1


chr6: 42776382-43502348
CRIP3, CUL7, GNMT,
0
0
4
1.22E−5
rs422717
0.2961
0.1982



KLC4, KLHDC3,



MEA1, MRPL2, PARC, PEAS,



PEX6, PPP2R5D, PRPH2, PTCRA,



PTK7, RPL7L1, SLC22A7,



SRF, TBCC, TNRC5, TTBK1,



UNQ1934, ZNF318


chr14: 39744450-39909619
BX248273
584047
584047
4
1.69E−5
rs6571989
0.1927
0.115


chr4: 189671181-189676119
AK095968
0
18764
2
1.78E−5
rs1606234
0.2961
0.4124


chr16: 6120908-6137836
A2BP1
0
110914
4
1.85E−5
rs12924882
0.1313
0.06915


chr2: 224812598-224841089
FAM124B
110570
110570
4
1.89E−5
rs4264554
0.3184
0.4353


chr2: 45599872-45895169
PRKCE, SRBD1
0
0
7
2.12E−5
rs17322265
0.3408
0.4576


chr10: 73083830-73084583
CDH23, KIAA1812
0
7287
2
2.45E−5
rs7087554
0.5894
0.4729


chr2: 74996818-75001224
AK125960
7650
7650
2
3.25E−5
rs3771781
0.3184
0.4319


chr20: 55079605-55083403
BMP7
95559
95559
2
4.44E−5
rs6127923
0.2318
0.1498


chr7: 4270929-4287047
SDK1
0
0
5
4.70E−5
rs895710
0.2905
0.4003


chr20: 15025030-15074507
C20orf133
0
50997
4
5.15E−5
rs6043091
0.2151
0.1367


chr8: 8804291-8808564
MFHAS1
15750
15750
3
6.80E−5
rs400404
0.3715
0.2726


chr1: 158072814-158076453
CR625159, RP11-
0
0
2
7.31E−5
rs10494349
0.2067
0.1312



190A12.4, SLAMF8


chr18: 25213638-25220939
CDH2
1202449
1202449
2
7.53E−5
rs9950880
0.2821
0.1943


chr3: 106535466-107174255
ALCAM, CBLB, Nbla00127
0
0
5
8.57E−5
rs13062596
0.4358
0.3328


chr6: 85682448-85684185
TBX18
151830
151830
2
9.07E−5
rs9444253
0.05028
0.1187


chr1: 20369369-20490791
FLJ32784, UBXN10
0
0
7
9.09E−5
rs7514144
0.2654
0.1808


chr10: 27709830-27728115
PTCHD3
0
0
7
9.77E−5
rs506659
0.4022
0.3026


chr12: 123259607-123275011
FAM101A
64652
64652
4
1.01E−4
rs7972182
0.3855
0.2874


chr13: 68136436-68138181
BC042673
195237
195237
2
1.09E−4
rs287355
0.2219
0.3213


chr16: 69996550-70004357
CALB2
14714
14714
2
1.09E−4
rs12102284
0.1676
0.1015


chr11: 25128661-25130289
LUZP2
67899
67899
2
1.10E−4
rs11028465
0.3352
0.2426


chr12: 49729991-49731928
DKFZp586A011, LETMD1
0
214
2
1.22E−4
rs4768959
0.1397
0.08033


chr4: 25702025-25756263
LOC389203
161493
161493
5
1.34E−4
rs2048507
0.257
0.1755


chr7: 13858986-13866233
ETV1
31148
31148
4
1.35E−4
rs12532319
0.09218
0.04617


chr1: 59987116-59993733
FLJ10986, RP11-242B9.1
0
2458
2
1.52E−4
rs11207520
0.3994
0.3026


chr6: 12070732-12073684
HIVEP1
47026
47026
2
1.54E−4
rs12193434
0.09777
0.05034


chr19: 40814281-40815243
MGC10433
0
134
2
1.55E−4
rs2285415
0.352
0.4559


chr6: 139472753-139477571
HECA
20371
20371
2
1.56E−4
rs17304375
0.2318
0.1549


chr12: 80435676-80444630
PPFIA2
0
59900
2
1.63E−4
rs2400955
0.4134
0.3159


chr12: 69717561-69720071
TSPAN8
85073
85073
2
1.63E−4
rs6581986
0.2905
0.2053


chr11: 102069786-102079454
MMP27
0
0
2
1.69E−4
rs17099394
0.1229
0.0687


chr3: 188168001-188168026
ST6GAL1
0
3593
2
1.71E−4
rs12495023
0.2765
0.1934


chr16: 71592439-71594134
ATBF1
0
40845
2
1.80E−4
rs8056528
0.3687
0.2754


chr8: 124729628-124734756
C8ORFK36
0
0
4
2.05E−4
rs4871402
0.06425
0.1325


chr22: 42908147-43159207
KIAA1644, PARVG
0
0
8
2.27E−4
rs80303
0.3994
0.5013


chr20: 44622587-44638545
NADC3, SLC13A3
0
0
5
2.49E−4
rs393990
0.09218
0.1664


chr1: 9459963-9467504
SLC25A33
54611
54611
2
2.57E−4
rs10746490
0.1453
0.2292


chr3: 7364204-7369287
GRM7
0
40865
4
2.96E−4
rs12491592
0.1536
0.0939


chr11: 46472478-46855347
ARHGAP1,
0
0
4
3.19E−4
rs2306029
0.3603
0.4593



CKAP5, CR612190, F2,



LRP4, MEGF7,



ZNF408, coagulation factor II


chr2: 181999958-182002374
AK125001
27433
27433
2
3.22E−4
rs16867404
0.09497
0.05008


chr7: 121184414-121193891
PTPRZ1
106504
106504
2
3.24E−4
rs1196493
0.5251
0.4266


chr3: 8378739-8381919
BC020876
0
129448
2
3.51E−4
rs359030
0.1648
0.1033


chr6: 25570803-25574868
LRRC16
0
0
2
3.61E−4
rs4320355
0.3799
0.2898


chr4: 131889955-131893245
BC041448
793982
793982
2
3.68E−4
rs2125639
0.2905
0.2094


chr15: 58159939-58162165
FOXB1
74505
74505
2
3.71E−4
rs7168491
0.1648
0.1035


chr6: 153354039-153355207
MTRF1L
0
0
2
3.81E−4
rs9322400
0.4469
0.3526


chr5: 172969660-172992688
FAM44B
0
0
4
3.83E−4
rs258873
0.2849
0.3798


chr6: 55433518-55442340
HMGCLL1
0
21202
2
3.86E−4
rs9382494
0.2179
0.3078


chr9: 15192417-15195014
C9orf52
0
0
2
3.87E−4
rs693196
0.3603
0.2723


chr4: 20207250-20210955
SLIT2
0
0
2
4.29E−4
rs573118
0.243
0.169


chr3: 284363-285747
CHL1
0
19083
2
4.31E−4
rs17273893
0.148
0.09081


chr11: 45198534-45198959
PRDM11
0
650
2
4.36E−4
rs12417962
0.06983
0.1351


chr3: 138891290-138892064
SOX14
74205
74205
2
4.37E−4
rs12637203
0.1844
0.12


chr8: 121001793-121010709
DEPDC6
0
0
2
4.41E−4
rs869340
0.324
0.2402


chr18: 74518781-74526115
SALL3
315148
315148
2
4.72E−4
rs2931060
0.1257
0.07381


chr4: 143965929-143971867
INPP4B
0
14945
2
4.81E−4
rs10000770
0.1788
0.1158


chr3: 115619659-115623404
ZBTB20
0
29017
2
5.19E−4
rs2718419
0.2598
0.1844


chr2: 65518304-65520560
FLJ16124
0
784
2
5.20E−4
rs1194849
0.3659
0.4614


chr5: 68236398-68245085
AK128486
55041
55041
2
5.27E−4
rs7718291
0.1313
0.2081


chr21: 27713547-27722878
BC043580
0
19690
2
5.51E−4
rs469709
0.1704
0.1095


chr3: 5866648-5866856
EDEM1
630006
630006
2
5.91E−4
rs2572690
0.1145
0.06602


chr16: 7470020-7474240
A2BP1
0
33910
2
5.93E−4
rs2191388
0.2709
0.1948


chr6: 88936647-88941540
CNR1
4366
4366
2
6.35E−4
rs9344757
0.2179
0.3041


chr4: 32018838-32029980
PCDH7
1265004
1265004
3
6.41E−4
rs2130904
0.2458
0.3343


chr1: 202125395-202130221
SNRPE
18492
18492
2
6.48E−4
rs12145634
0.1369
0.08346


chr7: 38733082-38736264
HVPS41, VPS41
0
0
2
6.67E−4
rs10255854
0.2179
0.3038


chr11: 120260865-120269428
GRIK4
0
5023
2
6.67E−4
rs12577638
0.2011
0.1356


chr11: 91278373-91283755
FAT3
444245
444245
2
6.97E−4
rs10501763
0.03911
0.09207


chr16: 20151678-20152281
GP2
77031
77031
2
7.14E−4
rs9921767
0.0419
0.09572


chr1: 221871750-221882793
CAPN2
84031
84031
3
7.29E−4
rs3856154
0.4134
0.3254


chr10: 130929688-130931369
MGMT
224087
224087
2
7.33E−4
rs538186
0.05307
0.1104


chr5: 170727300-170734557
NPM1
12168
12168
2
7.52E−4
rs7707008
0.1173
0.1894


chr10: 63071553-63080172
C10orf107
12553
12553
2
7.69E−4
rs10994852
0.1061
0.06051


chr21: 31427707-31432285
TIAM1
0
0
2
7.93E−4
rs2833297
0.2095
0.1435


chr4: 42434168-42438752
ATP8A1
80521
80521
2
8.09E−4
rs6812482
0.2709
0.1965


chr6: 161106705-161109328
PLG
12377
12377
2
8.78E−4
rs9295131
0.2151
0.2986


chr17: 12323188-12325534
AX747308, BC122562
68477
68477
2
9.02E−4
rs8069430
0.2584
0.186


chr17: 50125991-50127579
TOM1L1, tom1-like
205624
205624
2
9.20E−4
rs6504930
0.03371
0.08325


chr16: 77330083-77334376
WWOX
0
305933
2
9.21E−4
rs1364290
0.1844
0.1231


chr11: 5493737-5511794
HBG2, UBQLNL
0
0
4
9.28E−4
rs2047456
0.3436
0.2624


chr3: 70644812-70648510
LOC401072
96929
96929
2
9.91E−4
rs17790790
0.5196
0.4293


chr3: 1292289-1302261
CNTN6
0
0
2
1.06E−3
rs6799262
0.3571
0.2733


chr3: 64977356-64985877
BC040632
5173
5173
2
1.15E−3
rs1517927
0.2318
0.1643


chr5: 14818187-14825245
ANKH
0
0
2
1.15E−3
rs17251715
0.3955
0.3113









We next sought to replicate these SNPs and the direction of the difference in allele frequency, based on our replication cohort of 109 cases and 1114 controls with a genomic inflation factor of 1.04789 (FIG. 1B). Four SNPs displayed significance (P<0.05) and allele frequency in the same direction as the discovery cohort, including rs11207520, rs1194849, rs17790790, and rs4872262. The associated chromosomal region demonstrating replication that impacted characterized genes was 8p21.2, harboring ADAM28, ADAM7, ADAMDEC1, and STC1. When all SNPs contributing to the discovery region were queried in replication rather than just the most significant SNP, 8 (four additional) SNPs displayed significance (P<0.05) and allele frequency in the same direction as discovery. There were three additional regions with directly affected genes: the MHC, UBXN10, and SDK1 (Table 2); others did not overlap genes directly.







TABLE 2







Most Significant Associated Regions Based on Genotype Association





















Count
Dis-













SNPs
tance





P

F_A
F_U


Region

Top
From





Repli-
SNP
Repli-
Repli-


Genomic Span
Gene
1000
Exon
P
SNP
A1
F_A
F_U
cation
Replication
cation
cation






















chr6:
MHC
110
0
8.62E−10
rs3117426
T
0.3268
0.1907
0.0004
rs2156875
0.5734
0.4475


27236785-32521295


chr8:
ADAM28,
4
0
6.24E−6
rs4872262
A
0.0391
0.0107
0.0314
rs4872262
0.0321
0.0135


23746576-24681608
ADAM7,



ADAMDEC1,



STC1


chr7:
SDK1
5
0
4.70E−5
rs895710
T
0.2905
0.4003
0.0235
rs4720301
0.3991
0.4793


4270929-4287047


chr1:
FLJ32784,
7
0
9.09E−5
rs7514144
C
0.2654
0.1808
2.25E−8
rs6426636
0.4679
0.2857


20369369-20490791
UBXN10


chr1:
FGGY
2
2458
1.52E−4
rs11207520
G
0.3994
0.3026
0.0015
rs11207520
0.2569
0.1706


59987116-59993733


chr2:
FLJ16124
2
784
5.20E−4
rs1194849
G
0.3659
0.4614
0.0119
rs1876518
0.3853
0.4744


65518304-65520560





Bold underline: Known immunological function genes,


A1: allele 1 which is the minor allele,


F_A: Frequency of allele 1 in affected individuals,


F_U: Frequency of allele 1 in unaffected individuals.


See additional detail inTable 11.






We had access to a large IBD cohort that had been genotyped previouslyl9. Since this is also a heterogeneous immunological condition but without increased infectious susceptibility, it was considered that this group would be appropriate as a disease control group. Analysis of the 2,413 IBD cases and 6,197 controls revealed a number of signals in the IBD cohort that were distinct from both controls and the CVID cohort (including 19 CVID subjects who had been diagnosed with IBD). These included a signal in DEPDC6 (P<0.05) with allele frequency in the same direction as well as the MHC locus. In addition, TIAM1, SLAMF8, GRIK4, 6p21.1, PPFIA2, SLC25A33, A2BP1, and UBXN10 were also observed in the IBD cohort only (P<0.05). Importantly, however, the majority of associations in CVID identified in comparison to controls were not observed in the IBD cohort, suggesting that that they were not a general feature of an immunologic disease, but specific to the CVID phenotype. The non-overlapping loci between CVID and IBD included FLJ16124, ADAM28-ADAM7-ADAMDEC1-STC1, ANKH, FLJ109860-RP11-242B9.1, and WWOX, which are therefore more likely to lead to loss of B cell and antibody function.
We next performed a CNV analysis, where rare (<1%) CNVs were called, using PennCNV20 and SNP-based CNV frequency in cases and controls; these were compared for associations. We analyzed deletion and duplication CNV frequency of 311 cases and 2,766 controls (FIG. 2). To limit bias due to population stratification, the contribution of patients from the discovery Caucasian cluster defined in the genotype GWAS was required to establish confidence. We also monitored the contribution of each sample subgroup from the four different sample collection sites. We found 5 deletions and 11 duplications to be recurrent and significantly enriched in the patients as compared to controls (Table 3). In addition, a total of 15 regions were exclusive to CVID cases. Duplications of 2q23.1 associated with multiple exons of ORC4L were observed to be exclusive to 15 cases (P=8.66×10−16) (FIG. 3). We also observed large CNVs with at least 10 SNPs exclusive to single CVID cases totaling 84 deletions and 98 duplications in CNVs. (Table 4). No significant overrepresentation was observed in the controls in this frequency range. Of clinical relevance, 2 patients were newly identified with deletion in the 22q11 region suggesting an alternative presentation of the well-characterized PIDD associated with this microdeletion. Of 5 deletions and 23 duplications significantly enriched in CVID, 4 deletions and 21 duplications significant in CVID were not significant in IBD. 10 regions of homozygous deletion were observed exclusively in CVID cases (Table 5). To further prioritize resulting significant loci for potential functional impact, we calculated distance of CNVs from exons to highlight loci impacting exons directly, a method applied previously21. We examined overall CNV burden in cases compared to controls for significant results, exonic CNVs, and large (100 kb) rare (<1%) CNVs and found deletions to be significantly enriched in cases when considering CNV observations on all loci genome wide (Table 6). All but 5 of the 182 CNVs exclusive to the CVID cohort were intraexonic. To assess the reliability of our CNV detection method, we reviewed the BAF(genotype) and LRR(intensity) values of our Illumina data and experimentally validated all the significant CNVRs using an independent method, the Affymetrix Cytogenetics Whole-Genome 2.7M Array which provides high resolution with 400,103 SNP and 2,387,595 CN probes (FIG. 4).







