# Patent

## Title
Methods and compositions for the diagnosis of Cornelia de Lange Syndrome

## Abstract

Compositions and methods for the diagnosis of Cornelia de Lange Syndrome are disclosed.

## Background
This application claims priority under 35 U.S.C. §119(e) to U.S. provisional Application No. 60/567,756 filed May 3, 2004, the entire contents of which are incorporated by reference herein.


Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described, which was made in part with funds from the National Institutes of Health, Grant Numbers: 1 RO1 HD39323 and RO1 DK53104.


FIELD OF THE INVENTION
This invention relates to the fields of human genetics and molecular biology. More specifically, the invention provides compositions and methods useful for screening and diagnosing patients with Cornelia de Lange Syndrome.
BACKGROUND OF THE INVENTION
Several publications and patent documents are cited throughout 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.
Cornelia de Lange syndrome (CdLS [OMIM #122470]), which was recognized as a distinct entity over 70 years ago, is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper extremity malformations, hirsutism, cardiac defects, growth and cognitive retardation, and gastrointestinal abnormalities (Brachmann 1916; de Lange 1933). The distinctive facial features include synophrys, long eyelashes, depressed nasal bridge with an up-tilted nasal tip and anteverted nares, thin upper lip with down-turned corners of the mouth, and low-set, posteriorly-rotated ears. Abnormalities in the upper extremities range from subtle changes in the phalanges and metacarpal bones with small hands to oligodactyly and severe reduction defects. Gastrointestinal abnormalities include gastroesophageal reflux, intestinal malrotation, and pyloric stenosis. Additional relatively frequent features include hearing loss, ophthalmologic findings (ptosis, myopia), palatal abnormalities, genitourinary abnormalities (cryptorchidism, hypospadias), cardiac septal defects, and congenital diaphragmatic hernias. Growth retardation is an almost universal finding in CdLS and is typically of prenatal onset. Standard growth curves have been established for height, weight, and head circumference (Kline et al. 1993a). The mental retardation in CdLS is often severe, with a mean I.Q. of 53 (range 30-86) (Kline et al. 1993b). Many patients also demonstrate autistic-like behavior and self-injurious behavior (Jackson et al. 1993).
The clinical features seen in individuals with classic CdLS are striking and easily recognizable; however, there is marked variability and a milder phenotype has been consistently described (Ireland et al. 1993; Saul et al. 1993; Selicorni et al. 1993; Van Allen et al. 1993). Indeed, even the first reported descriptions of CdLS were markedly discrepant in phenotype: Brachmann described major upper limb reduction abnormalities (Brachmann 1916), while de Lange reported no limb reduction defects (de Lange 1933). This phenotypic variability and lack of a diagnostic marker have complicated the diagnosis and counseling for CdLS.
The prevalence of CdLS is estimated to be as high as 1 in 10,000 (Opitz 1985) and most cases appear to be sporadic. Pedigree analyses of several families demonstrate autosomal dominant inheritance with both maternal and paternal transmission (Robinson et al. 1985; Bankier et al. 1986; Halal and Silver 1992; Feingold and Lin 1993; Chodirker and Chudley 1994; Kozma 1996; Russell et al. 2001; McConnell et al. 2003). Assuming autosomal dominant inheritance, cases of apparently unaffected parents having multiple children with CdLS were hypothesized to be the result of germ line mosaicism (Beratis et al. 1971; Lieber et al. 1973; Fryns et al. 1987; Naguib et al. 1987; Krajewska-Walasek et al. 1995; Caksen et al. 2001). This hypothesis of germ line mosaicism was further supported by the identification of several families where an unaffected parent had multiple affected children through different partners (Krantz et al. 2001).
Due to the severity of the physical and cognitive impairment seen in CdLS, it has long been felt that an underlying chromosomal imbalance encompassing multiple genes may be etiologically responsible. Although several chromosomal rearrangements have been reported in the past in patients with CdLS, no consistent abnormalities have been identified (Kousseff et al. 1994). The identification of individuals with CdLS who carry de novo balanced translocations (Ireland et al. 1991) are of interest as they may be involved in disruption of the causative gene. The frequency of balanced translocations in the general population is estimated to be 1/500 (Bugge, M. 2000), so reports of a handful of apparently unrelated de novo translocations in children with CdLS, most of whom will have chromosomal analysis performed, may in fact be incidental.
Partial phenotypic overlap between individuals with CdLS and individuals with duplications of chromosome 3q (dup 3q syndrome) has been noted (Falek et al. 1966; Aqua M.S. et a. 1995; Ireland et al. 1995; Rizzu et al. 1997) and resulted in a locus assignment in OMIM for CdLS at 3q26 (See NCBI website at nlm. nih.gov/htbinpost/Omim/dispmim?122470). The identification of a de novo t(3;17)(q26.3;q23.1) translocation in a patient with a classic CdLS phenotype (Ireland et al. 1991) with the 3q breakpoint within the dup3q critical region further implicated this region. The 3q breakpoint has been cloned and a novel gene identified at the breakpoint however no mutations have been identified in this gene or in neighboring genes in individuals with CdLS (Smith et al. 1999; Tonkin et al., 2001). Linkage analysis to this region did not demonstrate cosegregation of chromosome 3q markers with the CdLS phenotype in all of the families studied (Krantz et al. 2001). These reports suggested that loci other than 3q26-27 may harbor a CdLS disease gene.
In light of all the foregoing, it is clear that a need exists for a new diagnostic marker for CdLS and methods of use thereof. Such a marker and methods can be used to advantage for genetic counseling and prenatal screening.
SUMMARY OF THE INVENTION
In accordance with the present invention, it has been discovered that NIPBL, when mutated, gives rise to Cornelia de Lange Syndrome (CdLS). Thus, NIPBL provides the first molecular diagnostic marker for this disorder. NIPBL is a large gene consisting of 47 exons spanning 188 kilobases of DNA with a transcript (mRNA) of 9505 base pairs. The gene was identified through a combination of genome-wide linkage analysis and fluorescence in situ hybridization (FISH) in families and individuals with CdLS. Mutations, (e.g., missense, splice site, frameshift and complex) are spread through out the gene, although there appears to be some clustering of mutations in exon 10. Most mutations identified are de novo, i.e., they occurred as a spontaneous event in the affected individual, although certain mutations have been found to run in families.
In one aspect of the invention, a method for diagnosing a patient as having an increased risk of developing Cornelia de Lange Syndrome (CdLS) is disclosed. An exemplary method entails providing a biological sample comprising DNA, or RNA from the individual and assessing the DNA or RNA for the presence or absence of a mutation in the NIPBL gene, wherein the presence of NIPBL gene mutation is correlated with the presence of CdLS in the individual being diagnosed. Suitable biological samples include, but are not limited to blood, saliva, amniotic fluid, and tissue.
In yet another embodiment of the invention, the protein product encoded by the NIPBL gene may be isolated and further assessed to determine whether the mutation, if present, results in an alteration in the amino acid sequence of the protein product. Accordingly, methods of expressing the nucleic acids encoding NIPBL mutated proteins in vitro and in host cells are provided herein. Such proteins and host cells are useful for screening test compounds which bind or modulate the activity of the mutated NIPBL molecules described herein.
A further aspect of the invention is based on the discovery that NIPBL plays a role in sister chromatid cohesion in humans. Accordingly, samples from patients suspected of having CdLS can be screened for precocious sister chromatid separation (PSCS) as described in Example 3. Such screening assays can be performed with or without the mutational screening described in Example 2. In yet another aspect of the invention, PSCS assays can be performed on cells isolated from CdLS patients in the presence and absence of a test compound to determine whether the test compound is capable of modulating the PSCS observed in CdLS patients. Agents which reduce PSCS may have efficacy in the treatment of CdLS.
Diagnostic probes useful in the methods of the invention are also disclosed herein. Suitable probes comprise about 10-200, more preferably about 10-100 and most preferably at least 10 contiguous bases from SEQ ID NOS: 1 or 2. Other exemplary probes suitable for identifying the mutations and polymorphisms described herein can be designed using the information provided in Tables 3, 4 and 5 are encompassed within the scope of the invention. Also provided herein is a kit for practicing the methods disclosed herein. An exemplary kit comprises the diagnostic probes described above useful for identifying alterations in the NIPBL gene, reagents useful for nucleic acid hybridization, polymerase chain reaction or in situ hybridization and suitable instructional materials.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Facial features and limb findings in mutation positive individuals with CdLS. Note the variability of features even among individuals with similar mutation types.
FIG. 2. High-resolution linkage analysis of 5p13 in the 12 CdLS families. All microsatellite markers used are indicated on the left (distances between markers are indicated in FIG. 3). Haplotypes for each individual are indicated below the individual identification numbers. Paternal alleles are on the left and maternal alleles are on the right. Alleles shared by affected individuals are indicated by gray shading. Family XX allowed for refinement of the distal boundary to marker D5S477 and of the proximal boundary to marker to D5S1376. Marker D5S426 which gave the initial positive LOD score in the genome-wide scan lies between markers D5S674 and D5S2101. The gene lies between markers D5S1994 and D5S 2021 (indicated in red).
FIG. 3. Identifying NIPBL as the CdLS disease gene. FIG. 3a) Chromosome 5p13 critical region. Ideogram of the 5p13 critical region with microsatellite markers indicated above the ideogram. Distance of markers from the p-terminal arm of chromosome 5, in megabases, are indicated in brackets. Arrows indicate refined critical region after high-resolution analysis and identification of obligate recombination events. BAC clones used for FISH analysis are indicated by their RP11 addresses. FISH analysis allowed for further narrowing of the critical region as indicated by arrows below the ideogram. An expanded view of the critical region is depicted. The genes contained within the defined critical region are indicated (from the July 2003 UCSC genome browser). FIG. 3b) Fluorescence in situ hybridization (FISH) using BACs from within the linkage defined chromosome 5p13 critical region. In all panels the chromosome 5p telomeric control probe is labeled in green and the 5q telomeric control probe is labeled in red. In the first panel BAC RP11-252F20 is labeled in green. Both signals from BAC RP11-252F20 are on chromosome 5 p (arrows) indicating that it is proximal to the translocation breakpoint. In the middle panel BAC RP11-14121, that contains the NIPBL gene, is labeled in green. There is signal on both of the chromosome 5p arms as well as on chromosome 13q (arrows) indicating that the probe was split on the translocated chromosome. In the right hand panel BAC RP11-317123 is labeled in red. One signal is present on the normal chromosome 5p arm and the other is present on 13q indicating that this probe is distal to the translocation breakpoint.
FIG. 4. Northern Blot Analysis of NIPBL. Analysis of NIPBL expression in adult and fetal tissues. Probes from BX5381 78-specific (NIPBL exons 2 and 3), IDN3 specific (NIPBL exons 46 and 47), and the overlap region of these 2 transcripts (NIPBL exon 10) were used giving identical results (not all data shown). An  ̃6 Kb and 1.9 Kb transcript were seen on all blots. Expression was ubiquitous however much lower levels of expression were seen in brain, colon and spleen, and high levels of expression seen in heart, skeletal muscle and liver.
FIG. 5. Expression of NIPBL in the developing mouse. FIGS. 5a-c) Embyronic day 9.5, whole mount in situ hybridization; FIGS. 5d-i) Embryonic day 10.5, vibratome sections (200 μm) of embryos processed for whole mount in situ hybridization. FIGS. 5a, d, g) Sense control; FIGS. 5b, e, h) mNIPBL; FIGS. 5c, f, i) fgf8 (positive control). In FIG. 5b, mNIPBL expression is observed widely throughout the embryo, especially in the limb buds and branchial arches (arrow, fore limb bud; arrowhead, first branchial arch). In FIG. 5c, FGF8 expression marks a portion of the surface ectoderm of the same structures (arrow and arrowhead as in FIG. 5b). At E10.5, sections through the forelimb bud show that mNIPBL expression is concentrated in the mesenchyme (asterisk in FIG. 5e marks ventral limb bud mesenchyme; dorsal mesenchyme is also stained), whereas fgf8 expression marks the apical ectodermal ridge (arrow in FIG. 5f). No significant differences in intensity between fore- and hindlimb buds were observed (data not shown) Coronal sections at the level of the head show expression of mNIPBL in the mesenchyme of both the lateral and medial nasal processes (labeled L and M, respectively, in FIG. 5h). In contrast, fgf8 hybridization marks the ectoderm surrounding the developing nasal pit. Bar in FIG. 5a=0.5 mm for FIGS. 5a-c; bar in g=0.5 mm for FIGS. 5d-i. 
FIG. 6. Evolutionary conservation of amino acid residues altered by missense mutations in NIPBL. A comparison of amino acids and the flanking sequences altered by 11 of the unique missense mutations in human (NIPBL) (SEQ ID NO: 118), rat (SEQ ID NO: 119), mouse (SEQ ID NO: 119) and Drosophila (SEQ ID NO: 120) is depicted. The mutated amino acid residue is shaded grey. Amino acid residue 2298 was mutated in three individuals- two had an R2298H change, and one had an R2298C change. The missense mutation M1K in the initation codon is not depicted since it is conserved in all species.
FIGS. 7A1-7Z2. The genomic sequence of the NIPBL gene. (SEQ ID NO: 1)
FIG. 8. The cDNA sequence encoded by the NIPBL gene. (SEQ ID NO: 2)
FIG. 9. The amino acid sequence of the protein encoded by the NIPBL gene. (SEQ ID NO: 3).
FIG. 10. Metaphase spreads in individuals with CdLS and unaffected controls. FIG. 10A. control metaphase spread stained with giemsa. FIG. 10B. Control C-stained metaphase. FIGS. 10C-E. Metaphase spread from 3 individuals with CdLS stained with giemsa demonstrating PSCS. Note separated sister chromatids and centromeres present in practically all sister chromatids. FIGS. 10F-H. C-stained metaphases from individuals with CdLS demonstrating the premature division and separation of the centromeres in the majority of sister chromatids. I. A metaphase form an individual with CdLS demonstrating apparent chromatid breaks (arrowheads).



DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, it has been discovered that mutations in NIPBL cause CdLS. Types of mutations identified in NIPBL include, missense, splice site, nonsense, and frameshift. Severe protein truncating mutations likely lead to haploinsufficiency of the NIPBL protein. Haploinsufficiency of NIPBL has been documented as a disease mechanism through the report of a child with classic features of CdLS who was stillborn but found prenatally to have a large cytogenetically visible deletion of chromosome 5p13.1-14.2 (Hulinsky et al. 2003). This deletion would be predicted to encompass the NIPBL gene. To our knowledge, there has not been any other reported cases of constitutional deletions of this region. The hypothesis of germ-line mosaicism was also validated by the identification of the same NIPBL mutation in affected siblings born to unaffected mutation-negative parents (Krantz et al. 2004).
The prevalence of NIPBL mutations in a large CdLS population and the correlation of specific mutations with phenotypic characteristics have not previously been formally addressed. The systematic molecular and cytogenetic evaluation of 120 individuals with CdLS for disruptions in the NIPBL gene is described herein. Mutations in 47% of tested probands have been identified and this cohort has been further assessed for genotype-phenotype correlations.
NIPBL is the human homolog of the Drosophila Nipped-B gene. Although its function in mammalian systems has not yet been elucidated, sequence homologs of Nipped-B in yeast (Scc2 and Mis4) are required for sister chromatid cohesion during mitosis, and a similar role was recently demonstrated for Nipped-B in Drosophila. In order to evaluate NIPBL's role in sister chromatid cohesion in humans, metaphase spreads on 90 probands (40 NIPBL mutation positive and 50 NIPBL mutation negative) with CdLS were evaluated for evidence of precocious sister chromatid separation (PSCS). We screened 50 metaphases from each proband and found evidence of PSCS in 41% (compared to 9% in control samples). These studies indicate that NIPBL may play a role in sister chromatid cohesion in humans as has been reported for its homologs in Drosophila and yeast.
I. Methods of Diagnosis
The present invention provides methods of identifying patients having a variant allele of a gene associated with the CdLS phenotype. The gene (NIPBL) is located in human chromosome 5 in the region conventionally designated p13.1 by reference to cytological markers and DNA. Specifically, the gene is within a segment of about 7.4 Mb spaning 5p13.1-13.3 and flanked by markers D5S477 distally and D5S1376 proximally and contained 58 putative genes. An allele of the gene present in persons not suffering from CdLS is arbitrarily designated as wildtype. A variant allele of the gene is associated with a phenotype of CdLS. Such genetic variants include, without limitation, nucleotide additions, deletions or substitutions relative to the wildtype allele. These genetic alterations are associated with a phenotype of CdLS, as defined above (see Example 1) in at least some individuals bearing the variant allele. The phenotype may result from a nucleotide change in the gene (addition, deletion or substitution) affecting expression of the gene by altering the kinetics of expression or the nature of the resulting expression product. For example, some genetic changes reduce transcription or translation of an expression product. Other changes result in a polypeptide having altered properties (cf. the sickle cell mutation). Still other changes introduce a premature stop codon thereby resulting in truncated expression product.
The genetic tests of the present invention provide a highly accurate assay for diagnosing CdLS. Physicians having the correct diagnosis in hand can then ensure that patients receive prophylactic or therapeutic treatment appropriate to the genetic and biochemical features of the disease.
The methods may also be used to advantage for in utero screening of fetuses for the presence of a variant NIPBL allele. Identification of such variations offers the possibility of gene therapy. For couples known to be at risk of giving rise to affected progeny, diagnosis can be combined with in vitro reproduction procedures to identify an embryo having wildtype NIPBL alleles before implantation. Screening children shortly after birth is also of value in identifying those having the variant gene. Early detection allows administration of appropriate treatment.
A. Mode of Inheritance
Evidence reveals that a CdLS susceptibility gene can be inherited in an autosomal dominant fashion. In 6 of the 7 familial cases, the NIPBL mutation appears to have resulted from germline mosaicism.
B. Diagnosis from Linked Polymorphic Markers
The invention further provides methods of diagnosing susceptibility to CdLS by detection of polymorphic markers linked to the NIPBL gene on human chromosome 5. Markers are linked if they occur within 50 cM from each other or the NIPBL gene. Preferably, markers occur within 15 cM and more preferably within 5 or 1 cM of the gene. The closer the polymorphic marker is to NIPBL locus, the less likely there is to be physical recombination between the two loci at meiosis. The polymorphic marker is usually outside the NIPBL gene, but also may occur within the gene. All human chromosomes are subdivided into regions by cytological and polymorphic markers. Example 1 shows that preferred markers include those mapped between D5S477 and D5S1376. Thus, these markers and other markers within about 5 cM are preferred for use in the methods of the present invention. Most preferred are markers which occur within the NIPBL gene itself. The claimed methods are utilized to determine which alleles of a linked polymorphic marker are present in the patient being diagnosed. For example, if the polymorphic marker is an RFLP, the alleles differ in the size of a restriction fragment. The determination is typically made by PCR amplification of a segment spanning the polymorphism and gel analysis of the amplification product. If one of the alleles present in the patient is known to be in phase with a variant NIPBL locus (i.e., present on the same chromosome), it is concluded with a high probability that the patient has a variant NIPBL gene and will have CdLS. The closer linked the polymorphic marker to NIPBL, the higher the probability that the patient has received the variant NIPBL gene. See Sutherland & Mulley, Clinical Genetics 37:2-11 (1990). Preferably, the methods analyze the presence of alleles of two polymorphic markers spaced on either side of the NIPBL gene and both in phase with the gene. Absent a rare double recombination event, the presence of both alleles signals the presence of the variant NIPBL gene.
The method described above requires knowledge that a particular allele of a marker is in phase with the variant form of the NIPBL gene. This information is acquired from analyzing the phenotype and polymorphic content of relatives of the patient in a family, some of whose members exhibit CdLS. The linkage and/or phase determinations are usually performed before analysis of DNA from the patient.
A phase determination requires at least two relatives of the patient who are of known phenotype for CdLS, at least one of the relatives having the disease and being informative for the marker. In practice, a relative having the disease is screened at several polymorphic markers to identify at least one marker in which the relative is heterozygous. The phase of this marker is then set by determining which alleles of the marker are present in a second relative of known phenotype. Strategies for setting phase in different families are described by Lazarou, Clinical Genetics 43:150-156 (1993). For example, consider two siblings, X (with disease) having alleles 1 and 2 of a marker linked to the disease, and Y (without disease) having alleles 3 and 4. It can be concluded that in this family, the 1 and 2 alleles are in phase with the variant NIPBL gene. As a further example, consider X (with disease) having alleles 1 and 2 and Y (with disease) having alleles 1 and 5. It is deduced that the 1, 2 and 5 alleles are in phase with the variant gene. Within a family, the allele of a closely linked marker that is in phase with the variant gene is usually the same in each affected family member because there is a low probability of recombination between the two loci. The more closely related the relatives to the patient, the more likely phase is to be conserved between the relatives and the patient. Thus, it is preferred that one of the relatives used in setting phase is a parent or sibling of the patient. Once phase has been determined for a family, multiple members of the family can be diagnosed without repeating the analysis. In general, the phase relationship between an allele of a polymorphic marker and a variant allele of the NIPBL gene is different in each family. However, certain alleles may be in linkage disequilibrium with the NIPBL gene. For such markers, the same allele is likely to be in phase with the variant allele of the NIPBL gene in any family. Thus, once such an allele is identified it is not necessary to set phase in every family to be tested.
C. Direct Assays for NIPBL Gene
Having localized the NIPBL gene as described infra, variations can be detected by more direct methods. These methods represent a special case of the methods described above in which the polymorphic marker being detected is a variation arising within the NIPBL gene.
1. Detection of Uncharacterized Variations
Hitherto uncharacterized variations in the NIPBL gene are identified and localized to specific nucleotides by comparison of nucleic acids from an individual with CdLS with an unaffected individual, preferably a relative of the affected individual. Comparison with a relative is preferred because the possibility of other polymorphic differences between the patient and person being compared, not related to the CdLS phenotype, is lower. Various screening methods are suitable for this comparison including, but not limited to, direct DNA sequencing, single strand conformation polymorphism analysis (SSCP), conformation shift gel electrophoresis (CSGE), heteroduplex analysis (HA), chemical cleavage of mismatched sequences (CCMS), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography (dHPLC), ribonuclease cleavage, carbodiimide modification, and microarray analysis. See Cotton, Mutation Res. 285:125-144 (1993). Comparison can be initiated at either cDNA or genomic level. Initial comparison is often easier at the cDNA level because of its shorter size. Corresponding genomic changes are then identified by amplifying and sequencing a segment from the genomic exon including the site of change in the cDNA. In some instances, there is a simple relationship between genomic and cDNA changes. That is, a single base change in a coding region of genomic DNA gives rise to a corresponding changed codon in the cDNA. In other instances, the relationship between genomic and cDNA changes is more complex. Thus, for example, a single base change in genomic DNA creating an aberrant splice site can give rise to deletion of a substantial segment of cDNA.
2. Detection of Characterized Changes
The preceding methods serve to identify particular genetic changes responsible for CdLS. In a small number of families, affected members have the same change. However, individuals from different families appear to have different changes in the NIPBL gene. In contrast, in cystic fibrosis, about seventy percent of individuals have the same mutation in the CFTR gene. Once a change has been identified within a family, and/or as occurring within a population of affected individuals at a significant frequency, individuals can be tested for that change by various methods. A sample of such changes is provided in Tables 4 and 5. These methods include direct sequencing, allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and artificial introduction of extension sites (see Cotton, supra). For example, the allele-specific detection method uses one oligonucleotide exhibiting a perfect match to a target segment of the NIPBL gene having the change and a paired probe exhibiting a perfect match to the corresponding wildtype segment. If the individual is homozygous wildtype, only the wildtype probe binds. If the individual is a heterozygous variant, both probes bind. If the individual is a homozygous variant, only the variant probe binds. Paired probes for several variations can be immobilized as an array and the presence of several variations can thereby be analyzed simultaneously. Of course, the methods noted above, for analyzing uncharacterized variations can also be used for detecting characterized variations.
II. Identification of the NIPBL Gene
In accordance with the present invention, a human gene, NIPBL, has been discovered, which when mutated, gives rise to Cornelia de Lange syndrome. Genome-wide exclusion analysis was performed in 12 CdLS families resulting in the identification of 4 candidate regions, with chromosome 5p13.1 giving the highest multipoint LOD score of 2.7. Within this region, mutations in one gene, NIPBL, were identified in 4 sporadic and two familial CdLS cases. The gene is widely expressed in human tissues and is the human homolog of the Drosphila Nipped-B gene. The product of the Nipped-B gene is a facilitator of enhancer-promoter communication and plays a role in Notch signaling and other developmental pathways in Drosophila. 
III. Expression Systems