TABLE 3





Most Significant Associated Regions Based on CNV Association







A) Deletions


















Count
Distance

Cases
Controls







CNVR Deletion
SNPs
From Exon
P Deletion
Del
Del
Gene
MSSM
Oxford
USF
Combined





chr11: 85365857-85381622
5
0
0.01
2
0
PICALM, PICALM variant
0
2
0
2








protein


chr20: 57735790-57741780
6
5902
0.026
3
4
PHACTR3
2
1
0
3


chr22: 17396663-18417315
270
0
0.029
2
1
ARVCF, COMT, TBX1,
2
0
0
2








others


chr4: 10256682-10264316
4
2915
0.029
2
1
CLNK
0
1
1
2


chr10: 46003146-46042543
8
0
0.029
2
1
DKFZp566K0524,
2
0
0
2








PTPN20A, PTPN20B










B) Duplications


















Count
Distance

Cases
Controls







CNVR Duplication
SNPs
From Exon
P Dup
Dup
Dup
Gene
MSSM
Oxford
USF
Combined





chr2: 148396730-148433180
6
0
8.66E−16
15
0
ACVR2A,
10
5
0
15








ORC4L


chr15: 35053039-35063531
6
23146
5.72E−13
19
10
MEIS2
17
2
0
19


chr7: 91789778-91801963
4
0
1.20E−09
12
4
ANKIB1
9
3
0
12


chr2: 163316298-163316595
3
17100
1.00E−07
7
0
KCNH7
6
1
0
7


chr7: 87180702-87191931
4
202
7.41E−07
7
1
RPIB9
5
2
0
7


chr19: 9256584-9277749
4
0
0.001
3
0
ZNF699
2
0
1
3


chr4: 39190766-39201960
6
0
0.004
3
1
UGDH
1
2
0
3


chr7: 18903915-18905725
4
23171
0.009
3
2
HDAC9
1
2
0
3


chr5: 170531269-170536993
4
374
0.01
2
0
DKFZp666P032,
1
1
0
2








RANBP17


chr7: 34685030-34686172
4
4517
0.01
2
0
AAA1, NPSR1
1
1
0
2


chr1: 170400944-170403742
4
26274
0.01
2
0
DNM3
0
1
1
2





Additional detail in Table 12.













TABLE 4





Large Genic CNVs Impacting Single CVID Cases not Observed in Controls




















Distance




Count
From



CNVR Del Singleton
SNPs
Exon
Gene










A) Deletions Impacting Immune Function Genes










chr4: 86650109-
209
0
ARHGAP24, BC038746, MAPK10


87684765





chr16: 75457204-
78
0
ADAMTS18, AK026469, BC035731, CLEC3A, KIAA1576, MON1B, WWOX


76988778





chr3: 74573112- 
66
0
CNTN3


74866049





chr19: 56625454-
64
0
LOC729767, SIGLEC12, SIGLEC6, SIGLEC8


56761712





chr4: 103671292-
51
0
AK093356, BDH2, CENPE, CENPE variant protein, CR604221, LOC133308, MANBA, NFKB1,


104700248


NHEDC1, UBE2D3, UNQ6308, ZCD2


chr14: 43997374-
45
0
AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3, KIAA0423, KLHL28, 


46152755


PRPF39


chr6: 29844580-
44
0
AK097625, BC035647, HLA-A, HLA-A*0226, HLA-G, HLA-G2.2, LOC554223, LOC642032,


30045812


NR_001317, NR_001318, NR_002139


chr2: 86695685-
40
0
ANAPC1, BC066991, CD8A, CD8B, DQ576041, LOC285074, MGC4677, PLGLB1, PLGLB2, RGPD1,


87945980


RGPD2, RMND5A, RNF103


chr6: 22380732- 
40
0
PRL


22525808





chr6: 31465923-
34
0
AF075059, AGER, AGPAT1, AIF1, AK057104, AL050203, APOM, ATP6V1G2, B3GALT4, BAT1,


33432505


BAT2, BAT3, BAT4, BAT5, BF, BRD2, BTNL2, C2, C4A, C4A variant protein, C6orf10, C6orf21, 





C6orf25, C6orf26, C6orf27, C6orf31, C6orf47, C6orf48, CFB, CLIC1, COL11A2, CREBL1, CS266662,





CSNK2B, CYP21A2, DAXX, DDAH2, DKFZp313H139, DKFZp547I194, DKFZp779M0311, DOM3Z,





EGFL8, EHMT2, FKBPL, G18.1a, G6E, G6e, G7c, G8, GPSM3, HCP5, HLA-DMA, HLA-DMB, HLA-





DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DPB2, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-





DQB2, HLA-DRA, HLA-DRB1, HLA-DRB5, HSD17B8, HSPA1A, HSPA1B, HSPA1L, LOC401252,





LSM2, LST1, LTA, LTB, LY6, G5B, LY6G5C, LY6G6C, LY6G6D, MCCD1, MEGT1, MICA, MICB,





MSH5, NCR3, NEU1, NFKBIL1, NG36/G9a, NOTCH4, NR_002742, NR_002745, NR_002812,





NR_002971, NR_003140, PBX2, PFDN6, PP199, PPT2, PRRT1, PSMB8, PSMB9, RAGE, RDBP, 





RGL2, RING1, RNF5, RP, RPS18, RXRB, SKIV2L, SLC39A7, SLC44A4, STK19, TAP1, TAP2,





TAPBP, TNF, TNFa, TNXB, VARS, VPS52, WDR46, ZBTB12, ZBTB22 


chr2: 70616413- 
30
0
ADD2, AF090102, ANKRD53, ATP6V1B1, CD207, CLEC4F, FIGLA, KIAA1155, MCEE, 


71523501


MPHOSPH10, N-Acetylglucosamine kinase, NAGK, NP220, OR7E91P, TEX261, TGFA, VAX2, 





ZNF638


chr8: 95257759-
28
0
AF086017, AX747981, C8orf38, CCNE2, CDH17, DPY19L4, FSBP, GEM, INTS8, KIAA1429,


96161427


RAD54B, RBM35A, TP53INP1, p53DINP1


chr1: 184639488-
24
0
C1orf27, PDC, PLA2G4A, PTGS2


185615061





chr4: 124486894-
21
0
AL833449, BC053945, SPRY1


125137357





chr1: 226522770-
20
0
KIAA1556, KIAA1639, OBSCN, TRIM11, TRIM17


226673588





chr21: 14806346-
17
0
HACS1, SAMSN1


14824849





chr7: 84542383-
16
0
SEMA3D


84633646





chr4: 162635366-
15
0
FSTL5


163113553





chr19: 48834611-
15
0
AK090553, AK098175, AK131520, AX748287, BC041923, BC045755, CADM4, CR593740,


49626859


DKFZp564H1322, FLJ12886, HZF19, HZF6, IRGC, KCNN4, LYPD5, PLAUR, ZNF155, ZNF221,





ZNF222, ZNF223, ZNF224, ZNF225, ZNF226, ZNF227, ZNF228, ZNF229, ZNF230, ZNF233, 





ZNF234, ZNF235, ZNF284, ZNF285A, ZNF404, ZNF45


chr4: 90603695-
14
0
AK123890, CR605611, KIAA1680, MGC48628, MMRN1, SNCA


91299621





chr6: 138020053-
14
0
AK124173, PERP, TNFAIP3


138532047





chr7: 30225447-
14
0
AK096056, AK096687, AK097240, AL137445, BC016976, BC041636, BX648714, C7orf24, CR595731,


30798217


CRH2R, CRHR2, FLJ22374, GARS, INMT, NOD1, NR_002186, ZNRF2


chr12: 55685282-
14
0
ARHGAP9, AVIL, B4GALNT1, BC019026, BC033961, BC073932, CDK4, CENTG1, CR625050,


56515644


CS444342, CTDSP2, CYP1, CYP27B1, DCTN2, DDIT3, DTX3, FAM119B, G43318, GEFT, GLI1,





INHBC, INHBE, KIAA0286, KIAA1002, KIF5A, LRP1, MARCH9, MARS, MBD6, METTL1, MYO1A,





NAB2, NDUFA4L2, NXPH4, OS9, PIP5K2C, R3HDM2, RGL1, SHMT2, SLC26A10, STAC3, STAT6,





TAC3, TSFM, TSPAN31, ZBTB39, ZNEUROK1


chr16: 86267434-
14
0
AK126852, AX747795, BANP, CA5A, DKFZp434G0522, FLJ00104, JPH3, KLHDC4, SLC7A5


86992013





chr2: 179266611-
13
0
AK123298, CR624402, FLJ39502, SESTD1, TTN


179839804





chr3: 95106214-
12
0
PROS1


95125111





chr2: 36745429-
11
0
AK001814, BC017652, CCDC75, CEBPZ, EIF2AK2, HEATR5B, KIAA1414, PRKD3, PRO1853, 


37640775


QPCT, STRN, SULT6B1, VIT


chr4: 151504432-
11
0
DKFZp686K03100, LRBA


151570906





chr11: 127094060-
11
0
AX747861, BC039676, DKFZp686D0662, ETS1, FLI1


128138864





chr1: 31912222-
10
0
AK096192, BAI2, BAI2, BC069257, BC111382, BSDC1, C1orf90, C1orf91, CCDC28B, COL16A1,


32647867


CR601100, DCDC2B, DKFZp564C2082, DKFZp686B09139, EIF3S2, HDAC1, IQCC, KHDRBS1, 





KPNA6, LCK, LOC339483, MARCKSL1, PTP4A2, SPOCD1, TMEM39B, TSSK3, TXLNA, UNQ548


chr3: 106447305-
10
0
ALCAM, CBLB, Nbla00127


107177994





chr6: 161278798-
10
0
AGPAT4, C6orf59, LPAAT-delta, MAP3K4, MTK1, PARK2, parkin


161959701





chr7: 24205709-
10
0
DFNA5, ICERE-1, KIAA0704, MPP6, NPY, OSBPL3


24955079










B) Deletions Not Impacting Known Immune Function Genes










chr3: 163785970-
94
0
BC073807, LOC647107


165636507





chr2: 78159130-
86
0
BC024248, BC030125


78549747





chr3: 7053179-
49
0
GRM7


7177909





chr11: 109142987-
48
0
AK094117, AK124179, ARHGAP20, DKFZp43410812, FDX1, KIAA1726, NR_001287, RDX


110423998





chr12: 27002588-
44
0
AK000807, ARNTL2, BC043511, DKFZp564O1863, FGFR1OP2, KIAA0965, KIAA1230, KLHDC5,


28111983


LOC728858, MDS023, MRPS35, PPFIBP1, PTHLH, REP15, STK38L, SURB7, TM7SF3


chr6: 97170276-
39
0
C6orf66, FHL5, GPR63, KIAA1900, KLHL32


97625708





chr6: 163188894-
35
33591
PACRG


163315053





chr16: 9624870-
35
0
ATF7IP2, DKFZp666E123, DQ587956, DQ595173, GRIN2A, U07199


10467886





chr7: 64220284-
34
0
AK057766, DQ596928, FLJ25037, LOC441242, ZNF92


64795508





chr5: 90035491-
33
0
GPR98, KIAA0686, VLGR1


90386715





chr3: 37578530-
32
0
ITGA9


37705809





chr6: 38413748-
32
0
BTBD9


38630608





chr20: 51844911-
28
0
NR_002189


51931244





chr15: 57055702-
26
0
BNIP2, CCNB2, DKFZp761D081, FAM81A, GCNT3, GTF2A2, LDHAL6B, MYO1E, RNF111


57917444





chr15: 54225066-
24
0
MNS1, TEX9


54550705





chr17: 3128134-
23
0
OR3A1, OR3A2, OR3A4


3194433





chr7: 7480856-
22
0
AK027125, COL28, COL28A1, FLJ20323, RPA3


7819889





chr10: 94222227-
22
0
EXOC6, HHEX, IDE, KIF11


94754640





chr14: 91502061-
21
0
TRIP11, Trip230


91569101





chr2: 42398844-
19
0
COX7A2L, EML4, HAAO, KCNG3, MTA3, OXER1


43092929





chr14: 26535543-
19
0
BX538073


27350537





chr14: 92744837-
19
0
BTBD7, C14orf130, COX8C, KIAA1409, PRIMA1


93367366





chr2: 14622841-
18
0
AX747684, FAM84A


14924411





chr12: 10589522-
17
0
KLRA1


10635183





chr3: 99229541-
16
0
CR749263, OR5AC2, OR5H1, OR5H14, OR5H15


99419735





chr2: 132326867-
15
0
FLJ41821


132449751





chr4: 9136858-
15
0
DKFZp586I2219, DQ581767, DQ583133, DQ584082, DQ585713, DQ589421, DRD5, SLC2A9, WDR1


9881191





chr4: 128805691-
15
0
INTU, SLC25A31


128920529





chr9: 16987947-
15
0
C9orf39, RP11-340N12.1


17478111





chr14: 59952568-
15
0
C14orf39, SIX6


60075378





chr18: 5682313-
15
0
AF301223, TTMA


5922670





chr19: 23304120-
15
0
AK022793, BC038574, BC043213, ZNF91


23389292





chr12: 39980210-
14
0
GLT8D3, PDZRN4


40809427





chr19: 19759149-
14
0
CR593334, CR614976, DKFZp761G18121, FLJ44894, NR_003128, ZNF253, ZNF486, ZNF506,


20368239


ZNF682, ZNF90, ZNF93


chr2: 98228529-
13
0
DKFZp434J0326, DKFZp451I0318, MGC26733


98252357





chr2: 168309981-
13
0
AL080192, B3GALT1, BC035245, LASS6, NOSTRIN, STK39


169429627





chr4: 22663503-
13
0
CR607430


22803604





chr5: 107695465-
13
0
AK021888, BC034788, FBXL17, FER, MAN2A1, PJA2


109245567





chr4: 20984915-
12
0
KCNIP4


21185852





chr5: 178315216-
12
0
ADAMTS2, BX648737, CR598488, GRM6, ZNF354C, ZNF454, mGluR6


178514465





chr17: 55759021-
12
0
APPBP2, BCAS3, C17orf64, L32131, PPM1D, USP32


56436171





chr18: 60137564-
12
0
BC036306


60597211





chr5: 38305919-
11
11209
EGFLAM


38317501





chr6: 12643097-
11
0
PHACTR1, RPEL


13219417





chr4: 69405358-
10
0
TMPRSS11E


69494103





chr9: 40497792-
10
0
AK024257


40617437





chr11: 102824056-
10
0
DYNC2H1


102856185





chr11: 124360372-
10
0
CCDC15


124420328





chr12: 47817725-
10
0
TUBA1A, TUBA1B, TUBA1C


47931507





chr14: 62671250-
10
0
GPHB5, PPP2R5E, RHOJ


62971495





chr15: 91046103-
10
0
CHD2, DKFZp781D1727, LOC400451


91378387







Distance




Count
From



CNVR Dup Singleton
SNPs
Exon
Gene










C) Duplications Impacting Immune Function Genes










chr19: 5773490-
827
0
ACP5, ACSBG2, ADAMTS10, AF019226, AF075036, AF161365, AK056073, AK097685, 


12007370


AK124717, ALKBH7, ANGPTL4, ANGPTL6, ANKRD25, ANKRD47, AP1M2, ARHGEF18, 





ASAH3, ATG4D, AX747405, AX747599, AX748210, AY203940, BC007593, BC014506, BC029904,





BC033124, BC039523, BC042816, BGR, C19orf39, C19orf45, C19orf52, C19orf59, C3, CAPS,





CARM1, CCL25, CD209, CD320, CD70, CDC37, CDKN2D, CLEC4G, CLEC4M, CLPP, CNN1,





COL5A3, CR598956, CRB3, CTXN1, DENND1C, DKFZp547H118, DKFZp564K0223,





DKFZp564O1762, DKFZp666A071, DKFZp667O2312, DKFZp761J1410, DKFZp761K0816,





DNM2, DNMT1, DOCK6, ECSIT, EDG5, EDG8, EIF3S4, ELAVL1, ELAVL3, ELOF1, EMR1, 





EMR4, EPOR, EVI5L, FBN3, FBXL12, FCER2, FLJ00153, FLJ11286, FLJ12949, FLJ20079,





FLJ22184, FLJ25758, FUT3, FUT5, FUT6, GPR108, GTF2F1, HNRPM, HSZFP36, ICAM1,





ICAM3, ICAM4, ICAM5, ILF3, INSR, KEAP1, KHSRP, KIAA0521, KIAA1395, KIAA1518,





KIAA1543, KIAA1588, KIAA1776, KIAA1978, LASS4, LDLR, LOC126075, LOC162993,





LOC388503, LOC401898, LOC440508, LOC55908, LPPR2, LRRC8E, MAP2K7, MARCH2, 





MBD3L1, MBD3L2, MCOLN1, MGC19604, MGC20983, MGC33407, MKK7, MLLT1, MRPL4,





MUC16, MYO1F, MYO1F variant protein, NDUFA11, NDUFA7, NRTN, OLFM2, OR1M1, OR2Z1,





OR7D2, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, P2RY11, PCP2, PDE4A, PEX11G, PIN1, 





PNPLA6, PPAN, PPAN-P2RY11, PRAM1, PRKCSH, PSPN, QTRT1, RAB11B, RAB3D, RANBP3,





RAVER1, RDH8, RETN, RFX2, RGL3, RPS28, SH2D3A, SLC25A23, SLC25A41, SLC44A2,





SMARCA4, SNAPC2, SPC24, STXBP2, TIMM44, TMED1, TNFSF14, TNFSF9, TRAPPC5,





TRIP10, TSPAN16, TUBB4, TYK2, UBL5, UNQ2443, UNQ501, VAV1, VMAC, XAB2, YIPF2,





ZNF121, ZNF177, ZNF266, ZNF317, ZNF358, ZNF414, ZNF426, ZNF433, ZNF439, ZNF440,





ZNF441, ZNF491, ZNF557, ZNF558, ZNF559, ZNF560, ZNF561, ZNF562, ZNF627, ZNF653, 





ZNF69, ZNF699, ZNF700, ZNF763, pp10122


chr14: 92664804-
448
0
AK125038, ASB2, BC016484, BC037859, BC038791, BDKRB2, BTBD7, BX247990, C14orf109,


95787550


C14orf130, C14orf132, C14orf139, C14orf142, C14orf152, C14orf48, C14orf49, CLMN, COX8C,





CR611440, DDX24, DICER1 FAM14A, FAM14B, GIG24, GLRX5, GSC, IFI27, KIAA0928,





KIAA1409, KIAA1622, MOAP1, NM_207443, NR_001459, NR_003002, OTUB2, P27, PRIMA1,





SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINA3, SERPINA4,





SERPINA5, SERPINA6, SERPINA9, TCL1A, TCL1B, TCL6, TCL6a1, TCL6d1, TML1


chr11: 7678446-
315
0
ADM, AK000908, AK055772, AK090613, ASCL3, AX747224, BC017787, BC027619, BC068088,


10337351


BC073899, BCA3, C11orf16, C11orf17, CMT4B2, CR598129, DKFZp451C1317, DKFZp686118166, 





EIF3S5, IPO7, KIAA0298, KIAA1766, LMO1, MGC10850, NLRP10, NRIP3, NR_002580, 





NR_002962, NR_002977, OR10A3, OR10A6, OR5E1P, OR5P2, OR5P3, OVCH2, RAB6IP1, RIC3, 





RPL27A, SBF2, SCUBE2, ST5, STK33, SWAP70, TMEM41B, TMEM9B, TUB, U80769, WEE1, 





ZNF143


chr13: 100600344-
197
0
AK093430, AK096424, AK125748, AX747578, BIVM, C13orf27, CR616826, DKFZp434L2319,


103567030


ERCC5, FGF14, ITGBL1, KDELC1, LOC196541, RP11-430M15.2-003, SLC10A2, TPP2,





UNQ1910, VGCNL1


chr14: 65357663-
159
0
CR594591, GPHN, MGC88374


66177823





chr4: 119628184-
158
0
AK000709, AK024248, AK097701, AK098126, BC035733, BC070391, CEP170L, DQ574659,


120591177


DQ575011, DQ575856, DQ576410, DQ582480, DQ599872, FABP2, KIAA0755, KIAA1627,





LOC401152, MYOZ2, Myopodin, SEC24D, SYNPO2, USP53


chr8: 106761674-
156
0
ABRA, ANGPT1, CR602836, Nbla00307, OXR1, ZFPM2


108458326





chr4: 113049611-
129
0
ALPK1, C4orf16, C4orf21, C4orf32, CR590073, DKFZp434C0927, FLJ00302, KIAA1527, 


113759418


LOC91431, NEUROG2, TIFA


chr15: 40145534-
90
0
CAPN3, GANC, KIAA0770, PLA2G4D, PLA2G4F, SNAP23, TMEM87A, VPS39, ZFP106


40608559





chr14: 43658461-
87
0
AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3, KIAA0423, KLHL28,


45064383


PRPF39


chr10: 67399581-
75
0
CTNNA3, DNAJC12, HERC4, LRRTM3, MYPN, SIRT1


69536663





chr3: 107041527-
71
0
CBLB


108030375





chr9: 137454076-
70
0
ABCA2, AF161442, AGPAT2, AK023162, AK054908, AK055547, AK090585, AK096249, 


139460602


AK098241, AK128153, AK128864, AL832276, ANAPC2, AX747706, AY952890, BC015688, 





BC032375, BC034456, BC042667, BC043225, BC061888, BC064596, BC092490, BC101937,





BTBD14A, C8G, C9orf116, C9orf139, C9orf140, C9orf142, C9orf163, C9orf167, C9orf75,





C9orf86, CAMSAP1, CARD9, CLIC3, COBRA1, CR614579, CR619051, DKFZp434B205, 





DKFZp564M173, DKFZp762I052, DPP7, EDF1, EGFL7, ENTPD2, ENTPD8, FAM69B, FBXW5,





FLJ20433, FLJ45224, FUT7, GBDR1, GLT6D1, GPSM1, GRIN1, HBE269, INPP5E, KCNT1, 





KIAA0310, KIAA0649, KIAA1062, KIAA1422, KIAA1984, LCN1, LCN10, LCN12, LCN6, 





LCN8, LCN9, LHX3, LOC389813, LOC389816, LOC401565, LOC441476, LOC728489,





MAMDC4, MAN1B1, MGC14327, MGC59937, MGC61598, MRP-S2, MRPS2, NDOR1, 





NOTCH1, NOXA1, NPDC1, NPTIIc, NR1, NR_002958, NR_002975, OBP2A, PAEP, PHPT1,





PMPCA, PTGDS, QSCN6L1, RNF208, RP11-100C15. 2, RP11-413M3.10, SDCCAG3, SLC34A3, 





SNAPC4, SOHLH1, SSNA1, TMEM141, TRAF2, TUBB2C, UAP1L1, UBADC1, UNQ2492, 





UNQ2541, UNQ747, hNMDAR1-3b, pp8875, ve-statin


chr18: 73989470-
60
0
AK056304, ATP9B, AX746671, BC016878, BC017654, BC040056, BX537710, C18orf22, CTDP1,


76116152


DKFZp434A042, KCNG2, NFATC1, PARD6G, PQLC1, SALL3, TXNL4A, ZNF508, hdim1+


chr9: 134048500-
54
0
ABO, ADAMTS13, ADAMTSL2, AJ011378, AK123314, AX748058, BARHL1, C9orf166, C9orf7,


135886847


C9orf9, C9orf96, C9orf98, CEL, CELL, CR592591, CR593670, DBH, DDX31, EEF1A1, FLJ46082,





GBGT1, GFI1B, GTF3C4, GTF3C5, KIAA1308, LOC389827, NR_002783, NTNG2, OBP2B,





RALGDS, REXO4, RPL7A, SARDH, SETX, SLC2A6, SURF1, SURF2, SURF4, SURF5, SURF6,





TSC1, TTF1 UNQ2513, VAV2, XPMC2H, vWF-CP


chr1: 2182293-
50
0
ACTRT2, AK021767, AK124865, ARHGEF16, BC114358, C1orf93, CR749717, DQ601700,


3539057


FAM79A, FLJ42875, HES5, MEGF6, MMEL1, MORN1, PANK4, PEX10, PLCH2, PRDM16, 





RER1, RP3-395M20.1, RP3-395M20.10, RP4-740C4.2, SKI, TNFRSF14, WDR8


chr1: 245537326-
49
0
AB120962, AK130400, BC034303, C1orf150, CIAS1, NLRP3, OR13G1, OR2B11, OR2C3, 


245976637


OR2G2, OR2G3, OR2W5, OR5AY1, OR6F1, ZNF496


chr14: 21582956-
47
0
AK093552, AK125397, AV1S3A1T, AV25S1, AV4S1, TCR-[alpha] V 33.1, TCR-alpha,


21976908


TCRA, TCRAV14.1a, TCRAVN1, TCRD, TRA@, V alpha immunoglobulin, av27s1,





hADV14S1, hADV23S1, hADV29S1, hADV38S2, hDV102S1


chr7: 157944418-
46
0
AX746826, BC041429, BC042556, FAM62B, KIAA1228, NCAPG2, PTPRN2, V1PR2, WDR60


158676623





chr19: 48498399-
46
0
CD177


48562727





chr22: 34946081-
41
0
APOL1, APOL2, AX747758, MYH9


35041308





chr1: 113185538-
40
0
AFARP1, AX748125, BC023568, BC037540, BC047723, BX648855, LRIG2, SLC16A1


113437683





chr2: 187993955-
39
0
CALCRL, CED-6, GULP1 TFPI


189286647





chr11: 382079-
38
0
AK094678, AK126635, AP2A2, AX747537, AX748330, BC031953, BC048998, BC066355, 


1386192


BRSK2, C11orf35, CD151, CEND1, CHID1, DEAF1, DKFZp434K249, DRD4, EFCAB4A, 





EPS8L2, HRAS, IRF7, KIAA0899, KIAA1542, LRDD, LRRC56, MG1, MUC5AC, MUC2,





MUC5AC, MUC5B, MUC6, MUCDHL, NR_002585, PDDC1, PEN11B, PKP3, PNPLA2, 





POLR2L, PTDSS2, RASSF7, RNH1, RPLP2, SCT, SIGIRR, SLC25A22, TALDO1, TMEM16J, 





TMEM80, TOLLIP, TSPAN4, tollip


chr12: 98815353-
37
0
ACTR6, ANKS1B, AX746635, BC048272, BC062763, DEPDC4, DKFZp434M0331, DQ579681, 


99473749


DQ583972, DQ595598, DQ598729, EB-1, GOLGA2L1, KIAA0701, NR1H4, SCYL2, SLC17A8, 





hArpX


chr4: 7568079-
36
0
ABLIM2, AFAP1, AJ431609, BC043614, KIAA1808, LOC389199, SORCS2


8105372





chr14: 23995185-
30
0
AK056368, CMA1


24062459





chr14: 103352857-
30
0
ADSS, ADSSL1, AK057986, AK094143, AKT1, AX721091, AX746996, BRF, BRF1, C14orf151,


104756910


C14orf173, C14orf2, C14orf78, C14orf79, CDCA4, CR602005, DKFZp434N0820, DKFZp434N178, 





DKFZp686J02145, GPR132, JAG2, KIAA0284, KIAA0771, KIF26A, LOC374569, LOC400258,





MGC23270, NUDT14, PLD4, PPP1R13B, SIVA1, TDRD9, TMEM179, UGPP


chr17: 73662981-
23
0
AFMID, BC036810, BIRC5, EPR-1, SYNGR2, TK1, UNQ464/PRO809, survivin-3B


73765190





chr13: 45066410-
22
0
AK095119, AK124928, C13orf18, CPB2, FLJ32682, KIAA0853, LCP1, LOC220416, LOC283514, 


45918722


RP11-139H14.4-001, RP11-351K3.2-001, SPERT, ZC3H13


chr19: 46758119-
21
0
CEACAM21, CEACAM4, UNQ3098


46863746





chr8: 6849317-
20
0
AF355799 DEFA3


6889488





chr20: 60320976-
17
0
AK056267, AK128329, AL137301, ARFGAP1, ARFRP1, AX747649, AY940852, BC002534,


62223928


BC025345, BC069708, BC127852, BHLHB4, BIRC7, C20orf11, C20orf135, C20orf149, C20orf151, 





C20orf166, C20orf195, C20orf20, C20orf58, C20orf59, CABLES2, CHRNA4, COL20A1, COL9A3, 





DIDO1, DNAJC5, EEF1A2, FLJ00084, FLJ00118, FLJ30313, GATA5, GMEB2, HRIHFB2281, 





KCNQ2, KIAA1088, KIAA1269, KIAA1510, LAMA5, LIME1, LOC198437, NPBWR2, 





NR_003244, NR_003245, NTSR1, OATP-E, OGFR, OK/SW-c1.69, OPRL1, PRIC285, PRPF6, 





PRR17, PTK6, RGS19, RP4-697K14.11, RPS21, RTEL1, SAMD10, SLC2A4RG, SLCO4A1, 





SOX18, SRMS, STMN3, Si-1-2-19, TCEA2, TCFL5, TNFRSF6B, TPD52L2, UCKL1, URKL1, 





YTHDF1, ZBTB46, ZGPAT, ZNF512B


chr2: 86924985-
16
0
CD8B, RGPD1, RMND5A


87026840





chr6: 128245227-
16
0
C6orf190, PTPRK


128538250





chr1: 111628132-
15
0
CHIA, RP11-165H20.1


111661435





chr7: 80041654-
13
0
CD36


80110504





chr12: 6085895-
11
0
VWF


6134080





chr12: 17854127-
11
0
CAPZA3, FLJ22655, PIK3C2G, PLCZ1, PLEKHA5


19338878










D) Duplications Not Impacting Known Immune Function Genes










chr8: 430424-
189
0
AK128400, BC022082, BC038783, C8orf42, DQ584928, ERICH1, LOC389607


977192





chr10: 2682656-
157
0
PFKP


3123648





chr19: 21575835-
152
0
BC030765, CR936832, FKSG70, ZNF100, ZNF208, ZNF257, ZNF43, ZNF492, ZNF676, 


22802826


ZNF99


chr6: 63118894-
116
0
GLULD1, LGS


64091007





chr2: 126935385-
98
0
GYPC


127317174





chr5: 169763949-
96
0
DKFZp666P032, GABRP, KCNIP1, RANBP17


170518986





chr6: 1977258-
92
0
AJ420566, AK023629, AK091028, CR598484, GMDS


2424512





chr1: 145196592-
65
0
BC036212, BCL9, CHD1L


145539979





chr3: 62813828-
63
0
BC043407, C3orf49, CADPS, FLJ44379, SYNPR


63789968





chr5: 172824934-
62
0
BC033564, FAM44B


173106866





chr3: 83912925-
54
0
BC068246


84849961





chr11: 106523381-
54
0
CWF19L2


106702850





chr7: 50911090-
50
0
COBL


51127086





chr9: 6557841-
41
0
AK098534, BC042976, GLDC, JMJD2C, KIAA0780


7007391





chr17: 793231-
40
0
ABR, NXN, TIMM22


917163





chr3: 148425802-
39
0
AK098763, ZIC1, ZIC4


149211699





chr13: 62536330-
36
0
AK057471, AK097490, AK098560, BC128161, NR_002171


64097876





chr4: 92669000-
34
0
GRID2, KIAA1680


94351161





chr3: 11041670-
33
0
SLC6A1


11154453





chr4: 141709869-
33
0
TBC1D9


141939520





chr13: 113032851-
30
0
ADPRHL1, GRTP1


113140550





chr2: 148947731-
29
0
KIAA1461, MBD5


149053581





chr3: 141649121-
28
0
CLSTN2, TRIM42


141911988





chr12: 20901315-
27
0
IAPP, LST-3TM12, LST3, SLCO1A2, SLCO1B1, SLCO1B3


21459060





chr12: 130676799-
27
0
EP400, KIAA1498, KIAA1818, MMP17, NR_002979, PUS1, SFRS8, ULK1


131128244





chr7: 34919485-
26
0
AJ011981, BC049371, BC084560, CR593784, CR595224, DPY19L1, DPY19L2P1, HERPUD2,


35862821


KIAA0877, SEPT7, TBX20


chr1: 30940148-
25
0
BC044253, LAPTM5, MATN1


31013899





chr4: 63433227-
25
0
SRD5A2L2


65099386





chr5: 12838251-
22
0
AY328033


13150705





chr6: 138653005-
22
0
KIAA1244


138759016





chr1: 103111860-
21
0
COL11A1


103157198





chr6: 34836231-
21
0
ANKS1A, C6orf107, SNRPC, TAF11


34976532





chr15: 27167056-
21
0
APBA2, BC043570, BC070492, BC071630, BC071855, DQ572986, DQ573498, DQ575284,


28188067


DQ575742, DQ577333, DQ578370, DQ578838, DQ582641, DQ582940, DQ590322, DQ592322, 





DQ595055, DQ596303, DQ596319, DQ597873, KIAA0574, NDNL2, TJP1, hXIIL


chr2: 3287465- 
20
0
TSSC1


3327227





chr10: 98101269-
19
0
TLL2, TMEM10


98152781





chr1: 79037280-
18
0
ELTD1


79973037





chr4: 68749536-
18
0
AK123556, TMPRSS11B, TMPRSS11E, UGT2A3, UGT2B10, UGT2B15, UGT2B17, UGT2B7,


70089270


YTHDC1


chr2: 27584444-
17
0
AK124439, BC041993, BC048132, BRE, C2orf16, CCDC121, GCKR, MRPL33, RBKS, SLC4A1AP,


28424525


SUPT7L, XAB1, ZNF512


chr2: 172356838-
17
0
HAT1, SLC25A12


172528561





chr2: 180123158-
16
0
ZNF533


180216026





chr4: 15529521-
15
0
FGFBP1


15552238





chr18: 14805527-
15
0
ANKRD30B


14905835





chr5: 1601764-
14
0
CR749689


1621442





chr10: 88722456-
14
0
AK091716, BC036645, BC047063, BC065757, BC082979, BC092519, CR609725, CR614919,


89266679


FAM35A, GLUD1, KIAA1975, KIAA2020, MINPP1


chr1: 225308870-
13
0
CDC42BPA


225407413





chr7: 74674968-
13
0
LOC441257, PMS2L14


74738702





chr1: 193808000-
12
0
KCNT2, SLICK


194531039





chr2: 165344115-
12
0
COBLL1, KIAA0977


165435694





chr6: 123847128-
12
0
TCBA1, TRDN


124726881





chr16: 26961662-
12
0
TNT


26994169





chr6: 102029392-
11
11862
GRIK2


102048325





chr8: 11397047-
11
0
BLK


11414199





chr8: 27689983-
11
0
ESCO2, PBK


27745887





chr2: 77709215-
10
0
BC024248, BC030125


78761206





chr11: 5444172-
10
11939
HBE1, HBG2, OR51B5


5454375





chr11: 19571323-
10
96377
NAV2


19595111





chr18: 68553733-
10
0
BC013370, BC034583, NETO1


69070964





chr19: 34552727-
10
0
AK094793


34574348





chr4: 2059850-
10
0
AB000464, AB000465, AB000466, ADD1, AK054619, BC010180, BC032331, C4orf10, C4orf15,