Identification of the NIPBL gene facilitates the production of the gene product. The cDNA fragment or any other nucleic acid encoding the NIPBL gene can be used to make an expression construct for the NIPBL gene. The expression construct typically comprises one or more nucleic acid sequences encoding the NIPBL gene operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous enhancer or promoter which is recognized by the host. The selection of an appropriate promoter, for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
The means of introducing the expression construct into a host cell varies depending upon the particular vector and targeted host cell. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra. A wide variety of host cells can be employed for expression of the NIPBL gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the NIPBL gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.
The NIPBL protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, N.Y. (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, N.Y. (1987); and Deutscher (ed), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.
The invention further provides transgenic nonhuman animals capable of expressing an exogenous NIPBL gene and/or having one or both alleles of an endogenous NIPBL gene inactivated. Expression of an exogenous NIPBL gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al., “Manipulating the Mouse Embryo, A Laboratory Manual,” Cold Spring Harbor Laboratory. Inactivation of endogenous NIPBL genes can be achieved by forming a transgene in which a cloned NIPBL gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244:1288-1292 (1989). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous NIPBL gene. Mice and other rodents are preferred animals. Such animals provide useful in vivo drug screening systems.
In addition to substantially full-length polypeptides expressed by the NIPBL gene, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the NIPBL gene product, including ligand binding, substrate for other molecules, dimer association, and the like. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
Polyclonal and/or monoclonal antibodies to the NIPBL gene product are also provided. Antibodies can be made by injecting mice or other animals with the NIPBL gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened by methods known in the art, as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1988), and Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with an epitope of the NIPBL gene product. These antibodies are useful in diagnostic assays for detection of the NIPBL gene product or a variant form thereof, or as an active ingredient in a pharmaceutical composition.
IV. Methods of Treatment
There are a number of drugs presently in use for treating the symptoms of CdLS. The present discovery that at least some subtypes of CdLS are associated with common genetic and presumably, biochemical features allows drug screening programs to be conducted in a group of patients having homogeneous disposition with respect to the NIPBL gene. Such a group is identified by the diagnostic methods discussed above. The provision of DNA encoding the NIPBL gene is also useful in developing new drugs and methods of treatment for CdLS. For example, variations in the NIPBL gene, including regulatory sequences, can be corrected by gene therapy. See Rosenberg, J. Clin. Oncol. 10:180-199 (1992). Gene therapy is preferably performed in utero rather than after birth, because of the undifferentiated nature of cells in a developing fetus. Exogenously supplied corrective genes integrate into the genomes of undifferentiated cells, and are subsequently distributed and expressed in entire tissues by the proliferation and differentiation of the ancestor cell.
The provision of the NIPBL gene product also allows screening for molecules that interact with the same and design of agents that agonize or antagonize this interaction. Such agents include monoclonal antibodies against the NIPBL gene product, fragments of the NIPBL gene product that compete with the full-length protein for binding, and synthetic peptides or analogs thereof selected from random combinatorial libraries. See, e.g., Ladner et al., U.S. Pat. No. 5,223,409 (1993) (incorporated by reference in its entirety herein). Therapeutic agents also includes transcription factors, and the like, which stimulate expression of the NIPBL gene.
V. Diagnostic Kits
The present invention also includes kits for the practice of the methods of the invention. The kits comprise a vial, tube, or any other container which contains one or more oligonucleotides or diagnostic probes, which hybridizes to a DNA segment within chromosome 5p13, which DNA segment is linked to the NIPBL gene. Preferably, the oligonucleotide hybridizes to a segment of chromosome 5 between markers D5S477 and D5S1376. Most preferably, the diagnostic probes will hybridize to at least one DNA molecule comprising an alteration as described in Table 4. Preferably, additional probes will be employed to identify a polymorphism set forth in Table 5. Some kits contain two such oligonucleotides, which serve as primers to amplify a segment of chromosome DNA. The segment selected for amplification can be a polymorphic marker linked to the NIPBL gene or a region from the NIPBL gene that includes a site at which a variation is known to occur. See Tables 3, 4 and 5. Some kits contain a pair of oligonucleotides for detecting precharacterized variations. For example, some kits contain oligonucleotides suitable for allele-specific oligonucleotide hybridization, or allele-specific amplification hybridization. The kits of the invention may also contain components of the amplification system, including PCR reaction materials such as buffers and a thermostable polymerase. In other embodiments, the kit of the present invention can be used in conjunction with commercially available amplification kits, such as may be obtained from GIBCO BRL (Gaithersburg, Md.) Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.), Schleicher & Schuell (Keene, N.H.), Boehringer Mannheim (Indianapolis, Ind.). The kits may optionally include positive or negative control reactions or markers, molecular weight size markers for gel electrophoresis, and the like. The kits usually include labelling or instructions indicating the suitability of the kits for diagnosing CdLS and indicating how the oligonucleotides are to be used for that purpose. The term “label” is used generically to encompass any written or recorded material that is attached to, or otherwise accompanies the diagnostic at any time during its manufacture, transport, sale or use.
Kits for performing PSCS assays may contain reagents suitable for isolating blood or lymphoblastoid cells from CdLS patients and culture media and reagents which promote the growth and viability of such cells. Such kits may also contain reagents (e.g., giemsa stain) for generating metaphase spreads from cells so cultured.
MODES OF PRACTICING THE INVENTION
1. Mutational Analysis/Conformation Sensitive Gel Electrophoresis (CSGE).
Conformation sensitive gel electrophoresis (CSGE) was carried out using standard protocols. See Examples I and II. Oligonucleotide primer sequences and PCR conditions used for amplification of all exons of the NIPBL gene are provided herein. PCR products corresponding to all altered migration patterns (shifts) were purified using QIAquick® PCR purification kit, QIAGEN Sciences) and sequenced on an ABI 377 sequencer.
2. Linkage Analysis
Determining linkage between a polymorphic marker and a locus associated with a particular phenotype is performed by mapping polymorphic markers and observing whether they co-segregate with the CdLS phenotype on a chromosome in an informative meiosis. See, e.g., Kerem et al., Science 245:1073-1080 (1989); Monaco et al., Nature 316:842 (1985); Yamoka et al., Neurology 40:222-226 (1990), and as reviewed in Rossiter et al., FASEB Journal 5:21-27 (1991). A single pedigree rarely contains enough informative meioses to provide definitive linkage, because families are often small and markers may be not sufficiently informative. For example, a marker may not be polymorphic in a particular family.
Linkage may be established by an affected sib-pairs analysis as described in Terwilliger & Ott, Handbook of Human Genetic Linkage (Johns Hopkins, Md., 1994), Ch. 26. This approach requires no assumptions to be made concerning penetrance or variant frequency, but only takes into account the data of a relatively small proportion (i.e., the SIB pairs) of all the family members whose phenotype and polymorphic markers have been determined. Specifically, the affected SIB pairs analysis scores each pair of affected SIBS as sharing (concordant) or not sharing (discordant) the same allelic variant of each polymorphic marker. For each marker, a probability is then calculated that the observed ratio of concordant to discordant SIB pairs would arise without linkage of the marker.
As described in Thompson & Thompson, Genetics in Medicine, 5th ed, 1991, W.B. Saunders Company, Philadelphia, in linkage analysis, one calculates a series of likelihood ratios (relative odds) at various possible values of 0, ranging from θ=0.0 (no recombination) to θ=0.50 (random assortment). Thus, the likelihood ratio at a given value of θ is (likelihood of data if aloci are linked at θ)/(likelihood of data if loci are unlinked). Evidence in support of linkage is usually expressed as the log10 of this ratio and called a “lod score” for “logarithm of the odds.” For example, a lod score of 5 indicates 100,000:1 odds that the linkage being observed did not occur by chance. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of θ. Available programs include LIPED, and MLINK (Lathrop, Proc. Nat. Acad. Sci. 81:3443-3446 (1984).
For any particular lod score, a recombination fraction may be determined from mathematical tables. See Smith et al., Mathematical tables for research workers in human genetics (Churchill, London, 1961) and Smith, Ann. Hum. Genet. 32:127-150 (1968). The value of θ at which the lod score is the highest is considered to be the best estimate of the recombination fraction, the “maximum likelihood estimate”.
Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of θ) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked. Similarly, by convention, a negative lod score of −2 or less is taken as definitive evidence against linkage of the two loci being compared. If there are sufficient negative linkage data, a locus can be excluded from an entire chromosome, or a portion thereof, a process referred to as exclusion mapping. The search is then focused on the remaining non-excluded chromosomal locations. For a general discussion of lod scores and linkage analysis, see, e.g., T. Strachan, Chapter 4, “Mapping the human genome” in The Human Genome, 1992 BIOS Scientific Publishers Ltd. Oxford.
The data can also be subjected to haplotype analysis. This analysis assigns allelic markers between the chromosomes of an individual such that the number of recombinational events needed to account for segregation between generations is minimized. Linkage may also be established by determining the relative likelihood of obtaining observed segregation data for any two markers when the two markers are located at a recombination fraction θ, versus the situation in which the two markers are not linked, and thus segregating independently.
3. Isolation and Amplification of DNA
Samples of patient, proband or family member genomic DNA is isolated from any convenient source including saliva, buccal cells, hair roots, blood, cord blood, amniotic fluid, interstitial fluid, peritoneal fluid, chorionic villus, and any other suitable cell or tissue sample with intact interphase nuclei or metaphase cells. The cells can be obtained from solid tissue as from a fresh or preserved organ or from a tissue sample or biopsy. The sample can contain compounds which are not naturally intermixed with the biological material such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
Methods for isolation of genomic DNA from these various sources are described in, for example, Kirby, DNA Fingerprinting, An Introduction, W.H. Freeman & Co. New York (1992). Genomic DNA can also be isolated from cultured primary or secondary cell cultures or from transformed cell lines derived from any of the aforementioned tissue samples.
Samples of patient, proband or family member RNA can also be used. RNA can be isolated from tissues expressing the NIPBL gene as described in Sambrook et al., supra. RNA can be total cellular RNA, mRNA, poly A+ RNA, or any combination thereof. For best results, the RNA is purified, but can also be unpurified cytoplasmic RNA. RNA can be reverse transcribed to form DNA which is then used as the amplification template, such that the PCR indirectly amplifies a specific population of RNA transcripts. See, e.g., Sambrook, supra, Kawasaki et al., Chapter 8 in PCR Technology, (1992) supra, and Berg et al., Hum. Genet. 85:655-658 (1990).
4. PCR Amplification
The most common means for amplification is polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,965,188 each of which is hereby incorporated by reference. If PCR is used to amplify the target regions in blood cells, heparinized whole blood should be drawn in a sealed vacuum tube kept separated from other samples and handled with clean gloves. For best results, blood should be processed immediately after collection; if this is impossible, it should be kept in a sealed container at 4° C. until use. Cells in other physiological fluids may also be assayed. When using any of these fluids, the cells in the fluid should be separated from the fluid component by centrifugation.