2960297


C4orf8, CR622423, GRK4, KIAA1643, MXD4, Mad4, NOL14, POLN, RNF4, SH3BP2, TETRAN,





TNIP2, ZFYVE28
















TABLE 5







Homozygous Deletions Observed Exclusive to Cases












Cases


Distance From


CNVR
Loss
Gene
Distance
Exon














chr3: 100430538-
2
CLCP1, DCBLD2
327315
327315


100430538






chr2: 118777060-
1
INSIG2
192993
192993


118778863






chr3: 146130742-
1
DQ595575
883692
883692


146141275






chr5: 29455914-
1
AK098570
247692
247692


29488308






chr5: 141998044-
1
FGF1
0
16953


142000692






chr6: 32525108-
1
HLA-DRA
4306
4306


32530999






chr6: 62122152-
1
G43499
251536
251536


62144592






chr6: 77159914-
1
IMPG1
320859
320859


77159914






chr6: 128018101-
1
C6orf190
46336
46336


128024702






chr12: 17277311-
1
LMO3, Nbla03267
625020
625020


17282429
















TABLE 6





Genome Global CNV Burden

















Significant Including Common CNV Exonic














Locus

Locus






Exonic

Exonic
Locus Non-





Significant
Locus Non-
Significant
Exonic





Case
Exonic Case
Control
Control





Enriched
Enriched
Enriched
Enriched
P
DifFreq





Deletion
21
61
8
67
0.022628
0.149431


Duplication
43
173
12
12
0.003336
−0.30093












Rare CNV Exonic















Case CNV

Control





Case CNV
Calls Non-
Control CNV
CNV Calls





Calls Exonic
Exonic
Calls Exonic
Non-Exonic
P
DifFreq





Deletion
527
1149
3970
10290
0.002138
0.036038


Duplication
813
1785
5539
5997
2.45E−55
−0.16722












Rare CNV >100 KB Exonic















Case CNV

Control





Case CNV
Calls Non-
Control CNV
CNV Calls





Calls Exonic
Exonic
Calls Exonic
Non-Exonic
P
DifFreq





Deletion
136
83
1012
653
0.768312
0.013197


Duplication
278
146
2263
546
2.46E−11
−0.14996












Samples w Rare CNV >100 KB (Impacting gene or not)















Case

Control





Case with
without
Control with
without





large rare
large rare
large rare
large rare





CNV
CNV
CNV
CNV
P
DifFreq





Deletion
131
180
1156
1610
0.951669
0.00329


Duplication
186
125
1391
1375
0.001504
0.095178









Finally, to address the heterogeneity of the CVID phenotype and comorbid clinical manifestations that affect subsets of patients, we compared CVID patients with common subphenotypes to patients without any additional comorbidities. Based on recent phenotypic characterization22, we established 16 distinct clinical subtracks within CVID to study: cancer, lymphoma, lymphadenopathy, nodular regenerative hyperplasia of the liver (NRH), lymphoid interstitial pneumonitis (LIP), bronchiectasis, biopsy proven granuloma, GI enteropathy, malabsorption, splenectomy, cytopenias, organ specific autoimmunity (OSAI), low IgM (<50 mg/dL), low IgA (<10 mg/dL), low B cells (CD19+ cells <1%), and young age of symptom onset (<10 yrs) (FIG. 5 and Table 7). The case and control labels within the CVID cohort were based on previously defined criteria. Differences in genotype frequencies were assigned significance and regions with multiple significant SNPs were then scored. Significant SNP associations were made for all subsets (Table 8 and FIG. 1C). The most significant association was observed with NRH on AK096081-AK124028 (P=2.29×10−10). Lymphoma was associated with KIAA0834, PFTK1 and HAVCR1, with corresponding P values ranging from P=1.69×10−8-3.62×10−8. An association was also observed between LIP and FGF14 and ZNF81, with corresponding P values of P=5.76×10−8 and P=7.70×10−8, respectively. OSAI also associated with SNX31 (P=6.89×10−8) and low IgM levels (<50 mg/dL) in the case subjects were associated with LDLRAP1 (P=6.02×10−8). Patients with CVID manifesting enteropathy and their resulting subphenotype significance were additionally queried against the IBD significance observations to assess if specific loci contributed to a common etiology. The SNP rs12889533 was significant in both CVID+ enteropathy and IBD with allele frequency difference in the same direction (Table 9).







TABLE 7







CVID Subphenotypes and Sample Sizes











CVID





Subphenotype
Cases
Controls















Cancer
13
249



Lymphoma
9
253



Lymphadenopathy
30
232



NRH
13
249



LIP
10
252



Bronchiectasis
78
184



Granuloma
34
228



GIEnteropathy
19
243



Malabsorption
13
249



Splenectomy
40
222



Cytopenias
27
235



OSAI
75
187



Low IgM (<50 mg/dL)
206
56



Low IgA (<10 mg/dL)
160
102



CD19 (<1%)
11
251



AgeOnsetSx (<10 yrs)
42
220

















TABLE 8







Most Significant Associated Regions Based on Subphenotype Genotype


Association




























Dis-













tance



Significant






Count

Dis-
From



Region
P-value
SNP
A1
F_A
F_U
OR
SNPs
Gene
tance
Exon
Subphenotype





















chr1: 68153970-68161019
2.29E−10
rs1926283
C
0.2308
0.01807
16.3
3
AK096081,
0
33408
NRH










AK124028





chrX: 132092989-132095451
5.50E−10
rs5977837
C
0.2778
0.02174
17.31
2
TFDP3
82912
82912
Lymphoma


chr10: 58755852-58767334
1.36E−09
rs16910534
T
0.2222
0.01383
20.37
3
IPMK
858286
858286
Lymphoma


chr20: 55901032-55985359
3.21E−09
rs8124301
T
0.4231
0.07631
8.877
5
C20orf85
174030
174030
Malabsorption


chr7: 90245135-90245280
1.69E−08
rs975004
G
0.3333
0.03953
12.15
2
KIAA0834,
0
12544
Lymphoma










PFTK1





chr5: 156402979-156407976
3.62E−08
rs10038271
T
0.7222
0.1877
11.25
2
HAVCR1
0
651
Lymphoma


chrX: 22422160-22428888
5.71E−08
rs5925651
A
0.3462
0.1386
3.291
3
ZNF645
219665
219665
Bronchiectasis


chr13: 101641990-101673539
5.76E−08
rs1336698
G
0.65
0.168
9.197
6
FGF14
0
69139
LIP


chr1: 25735669-25753653
6.02E−08
rs2065970
G
0.06553
0.2411
4.529
5
LDLRAP1
0
0
Low IgM


chrX: 143116309-143119321
6.77E−08
rs6649722
A
0.3
0.0377
10.94
3
UBE2NL
320286
320286
LIP


chr8: 101728102-101728344
6.89E−08
rs7815950
G
0.2133
0.05615
4.559
2
SNX31
0
2334
OSAI


chr8: 13834782-13842376
7.62E−08
rs2682665
C
0.1818
0.01394
15.71
3
SGCZ
149368
149368
Low B cells


chrX: 47642545-47651320
7.70E−08
rs12387999
A
0.2
0.016
15.38
2
ZNF81
0
2262
LIP


chrX: 152295068-152303019
9.84E−08
rs5987017
A
0.2143
0.04773
5.442
4
ZNF275
24829
24829
Young age


chr12: 2531014-2538733
1.21E−07
rs4765961
C
0.4737
0.142
5.439
3
CACNA1C
0
0
GI Enteropathy


chrX: 30733771-30736604
1.44E−07
rs11095197
T
0.1469
0.3431
3.034
2
MAP3K7IP3
18876
18876
Low IgA


chrX: 37803525-37832251
1.54E−07
rs5918500
C
0.4878
0.2123
3.533
3
SYTL5
0
0
Young age


chrX: 53035117-53039801
1.90E−07
rs10127016
G
0.3833
0.125
4.351
2
TMEM29
0
1409
Lymphade-













nopathy


chr7: 107306707-107314113
2.16E−07
rs9690688
A
0.3636
0.06375
8.393
2
DLD
4709
4709
Low B cells


chr21: 18188480-18192815
2.84E−07
rs7280675
G
0.1923
0.02008
11.62
2
CHODL
2636
2636
NRH


chr1: 55704352-55706323
2.85E−07
rs356086
G
0.1111
0.00395
31.5
2
FLJ45337
112975
112975
Lymphoma


chr21: 45476504-45476918
3.23E−07
rs4592938
A
0.1154
0.00602
21.52
2
C21orf89
1777
1777
NRH


chr14: 23168471-23169215
4.57E−07
rs222723
C
0.6154
0.1968
6.531
2
DHRS2
0
0
Cancer


chr2: 109118795-109120561
4.62E−07
rs375099
C
0.25
0.02976
10.87
2
POSH2
0
5794
LIP


chr12: 30583074-30598457
4.82E−07
rs1905675
C
0.2
0.5453
0.209
2
IPO8
74732
74732
Lymphade-













nopathy


chrX: 39933689-39956544
5.18E−07
rs2948491
A
0.18
0.04545
4.61
4
BCOR
12163
12163
OSAI


chr14: 64723761-64783067
8.09E−07
rs4299072
A
0.1667
0.02802
6.938
4
BX161428
0
0
Lymphade-













nopathy


chr7: 117377201-117382435
9.94E−07
rs17140937
C
0.2692
0.04418
7.971
2
CTTNBP2
76404
76404
Malabsorption





OR: Odds Ratio Additional detail in Table 13.













TABLE 9







CVID Patients with GI Enteropathy vs CVID Patients without Genotype


Association Replication in IBD Based on Single SNP Significance






















P_CVID +













Dis-
GIEnteropathy











SNP
Gene
tance
(IBD)
Allele1
F_A
F_U
OR
P_IBD
Allele1
F_A
F_U
OR






















rs6532122
GPRIN3
3497
0.001204
A
0.2632
0.0947
3.42
9.90E−8
A
0.0786
0.1072
0.7109


rs17041264
COLQ
196
0.000368
T
0.1579
0.0350
5.17
3.15E−6
T
0.0049
0.0135
0.3608


rs12889533
C14orf143
19903
0.000561
T
0.1316
0.4156
0.21
1.28E−4
T
0.3563
0.3878
0.8736





rs12889533 replicates with allele frequency in the same direction while rs6532122 and rs17041264 are significant in the opposite direction of allele frequency.






Finally, to identify potential functional biases specific to CVID, we evaluated clustering into specific INTERPRO categories using DAVID23: Database for Annotation, Visualization, and Integrated Discovery. We found two significant INTERPRO categories with genes contributing from several of our different analysis methods. First, “Interleukin 1/heparin-binding growth factor” P=9.60×10−3 including FGF1, FGF23, FGF6, and FGF14 from subphenotype analysis of lymphadenopathy, cytopenias, LIP, and low IgA. Secondly, “Immunoglobulin I-set” P=5.40×10−2 including CHL1, CNTN4, LINGO2, SDK1 from overall GWAS analysis, cytopenias subphenotype, and duplication CNV.
Discussion
CVID as a clinical phenotype was described more than 50 years ago, but aside from a small number of recessively inherited genes in a few families, and the more prevalent but poorly understood contribution of mutations in TNFRSF13B5,6,7,24, other causes have remained obscure. CVID has thus been hypothesized to represent a diverse collection of genetic lesions resulting in a similar immunologic phehotype. The MHC region has been associated with a myriad of complex diseases25 including immune-related conditions26 and CVID8,9. MHC was robustly associated with this CVID in our study, confirming the prior work that was not performed at the genome-wide level. However, the SNP genotype association analysis presented here also revealed novel genes, with the most significant CVID associations outside of the MHC region with a locus encompassing ADAM28, ADAM7, ADAMDEC1 and STC1 (P=6.24×10−6). The ADAM family proteins are zinc metalloproteases involved in diverse biologic processes, including immune responses. Interestingly, the metallopeptidase MMP27 was also associated with CVID (P=1.69×10−4). Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix to promote routine physiological processes, but may also facilitate disease pathogenesis. Related genes have demonstrated immunological function involved in the regulation of cytokine release, Th2 immune responses and specific inflammatory processes. ADAM28 is also known as the lymphocyte metalloprotease MDC-L, expressed on the lymphocyte cell surface27. As such, it has been defined as a ligand for α4β1 integrin which enables the adhesion of other leukocytes expressing this integrin. STC1 in the same region, is a gene that encodes stanniocalcin 1 protein, involved in regulation of calcium including in antioxidant pathways of marcophages28. In this light, calcium regulation within immune cells has been previously identified as aberrant in certain CVID patient samples29. UBXN10 may be a compelling candidate potentially involved in immunopathogenesis of primary antibody failure in that it encodes an Ubiquitin-like protein, which in addition to phosphorylation, has been shown to regulate NF-κB activity30. SDK1 (Sphingosine-dependent protein kinase-1) is important in the survival of alveolar macrophages31. DEPDC6 is a negative regulator of mTORC signaling pathways and RNA expression levels were found to be significantly different between those mice resistant to H5N1 influenza virus when compared to those that were susceptible32. No significant associations in the region of TACI were observed, and subjects with TACI mutations were not separately identified. We reviewed the reported amino acid changes and looked up their corresponding non-synonymous SNP IDs (Table 10). No imputation reference files including these SNPs were available to infer genotypes.







TABLE 10







TACI Protein TNFRSF13B Gene Amino Acid Variant and


Corresponding nsSNP rs ID















Clinical 






Report



DNA


(bold-in


nsSNP
position
Alleles
AA Change
reports)















rs34562254
16783716
C/T
P (CCC) → 
L (CTC)
P251L


rs72553886
16783732
G/T
V (GTC) → 
F (TTC)
V246F


rs56063729
16783809
A/G
V (GTG) → 
A (GCG)
V220A


rs56248318
16784408
A/C
Q (CAG) → 
H (CAT)
Q196H


rs72553885
16784417
A/C
C (TGC) → 
* (TGA)
C193X


rs72553883
16784454
A/C
A (GCG) → 
E (GAG)
A181E


rs72553882
16784504
A/C
Y (TAC) → 
* (TAA)
Y164X


rs72553881
16784541
A/G
G (GGG) → 
E (GAG)
G152E


rs72553880
16792777
A/G
A (GCT) → 
T (ACT)
A149T


rs72553879
16792911
A/G
C (TGT) → 
Y (TAT)
C104Y


rs34557412
16792912
C/T
C (TGT) → 
R (CGT)
C104R


rs72553877
16792962
A/T
I (ATC) → 
N (AAC)
I87N


rs72553876
16792986
A/G
Y (TAT) → 
C (TGT)
Y79C


rs55916807
16793007
C/T
R (CGC) → 
H (CAC)
R72H


rs67951770
16796563
C/G
D (GAT) → 
H (CAT)
D41H











rs67951769
16796563
—/G
frameshift
D41fx












rs72553874
16796566
C/T
W (TGG) →
R (CGG)
W40R











rs72553884
16784425-
—/G
Frameshift
D191G



16784424





rs72553878
16792925-
—/T
Frameshift
Q99H



16792924





rs34182967
16793004-
—/C
Frameshift
K73R



16793003





rs72553875
16793018-
—/A
Frameshift
S68S



16793017
















TABLE 11





Most Significant Associated Regions Based on Genotype Association


























Count






P




SNPs
Distance





Republication


Region

Top
From





(Discovery


Genomic Span
Gene
1000
Exon
BestP
BestSNP
Allele1
F_A
F_U
SNP)





chr6: 27236785-32521295
MHC
110
0
8.62E−10
rs3117426
T
0.3268
0.1907
0.3634


chr8: 23746576-24681608
ADAM28,
4
0
6.24E−6
rs4872262
A
0.0391
0.0107
0.03135



ADAM7, ADAMDEC1,











STC1










chr4: 189671181-189676119
AK095968
2
18764
1.78E−5
rs1606234
T
0.2961
0.4124
0.4646


chr10: 73083830-73084583
CDH23
2
7287
2.45E−5
rs7087554
A
0.5894
0.4729
0.4591


chr7: 4270929-4287047
SDK1
5
0
4.70E−5
rs895710
T
0.2905
0.4003
0.4551


chr1: 20369369-20490791
FLJ32784,
7
0
9.09E−5
rs7514144
C
0.2654
0.1808
0.3018



UBXN10










chr10: 27709830-27728115
PTCHD3
7
0
9.77E−5
rs506659
T
0.4022
0.3026
0.9879


chr1: 59987116-59993733
FGGY
2
2458
1.52E−4
rs11207520
G
0.3994
0.3026
0.00148


chr12: 80435676-80444630
PPFIA2
2
59900
1.63E−4
rs2400955
T
0.4134
0.3159
0.8687


chr11: 102069786-102079454
MMP27
2
0
1.69E−4
rs17099394
T
0.1229
0.0687
0.6947


chr16: 71592439-71594134
ZFHX3
2
40845
1.80E−4
rs8056528
C
0.3687
0.2754
0.3698


chr8: 124729628-124734756
KLHL38
4
0
2.05E−4
rs4871402
A
0.0643
0.1325
0.7201


chr3: 7364204-7369287
GRM7
4
40865
2.96E−4
rs12491592
A
0.1536
0.0939
0.5051


chr3: 8378739-8381919
BC020876
2
129448
3.51E−4
rs359030
C
0.1648
0.1033
0.1446


chr6: 25570803-25574868
LRRC16A
2
0
3.61E−4
rs4320355
T
0.3799
0.2898
0.2219


chr6: 153354039-153355207
MTRF1L
2
0
3.81E−4
rs9322400
T
0.4469
0.3526
0.454


chr3: 284363-285747
CHL1
2
19083
4.31E−4
rs17273893
T
0.148
0.0908
0.6342


chr8: 121001793-121010709
DEPDC6
2
0
4.41E−4
rs869340
C
0.324
0.2402
0.7402


chr2: 65518304-65520560
FLJ16124
2
784
5.20E−4
rs1194849
G
0.3659
0.4614
0.01381


chr21: 27713547-27722878
BC043580
2
19690
5.51E−4
rs469709
A
0.1704
0.1095
0.9615



















F_A
F_U








Republication
Republication







Region
(Discovery
(Discovery
BestP
BestSNP
F_A
F_U



Genomic Span
SNP)
SNP)
Replication
Replication
Replication
Replication






chr6: 27236785-32521295
0.1697
0.1468
0.0004
rs2156875
0.5734
0.4475



chr8: 23746576-24681608
0.0321
0.01346
0.0314
rs4872262
0.0321
0.0135



chr4: 189671181-189676119
0.4266
0.4524
0.4646
rs1606234
0.4266
0.4524



chr10: 73083830-73084583
0.5092
0.4829
0.4591
rs7087554
0.5092
0.4829



chr7: 4270929-4287047
0.3578
0.3835
0.0235
rs4720301
0.3991
0.4793



chr1: 20369369-20490791
0.1560
0.1311
2.25E−8
rs6426636
0.4679
0.2857



chr10: 27709830-27728115
0.2798
0.2793
0.6291
rs493965
0.2844
0.3001



chr1: 59987116-59993733
0.2569
0.1706
0.0015
rs11207520
0.2569
0.1706



chr12: 80435676-80444630
0.3899
0.3842
0.5347
rs10746192
0.4306
0.4526



chr11: 102069786-102079454
0.0734
0.06643
0.6947
rs17099394
0.0734
0.0664



chr16: 71592439-71594134
0.3670
0.3368
0.3698
rs8056528
0.367
0.3368



chr8: 124729628-124734756
0.1009
0.1088
0.3161
rs7463896
0.1697
0.1979



chr3: 7364204-7369287
0.0963
0.08318
0.1237
rs7617297
0.3073
0.2592



chr3: 8378739-8381919
0.1147
0.08535
0.1139
rs358994
0.1055
0.0754



chr6: 25570803-25574868
0.2844
0.2469
0.1896
rs301396
0.445
0.3993



chr6: 153354039-153355207
0.3945
0.3688
0.454
rs9322400
0.3945
0.3688



chr3: 284363-285747
0.0963
0.08678
0.6342
rs17273893
0.0963
0.0868



chr8: 121001793-121010709
0.3073
0.2966
0.682
rs4871793
0.3113
0.2978



chr2: 65518304-65520560
0.3899
0.4771
0.0119
rs1876518
0.3853
0.4744



chr21: 27713547-27722878
0.0963
0.09532
0.6677
rs17631106
0.0688
0.0769





Bold underline: Known immunological function Gene













TABLE 12





Most Significant Associated Regions Based on CNV Association







A) Deletions



















Distance











Count
From
P
Cases
Controls







CNVR Deletion
SNPs
Exon
Deletion
Del
Del
Gene
MSSM
Oxford
USF
Combined





chr11: 85365857-85381622
5
0
0.01
2
0
PICALM, PICALM variant
0
2
0
2








protein






chr20: 57735790-57741780
6
5902
0.026
3
4
PHACTR3
2
1
0
3


chr22: 17396663-18417315
270
0
0.029
2
1
ARVCF, C22orf25,
2
0
0
2








C22orf29, CDC45L,












CLDN5, CLTCL1,












COMT, CR618542,












CR625276, DGCR14,












DGCR2, DKFZp761P1121,












DKFZp781E0833,












GNB1L, GP1BB, GSCL,












HIRA, KIAA1647,












L77561, LOC128977,












MRPL40, SEPT5,












SLC25A1, TBX1,












TRXR2A, TSSK2,












TXNRD2, U84523,












UFD1, UFD1L






chr4: 10256682-10264316
4
2915
0.029
2
1
CLNK
0
1
1
2


chr10: 46003146-46042543
8
0
0.029
2
1
DKFZp566K0524,
2
0
0
2








PTPN20A, PTPN20B










B) Duplications



















Distance










CNVR
Count
From

Cases
Controls







Duplication
SNPs
Exon
P Dup
Dup
Dup
Gene
MSSM
Oxford
USF
Combined





chr2: 148396730-148433180
6
0
8.66E−16
15
0
ACVR2A, ORC4L
10
5
0
15


chr15: 35053039-35063531
6
23146
5.72E−13
19
10
MEIS2
17
2
0
19


chr10: 53044735-53045426
4
79826
9.63E−13
12
0
PRKG1
12
0
0
12


chr8: 77719805-77720240
3
27377
1.05E−09
10
1
BC037827
10
0
0
10


chr7: 91789778-91801963
4
0
1.20E−09
12
4
ANKIB1
9
3
0
3


chr2: 163316298-163316595
3
17100
1.00E−07
7
0
KCNH7
6
1
0
7


chr7: 87180702-87191931
4
202
7.41E−07
7
1
RPIB9
5
2
0
7


chr12: 72825720-72832667
3
0
3.04E−06
7
2
BC061638
7
0
0
7


chr2: 203004035-203017311
5
20466
6.58E−05
6
3
BMPR2
6
0
0
6


chr19: 9256584-9277749
4
0
0.001
3
0
ZNF699
2
0
1
0


chr4: 39190766-39201960
6
0
0.004
3
1
UGDH
1
2
0
3


chr9: 112207473-112207708
3
553
0.004
3
1
SVEP1
3
0
0
3


chr7: 18903915-18905725
4
23171
0.009
3
2
HDAC9
1
2
0
3


chr13: 64053258-64056530
15
505114
0.01
2
0
AK057471, AK098560,
2
0
0
2








BC128161






chr14: 35408995-35422074
5
0
0.01
2
0
BRMS1L
0
2
0
2


chr5: 170531269-170536993
4
374
0.01
2
0
DKFZp666P032,
1
1
0
2








RANBP17






chr4: 91413829-91415037
3
33381
0.01
2
0
KIAA1680, MGC48628
2
0
0
2


chr7: 34685030-34686172
4
4517
0.01
2
0
AAA1, NPSR1
1
1
0
2


chr3: 176427638-176429297
7
5118
0.01
2
0
NAALADL2
2
0
0
2


chr1: 170400944-170403742
4
26274
0.01
2
0
DNM3
0
1
1
2


chr2: 167031393-167045981
4
0
0.01
2
0
Na+ channel, SCN7A
2
0
0
2


chr9: 28683005-28687679
3
21420
0.01
2
0
LINGO2
2
0
0
2


chr9: 105860023-105868928
9
27434
0.01
2
0
OR13C3, OR13C4,
2
0
0
2








OR13C5, OR13C8,












OR13F1, SMC2, hCAP-E
















TABLE 13







Most Significant Associated Regions Based on Subphenotype Genotype


Association



























Distance









Count


From


Significant Region
P-value
SNP
A1
F_A
F_U
OR
SNPs
Gene
Distance
Exon










A) Cancer

















chr14: 23168471-23169215
4.57E−7
rs222723
C
0.6154
0.1968
6.531
2
DHRS2
0
0


chr6: 140225308-140225386
1.53E−6
rs12111348
T
0.4231
0.1064
6.157
2
BC039503
2009
2009







B) Lymphoma

















chrX: 132092989-132095451
5.50E−10
rs5977837
C
0.2778
0.02174
17.31
2
TFDP3
82912
82912


chr10: 58755852-58767334
1.36E−9
rs16910534
T
0.2222
0.01383
20.37
3
IPMK
858286
858286


chr7: 90245135-90245280
1.69E−8
rs975004
G
0.3333
0.03953
12.15
2
KIAA0834, PFTK1
0
12544


chr5: 156402979-156407976
3.62E−8
rs10038271
T
0.7222
0.1877
11.25
2
HAVCR1
0
651


chr1: 55704352-55706323
2.85E−7
rs356086
G
0.1111
0.003953
31.5
2
FLJ45337
112975
112975


chr1: 106430611-106438079
1.55E−6
rs11184786
T
0.3333
0.05336
8.87
2
BC043293
467531
467531







C) Lymphadenopathy

















chrX: 53035117-53039801
1.90E−7
rs10127016
G
0.3833
0.125
4.351
2
TMEM29
0
1409


chr12: 30583074-30598457
4.82E−7
rs1905675
C
0.2
0.5453
0.2085
2
IPO8
74732
74732


chr14: 64723761-64783067
8.09E−7
rs4299072
A
0.1667
0.02802
6.938
4
BX161428
0
0


chr5: 141916656-141926115
1.13E−6
rs17706715
A
0.2333
0.05603
5.127
2
FGF1
27191
27191


chr11: 101265604-101266102
1.55E−6
rs17097290
T
0.15
0.02371
7.267
2
ANGPTL5
513
513







D) Nodular regenerative hyperplasia of the liver (NRH)

















chr1: 68153970-68161019
2.29E−10
rs1926283
C
0.2308
0.01807
16.3
3
AK096081,
0
33408










AK124028




chr21: 18188480-18192815
2.84E−7
rs7280675
G
0.1923
0.02008
11.62
2
CHODL
2636
2636


chr21: 45476504-45476918
3.23E−7
rs4592938
A
0.1154
0.006024
21.52
2
C21orf89
1777
1777


chr8: 143046590-143055893
1.18E−6
rs12676273
G
0.3462
0.07229
6.794
3
TSNARE1
235455
235455


chrX: 27453340-27462759
1.29E−6
rs5971431
C
0.3077
0.05823
7.188
3
AK057304
55692
55692







E) LIP

















chr13: 101641990-101673539
5.76E−8
rs1336698
G
0.65
0.168
9.197
6
FGF14
0
69139


chrX: 143116309-143119321
6.77E−8
rs6649722
A
0.3
0.0377
10.94
3
UBE2NL
320286
320286


chrX: 47642545-47651320
7.70E−8
rs12387999
A
0.2
0.016
15.38
2
ZNF81
0
2262


chr2: 109118795-109120561
4.62E−7
rs375099
C
0.25
0.02976
10.87
2
POSH2
0
5794


chrX: 30122360-30125674
2.52E−6
rs5927496
C
0.7
0.2341
7.633
2
MAGEB2
17927
17927







F) Bronchiectasis

















chrX: 22422160-22428888
5.71E−8
rs5925651
A
0.3462
0.1386
3.291
3
ZNF645
219665
219665


chr2: 200959722-201175137
2.39E−6
rs13019534
G
0.5064
0.2908
2.503
2
AOX1, DNAPTP6,
0
0










KCTD18, SGOL2




chr8: 15782386-15788801
2.92E−6
rs1563297
A
0.5321
0.3152
2.47
2
TUSC3
116020
116020


chr19: 51765478-51768118
6.30E−6
rs6509286
C
0.4167
0.2228
2.491
2
AK094504
0
27862


chrX: 68569536-68573727
1.04E−5
rs7056340
A
0.3141
0.1467
2.663
2
TMEM28
68076
68076


chr6: 32508322-32510683
1.12E−5
rs2027856
T
0.02564
0.163
0.1351
2
HLA-DRA
4942
4942







G) Granuloma

















chr11: 12182328-12186013
4.83E−6
rs16910765
T
0.2206
0.05921
4.497
3
KIAA0750, MICAL2,
0
0










MICAL2












PV1, MICAL2PV2




chr1: 33674005-33683227
1.26E−5
rs12751162
A
0.3824
0.1601
3.248
2
PHC2
4803
4803


chr6: 88453628-88454474
2.04E−5
rs2250276
G
0.1765
0.04386
4.671
2
AKIRIN2
0
5428


chrX: 27453340-27462759
3.18E−5
rs5971431
C
0.1912
0.05263
4.255
2
AK057304
55692
55692







H) GI Enteropathy

















chr12: 2531014-2538733
1.21E−7
rs4765961
C
0.4737
0.142
5.439
3
CACNA1C
0
0


chr10: 26643326-26646318
2.90E−6
rs7903552
G
0.3684
0.107
4.869
2
GAD2
9833
9833


chr5: 11592622-11597026
6.26E−6
rs2727602
T
0.6053
0.2613
4.334
2
CTNND2
0
21030


chr21: 42551877-42552623
9.95E−6
rs3787986
T
0.3684
0.1152
4.479
4
ABCG1
0
0


chr9: 4033590-4039110
1.13E−5
rs676472
C
0.2895
0.07613
4.944
3
GLIS3
0
68391


chr4: 33014145-33017023
1.43E−5
rs6846113
A
0.1053
0.01029
11.32
2
AK093205
552926
552926







I) Malabsorption

















chr20: 55901032-55985359
3.21E−9
rs8124301
T
0.4231
0.07631
8.877
5
C20orf85
174030
174030


chr7: 117377201-117382435
9.94E−7
rs17140937
C
0.2692
0.04418
7.971
2
CTTNBP2
76404
76404


chr11: 124071934-124079438
1.25E−6
rs1784539
G
0.6154
0.2068
6.136
2
SPA17
2037
2037


chr10: 30255454-30255817
5.18E−6
rs11007812
T
0.3077
0.06426
6.472
2
CR626438
85918
85918


chr12: 31276546-31281817
6.76E−6
rs12819069
C
0.3846
0.09839
5.727
2
OVOS2
26191
26191


chr8: 6374232-6376048
8.56E−6
rs2515477
T
0.4231
0.1185
5.456
2
ANGPT2, MCPH1
0
1213







J) Splenectomy

















chr9: 130092787-130095025
1.03E−6
rs7026795
A
0.425
0.1802
3.363
2
C9orf119
1698
1698


chr3: 72044083-72052222
1.11E−6
rs7648163
C
0.55
0.2748
3.226
2
AK097190
115138
115138


chr1: 73821234-73888127
2.23E−6
rs4606267
G
0.325
0.1194
3.552
3
BC041341
244086
244086


chr9: 70762083-70815910
2.46E−6
rs2993008
T
0.225
0.06306
4.313
2
AK057188, PIP5K1B
0
0


chr8: 137069094-137070609
3.13E−6
rs6985828
C
0.0625
0.002252
29.53
2
KHDRBS3
340064
340064


chr15: 66415767-66419741
3.78E−6
rs6494736
C
0.175
0.04054
5.02
2
ITGA11
0
0


chr7: 141314419-141314653
2.60E−5
rs11761774
A
0.475
0.2455
2.781
2
TAS2R38
4247
4247







K) Cytopenias

















chr12: 3711033-4573405
4.28E−6
rs241964
C
0.6111
0.3
3.667
6
C12orf4, C12orf5,
0
0