Tissues should be roughly minced using a sterile, disposable scalpel and a sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures for removing paraffin from tissue sections are described in a variety of specialized handbooks well known to those skilled in the art.
To amplify a target nucleic acid sequence in a sample by PCR, the sequence must be accessible to the components of the amplification system. One method of isolating target DNA is crude extraction which is useful for relatively large samples. Briefly, mononuclear cells from samples of blood, amniocytes from amniotic fluid, cultured chorionic villus cells, or the like are isolated by layering on sterile Ficoll-Hypaque gradient by standard procedures. Interphase cells are collected and washed three times in sterile phosphate buffered saline before DNA extraction. If testing DNA from peripheral blood lymphocytes, an osmotic shock (treatment of the pellet for 10 sec with distilled water) is suggested, followed by two additional washings if residual red blood cells are visible following the initial washes. This will prevent the inhibitory effect of the heme group carried by hemoglobin on the PCR reaction. If PCR testing is not performed immediately after sample collection, aliquots of 106 cells can be pelleted in sterile Eppendorf tubes and the dry pellet frozen at −20° C. until use.
The cells are resuspended (106 nucleated cells per 100 μl) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KCl 1.5 mM MgCl2, 0.5% Tween 20, 0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubating at 56° C. for 2 hr, the cells are heated to 95° C. for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool). If gross aggregates are present, another cycle of digestion in the same buffer should be undertaken. Ten μl of this extract is used for amplification. When extracting DNA from tissues, e.g., chorionic villus cells or confluent cultured cells, the amount of the above mentioned buffer with proteinase K may vary according to the size of the tissue sample. The extract is incubated for 4-10 hrs at 50°-60° C. and then at 95° C. for 10 minutes to inactivate the proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration.
When the sample contains a small number of cells, extraction may be accomplished by methods as described in Higuchi, “Simple and Rapid Preparation of Samples for PCR”, in PCR Technology, Ehrlich, H. A. (ed.), Stockton Press, N.Y., which is incorporated herein by reference. PCR can be employed to amplify target regions chromosome 1 in very small numbers of cells (1000-5000) derived from individual colonies from bone marrow and peripheral blood cultures. The cells in the sample are suspended in 20 μl of PCR lysis buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use. When PCR is to be performed, 0.6 μl of proteinase K (2 mg/ml) is added to the cells in the PCR lysis buffer. The sample is then heated to about 60° C. and incubated for 1 hr. Digestion is stopped through inactivation of the proteinase K by heating the samples to 95° C. for 10 min and then cooling on ice.
A relatively easy procedure for extracting DNA for PCR is a salting out procedure adapted from the method described by Miller et al., Nucleic Acids Res. 16:1215 (1988), which is incorporated herein by reference. Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na2 EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase K and 150 μl of a 20% SDS solution are added to the cells and then incubated at 37° C. overnight. Rocking the tubes during incubation will improve the digestion of the sample. If the proteinase K digestion is incomplete after overnight incubation (fragments are still visible), an additional 50 μl of the 20 mg/ml proteinase K solution is mixed in the solution and incubated for another night at 37° C. on a gently rocking or rotating platform. Following adequate digestion, one ml of a 6M NaCl solution is added to the sample and vigorously mixed. The resulting solution is centrifuged for 15 minutes at 3000 rpm. The pellet contains the precipitated cellular proteins, while the supernatant contains the DNA. The supernatant is removed to a 15 ml tube that contains 4 ml of isopropanol. The contents of the tube are mixed gently until the water and the alcohol phases have mixed and a white DNA precipitate has formed. The DNA precipitate is removed and dipped in a solution of 70% ethanol and gently mixed. The DNA precipitate is removed from the ethanol and air-dried. The precipitate is placed in distilled water and dissolved.
Kits for the extraction of high-molecular weight DNA for PCR include a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, La Jolla, Calif.), TurboGen Isolation Kit (Invitrogen, San Diego, Calif.), and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the present invention.
The concentration and purity of the extracted DNA can be determined by spectrophotometric analysis of the absorbance of a diluted aliquot at 260 nm and 280 nm. After extraction of the DNA, PCR amplification may proceed. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
In a particularly useful embodiment of PCR amplification, strand separation is achieved by heating the reaction to a sufficiently high temperature for an sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Pat. No. 4,965,188, incorporated herein by reference). Typical heat denaturation involves temperatures ranging from about 80° C. to 105° C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, the enzyme RecA has helicase activity in the presence of ATP. The reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference).
Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering systems. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis.
In some cases, the target regions may encode at least a portion of a protein expressed by the cell. In this instance, mRNA may be used for amplification of the target region. Alternatively, PCR can be used to generate a cDNA library from RNA for further amplification, the initial template for primer extension is RNA. Polymerizing agents suitable for synthesizing a complementary, copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Thermus thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer Cetus, Inc. Typically, the genomic RNA template is heat degraded during the first denaturation step after the initial reverse transcription step leaving only DNA template. Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus and commercially available from Perkin Elmer Cetus, Inc. The latter enzyme is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for using Taq polymerase are known in the art and are described in Gelfand, 1989, PCR Technology, supra.
5. Allele Specific PCR
Allele-specific PCR differentiates between chromosome 5 target regions differing in the presence or absence of a variation or polymorphism. PCR amplification primers are chosen which bind only to certain alleles of the target sequence. Thus, for example, amplification products are generated from those chromosome 5 sets which contain the primer binding sequence, and no amplification products are generated in chromosome 5 sets without the primer binding sequence. This method is described by Gibbs, Nucleic Acid Res. 17:12427-2448 (1989).
6. Allele Specific Oligonucleotide Screening Methods
Further diagnostic screening methods employ the allele-specific oligonucleotide (ASO) screening methods, as described by Saiki et al., Nature 324:163-166 (1986). Oligonucleotides with one or more base pair mismatches are generated for any particular allele. ASO screening methods detect mismatches between variant target genomic or PCR amplified DNA and non-mutant oligonucleotides, showing decreased binding of the oligonucleotide relative to a mutant oligonucleotide. Oligonucleotide probes can be designed that under low stringency will bind to both polymorphic forms of the allele, but which at higher stringency, bind to the allele to which they correspond. Alternatively, stringency conditions can be devised in which an essentially binary response is obtained, i.e., an ASO corresponding to a variant form of the NIPBL gene will hybridize to that allele, and not to the wildtype allele.
7. Ligase Mediated Allele Detection Method
Target regions of a patients can be compared with target regions in unaffected and affected family members by ligase-mediated allele detection. See Landegren et al., Science 241:1077-1080 (1988). Ligase may also be used to detect point mutations in the ligation amplification reaction described in Wu et al., Genomics 4:560-569 (1989). The ligation amplification reaction (LAR) utilizes amplification of specific DNA sequence using sequential rounds of template dependent ligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990).
8. Denaturing Gradient Gel Electrophoresis
Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. DNA molecules melt in segments, termed melting domains, under conditions of increased temperature or denaturation. Each melting domain melts cooperatively at a distinct, base-specific melting temperature (Tm). Melting domains are at least 20 base pairs in length, and may be up to several hundred base pairs in length.
Differentiation between alleles based on sequence specific melting domain differences can be assessed using polyacrylamide gel electrophoresis, as described in Chapter 7 of Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W.H. Freeman and Co, N.Y. (1992), the contents of which are hereby incorporated by reference.
Generally, a target region to be analyzed by denaturing gradient gel electrophoresis is amplified using PCR primers flanking the target region. The amplified PCR product is applied to a polyacrylamide gel with a linear denaturing gradient as described in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139 (1988), the contents of which are hereby incorporated by reference. The electrophoresis system is maintained at a temperature slightly below the Tm of the melting domains of the target sequences.
In an alternative method of denaturing gradient gel electrophoresis, the target sequences may be initially attached to a stretch of GC nucleotides, termed a GC clamp, as described in Chapter 7 of Erlich, supra. Preferably, at least 80% of the nucleotides in the GC clamp are either guanine or cytosine. Preferably, the GC clamp is at least 30 bases long. This method is particularly suited to target sequences with high Tm's. Generally, the target region is amplified by the polymerase chain reaction as described above. One of the oligonucleotide PCR primers carries at its 5′ end, the GC clamp region, at least 30 bases of the GC rich sequence, which is incorporated into the 5′ end of the target region during amplification. The resulting amplified target region is run on an electrophoresis gel under denaturing gradient conditions as described above. DNA fragments differing by a single base change will migrate through the gel to different positions, which may be visualized by ethidium bromide staining.
9. Temperature Gradient Gel Electrophoresis
Temperature gradient gel electrophoresis (TGGE)is based on the same underlying principles as denaturing gradient gel electrophoresis, except the denaturing gradient is produced by differences in temperature instead of differences in the concentration of a chemical denaturant. Standard TGGE utilizes an electrophoresis apparatus with a temperature gradient running along the electrophoresis path. As samples migrate through a gel with a uniform concentration of a chemical denaturant, they encounter increasing temperatures. An alternative method of TGGE, temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses a steadily increasing temperature of the entire electrophoresis gel to achieve the same result. As the samples migrate through the gel the temperature of the entire gel increases, leading the samples to encounter increasing temperature as they migrate through the gel. Preparation of samples, including PCR amplification with incorporation of a GC clamp, and visualization of products are the same as for denaturing gradient gel electrophoresis.
10. Single-Strand Conformation Polymorphism Analysis
Target sequences or alleles at the NIPBL locus can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86:2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. Thus, electrophoretic mobility of single-stranded amplification products can detect base-sequence difference between alleles or target sequences.
11. Chemical or Enzymatic Cleavage of Mismatches
Differences between target sequences can also be detected by differential chemical cleavage of mismatched base pairs, as described in Grompe et al., Am. J. Hum. Genet. 48:212-222 (1991). In another method, differences between target sequences can be detected by enzymatic cleavage of mismatched base pairs, as described in Nelson et al., Nature Genetics 4:11-18 (1993). Briefly, genetic material from a patient and an affected family member may be used to generate mismatch free heterohybrid DNA duplexes. As used herein, “heterohybrid” means a DNA duplex strand comprising one strand of DNA from one person, usually the patient, and a second DNA strand from another person, usually an affected or unaffected family member. Positive selection for heterohybrids free of mismatches allows determination of small insertions, deletions or other polymorphisms that may be associated with CdLS.
12. Non-PCR Based DNA Diagnostics