CCND2, DYRK4,












EFCAB4B,












FGF23, FGF6,












PARP11, RAD51AP1




chr8: 2721609-2723174
3.52E−5
rs341672
C
0.2593
0.0812
3.961
2
CSMD1, KIAA1890
59615
59615


chr3: 2628577-2629597
0.000337
rs1020997
G
0.2407
0.4979
0.3198
2
CNTN4
0
40335


chr2: 1783619-1783961
0.000426
rs6548056
G
0.1852
0.434
0.2963
2
MYT1L
0
514


chr11: 2240035-2241166
0.000533
rs17659078
A
0.4444
0.2286
2.699
2
ASCL2
5138
5138


chr10: 1236883-1237022
0.000603
rs10794730
T
0.4815
0.2596
2.649
2
ADARB2
0
583


chr9: 1588885-1595735
0.000866
rs1923928
T
0.1852
0.4191
0.315
2
SMARCA2
409607
409607


chr1: 4222578-4225577
0.000884
rs966321
C
0.2593
0.4979
0.353
2
AX748168
146394
146394







L) organ specific autoimmunity (OSAI)

















chr8: 101728102-101728344
6.89E−8
rs7815950
G
0.2133
0.05615
4.559
2
SNX31
0
2334


chrX: 39933689-39956544
5.18E−7
rs2948491
A
0.18
0.04545
4.61
4
BCOR
12163
12163


chr3: 109180117-109186346
6.27E−6
rs709477
A
0.2733
0.4893
0.3926
2
BC101231
50156
50156


chr1: 37438130-37446852
6.96E−6
rs6426015
A
0.2667
0.1096
2.953
3
GRIK3
165699
165699


chr15: 84701881-84720707
1.19E−5
rs1431234
C
0.3
0.5108
0.4105
2
AGBL1
0
20894







M) Low IgM (<50 mg/dL)

















chr1: 25735669-25753653
6.02E−8
rs2065970
G
0.06553
0.2411
4.529
5
LDLRAP1
0
0


chr4: 59393803-59398745
1.77E−5
rs2899130
A
0.2257
0.4286
2.573
2
BC034799
1366894
1366894


chr5: 76127981-76128963
2.17E−5
rs615986
T
0.4927
0.2679
0.3767
2
F2RL1
21647
21647







N) Low IgA (<10 mg/dL)

















chrX: 30733771-30736604
1.44E−7
rs11095197
T
0.1469
0.3431
3.034
2
MAP3K7IP3
18876
18876


chrX: 145696682-145751200
3.48E−6
rs6626815
C
0.5656
0.3578
0.4279
5
CXorf1
977620
977620


chr15: 47024768-47025722
6.75E−6
rs17469978
C
0.2313
0.4167
2.375
2
SHC4
0
16198


chrX: 32356174-32388539
1.25E−5
rs699457
G
0.2656
0.1078
0.3342
2
DMD
0
0


chr1: 3915418-3917931
2.69E−5
rs10737395
A
0.1
0.2353
2.769
2
AK124708
236
236


chr13: 101257602-101258342
3.22E−5
rs1322702
A
0.04375
0.1471
3.768
2
FGF14
0
60734







O) Low B cells (CD19+ cells <1%)

















chr8: 13834782-13842376
7.62E−8
rs2682665
C
0.1818
0.01394
15.71
3
SGCZ
149368
149368


chr7: 107306707-107314113
2.16E−7
rs9690688
A
0.3636
0.06375
8.393
2
DLD
4709
4709


chr9: 33103066-33103970
1.40E−6
rs12379501
T
0.3182
0.05578
7.9
2
B4GALT1
0
0


chr13: 37798435-37846851
2.25E−6
rs4943583
G
0.2273
0.02988
9.549
7
UFM1
0
0







P) Young age of symptom onset (<10 yrs)

















chrX: 152295068-152303019
9.84E−8
rs5987017
A
0.2143
0.04773
5.442
4
ZNF275
24829
24829


chrX: 37803525-37832251
1.54E−7
rs5918500
C
0.4878
0.2123
3.533
3
SYTL5
0
0


chr19: 7505735-7508421
2.31E−6
rs604959
C
0.4762
0.2273
3.091
2
PNPLA6
0
0









The CNV association analysis also uncovered novel genes associated with either the immunopathogenesis of antibody deficiency or the development of specific complications of CVIDs. In fact, 84 CNV deletions and 98 duplications were identified in one or more CVID patients but were not found in any controls. Most were intra-exonic and thus likely to exert impact. Some of the genes potentially affected by the identified CNV were also discovered in the GWAS part of this study. Many others have direct or potential relevance to the immune system and many were unique to individual patients, thus underscoring the great mechanistic diversity that is likely to underlie this collection of disorders, also reflected in the variability in clinical presentation and disease natural history1. Among those, we noted a highly significant number of subjects with duplications in ORC4L, a gene previously associated with B-Cell lymphoproliferative disorders33. This gene is essential for initiation of DNA replication, and potentially in rapidly proliferating immune cells.
We then performed a SNP genotype association to the particular features of CVID to define potential common mechanistic threads amongst the specific clinical and immumological variants to enable the prediction of CVID clinical phenotypes. Significant individual associations were made with all CVID variables studied (Table 8) including cancer (1), lymphoma (5), lymphadenopathy (3), NRH (3), LIP (4), bronchiectasis (1), granulomatous disease (0), enteropathy (1), malabsorption (2), splenomegaly (0), cytopenias (0), autoimmunity (2), low IgM (1), low IgA (1), low B cells (2), and early age of onset (2). In this regard, PFTK1 is a member of the CDC2-related protein kinase family found constitutively expressed at high levels in B cell lymphomas34, and also found associated with lymphoma in the studied subjects with this complication. Interestingly, HAVCR1, allele frequency also found enriched in the same subjects, plays a role in Th cell development and the regulation of asthma and allergic diseases35. FGF14, associated with LIP and low IgA, is a member of the fibroblast growth factor (FGF) family, which has crucial roles in embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion36. SNX31, associated with organ-specific autoimmunity, is a sorting nexin which may be involved in protein trafficking; SNX family proteins are subunits required for CD28 mediated T cell costimulation37. LDLRAP1, associated with lower serum IgM, encodes a cytosolic protein which contains a phosphotyrosine binding (PTD) domain which interacts with the cytoplasmic tail of the LDL receptor, and is associated with hypercholesterolaemia. In addition to the potential of these findings to provide mechanistic insight into how these subphenotypes arise, they may also allow for the prediction of associated comorbidity at the time of diagnosis. This has the potential to greatly improve the clinical management of the CVIDs.
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    5. Salzer U et al. Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat. Genet. 2005 August; 37(8):820-8.
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    12. van Zelm M C et al. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency. J Clin Invest. 2010 April; 120(4):1265-74. doi: 10.1172/JCI39748. Epub 2010.
    13. van Zelm M C et al. An antibody-deficiency syndrome due to mutations in the CD19 gene. N Engl J. Med. 2006 May 4; 354(18):1901-12.
    14. Kanegane H et al. Novel mutations in a Japanese patient with CD19 deficiency. Genes and Immunity (2007) 8, 663-670; doi:10.1038/sj.gene.6364431; published online 20 Sep. 2007.
    15. Kuijpers T W, et al. J Clin Invest. 2010 January; 120(1):214-22. Epub 2009 Dec. 21.CD20 deficiency in humans results in impaired T cell-independent antibody responses.
    16. Conley M E, Notarangelo L D, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol. 1999 December; 93(3):190-7.
    17. Price A L et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat. Genet. 2006 August; 38(8):904-9. Epub 2006 Jul. 23.
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    24. Zhang L et al. Transmembrane activator and calcium-modulating cyclophilin ligand interactor mutations in common variable immunodeficiency: clinical and immunologic outcomes in heterozygotes. J Allergy Clin Immunol. 2007 November; 120(5):1178-85.
    25. Shiina T, Inoko H, Kulski J K. An update of the HLA genomic region, loci information and disease associations. Tissue Antigens 2004, 64:631-649.
    26. International MHC and Autoimmunity Genetics Network et al. Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases. Proc Natl Acad Sci USA. 2009 Nov. 3; 106(44):18680-5.
    27. Bridges L C et al. The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1. J Biol. Chem. 2002 Feb. 1; 277(5):3784-92. Epub 2001 Nov. 27.
    28. Sheikh-Hamad D. Mammalian stanniocalcin-1 activates mitochondrial antioxidant pathways: new paradigms for regulation of macrophages and endothelium. Am J Physiol Renal Physiol. 2010 February; 298(2):F248-54. Epub 2009 Aug. 5.
    29. Foerster C. et al. B Cell Receptor-Mediated Calcium Signaling Is Impaired in B Lymphocytes of Type Ia Patients with Common Variable Immunodeficiency. J. Immunol. 2010.
    30. Karin M, Ben-Neriah Y. Phosphorylation Meets Ubiquitination: The Control of NF-κB Activity. Annual Review of Immunology Vol. 18: 621-663 (April 2000).
    31. Monick M M et al. Cooperative pro survival activity by ERK and Ala in human alveolar macrophages is dependent on high levels of acid ceramidase activity. J. Immunol. 2004 Jul. 1; 173(1):123-35.
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    36. Broadley K N et al. Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair. Lab Invest. 1989 November; 61(5):571-5.
    37. Badour K et al. Interaction of the Wiskott-Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells. Proc Natl Acad Sci USA. 2007 Jan. 30; 104(5):1593-8. Epub 2007 Jan. 22.
    38. Bonilla F A et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005 May; 94(5 Suppl 1):S1-63.
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Example 2
Prediction of CVID Status and Development of Complications Based on a Targeted SNP Panels
A Support Vector Machine (SVM) is a decision-based prediction algorithm which can classify data into several groups. SVM is based on training data mapped to a higher dimensional space and separated by a plane defining the two classes of data, followed by a testing stage where status is predicted based on the model and compared to known status to test the accuracy (V. Vapnik. The Nature of Statistical Learning Theory. Springer-Verlag, New York, N.Y., 1995). The implementation of SVM used is LIBSVM software (Chih-Chung Chang and Chih-Jen Lin, LIBSVM: a library for support vector machines, 2001. Software available at http://www.csie.ntu.edu.tw/ ̃cjlin/libsvm; C. W. Hsu, C. C. Chang, C. J. Lin. A practical guide to support vector classification, 2003). Genome-wide SNP microarrays provide 610,000 genotypes which tag the genome in an unbiased way without need for prior hypothesis concerning candidate genes. Classical genome-wide association methods evaluate single SNP significance for difference in allele frequency. Although successful, the reality that many common complex diseases are polygenic in etiology with epistasis of multiple gene combinations makes an integrative model utilizing more of the data content more incisive. The application of SVM to genome-wide SNP data provides a robust framework for clinical disease prediction to evaluate risk and enact preventative measures, as we have demonstrated previously for type 1 diabetes (Wei Z, et al., From disease association to risk assessment: an optimistic view from genome-wide association studies on type 1 diabetes. PLoS Genet. 2009 October; 5(10):e1000678. Epub 2009 Oct. 9).
There are two components to the data: the target value which is the classification of the subject and the corresponding attributes which are more granular characteristics. In the application to CVID, we sought to predict target Values (Class Labels): CVID (yes/no), Bronchiectasis (yes/no), and OSAI (yes/no). This prediction is based on the attributes (features or observed variables): Genotypes for the top 1,000 SNPs previously identified from case:control genome-wide association (top 0.16% of original data set and all P<0.0015).
Phenotype labels were scaled to +1 for affected and −1 for unaffected. Genotype data is scaled with AA=0, AB=0.5 and BB=1. Data is mapped to high dimensional space by the LIBSVM algorithm to differentiate populations. Iterations of a coarse grid are followed by finer grids to avoid excessive time expenditure of complete grid search. The SVM is based principally on two variables: cost C (the penalty parameter of SVMs) and γ=1/number of variables. The SVM type in our application was C-SVM classification which runs through iterations of minimizing error functions while maintaining a hyperplane with maximum margin to avoid overfitting (FIG. 6). The kernal function maps attribute data into a higher dimensional space to improve distinguishing characteristics. The kernel type in this application was a radial basis function (RBF) which is based on distance from an origin. Once the SVM model is established through training and validated for accuracy with an independent dataset in testing, additional data can be mapped and prediction made based on the model.
The Support vector machine (SVM) approach is a machine learning approach that can map combinations of genotypes in high dimensional space. The top 1000 significant SNPs from the discovery and replication case:control genotype association studies we used as observed characteristics to form a designation decision between CVID (common variable immunodeficiency) case and an individual not affected by CVID (Table 14). The dataset available was separated into a training set of 179 cases and 1,917 controls which the SVM model is based on. In this training set, the case/control label and genotype attributes guide model formation. In the testing set of independent 109 cases and 1,114 controls, only the genotype attributes are supplied to the model established during training which then distinguishes cases or controls based on the genotype profile. The simple diagnostic yes or no question is: based on this person's genotypes do they have CVID? The prediction accuracy turned out well: training data: 98.7% and testing data: 90.5% (FIG. 7).
Similarly, the top 1000 significant SNPs for Bronchiectasis (Table 15) and OSAI (organ specific autoimmunity) (Table 16) as input to the SVM. Training: 107 no Bronchiectasis and 59 Bronchiectasis. Testing: 77 no Bronchiectasis and 19 Bronchiectasis. The model had 100% accuracy in the training stage and 97.9% accuracy in testing stage (FIG. 8). Evaluating our cohort for OSAI yielded Training: 117 no OSAI and 49 OSAI. Testing: 70 no OSAI and 26 OSAI. The model had 100% accuracy in the training stage and 99.0% accuracy in testing stage (FIG. 9).
To demonstrate poor model accuracy based on the same SVM methods, we ran the training stage with randomized testing data. The Bronchiectasis model applied to random affected labels and random genotypes resulted in a prediction accuracy of 27.1%. The Bronchiectasis model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 77.1%. The OSAI model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 67.7%. Given these markedly poorer accuracy results from randomized data compared to our observed data bolsters confidence that our high success rate is less likely due to a confounding factor, bias, or error.
This study represents the first genome-wide population based study of CVID. The use of the relatively large cohorts assembled here was essential, both to discover and to confirm the findings and demonstrates the potential of genome-wide association in complicated polygenic rare diseases. This type of unbiased study has discovered many novel targets that may underlie the development of CVID and provide clues to the pathogenesis of the heterogeneous clinical complications and subtypes of CVID, providing a solid foundation for further studies to understand the mechanism, interplay, and clinical manifestations of CVID, with the immediate potential of improving clinical management. Finally the great diversity identified with regards to unique CNV substantiates the hypothesis that CVIDs are a collection of diverse mechanisms leading to complex phenotypes.