The identification of a DNA sequence linked to NIPBL can made without an amplification step, based on polymorphisms including restriction fragment length polymorphisms in a patient and a family member. Hybridization probes are generally oligonucleotides which bind through complementary base pairing to all or part of a target nucleic acid. Probes typically bind target sequences lacking complete complementarity with the probe sequence depending on the stringency of the hybridization conditions. The probes are preferably labeled directly or indirectly, such that by assaying for the presence or absence of the probe, one can detect the presence or absence of the target sequence. Direct labeling methods include radioisotope labeling, such as with 32p or 35S. Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags. Visual detection methods include photoluminescents, Texas red, rhodamine and its derivatives, red leuco dye and 3,3′,5,5′-tetramethylbenzidine (TMB), fluorescein, and its derivatives, dansyl, umbelliferone and the like or with horse radish peroxidase, alkaline phosphatase and the like.
Hybridization probes include any nucleotide sequence capable of hybridizing to the 5p13 region of chromosome 5, and thus defining a genetic marker linked to NIPBL, including a restriction fragment length polymorphism, a hypervariable region, repetitive element, or a variable number tandem repeat. Hybridization probes can be any gene or a suitable analog. Further suitable hybridization probes include exon fragments or portions of cDNAs or genes known to map to the p13 region of chromosome 5. Other suitable probes include portions of introns or intron/exon spanning regions from genomic fragments of chromosome 5, or portions of spacer DNA, i.e., DNA between genes that is not intronic.
Preferred tandem repeat hybridization probes for use according to the present invention are those that recognize a small number of fragments at a specific locus at high stringency hybridization conditions, or that recognize a larger number of fragments at that locus when the stringency conditions are lowered.
The following definitions are provided to facilitate an understanding of the present invention:
Cornelia de Lange Syndrome refers to a dominantly inherited disorder with characteristic facial appearance, limb defects and growth and cognitive retardation.
The term “LOD score” refers to a number used in genetic linkage studies; logarithm (base 10) of the odds in favor of genetic linkage.
The term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term “complementary to” is used herein to mean that the complementary sequences is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”. Hybridization probes may be DNA or RNA, or any synthetic nucleotide structure capable of binding in a base-specific manner to a complementary strand of nucleic acid. For example, probes include peptide nucleic acids, as described in Nielsen et al., Science 254:1497-1500 (1991).
“Linkage” describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and is measured by percent recombination (also called recombination fraction, or θ) between the two genes, alleles, loci or genetic markers.
“Centimorgan” is a unit of genetic distance signifying linkage between two genetic markers, alleles, genes or loci, corresponding to a probability of recombination between the two markers or loci of 1% for any meiotic event.
“Linkage disequilibrium” or “allelic association” means the preferential association of a particular allele, locus, gene or genetic marker with a specific allele, locus, gene or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population.
An “oligonucleotide” can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means.
The term “primer” refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term “primer” may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding one or both ends of the target region to be amplified. For instance, if a region shows significant levels of polymorphism or mutation in a population, mixtures of primers can be prepared that will amplify alternate sequences. A primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in an ELISA), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. A label can also be used to “capture” the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
“Penetrance” is the percentage of individuals with a defective gene who show some symptoms of a trait resulting from that defect. Expressivity refers to the degree of expression of the trait (e.g., mild, moderate or severe).
“Polymorphism” refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%. A polymorphic locus may be as small as one base pair. Polymorphic markers suitable for use in the invention include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, and other microsatellite sequences.
“Restriction fragment length polymorphism” (RFLP) means a variation in DNA sequence that alters the length of a restriction fragment as described in Botstein et al., Am. J. Hum. Genet. 32:314-331 (1980). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. For example, the DNA sequence GAATTC are the six bases, together with its complementary strand CTTAAG which comprises the recognition and cleavage site of the restriction enzyme EcoRI. Replacement of any of the six nucleotides on either strand of DNA to a different nucleotide destroys the EcoRI site. This RFLP can be detected by, for example, amplification of a target sequence including the polymorphism, digestion of the amplified sequence with EcoRI, and size fractionation of the reaction products on an agarose or acrylamide gel. If the only EcoRI restriction enzyme site within the amplified sequence is the polymorphic site, the target sequences comprising the restriction site will show two fragments of predetermined size, based on the length of the amplified sequence. Target sequences without the restriction enzyme site will only show one fragment, of the length of the amplified sequence. Similarly, the RFLP can be detected by probing an EcoRI digest of Southern blotted DNA with a probe from a nearby region such that the presence or absence of the appropriately sized EcoRI fragment may be observed. RFLP's may be caused by point mutations which create or destroy a restriction enzyme site, VNTR's, dinucleotide repeats, deletions, duplications, or any other sequence-based variation that creates or deletes a restriction enzyme site, or alters the size of a restriction fragment.
“Variable number of tandem repeats” (VNTR's) are short sequences of nucleic acids arranged in a head to tail fashion in a tandem array, and found in each individual, as described in Wyman et al., Proc. Nat. Acad. Sci. 77:6754-6758 (1980). Generally, the VNTR sequences are comprised of a core sequence of at least 16 base pairs, with a variable number of repeats of that sequence. Additionally, there may be variation within the core sequence, Jefferys et al., Nature 314:67-72 (1985). These sequences are highly individual, and perhaps unique to each individual. Thus, VNTR's may generate restriction fragment length polymorphisms, and may additionally serve as size-based amplification product differentiation markers. “Microsatellite sequences” comprise segments of at least about 10 base pairs of DNA consisting of a variable number of tandem repeats of short (1-6 base pairs) sequences of DNA(Clemens et al., Am. J. Hum. Genet. 49:951-960 1991). Microsatellite sequences are generally spread throughout the chromosomal DNA of an individual. The number of repeats in any particular tandem array varies greatly from individual to individual, and thus, microsatellite sequences may serve to generate restriction fragment length polymorphisms, and may additionally serve as size-based amplification product differentiation markers.
A “marker” is referred to as fully “informative” for a particular individual if the configuration of alleles observed in the family allow for the unambiguous determination of parental origin of the alleles of a child. For example, if the mother has a “1” and “2” allele, while the father has a “3” and “4” allele, then it is possible to unambiguously assign the parental origin of alleles in each of the four possible combinations in the children (1-3, 1-4, 2-3, 2-4). A marker is partially informative when unambiguous determination of parental origin is possible for only certain children. For example, if both parents have a “1” and “2” allele, then the parental origins of the alleles may be unambiguously determined for children with the genotypes 1-1 and 2-2, but not for the children with the genotype 1-2. If one parent is homozygous for a marker, the marker will be only partially informative, and the inheritance from that parent cannot be traced. If the marker is homozygous in both parents, the marker is fully uninformative for the transmission from them to their children, even though their children may be heterozygous and thus informative for the transmission of that marker to the next generation.
A “mutation” is any alteration in the NIPBL gene which alters the function or expression the NIPBL gene product.
The following examples are provided to illustrate embodiments of the present invention. They are not intended to limit the invention in any way.
EXAMPLE 1
Characterization of NIPBL Expression
As mentioned above, the Cornelia de Lange syndrome (CdLS [MIM #122470]) is a multisystem developmental disorder characterized by facial dysmorphia, upper extremity malformations, hirsutism, cardiac defects, growth and cognitive retardation, and gastrointestinal abnormalities. Both missense and protein truncating mutations in NIPBL, the human homolog of the Drosophila Nipped-B gene, have recently been reported to cause CdLS. See U.S. Provisional Application No. 60/567,756 incorporated by reference herein.
The Drosophila Nipped-B protein facilitates long-range enhancer-promoter interactions and plays a role in Notch signaling and other developmental pathways as well as being involved in mitotic sister chromatid cohesion. The present example describes the spectrum and distribution of NIPBL mutations in a large, well-characterized cohort of individuals with CdLS. Mutations were found in 56 of 120 (47%) unrelated individuals with sporadic or familial CdLS. Statistically significant phenotypic differences between mutation-positive and mutation-negative individuals were identified. Analysis also suggested a trend towards a milder phenotype in individuals with missense mutations as compared to other types of mutations.
The materials and methods set forth below are provided to facilitate the practice of Examples 1, 2 and 3.
Cornelia de Lange Syndrome Patients.
All patients and family members were enrolled in the study under an IRB-approved protocol of informed consent at The Children's Hospital of Philadelphia. Clinical dysmorphologists (I.D.K., A.D.K. and/or L.G.J.) with experience with CdLS evaluated all subjects. Clinical histories and photographs were obtained routinely for all probands, as well as for any other affected family members. Clinical records were reviewed for the presence of other CdLS-associated anomalies, such as: deafness, cleft palate, cardiac, ophthalmologic, gastrointestinal, genitourinary, and renal anomalies. For the purposes of the genotype-phenotype studies only probands were included and not affected family members. This may result in a bias towards the more severe phenotype, however as familial recurrences are extremely rare it was not possible to perform a separate analysis on the small number of affected family members. While all probands had characteristic facial features as part of their inclusion criteria into the study, we chose to further stratify the CdLS cohort based on the severity of three phenotypic parameters: limb differences, growth, and cognitive functioning (summarized in Table 1).
Limb malformations were classified by the presence or absence of reduction defects in the upper extremities as follows: Class I: mild, no reduction defect; Class II: moderate, partial reduction defect/oligodactyly (>2 digits on each hand); Class III: severe, reduction defect (≦2 digits on either hand). A score for severity of the physical growth parameters was calculated by averaging the percentiles for weight, height, and head circumference that were plotted on sex- and age-standardized growth curves for individuals with CdLS (Kline et al. 1993a). Growth parameters were classified as follows: Class I: mild, average growth parameters >75th centile on CdLS growth curves; Class II: moderate, average growth parameters 25th-75th centile on CdLS growth curves; Class III: severe, average growth parameters <25th centile on the CdLS growth curves. Cognitive functioning was the most difficult parameter to standardize as most individuals with CdLS enrolled in the study had not received formal developmental evaluations as well as the inherent difficulty of comparing developmental abilities in individuals of varying ages. We used a classification of developmental/cognitive abilities based on deviation from age-appropriate standards as follows: Class I: mild, motor milestones less than 2 years delayed from normal standards, development of speech and communication skills in older individuals; Class II: moderate, delay in reaching motor milestones greater than 2 years behind normal developmental standards, limited speech and communication; Class III: severe to profound delay in achieving motor milestones and without meaningful communication. Clinical stratification of all probands was performed without knowledge of mutational status.