TABLE 14





SNP Panel of 1994 SNPs for classifying CVID case versus individual without CVID
























rs3117426
rs2239914
rs9382495
rs139234
rs11132547
rs1391772
rs2107202
rs3763338
rs649647
rs7677584


rs2523535
rs366178
rs4269167
rs17631106
rs11135816
rs1400438
rs2112040
rs3763822
rs6504204
rs7690236


rs3130931
rs17304375
rs3130933
rs13019329
rs11138566
rs1411439
rs2113616
rs3766566
rs6505069
rs7716554


rs200968
rs3129871
rs9295768
rs7085343
rs11143520
rs1419183
rs2121289
rs3772054
rs6510040
rs7761966


rs879882
rs1634719
rs967005
rs12270763
rs11157606
rs1421240
rs2137512
rs3775574
rs6516091
rs7762279


rs3130564
rs4654849
rs2395174
rs12447026
rs11180576
rs1422122
rs214833
rs3781216
rs6538408
rs7772160


rs7767008
rs2400955
rs2931060
rs10940184
rs11183395
rs1438935
rs2153875
rs3788111
rs6549225
rs7774197


rs720831
rs6581986
rs10000770
rs139240
rs11206396
rs1443365
rs215393
rs3788317
rs6555424
rs7774567


rs4713208
rs2429485
rs10020322
rs3802888
rs11237730
rs1444467
rs2159318
rs3796504
rs656070
rs7781284


rs9266689
rs3129975
rs12636521
rs17790790
rs11247770
rs1445358
rs2168963
rs3801332
rs6577933
rs7789182


rs13194504
rs17099394
rs139136
rs4871793
rs1157548
rs1447398
rs217180
rs3804350
rs6583700
rs7808907


rs6926142
rs2395173
rs9380006
rs2279529
rs11669334
rs1452077
rs2175835
rs3810925
rs6586116
rs7815122


rs4872262
rs12495023
rs10476080
rs2902858
rs11698275
rs1461713
rs220309
rs3814585
rs6590124
rs7822769


rs12680982
rs10259703
rs358994
rs4720301
rs11717880
rs1466633
rs2213215
rs3818409
rs6656611
rs7834603


rs6456785
rs2505323
rs12573587
rs17372123
rs11786911
rs1477246
rs2236861
rs3847985
rs6657001
rs783756


rs2517532
rs2505327
rs139135
rs6679430
rs11819553
rs1481150
rs2237236
rs3863380
rs6688151
rs7838893


rs2156875
rs2484173
rs3095340
rs6485702
rs1182865
rs1497431
rs2242660
rs3913305
rs6688807
rs7857243


rs3130501
rs3132685
rs11087123
rs3134792
rs11848008
rs149990
rs2256726
rs3935608
rs6694182
rs7857474


rs422717
rs2523608
rs9266440
rs1377611
rs11862051
rs1523138
rs2262808
rs4082339
rs6727285
rs7867936


rs393034
rs3094122
rs2718419
rs9380120
rs11873322
rs1525745
rs2277210
rs4082846
rs6737948
rs7871753


rs3117326
rs8056528
rs1194849
rs6799262
rs11906
rs1544488
rs2292263
rs4146035
rs6743415
rs7887141


rs3129882
rs879484
rs7718291
rs12495026
rs11915523
rs1545692
rs2292307
rs4147525
rs6759577
rs7904468


rs3130837
rs2517549
rs12458578
rs2523989
rs11924324
rs1566579
rs2295330
rs420095
rs6764451
rs791033


rs8321
rs10734912
rs6426636
rs7945342
rs11933720
rs1569583
rs2299395
rs4236541
rs6794896
rs7928480


rs6571989
rs2523987
rs469709
rs950210
rs1194491
rs1571272
rs2306810
rs4238992
rs6807774
rs7937011


rs9261290
rs3130932
rs9329172
rs1406977
rs11959994
rs1574342
rs2343468
rs4320356
rs6865181
rs7939415


rs858985
rs2210313
rs13104213
rs720465
rs11963652
rs1582761
rs2358483
rs4324798
rs6877998
rs7964472


rs1606234
rs3135338
rs1876518
rs11870286
rs11977312
rs1592593
rs2373146
rs4353961
rs6886088
rs7970177


rs3129791
rs3770266
rs2572690
rs9257802
rs11998428
rs16181
rs2389789
rs4384353
rs6903608
rs8015024


rs12924882
rs149900
rs2191388
rs2517403
rs12001157
rs1619092
rs2406233
rs4409432
rs6904596
rs8015785


rs4264554
rs6457327
rs3094127
rs7103569
rs12047230
rs1629896
rs2425628
rs4486000
rs6910095
rs8020688


rs17322265
rs4871402
rs386843
rs12374025
rs12049377
rs1652500
rs2427399
rs4497887
rs6917366
rs8038569


rs1453666
rs2517448
rs17099425
rs10746192
rs12060746
rs1654774
rs2448396
rs4512403
rs6917419
rs8050560


rs7087554
rs2902982
rs310319
rs10254238
rs120960
rs1658102
rs2455826
rs4525114
rs6921388
rs8055387


rs853676
rs493965
rs2733393
rs139235
rs1209633
rs16891725
rs2473700
rs4531492
rs6923139
rs8057776


rs3771781
rs80303
rs1539120
rs3749530
rs12146261
rs16899857
rs2492460
rs454127
rs6928830
rs8061733


rs203888
rs9295730
rs2844494
rs2844635
rs12147922
rs16908361
rs2497295
rs4541737
rs6933251
rs8068923


rs3094551
rs200969
rs133826
rs1517927
rs12148329
rs16949286
rs2514861
rs4578658
rs6938076
rs8077733


rs1634718
rs1028308
rs9344757
rs17251715
rs12149564
rs16956295
rs2517646
rs4596286
rs6939576
rs8081687


rs3094550
rs3130350
rs1214751
rs9313540
rs12188351
rs16957467
rs2523454
rs4617771
rs6947841
rs8100750


rs6127923
rs2254556
rs2130904
rs4354950
rs12190473
rs16959820
rs2535319
rs464904
rs6959814
rs8105356


rs1064191
rs17293544
rs2730306
rs6785581
rs12201890
rs16971464
rs2554675
rs4671614
rs6960076
rs8109860


rs2517452
rs393990
rs12145634
rs2454873
rs12204145
rs17043526
rs2555575
rs4689343
rs6961642
rs8138344


rs9257809
rs16851846
rs4350445
rs5009448
rs12238437
rs17052298
rs2560623
rs4692098
rs6972083
rs821107


rs895710
rs1533476
rs606448
rs10018912
rs12258095
rs17090892
rs258229
rs4695718
rs697614
rs835361


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rs7962880
rs6542736
rs10984490
rs12827322
rs17627259
rs2703960
rs4864553
rs726558
rs9809773


rs315995
rs6714893
rs9844737
rs1100185
rs12882217
rs17654772
rs274646
rs4865815
rs7272481
rs982511


rs12155045
rs7171256
rs1316471
rs11014455
rs12885228
rs1765886
rs2755176
rs4885371
rs728034
rs9835081


rs6128300
rs1003802
rs236410
rs11022509
rs1289501
rs17685418
rs2763419
rs4901523
rs729154
rs9848911


rs11666885
rs13214001
rs473585
rs11033019
rs12982321
rs17692241
rs2802884
rs4915723
rs729648
rs9861236


rs2685515
rs316021
rs2420175
rs11059821
rs12990534
rs17709684
rs2817200
rs4920566
rs731809
rs9874349


rs6748817
rs4972265
rs3886771
rs11128687
rs12996858
rs17726892
rs2821236
rs4921593
rs7321884
rs988158


rs2279351
rs1650817
rs2025633
rs11131055
rs13023918
rs17734487
rs2822850
rs4933679
rs734434
rs988179


rs11059258
rs7265772
rs6012463
rs11143230
rs13035689
rs17761855
rs2825673
rs494469
rs7349465
rs9904523


rs4130446
rs7724262
rs3849699
rs11165460
rs1304065
rs17796406
rs2852424
rs4950989
rs739107
rs9912125


rs7084512
rs251039
rs12188691
rs11186642
rs13081891
rs17817943
rs2877498
rs4976254
rs740096
rs9913538


rs627830
rs2057904
rs9325837
rs11194816
rs13086978
rs17829645
rs2887022
rs4978774
rs7415038
rs9919607


rs2052701
rs12608393
rs2010464
rs11218881
rs13128635
rs178377
rs2904295
rs4981149
rs7428676
rs9966000


rs1475784
rs2475793
rs13394087
rs1124359
rs13132576
rs1790024
rs2914079
rs4984626
rs752278
rs9989335


rs6419277
rs2455308
rs11237690
rs11250154
rs13142179
rs1833755
rs2924264
rs500293
rs7539255



rs6769238
rs1808378
rs2152174
rs1146229
rs13189435
rs1861089
rs2940930
rs536299
rs7544671



rs7306734
rs4521737
rs4947602
rs1152986
rs13203110
rs186493
rs296200
rs542988
rs7553961



rs3125050
rs547491
rs6437079
rs11547160
rs13224666
rs1866714
rs2962270
rs556439
rs7554157



rs2174559
rs1477602
rs7609626
rs1156250
rs13229792
rs1885831
rs2972418
rs568789
rs7555638



rs6661074
rs7792461
rs722938
rs11576361
rs13238853
rs1889356
rs2974617
rs587263
rs7574670
















TABLE 15





SNP Panel of 1000 SNPs for classifying CVID case with Bronchiectasis versus CVID case


without Bronchiectasis
























rs3101219
rs1866236
rs9837945
rs7700961
rs6950759
rs7021834
rs6590583
rs8011776
rs6508240
rs5925649


rs4908463
rs1349159
rs13083177
rs297884
rs2191714
rs1536864
rs6590584
rs7174078
rs2120663
rs5925651


rs1725262
rs1448917
rs960353
rs7735038
rs2330055
rs11142904
rs6590694
rs11858397
rs16946189
rs5926027


rs10864368
rs12612313
rs7629105
rs1050975
rs11238193
rs7037278
rs546381
rs17227996
rs1350552
rs5926032


rs2071414
rs6436652
rs6443582
rs1890366
rs296308
rs2378383
rs11054372
rs12439272
rs621834
rs7882511


rs12034719
rs9288624
rs1522262
rs1933650
rs11766378
rs7025226
rs2071163
rs4924613
rs2433396
rs1527808


rs2982380
rs1515928
rs1994855
rs6904644
rs10499751
rs12238088
rs2239176
rs1712426
rs2847593
rs5943997


rs17038468
rs2556097
rs12487104
rs1331489
rs10270164
rs10491791
rs901528
rs3825786
rs7241842
rs12557438


rs1812242
rs1561466
rs4602335
rs1571827
rs13240999
rs7874528
rs10841413
rs12439521
rs16977580
rs16988109


rs4970535
rs13033944
rs4472002
rs9391956
rs12698917
rs17428350
rs2417862
rs7178085
rs1942159
rs12014989


rs12126652
rs830994
rs4234788
rs3812179
rs258702
rs7871887
rs2203494
rs11855605
rs3753067
rs1419850


rs12131682
rs831007
rs7435615
rs3812178
rs2888019
rs10818955
rs11045417
rs2306335
rs11152012
rs5973335


rs272561
rs831011
rs4697075
rs7760489
rs1557785
rs1413294
rs1861710
rs1993865
rs11663936
rs728149


rs1051648
rs6707969
rs2167955
rs966840
rs1921598
rs2114012
rs10492367
rs1026695
rs10516006
rs7064305


rs3444
rs16862458
rs5002502
rs13196069
rs1526491
rs3847303
rs7307357
rs4776191
rs17638216
rs5972346


rs6667593
rs6433615
rs7665747
rs12665403
rs7781914
rs328875
rs2388962
rs1904109
rs7251154
rs2124748


rs2354463
rs13032587
rs12186184
rs1905212
rs2188508
rs7852829
rs10844021
rs17183491
rs10416824
rs228379


rs10493140
rs17362588
rs1377347
rs9476488
rs17166818
rs1385143
rs1666235
rs3809539
rs6508999
rs2180648


rs10888636
rs2290517
rs13116757
rs9358858
rs1015882
rs4979619
rs10743775
rs1865930
rs7254214
rs5917579


rs2806405
rs12465639
rs7349609
rs6904130
rs2158137
rs12236795
rs12319134
rs11858355
rs1594895
rs1800321


rs2764687
rs10204810
rs4527518
rs2524099
rs10275909
rs7020797
rs995342
rs12914140
rs2217672
rs5917584


rs12728521
rs4667046
rs10034992
rs9501626
rs7777145
rs10481656
rs1389134
rs8032153
rs8113456
rs11266207


rs17124275
rs785240
rs1000226
rs3135338
rs3735258
rs7049083
rs1498712
rs11639228
rs6509286
rs5917593


rs4512683
rs12613687
rs2622604
rs2027856
rs234
rs7041855
rs10877894
rs4436753
rs689292
rs5964215


rs11208446
rs6740981
rs1708670
rs2395173
rs17356935
rs1249904
rs11174573
rs4534816
rs3746660
rs2239455


rs12407601
rs10445792
rs583908
rs154978
rs7455060
rs2225067
rs3936640
rs6497041
rs6084506
rs10854983


rs6663109
rs4673837
rs4693223
rs958423
rs10272242
rs10818337
rs3864455
rs11635007
rs998132
rs5919551


rs7522367
rs6739563
rs10049681
rs804829
rs6466735
rs10984619
rs4763120
rs768399
rs945767
rs5919560


rs11208834
rs11687313
rs10516451
rs1109798
rs4726499
rs11790238
rs11175217
rs8024991
rs6139278
rs5936698


rs1415974
rs4672726
rs4833233
rs3807045
rs2001942
rs6478430
rs962415
rs12591805
rs6076598
rs7056340


rs3738168
rs2043769
rs1155135
rs2257082
rs12703419
rs1860665
rs10506554
rs13380379
rs6516091
rs6625489


rs787492
rs13019534
rs1480902
rs941967
rs855740
rs2297454
rs10506762
rs4965678
rs6054459
rs5936708


rs7541725
rs12053340
rs1383518
rs2268718
rs1111467
rs10818649
rs919993
rs7177883
rs2206423
rs1327347


rs6691042
rs1527944
rs13125153
rs4391265
rs1079060
rs10988617
rs2468358
rs9944290
rs2050104
rs5936488


rs617196
rs1527947
rs12503254
rs1377392
rs2952648
rs11103479
rs7300004
rs11640138
rs8120907
rs5936735


rs263463
rs16836294
rs1456266
rs10948750
rs11777063
rs10905076
rs10777720
rs415595
rs2223565
rs6418420


rs263495
rs17450826
rs951850
rs9342394
rs6558946
rs12781427
rs3782518
rs949429
rs1033470
rs5936510


rs12409961
rs12472818
rs4569797
rs701699
rs7816614
rs10752197
rs2293055
rs12918743
rs4813897
rs5980665


rs2295330
rs16858496
rs939689
rs493265
rs1526371
rs7092558
rs11615235
rs7404377
rs6040808
rs5936518


rs11184300
rs12464171
rs6536370
rs9351817
rs2527748
rs7897007
rs4760566
rs238848
rs2009018
rs5936520


rs11184318
rs10933001
rs6858744
rs199623
rs13254568
rs11816696
rs2129663
rs2521477
rs6110058
rs5936795


rs2765283
rs12479209
rs2047633
rs12207816
rs7836269
rs12254001
rs7134809
rs2910848
rs17272940
rs5936796


rs7516108
rs1400795
rs13140600
rs16881643
rs2736020
rs749631
rs7300570
rs8045557
rs6079923
rs5936524


rs401904
rs17371273
rs2706740
rs10485242
rs1563297
rs2793345
rs12422489
rs763763
rs6046129
rs3811372


rs1748383
rs6711657
rs2706727
rs16881894
rs2720611
rs7071467
rs1334958
rs3935714
rs7267722
rs749789


rs7523184
rs12463466
rs2660404
rs10944450
rs4831774
rs7911971
rs9510802
rs257868
rs6049003
rs7879974


rs1986860
rs7355380
rs6841955
rs16881936
rs10503553
rs4256883
rs4771142
rs9926100
rs6121337
rs6615046


rs4233394
rs11685503
rs2082317
rs7754169
rs822249
rs2138566
rs1336628
rs7199018
rs228838
rs12012447


rs4540634
rs2645779
rs11729502
rs2518321
rs822304
rs10822031
rs4771033
rs7188866
rs6126431
rs5958896


rs1986385
rs1705805
rs11725347
rs12212740
rs11203812
rs10996997
rs903777
rs8049176
rs6069746
rs6621735


rs2766085
rs17709863
rs6818908
rs1858234
rs11204097
rs10762038
rs1952994
rs8047279
rs438363
rs5985075


rs12062372
rs356418
rs11733679
rs4371861
rs12545707
rs2894317
rs391933
rs16959476
rs160396
rs5942366


rs10800150
rs6769422
rs10024068
rs6913350
rs7841346
rs890687
rs398655
rs12449237
rs1151621
rs5942534


rs10918281
rs9875884
rs7698980
rs1933755
rs2942202
rs1550023
rs9603418
rs4458015
rs6062292
rs5941436


rs4472738
rs11129323
rs2280664
rs9492657
rs2685320
rs1684543
rs4346095
rs8057941
rs8129104
rs1570995


rs6680341
rs1015856
rs2692596
rs2249821
rs11136019
rs4547043
rs9526255
rs17296283
rs443823
rs6522689


rs10489199
rs9837352
rs1288536
rs2807278
rs7833986
rs2497304
rs4534729
rs13338146
rs2407255
rs638376


rs2205851
rs33454
rs6849285
rs2024692
rs7822429
rs10736156
rs17069883
rs17712938
rs2825809
rs12833867


rs232293
rs3821477
rs11724261
rs9403904
rs4397440
rs10884480
rs9534596
rs11648482
rs2825817
rs6644233


rs1933539
rs971866
rs957792
rs4870376
rs7824497
rs10748948
rs1323537
rs8055798
rs2825819
rs12556735


rs6593937
rs715382
rs1444127
rs9322536
rs924740
rs10749189
rs9535080
rs16955356
rs2826213
rs5910956


rs12061474
rs7647357
rs16892616
rs12174424
rs1545824
rs690870
rs777771
rs2317835
rs236044
rs4373692


rs11579791
rs6810180
rs4869452
rs2770104
rs2128105
rs7073467
rs9598119
rs378138
rs5992088
rs204330


rs10489951
rs6445254
rs6883671
rs1118242
rs7832751
rs3011399
rs2137512
rs1946482
rs2535707
rs203490


rs892975
rs11130981
rs1466011
rs9654570
rs7015186
rs3011408
rs7988149
rs8081951
rs361893
rs16309


rs6426156
rs1283532
rs2910468
rs751873
rs6472384
rs7070793
rs3013580
rs7219019
rs849357
rs201637


rs4653662
rs7373042
rs2910469
rs9456433
rs11778612
rs1898463
rs4884738
rs17762680
rs1297593
rs149908


rs973252
rs13100924
rs1423131
rs4368835
rs17824659
rs11248595
rs7997966
rs7215084
rs1807510
rs2046394


rs2808611
rs6548962
rs2910475
rs10214534
rs6472770
rs11244515
rs9317784
rs7503953
rs738456
rs683335


rs2808614
rs7610023
rs2961838
rs2457571
rs7461233
rs4962566
rs2325194
rs4792143
rs392277
rs546562


rs11122322
rs6548984
rs1862526
rs9365646
rs5017296
rs12098358
rs1446385
rs4393623
rs5752634
rs5932370


rs2814536
rs2136715
rs1309824
rs9365647
rs3735912
rs7124824
rs1249772
rs12451200
rs427736
rs5932394


rs2644441
rs4855469
rs10940096
rs9355453
rs13272532
rs903209
rs1334825
rs8067882
rs5762430
rs6637420


rs1874400
rs6549411
rs2937715
rs9355454
rs17700155
rs2283257
rs1112466
rs1555142
rs470105
rs6529359


rs3011577
rs9310222
rs10064609
rs916368
rs9969608
rs2270024
rs2325364
rs8073784
rs5762461
rs5932624


rs4443876
rs2322165
rs13164793
rs6915442
rs4602925
rs4569005
rs9600257
rs2075053
rs5750726
rs1324149


rs4658702
rs2322162
rs33003
rs221725
rs7823271
rs869377
rs9573480
rs11079868
rs4821862
rs5975531


rs12407427
rs11718674
rs6874731
rs221723
rs6983626
rs11030196
rs9565288
rs747039
rs2014881
rs5954074


rs1039010
rs11921763
rs4703545
rs7789861
rs1550856
rs4923526
rs4635230
rs17637472
rs2076101
rs12687312


rs384526
rs12714781
rs2656984
rs12540575
rs3019885
rs924714
rs9517921
rs11869714
rs5758991
rs644210


rs6542651
rs6765942
rs1505000
rs4719266
rs10464861
rs1216155
rs11069386
rs739924
rs9615362
rs5919819


rs2872977
rs1319094
rs448840
rs6460807
rs7816758
rs10897108
rs9805437
rs11651374
rs9627450
rs5965846


rs934615
rs7616034
rs7733888
rs6970647
rs4593504
rs4930431
rs12428930
rs9892374
rs4459004
rs1337635


rs2110965
rs11718781
rs1449227
rs136
rs12674562
rs7118125
rs9521350
rs9907787
rs1026162
rs596987


rs7562836
rs10049211
rs4869255
rs142
rs1768869
rs7946537
rs12886280
rs11653824
rs4641225
rs580628


rs13427136
rs11128275
rs1974789
rs135
rs10511649
rs567236
rs11156803
rs17462688
rs7064826
rs995895


rs7601672
rs6808996
rs10062935
rs7783383
rs7045369
rs487345
rs12884688
rs11870700
rs1266320
rs1547727


rs10490207
rs9869897
rs10478040
rs4722417
rs10757394
rs12807746
rs1058010
rs11655981
rs6641094
rs6877


rs9309192
rs4680967
rs256249
rs6966737
rs10966093
rs7481199
rs1952198
rs7220430
rs1731475
rs12008689


rs10184594
rs1561162
rs329312
rs38488
rs824230
rs621310
rs11625855
rs7230740
rs1731470
rs1882713


rs11885364
rs729942
rs329317
rs10272887
rs928484
rs11021014
rs11159087
rs1402630
rs5955621
rs5925038


rs13427078
rs959132
rs2069882
rs739981
rs1115553
rs11021018
rs10483899
rs1940615
rs5909069
rs17253949


rs6545738
rs635268
rs17169344
rs7790537
rs3780169
rs1791459
rs1112573
rs12185468
rs6633148
rs5970118


rs1503236
rs6439419
rs2347577
rs10250368
rs17520103
rs1625749
rs12432856
rs9947725
rs2283712
rs5924705


rs6704741
rs1466827
rs10040966
rs12701041
rs495259
rs11216930
rs8008554
rs930926
rs5955648
rs5925082


rs1517862
rs2052832
rs152523
rs2392151
rs605683
rs17748
rs17259779
rs8083849
rs2122
rs4828733


rs11887934
rs347965
rs6881655
rs2598105
rs662975
rs11216943
rs1286496
rs12961249
rs6633552
rs2071122


rs4549098
rs10513320
rs1025489
rs12701773
rs567490
rs4936507
rs12893100
rs206416
rs16982185
rs3747309


rs7558702
rs391398
rs302401
rs2119053
rs985654
rs7130937
rs17099646
rs3816822
rs6653666
rs5940453


rs1459166
rs6793037
rs10516032
rs4720500
rs11142769
rs4282990
rs4906007
rs11083004
rs5925647
MitoG12373A
















TABLE 16





SNP Panel of 1000 SNPs for classifying CVID case with OSAI (organ specific autoimmunity)


versus CVID case without OSAI
























rs2297829
rs16838906
rs4274923
rs7718152
rs3912067
rs2927572
rs6589426
rs1012016
rs2850451
rs5951587


rs2275875
rs1876110
rs9291598
rs10059157
rs7805441
rs1536950
rs11214983
rs7156281
rs9949880
rs5926216


rs6699993
rs10489999
rs187196
rs7719533
rs4730345
rs957903
rs7116557
rs1840790
rs11150900
rs12011998


rs11580688
rs7562525
rs4351004
rs2400176
rs2371366
rs784790
rs7942551
rs1379230
rs11661755
rs6653559


rs1005301
rs1431900
rs10025742
rs17640419
rs17156818
rs10759209
rs4936297
rs12885991
rs12605082
rs5985835


rs12023073
rs1406665
rs10939265
rs2277025
rs2106277
rs7031681
rs7112940
rs1587351
rs12461534
rs3997122


rs533808
rs1147148
rs2687968
rs412805
rs2213979
rs10978723
rs2298767
rs4774966
rs1428752
rs5985846


rs2997447
rs1226906
rs2249563
rs394378
rs6972034
rs7871882
rs743632
rs11632793
rs10413521
rs1921501


rs3008418
rs1147155
rs2732063
rs10516028
rs17153071
rs784683
rs4937230
rs7179813
rs3786521
rs5985847


rs12069719
rs438027
rs1706023
rs868103
rs11772805
rs784676
rs7933438
rs11637130
rs6509637
rs1500727


rs2783711
rs4476347
rs1812922
rs4640812
rs1404228
rs784667
rs7948482
rs11637277
rs1366257
rs7065783


rs17257107
rs10203019
rs10518221
rs10038486
rs7777183
rs7034018
rs10790952
rs2917821
rs2561008
rs5971516


rs1325281
rs1010491
rs6857026
rs248230
rs7797250
rs7034341
rs11221388
rs12915827
rs7245993
rs2692983


rs12136144
rs6707140
rs4271959
rs9393002
rs10488631
rs13287637
rs7115037
rs8025474
rs11881864
rs2692992


rs297802
rs1446928
rs11727192
rs9379231
rs12531711
rs10306141
rs4937653
rs11071986
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Example 3
Exome Sequencing Identifies Missense IRF2BP2 Mutation in a Family with Autosomal Dominant Common Variable Immunodeficiency (CVID)
Common Variable Immunodeficiency (CVID) is among the most frequently diagnosed forms of primary immunodeficiency disorders (PIDD), and is the most frequent requiring clinical intervention. Defined clinically by low quantity of two immunoglobulin classes (including IgG) and poor specific antibody production, it has been thought of as an “umbrella diagnosis” due to the heterogeneity of its onset and co-morbidities, including autoimmune disease and risk of malignancy. Though thought to be polygenic, roughly 10% of CVID is familial, and in recent years, 11 gene causes and/or associations with CVID have been described (Table 17).







TABLE 17







CVID Gene Associations









Function
Gene
Ref.





BCR function
CD19
Van Zelm, MC, et al. NEJM 2006



CD20
Kuijpers TW, et al. JCI 2010



CD21
Thiel J, et al. JACI 2012



CD81
Van Zelm MC, et al. JCI 2010.


B-cell co-stimulation
TACI
Martinez-Gallo M, et al. JACI 2013



ICOS
Grimbacher B, et al. Nat Immunol 2003



BAFFR
Warnatz H, et al. Proc Natl Acad Sci 2009



CD27
Van Montfrans J, et al. JACI 2012 


DNA repair/VDJ recombination
MSH5
Sekine H, et al. Proc Natl Acad Sci 2007


B-cell survival/differentiation
LRBA
Lopez-Herrera G, et al. Am J Hum Genet 2012


TCR/BCR signaling
PLCγ2
Ombrello MJ, et al. NEJM 2012









As discussed above in the previous examples, a recent multi-institutional genome-wide array study of CVID showed unique associations with specific single nucleotide polymorphisms (SNP) and copy number variants (CNV), with intraexonic duplications in ORC4L being most highly associated with disease. Beyond individual associations, CVID has a unique pattern of SNP and CNV, as suggested by the successful use of a Support Vector Machine (SVM) algorithm to identify this pattern in CVID patients and controls. SVM can be trained with a variety of data, and produces a “hyperplane” for subsequent classification. In the studies described above, the CVID SVM hyperplane successfully classified cases with an accuracy of 91%, PPV 100%, and NPV 96%.
Though the use of SVM supports the polygenic nature of CVID, a remaining question was whether monogenic CVID would lack the genetic fingerprint of the more common polygenic disease. If this were the case, SVM classification of microarray data would incorrectly classify these individuals as not affected, and could therefore be a useful screening method for monogenic CVID, most notably in familial cases. Accordingly additional studies were undertaken.
Patients
Patients were enrolled in research protocols approved by the host institutional review board to allow genetic analysis and clinical data collection.
Clinical History
The proband is a young woman who experienced recurrent sinopulmonary infections beginning in early adolescence. Evaluation at age 19 years showed IgG 620 mg/d1 (normal range 635-1775 mg/dL), undetectable IgM and IgA (normal ranges 71-237 and 70-486 mg/dL respectively), and poor specific antibody production following vaccination to S. pneumoniae (1 of 14 serotypes >1.3 mcg/ml) and N. meningitidis (0 of 4 strains protective). Lymphocyte subsets were normal in number with CD19+388 cell/mcl (normal range 100-570), though switched memory B-cells (CD19+IgD−CD27+) were low at 0.4% (normal >0.5%). She has been maintained on subcutaneous immunoglobulin with good control of infections, and has no autoimmune disease.
Her family history is notable for CVID in both her father as well as her older brother (FIG. 11). Her father also has a history of psoriasis, and her brother has type I diabetes. Her paternal grandparents, aunts, and their families are unaffected, as is her maternal family.
Genetic Studies
High throughput SNP genotyping was performed with the Infinitium HumanHap610 Beadchip, and the PennCNV algorithm was used for CNV calls. A support vector machine algorithm was trained with data from 179 CVID cases and 1917 controls, utilizing the 658 most significantly associated variants from the 2011 study, and subsequently tested on the patient.
Whole-exome sequencing using the Agilent SureSelect Human All Exon 50 Mb kit was performed on the patient and her family. Variants were matched to disease segregation (which suggested a heterozygous, autosomal dominant pattern), and further narrowed by exclusion of synonymous mutations, in silico analysis of mutation impact, exclusion of SNPs in public databases (1000 Genomes and NHLBI 5400 exomes Project), tissue expression pattern (BioGPS.org), and ties to known immunologic pathways.
RT-PCR and Immunoblotting
RNA was isolated from whole blood obtained from the patient and controls via Trizol reagent (Applied Biosystems, Grand Island, N.Y.) and RNEasy kit (Qiagen, Germantown, Md.). cDNA was produced via high capacity Reverse Transcriptase kit (Applied Biosystems), and custom cDNA primers for IRF2BP2 (both total and isoform 2) and GAPDH were created. RT-PCR performed via SYBR Green core reagents on a QPCR 7900HT system. Gene dose was calculated via DDCT method.
Protein immunoblotting was performed on lymphoblasts derived from peripheral blood mononuclear cells via treatment with phytohemagglutanin (2 ug/ml) and IL-2 (500 IU/ml). Antibodies against IRF2BP2 and TATA binding protein (Abeam, Cambridge, Mass.) were used.
Support Vector Machine Hyperplane Classification
Analysis of the top 658 SNP/CNVs by the pre-trained CVID SVM algorithm predicted that the patient was not affected (FIG. 11).
Whole Exome Sequencing
Sequencing of the proband revealed 12 non-synonymous, rare variants, of which 5 were predicted to be damaging via in silico analysis (SIFT, polyphen2). Three variants (AMBP, IRF2BP2, and PIK3C2G) had been tied to immunologic pathways. Sanger sequencing of IRF2BP2 in the proband and family confirmed that a heterozygous S535N mutation was present in all affected family members, and none of those who were unaffected. This mutation is in the RING domain of the protein, which has been described as the region of interaction with IRF2 and NFAT1 [3]. See FIG. 12.
RT-PCR of IRF2BP2
Quantitative PCR of IRF2BP2 from patient and control peripheral blood showed a lower transcript quantity in the patient versus normal control (FIG. 13). In the normal control, Isoform 1 was predominant, with comparatively less transcripts of Isoform 2 (which differs by exon 1 length). In the patient, isoform expression was roughly equal.
These results reveal that learning algorithms such as SVM provide a valuable method of screening for monogenic diseases within polygenic populations by searching for differences in the genetic pattern of SNP and rare CNV. In the field of PIDD, this is particularly useful for screening for monogenic causes of early-onset autoimmune disease such as type I diabetes or inflammatory bowel disease.
Moreover, these studies reveal that the presence of a SNP in IRF2BP2 on chromosome 1 at position 234742995 is indicative of a new CVID-associated candidate gene.
Example 4
The information herein above can be applied clinically to patients for diagnosing an increased susceptibility for developing CVID, and therapeutic intervention. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. Diagnostic compositions, including microarrays, and methods can be designed to identify the genetic alterations described herein in nucleic acids from a patient to assess susceptibility for developing CVID. This can occur after a patient arrives in the clinic; the patient has blood drawn, and using the diagnostic methods described herein, a clinician can detect one or more indicative SNPs or CNVs described in the Tables hereinabove. The information obtained from the patient sample, which can optionally be amplified prior to assessment, will be used to diagnose a patient with an increased or decreased susceptibility for developing CVID. Kits for performing the diagnostic method of the invention are also provided herein. Such kits comprise a microarray comprising at least one of the SNPs provided herein in and the necessary reagents for assessing the patient samples as described above.
The identity of CVID-involved genes and the patient results will indicate which variants are present, and will identify those that possess an altered risk for developing CVID. The information provided herein allows for therapeutic intervention at earlier times in disease progression that previously possible. For example, agents such as STA-5326, sotatercept, PF3446962, PEG-interleukin-2 and B-lymphocyte stimulators can be administered as appropirate.
Also as described herein above, several new genes involved in CVID pathogenesis have been identified which provide novel targets for the development of new therapeutic agents efficacious for the treatment of CVID.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope of the present invention, as set forth in the following claims.

## Claims

1. A method for treating common variable immunodeficiency (CVID) in a human subject, comprising:
a) obtaining genotype sequence information from sequencing a nucleic acid from said subject;
b) detecting in said information the presence of at least one CVID-associated copy number variation (CNV), wherein said at least one CVID-associated CNV:
is selected from the group consisting of deletions comprising: chr11: 85365857-85381622; chr20: 57735790-57741780; chr22: 17396663-18417315; chr4: 10256682-10264316; chr10: 46003146-46042543; or
is selected from the group consisting of duplications comprising: chr2: 148396730-148433180; chr15: 35053039-35063531; chr7: 91789778-91801963; chr2: 163316298-163316595; chr7: 87180702-87191931; chr19: 9256584-9277749; chr4: 39190766-39201960; chr7: 18903915-18905725; chr5: 170531269-170536993; chr7: 34685030-34686172; chr1: 170400944-170403742;
c) correlating the presence of the detected CVID-associated CNV with a diagnosis of CVID in the subject; and
d) administering to said human subject a therapeutically effective amount of at least one pharmaceutical agent useful for the treatment of CVID symptoms.

2. The method of claim 1, wherein said symptoms are gastrointestinal and said agent is an inhibitor of IL-12 production.

3. The method of claim 1, wherein said agent is pegylated IL-12.

4. The method of claim 1, wherein said agent is Sotatercept.

5. The method of claim 1, wherein said agent is apilimod mesylate.

6. The method of claim 1, wherein said genotype sequence information is determined using a process selected from the group consisting of: detection of specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, allele-specific hybridization analysis, single base primer extension reaction, and sequencing of an amplified polynucleotide.

7. The method of claim 1, wherein said CVID includes a subphenotype selected from the group consisting of: cancer, lymphoma, lymphadenopathy, nodular regenerative hyperplasia of the liver (NRH), lymphoid interstitial pneumonitis (LIP), bronchiectasis, biopsy proven granuloma, GI enteropathy, malabsorption, splenectomy, cytopenias, organ specific autoimmunity (OSAI), low IgM (<50 mg/dL), low IgA (<10 mg/dL), low B cells (CD19+ cells <1%), and young age of symptom onset (<10 yrs).