TABLE 1







Phenotypic Classifications











1 (Mild)
2 (Moderate)
3 (Severe)














Limb Reduction
No reduction
Partial reduction
Severe reduction



defect
defect,
defect (≦2 digits on




oligodactyly (>2
either hand)




digits on each




hand)


Development and
Motor milestones <
Motor milestones >
Profound delay in


Cognitive Abilities
2 yrs delayed;
2 yrs delayed;
achieving motor



speech and
limited speech and
milestones and lack



communication
communication
of meaningful



skills present

communication


Growth (Average of
>75th percentile
>25th and <75th
<25th percentile


Percentiles for Weight,

percentile


Height, & Head


Circumference Plotted


on CdLS Standard


Growth Curves)









Genome-Wide Linkage Analysis.

Linkage studies were performed using the ABI linkage mapping set version 2 consisting of 400 fluorescently-labeled polymorphic markers spaced at approximately 10 cM intervals throughout the genome. Marker allele frequencies used in the lod-score analysis were estimated based on alleles observed in the families' founders. Model based two point and multipoint linkage analysis on data from the whole genome scan and from the fine mapping of chromosomes 2, 5, 10, and 14 in all families were carried out by means of the GENEHUNTER computer program version 2.0 (GH2) (Kruglyak et al. 1996). For the purpose of lod-score analysis, we assumed the disease to follow an autosomal dominant mode of inheritance with disease allele frequency of 0.00001. In order to account for the possibility that the disease in families with unaffected parents was due to germline mosaicism in one of the parents, all unaffected individuals (parents and sibs) that were available for genotyping were coded as unknown at the disease phenotype. In this way, we did not have to make any assumption about the unknown penetrance of the putative CdLS gene mutation. However, marker genotype information from unaffected sibs was retained, when available, and used to reconstruct phase for haplotyping. Marker maps used in multipoint linkage analysis were sex averaged genetic maps from the Center for Medical Genetics of the Marshfield Clinic Research Foundation.
Fluorescence In Situ Hybridization (FISH) Analysis
Fluorescence in situ hybridization (FISH) studies were performed on metaphase chromosomes prepared from peripheral blood lymphocytes using standard techniques (Krantz et al. 1997). FISH was performed with the NIPBL-containing BAC RP1 1-14121 (ACO18853.3) (CHORI BACPAC Resources, Oakland, Calif.) on 28 mutation-negative individuals (4 familial, 24 sporadic) to evaluate for the possibility of a large but submicroscopic deletion encompassing the NIPBL gene. BAC DNA was isolated (Perfect Prep Plasmid XL, Eppendorf, Hamburg, Germany) and labeled by nick translation in the presence of Spectrum Red dUTP (Vysis, Downers Grove, Ill.). The labeled BAC probe was dissolved in LSI/WCP Hybridization Buffer (Vysis, Downers Grove, Ill.); 10 μg of Human Cot-I DNA® (Invitrogen Corp., Carlsbad, Calif.) was added per 1 μg of labeled BAC RP1 1-14121 probe. TelVysion® Probe 5p and/or 5q (Vysis, Downers Grove, Ill.) (as per manufacturer instructions) and 100 ng of labeled BAC probe per micoscope slide were co-denatured under a coverslip for 2 min on a 75° C. slide warmer and hybridized at 37° C. for  ̃16 hours in a humid chamber. Slides were subjected to two post hybridization washes; wash one (0.4×SSC, 0.3% NP-40) at 73° C. for 2 minutes and wash two (2×SSC, 0.1% NP-40) at room temperature for 1 minute, and counter stained with DAPI II (Vysis, Downers Grove, Ill.). A Nikon microscope, equipped with the appropriate filters, was used to visualize each slide. CytoVision® application software version 3.1 build 10 (Applied Imaging, Santa Clara, Calif.) and a CCD camera were used to capture FISH images.
In Situ Hybridization in the Developing Mouse
A probe to mouse NIPBL was generated by PCR from an EST clone using oligonucleotide primers 5′-CCGCTCGAGGATTCAAACGCTTCATCA-3′(SEQ ID NO: 4) and 5′-AGGATGGGAATATGGCATGTA-3′ (SEQ ID NO:5), which yielded a 389 bp product corresponding to the mouse homologue of the last 190 bp of exon 10 and all of exon 11 of human NIPBL. This was subcloned into pCRII-TOPO (Invitrogen) for generation of antisense and sense digoxigenin-labeled cRNA probes. An Fgf8 probe (positive control) was generated from a 422bp NcoI-PstI fragment of the Fgf8 cDNA (bp 59-481 of GenBank Z48746) cloned into pBluescript. CD-1 (Charles River) mouse embryos were dissected at 9.5 and 10.5 days of gestation, and fixed and processed for whole mount in situ hybridization, with detection using alkaline phosphatase-conjugated, sheep-anti-digoxigenin antibodies, and BCIP-NBT as the chromagenic substrate (Kawauchi et al. 1999).
Northern Blot Analysis.
Poly A+ RNA Northern blots of multiple adult human tissues (Human 12-Lane Multiple Tissue Northern (MTNTM) Blot BD Biosciences Clontech) and human fetal tissues (MessageMapTM Northern Blot, Stratagene) were hybridized with a 301 base pair probe from BX5381 78-specific cDNA sequence (NIPBL exon 2 and 3) (amplified from the following primers: forward: TGTTTGGGAAATGGGAAGTAA (SEQ ID NO: 6), reverse: TGTTGATACCTGGTTGAGGCTA (SEQ ID NO:7)) a 344 base pair probe from IDN3-specific cDNA sequence (NIPBL exon 46 and 47) (amplified from the following primers: forward: GAAGAAGGGGAGGTTTCAGC (SEQ ID NO: 8), reverse: GTCCAGGAGCCACTGTAGGA (SEQ ID NO:9)), and a 252 base pair probe from a region of overlap between the 2 putative transcripts (NIPBL exon 10) (amplified from the following primers: forward: TGAGAGCAGAACAACTGAATGC (SEQ ID NO:10), reverse: TGGCTTTCCAGAATCCCTCC (SEQ ID NO:11)). BD SpotLightTM Random Primer Labeling Kit (BD Bioscience Clontech) was used for labeling probes and SpotLightTM Chemiluminescent Hybridization & Detection Kit (BD Bioscience Clontech) was used for hybridization and visualization. Experiments were duplicated using Ready-to-goTM DNA labeling beads (−dCTP) (Amersham) with P32 dCTP and purified on ProbeQuanTM G-50 microcolumns (Amersham), and blots were blocked with yeast tRNA and herring sperm DNA. Visualization of signal was carried out by exposure to autoradiograph film for 1-5 minutes (chemiluminescent) and 1-4 hours (P32).







TABLE 2







Results of linkage analysis for markers with highest two point lod-scores from five positive regions after genome-wide scan









Total lod-score










Size of














Lod-scores by family
9
12
linked
~# of
























Marker
cM
I
II
III
VI
VII
XIII
XIV
XV
XVII
XX
XXI
XXIV
families
families
Region
genes



























D2S125
260.6
0.30
0.60
0.30
0
−0.03
0.30
0
0.30
0.30
0
0.30
0.18
2.08
2.55
11 Mb
160


D5S426
52.0
0
0
0.30
0.30
0.28
0.30
−0.30
0.30
0.30
0
0.18
0
1.49
1.66
14 Mb
55


D10S1653
40.4
0
0.60
0.30
0
0.13
0.30
0.18
0.18
0.30
0
0
−0.30
1.98
1.68
21 Mb
100


D14S74
87.4
0.30
0.60
0
0.30
0.26
0
0
0.18
0.30
0.18
−.30
0
1.94
1.82
 8 Mb
90


D17S938
14.7
0.60
0.60
0
0.18
0
0.30
0
0
0
0.18
0
−4.40
1.08
−3.14
 9 Mb
170









Mutational Analysis

Genomic DNA was isolated from peripheral blood lymphocytes (Gentra, Minneapolis, Minn.). Parental DNA was available for 41 of 48 (85%) patients with sporadic CdLS who had NIPBL mutations. DNA from both parents was available in 25/48 patients (52%), and DNA from only one parent was available in 16/48 patients (33%). The entire NIPBL coding region (exons 2-47) was screened for mutations. Primer sequences, annealing temperatures, and sizes of PCR products are listed in Supplementary table 1 (available online). Primer pairs were designed to amplify exons, exon/intron boundaries, and short flanking intronic sequences. Larger exons were subdivided to allow for optimal product lengths. All PCR reactions were performed in a 25-μl reaction volume containing 75 ng genomic DNA, 1 U AmpliTaq Gold (Applied Biosystems, Foster City, Calif.), 20 pmol each primer, 75 μM each dNTP, 10×PCR Buffer II (Applied Biosystems, Foster City, Calif.), and 1.0 mM or 1.5 mM MgCl2 (Applied Biosystems, Foster City, Calif.). Cycling parameters were as follows: 36 cycles of 94° C. for 30 s; 51-60° C. for 45 s, and 72° C. for 30 s; and 72° C. for 5 min (last ex Amplifications for exons 6, 11, 21, 26, 30, 44, and 45 were performed using 10 cycles of 95° C. for 30 s; 51-62° C. for 30 s, and 72° C. for 35 s followed by 25 cycles of s; 51-62° C. for 30 s, and 72° C. for 45 s increasing by 5 s for each cycle See Table 3. Mutational analysis of the amplimers was performed by means of conformation sensitive gel electrophoresis (CSGE), using standard protocols (Ganguly et al. 1993). PCR products corresponding to all altered migration patterns (shifts) on CSGE were purified using QIAquickTM PCR purification kit (QIAGEN Sciences, Valencia, Calif.) and sequenced bidirectionally on an ABI 377 sequencer.







TABLE 3







Primers and Conditions used to Amplify NIPBL Coding Sequence















SEQ

SEQ
Length



Exon
Forward
ID NO:
Reverse
ID NO:
(bp)
Conditions





 2
ACTGGGTTGTTGTGAGAACTG
 (12)
GCATTTCAGTTGCTATTTCTG
 (13)
470
  1.5 mM MgCl2 55° C.


 


 3
TTAGGAAGAGGAGGAATGCC
 (14)
CTGAAATAAAACCAGGAATACGG
 (15)
387
  1.5 mM MgCl2 55° C.


 


 4
TGGGGGACAAGAGTGAGACTTC
 (16)
GCATAAACATCGCATTCCTGATAG
 (17)
532
  1.5 mM MgCl2 55° C. + DMSO


 


 5
AAGGACACTTTACTGTTAGAAGAA
 (18)
GCAAATGCAAAGTGGATTACT
 (19)
301
  1.5 mM MgCl2 55° C.


 


 6
CAGTCAGATTTCAAGGAATAGCG
 (20)
CTCCTTTCACCTCCTAAAATGAC
 (21)
429
* 1.5 mM MgCl2 58° C.


 


 7
AACTAGTCAGTACATGAGTATCTG
 (22)
GAAATGGAAATACTAGGTTATATG
 (23)
369
  1.5 mM MgCl2 60° C.


 


 8
CAAGAAGAAAACAGGAAAGTGC
 (24)
CTGCTTTAGGAAGTCTGAGTTCT
 (25)
325
  1.5 mM MgCl2 55° C.


 


 9A
GTGAAACCACCACAACTG
 (26)
TGAGCAGCATTTAGTGGGC
 (27)
429
  1.5 mM MgCl2 55° C.


 


 9B
CAGGACAGACTTCAAAAACACC
 (28)
CCAAATCTCATATAGTTGTTTCAG
 (29)
512
  1.5 mM MgCl2 55° C.


 


10A
TTGCATTTGCATTTTACTCCA
 (30)
GTGTCTCAGGATGGTTTTCTGG
 (31)
428
  1.5 mM MgCl2 58° C.


 


10B
TACGGGAAATGGGTCAAGGC
 (32)
AGGCTCAACTATGGTGCTCTCG
 (33)
424
  1.5 mM MgCl2 55° C. + DMSO


 


10C
TGAGAGCAGAACAACTGAATGC
 (34)
TGGCTTTCCAGGAATCCCTCC
 (35)
352
  1.5 mM MgCl2 55° C. + DMSO


 


10D
AGGTGAGAGCCGCCCTGAAACTC
 (36)
CACGAGGACTGTCAGGTCTTGA
 (37)
467
  1.5 mM MgCl2 55° C. + DMSO


 


10E
TGAATCAGGGGACTCAAGGG
 (38)
AGGGAACTTCTTGATTTGTCCTC
 (39)
468
  1.5 mM MgCl2 55° C. + DMSO


 


10F
AGGAGCTAAGCCTGTAGTTGTG
 (40)
CTTGAGTAGTGGGTGGGGAAGA
 (41)
349
  1.0 mM MgCl2 55° C. + DMSO


 


11
TGTCACTTTAGGGTTAAGAGT
 (42)
GACTGTGCTTTTGCTAAACCC
 (43)
439
* 1.5 mM MgCl2 52° C.


 


12
CACTGAATTTCCTAGACCCTATG
 (44)
ATCACTGCACATAGAAACTAAG
 (45)
464
  1.5 mM MgCl2 55° C. + DMSO


 


13/14
GTTTCTATGTGCAGTGATTATCG
 (46)
GATTTCAAGGTAGGACACATCAC
 (47)
483
  1.5 mM MgCl2 58° C.


 


15
ATTCAGGGTTTACTTGAGGTT
 (48)
AGTCCATGCCTCTTTCAATGCAG
 (49)
486
  1.5 mM MgCl2 58° C.


 


16
AGTCATTTAGGGTCGTTGAGT
 (50)
GCATGGGAAGAGATTAATGAC
 (51)
449
  1.5 mM MgCl2 58° C.


 


17
CATCATAACACTTTTCCACCAG
 (52)
TGGTGCCATTTTAAGTCCTAT
 (53)
415
  1.5 mM MgCl2 55° C.


 


18
CTTCCAGGTTCTGTAGCTAGA
 (54)
GAGTTTGGAATTTACACTACATT
 (55)
483
  1.5 mM MgCl2 55° C.


 


19
TGCTAACGTGCTTTGAGGATG
 (56)
TAGTCCTTAGATTGAAATGAATG
 (57)
393
  1.5 mM MgCl2 55° C.


 


20
GAGCAGCTTACCTTAGATACTGA
 (58)
ATGCTGTTCTGATGTAACTGCC
 (59)
363
  1.5 mM MgCl2 60° C.


 


21
GGCAAAACACAGTATCGTGAAAC
 (60)
GATCGCGCCACTGCACTC
 (61)
389
* 1.0 mM MgCl2 55° C.


 


22
TAGTGTGCTAATTTTGGCTTCT
 (62)
ATTCAAGGTTCAGATTATGGC
 (63)
350
  1.5 mM MgCl2 60° C.


 


23
CAATTTCAATCATGTTGGTAGAC
 (64)
GTGTACAGTTATGCACATGC
 (65)
359
  1.0 mM MgCl2 52° C. + DMSO


 


24
ACAGTTGAGCCTGCATATTTA
 (66)
ACCATTCAGAAGTCCCTGTTA
 (67)
594
  1.5 mM MgCl2 55° C.


 


25
AAGGCAAACTTCAGCTATCAA
 (68)
CCTCTTCATCATGCTACCTCC
 (69)
366
  1.5 mM MgCl2 58° C.


 


26
TGTATTCCTGTAATGTGAGCACTC
 (70)
TCATCCTGCAACAAAAAGTCA
 (71)
413
* 1.5 mM MgCl2 58° C.


 


27
ACCACACCTTCTCAGTTTAGCA
 (72)
CTCACAAGCATCCAGAATCAG
 (73)
297
  1.5 mM MgCl2 55° C.


 


28/29A
ACGAAAGGCTCCAAAGTATG
 (74)
ACTGCTGCTTCTCGGACAC
 (75)
473
  1.5 mM MgCl2 52° C. + DMSO


 


28/29B
GTCTGAGGTTGTTGCTGTAGA
 (76)
ATGATATTGCAAGGGCTATTC
 (77)
423
  1.5 mM MgCl2 55° C.


 


30
TTCTAGTCTTGTGTCCAGGGC
 (78)
ATCAACATTTAGGTGCAATAA
 (79)
462
* 1.5 mM MgCl2 55° C.


 


31
TCCTGGCAGTTTGTGTTTTG
 (80)
CTGGAGGAATAGGAAAATCTCAG
 (81)
470
  1.5 mM MgCl2 55° C. + DMSO


 


32
GTTCTGTAACGTTGGTAAATGGT
 (82)
GGTTCTTTTAAATCATACAGTCCA
 (83)
321
  1.5 mM MgCl2 58° C.


 


33
ACCTTAGGTCTTACACAGCAA
 (84)
TGTGCTCAACTAGGTTATCAAC
 (85)
362
  1.5 mM MgCl2 60° C.


 


34
TTGAGGCCTATACTGGACCTA
 (86)
GGTTGACGCATGTGAACTCTA
 (87)
333
  1.5 mM MgCl2 60° C.


 


35
TAACTGGACCTTTACGTGCAA
 (88)
GCTCACACAATGTTGCACTAC
 (89)
423
  1.5 mM MgCl2 55° C.


 


36
TGGCATGACTGTAAGCACTCA
 (90)
AGAGGACCACGGTGGATAATC
 (91)
381
  1.5 mM MgCl2 60° C. + DMSO


 


37
TGGTGGCACACGACTGTAATCC
 (92)
TCATCCTGGGTCACTACTGTCAT
 (93)
467
  1.5 mM MgCl2 60° C.


 


38
CTGATACTTTGAATGCCACTG
 (94)
CACCAAATCCTACTGCTAATA
 (95)
378
  1.5 mM MgCl2 55° C.


 


39
CTCTAGGTAAGGCCACCAGCAT
 (96)
TAGACCTCAGCATAAGGACTGC
 (97)
466
  1.5 mM MgCl2 55° C.


 


40
CAGATTAAGAACCATTGAGCC
 (98)
GCAGTAATCATAACCCAAGAG
 (99)
492
  1.5 mM MgCl2 58° C.


 


41
AGTGTGAGAATGCTTTATGTT
(100)
ATTATGAATGTGGGCAGAGCA
(101)
474
  1.5 mM MgCl2 55° C.


 


42
ATGAAGCTAGCCTCAGAATGT
(102)
CAAAATTTCCCCTTCACTTCTGA
(103)
472
  1.5 mM MgCl2 58° C.


 


43
GTGAGGTGAAAGTGCCCTGTA
(104)
TCCCAAGTCAAGTATTGCCCAG
(105)
401
  1.5 mM MgCl2 52° C.


 


44
CAAGCTGTTGAATGGAGCATAC
(106)
CATGAGCCACCACACCCAGC
(107)
434
* 1.0 mM MgCl2 58° C.


 


45
TCCAAATACGTTGTTTCCATAG
(108)
TCAATGTGAAGGAGATAGTTAT
(109)
329
* 1.5 mM MgCl2 51° C.


 


46
CCACACCAAACTACTGCCATAG
(110)
CATTTTACGTAATACGCTGCG
(111)
334
  1.0 mM MgCl2 60° C.


 


47A
GTCACGGTGCGTCTCATTGC
(112)
TAGTGTCTACCCAAGGCACCA
(113)
395
  1.5 mM MgCl2 58° C.


 


47B
GGCTTCAGTGTTCAGTGGATG
(114)
TTTGCCCAACATTTCCTTC
(115)
364
  1.5 mM MgCl2 58° C.


 


47C
TGAAGAGTAAGTGGAACCTGG
(116)
GCTAAAGAAAGCCATCCGC
(117)
274
  1.5 mM MgCl2 55° C.





* Denotes GC-rich PCR cycle.






Genotype-Phenotype Correlations

Genotype-phenotype correlations were assessed using contingency table analysis. This was performed for the three categories (mild, moderate, and severe) for each phenotypic parameter (limb defect, growth, and development) versus presence or absence of a mutation in NIPBL, and versus missense or other types of mutations. For the mutation-positive versus mutation-negative analysis the chi-square test with 2 degrees of freedom was used. For the missense versus other types of mutations analysis Fisher's exact test was used. The significance threshold was set at P≦0.05.
Chromosomal Analysis and Evaluation for PSCS
Metaphase spreads were prepared for the 90 CDLS probands and 90 non-CdLS controls from either whole blood cultured in RPMI 1640 with 15% fetal bovine serum and phytohemagglutinin for 72 hours or lymphoblastoid cell lines transformed with Epstein-Barr Virus and harvested during the log phase. Metaphases were arrested with 0.8 ug/ml Colchicine (SIGMA-ALDRICH) for 20 minutes at 37° C., hypotonized with 0.075M KCL at room temperature and fixed with 3 parts methanol: 1 part acetic acid. The slides were stained with Wright's Stain (Fisher Scientific). Ten proband slides were C-banded. A minimum of 50 metaphases were microscopically examined and scored for PSCS. PSCS was diagnosed when the sister chromatids were completely separated and no connection at the Centromere was seen (Plaja et al., 2003). A metaphase was scored as positive for PSCS if all or the majority of sister chromatids in the metaphase spread demonstrated sister chromatid separation. A positive PSCS score was recorded for any individual with at least one metaphase per slide demonstrating PSCS.
GenBank Accession Numbers.
Human BX538178 (BX538178), Human IDN3 (NM—133433), Saccharomyces cerevisiae sister chromatid cohesion protein 2 (Q04002), Mouse IDN3 (BG070859; XM—127929), RatIDN3 (XM—238213). NIPBL (BKO05151).
Results
CdLS is a dominantly inherited disorder with characteristic facial appearance, limb defects, and growth/cognitive retardation. See FIG. 1. A genome-wide linkage analysis was initially carried out in 9 CdLS families with more than one affected family member. Under a model of genetic homogeneity, we used a linkage exclusion mapping approach, excluding all markers in which the affected individuals in one or more families demonstrated non-sharing of both parental alleles when both parents were unaffected, or of the allele transmitted by the affected parent. This analysis resulted in the identification of 5 regions containing one or more markers with positive lod-scores in the 9 families (chromosomes 2q37, 5p13, 10p13, 14q24, and 17p13) (Table 2). With the identification of 3 additional CdLS families, these 5 regions were analyzed in the initial cohort of 12 families. This resulted in negative lod-scores for D17S938 in one of the families and exclusion of chromosome 17. All other markers still gave positive total lod-scores (Table 2).
Fine mapping was performed in all families with additional markers at a 1-1.5 cM average density to the defined regions on chromosome 2, 5, 10, and 14. While multipoint linkage analysis did not improve the odds for linkage to chromosome 2, 10, or 14, it did result in a maximum LOD score of 2.7 for chromosome 5p13, which was the highest score for the entire genome analysis. The critical region on chromosome 5p13 was refined by obligate recombination events to an  ̃7.4 Mb region spanning 5p13.1-13.3 and flanked by markers D5S477 distally and D5S1376 proximally (FIG. 2) and contained 58 putative genes (FIG. 3a).
Based upon the results of this analysis, other corroborating evidence was sought to target one or more of the 4 candidate regions. A child with classic features of CdLS and a balanced de novo t(5;13)(p13.1;q12.1) had been identified by our group in the past and another child with classic features of CdLS and a de novo chromosome 5p13.1 -p14.2 deletion (the only reported case of a constitutional deletion of 5p13.2) was recently described. These cases supported the association of 5p13 with CdLS. Refinement of the 5p breakpoint in the translocation patient was undertaken (samples were not available on the 5p deleted child who died shortly after birth).
Fluorescence in situ hybridization (FISH) using clones from within the minimal critical region on 5p13 was performed on the t(5;13)(p13.1;q12.1) child (FIG. 3b). Due to sample limitations we were initially unable to identify a clone that spanned the translocation breakpoint, however we were able to narrow the critical region to a 1.1 Mb interval containing 11 putative genes (FIG. 3a). Mutational analysis of the first 3 exons of all 11 genes was undertaken by conformation-sensitive gel electrophoresis (CSGE). Mutations in two overlapping transcripts BX538178 (3653 bp mRNA) and IDN3 (8124 bp mRNA) were identified (FIG. 3a). The identification of mutations in both of these transcripts (in BX5381 78-specific sequence, IDN3-specific sequence and in the overlap region), and their exact sequence identity over a 2259 bp region of overlap suggested that they were part of a larger transcript that we refer to as NIPBL.
Expression patterns were studied by Northern blot and in situ analyses. Northern blots of both fetal and adult samples for multiple probes demonstrated  ̃6 Kb and 1.9 Kb transcripts and, in fetal samples, additional bands of 9.5 Kb and 7.2 Kb (FIG. 4). The presence of multiple transcripts suggests alternative splicing for this gene. Transcripts of the mouse homolog of NIPBL (mNIPBL) can be detected widely at days 9.5 and 10.5 of gestation (FIG. 5), with notable accumulations in limb bud, branchial arch and craniofacial mesenchyme. These data show that, in mouse, NIPBL is highly expressed in regions involved in patterning of, among other things, the skeleton and soft tissues of the limbs, jaw and face.
EXAMPLE 2
Mutational Analysis
The study population consisted of 120 propositi with CdLS, including 106 sporadic and 14 familial cases. Linkage to the NIPBL locus at 5p13.1 is described in the 12 families analyzed in Example I, with the identification of mutations in NIPBL in 2 of these families (Krantz et al. 2004). In one family, a missense mutation in the first codon (MIK) was identified in 3 affected half-siblings who each had a different father. The mutation was not present in DNA extracted from lymphocytes in their mother or in the 2 fathers on whom samples were available. In the second family, a splice site mutation (6763+5G>T) in the intron between exons 39 and 40 was identified in 2 affected siblings but not in DNA isolated from lymphocytes in either parent. The study population also included 4 previously reported, unrelated CdLS patients with unique, de novo mutations in NIPBL (Krantz et al. 2004).
Spectrum of NIPBL Mutations Detected
The 120 propositi with CdLS were screened for NIPBL coding-region mutations. Exons 2-47 and flanking intron sequences were PCR amplified and analyzed by means of CSGE. All products with variant migration profiles (band shifts) on CSGE were sequenced bidirectionally. NIPBL mutations were identified in 56 patients (7 familial; 49 sporadic) (47%) (Table 4, FIG. 1). Fifty-one different mutations were identified and comprised 21 frameshifts, 12 missense, 10 nonsense, and 9 splice site mutations. All but 4 (8%) mutations identified were private: a 2-bp deletion of exon 10 (2479delAG) in 2 unrelated patients; a nonsense mutation (R1536X) of exon 22 in 3 unrelated patients; a splice site mutation in the intron upstream of exon 35 (6109-3T>C) in 2 unrelated patients; and a missense mutation (R2298H) of exon 40 in 2 unrelated patients Forty-four different mutations were identified in 49 of the 106 individuals (46%) with sporadic CdLS. Fourteen (32%) of the 44 mutations were small deletions. Six (14%) of the mutations were insertions. Of these, five were single-base-pair insertions, and one was a complex deletion/insertion mutation with net insertion of 3 base pairs. All deletions and insertions led to frameshifts that resulted in a prematurely truncated protein product. The deletion, 2479delAG in exon 10, was seen in 2 unrelated sporadic cases. Nine (20%) of the different mutations had single-base-pair changes that led to immediate stop codons. Four (44%) of the nonsense mutations were found in exon 10. The nonsense mutation (RI 536X) of exon 22 was identified in 3 unrelated patients with sporadic CdLS. In one sporadic patient with a de novo t(14q;21q)(q32;q11) (Wilson et al. 1983), a nonsense mutation, S1459X in exon 20 of NIPBL was identified and was not present in either of her parents suggesting that the balanced de novo translocation may represent an unrelated event.
Seven patients (16%) with sporadic CdLS had different mutations predicted to lead to alterations in splicing. The six splice site mutations were not identified in available parental samples (6 mothers, 3 fathers), or in the 150 control patient samples.
Ten (23%) of the 44 mutations identified in patients with sporadic CdLS resulted in the substitution of a single amino acid. These substitutions were predicted to result in missense mutations by three criteria: absence in parental samples; absence in control samples; and evolutionary conservation of the altered amino acid. One missense mutation, R2298H in exon 40, was identified in 2 unrelated patients. The missense mutations identified included: A1246G (exon 15), L1312P (exon 17), R1789L (exon 28), D1803V (exon 28), R2298C (exon 40), R2298H (exon 40), G2312R (exon 40), G2381A (exon 42), A2390T (exon 42), and Y2440H (exon 43). These amino acids were, in general, highly conserved throughout evolution (FIG. 6). These mutations were not identified in available parental samples or in the 150 control patient samples.
In one apparent familial case of CdLS (previously reported as family XII) (Krantz et al. 2001), which was excluded from subsequent linkage analyses as it showed an atypical inheritance pattern with 2 affected first male cousins born to unaffected sisters, the two affected males were found to carry different de novo mutations in NIPBL. In one child an A1246G missense change in exon 15 was identified while in his affected cousin a 7861-1 G>C splice site change was identified in the intron upstream of exon 46. Neither mutation was identified in either of the two sets of parents nor in the other cousin. The maternal 5p13 regions flanking NIPBL (including intragenic SNP markers) in the affected individuals were not shared (data not shown). Paternity was confirmed in both cases.







TABLE 4







Summary of Mutations in NIPBL Identified in CdLS Probands













Exon
Mutation
Type
Effect on Protein
Father
Mother
Number
















2
2 T > A; M1K
missense
no initiating methionine
negative
Negative
Familial



64 + 1A > G
Splice site

NA
Negative
1


3
150delG
frameshift
Truncates protein 27 aa
negative
Negative
1





downstream



65 − 5A > G
Splice site

negative
Negative
1



199del10;
complex
Truncates protein 9 aa into
NA
NA
1



199ins13

insertion



(atcaacaggtgac) =



SEQ ID NO: 121


7
611 − 2A > G
Splice site

NA
Negative
1



742delCT
frameshift
Truncates protein 8 aa downstream
NA
NA
1


9
961delA
frameshift
Truncates protein 7 aa downstream
NA
NA
1



R479X
nonsense
Truncates protein
negative
Negative
1


10
1546insG
frameshift
Truncates protein 5 aa downstream
negative
Negative
1



1669insC
frameshift
Truncates protein 11 aa
NA
negative
1





downstream



1902insA
frameshift
Truncates protein 2 aa downstream
negative
negative
1



2479delAG
frameshift
Truncates protein 2 aa downstream
negative
negative
2



2520delT
frameshift
Truncates protein 6 aa downstream
negative
negative
1



2969delG
frameshift
Truncates protein 1 aa downstream
NA
NA
1



3023delTGTCT
frameshift
Truncates protein 2 aa downstream
NA
negative
1



3057delTAGA
frameshift
Truncates protein 23 aa
negative
negative
1





downstream



3060delAGAG
frameshift
Truncates protein 22 aa
negative
negative
1





downstream



R797X
nonsense
Truncates protein
NA
negative
1



R832X
nonsense
Truncates protein
negative
NA
1



E977X
nonsense
Truncates protein
negative
negative
1



S1024X
nonsense
Truncates protein
NA
negative
1


15
3736C > G; A1246G
missense

negative
negative
1


17
3969insG
frameshift
Truncates protein 6 aa downstream
negative
negative
1



3935T > C; L1312P
missense

NA
negative
1


18
S1398X
nonsense
Truncates protein
NA
negative
1


20
S1459X
nonsense
Truncates protein
negative
negative
1


21
4556delAAAAA
frameshift
truncates protein 1 aa downstream
negative
negative
1


22
R1536X
nonsense
truncates protein
negative (1)
negative (2)
3






NA (2)
NA (1)



4567delC
frameshift
truncates protein 1 aa downstream
NA
NA
1


26
R1723X
nonsense
truncates protein
negative
negative
Familial


27
R1758X
nonsense
truncates protein
negative
negative
1


28
5366G > T; R1789L
missense

NA
NA
1



5408A > T; D1803V
missense

NA
negative
1


29
5567G > C; R1856T
missense

negative
negative
Familial



5574 + 1G > T
Splice site

negative
negative
1


35
6109 − 3T > C
Splice site

NA (2)
negative (1)
2


39
6763 + 5G > T
Splice site

negative
negative
Familial


40
6892C > T; R2298C
missense

NA
negative
1



6893G > A; R2298H
missense

negative
negative
2


42
6934G > C; G2312R
missense

negative
negative
1



7151delAAGAC
frameshift
truncates protein 3 aa downstream
NA
negative
Familial



7210delC
frameshift
truncates protein 21 aa downstream
NA
negative
1


43
7142G > C; G2381A
missense

NA
negative
1



7168G > A; A2390T
missense

negative
negative
1



7318T > C; Y2440H
missense

negative
negative
1



7321 + 4 A > G
Splice site

negative
positive
Familial


44
7431delG
frameshift
truncates protein 30 aa downstream
NA
negative
1


45
7780delC
frameshift
truncates protein 16 aa downstream
negative
negative
Familial



7825insG
frameshift
truncates protein 22 aa downstream
negative
negative
1


46
7861 − 1G > C
Splice site

negative
negative
1









Mutations were identified in 7 of 14 (50%) familial cases of CdLS. Mutations in NIPBL were previously reported in 2 of these families: a missense mutation in the first codon (M1K) was identified in the 3 affected siblings, all of whom had different fathers, and was not present in the mother or two fathers available for testing (in all familial cases mutational analysis of parental samples was performed on DNA extracted from lymphocytes, and mosaicism in other tissues cannot be excluded); and a splice site mutation (6763+5G>T) segregated with the CdLS phenotype in a family with 2 affected siblings and not in either parent (Krantz et al. 2004). Four of the 5 remaining mutation-positive families had previously been linked to the NIPBL locus (Krantz et al. 2004). A nonsense mutation, R1723X, in exon 26 was identified in the first family with 2 affected brothers; neither of the parents nor the unaffected brother have the mutation. In the second family the two affected siblings share a unique 5-bp deletion, 7151delAAGAC in exon 42, resulting in protein truncation 3 amino acids downstream. The mother did not carry the change and there was no sample available for testing from the father. An affected brother and sister in the third family share a single base pair deletion, 7780delC of exon 45; this deletion results in premature protein truncation 16 amino acids downstream. Neither parent carried this mutation. In the fourth family with 4 affected siblings and a mildly affected mother a splice site mutation, 7321+4 A>G in exon 43, was identified in the 2 affected siblings from whom samples were available, as well as in the affected mother. In the final family, not included in the initial linkage studies as the affected female sibling of the proband was deceased and no sample was available, a missense mutation R1856T in exon 29, was identified in the affected male child, but was not present in either parent or in an unaffected sibling. Paternity was confirmed in all familial cases (as part of the genome-wide and high-resolution linkage analysis using multiple polymorphic markers) where a paternal sample was available.
None of the 51 different mutations were observed in 150 ethnically-matched control subjects.
Forty-two sequence variants that are likely to represent neutral polymorphisms were observed in subjects with CdLS, unaffected family members, and/or control individuals (Table 5). Three of the polymorphisms (N674S, N1994S, and 11206V) in the coding region of NIPBL led to an altered amino acid residue, while 3 (D817D, L1591L, and S1958S) were silent. Thirty-six polymorphisms were identified in intronic sequences flanking the exons.







TABLE 5







Polymorphisms in NIPBL Identified in Affected


Individuals, Family Members and/or Controls










Polymorphism
Exonic Localization







230 + 61 C > A*
Exon 3



611 + 102 A > G#
Exon 6



2021 A > G; N674S*
Exon 10



2451 C > T; D817D#
Exon 10



3575 − 14 A > G*
Exon 14



3616 A > G; I1206V
Exon 14



3855 + 52 A > G*
Exon 16



4088 + 53 T > C#
Exon 17



3586 − 59 G > A
Exon 17



4239 + 53 T > C#
Exon 18



4239 + 152 C > G#
Exon 18



4240 − 48 C > T#
Exon 19



4321 − 96 C > T
Exon 20



4321 − 35 T > C#
Exon 20



4560 + 77 A > G*
Exon 21



4560 + 108 del T*
Exon 21



4561 − 9 T > A#
Exon 22



4561 − 106 C > T#
Exon 22



4634 + 24 G > A#
Exon 22



4773 G > T; L1591L#
Exon 23



4777 − 108 del A*
Exon 24



4921 − 58 G > A*
Exon 25



5575 − 193 T > C*
Exon 30



5575 − 92 G > C
Exon 30



5575 − 18 G > C#
Exon 30



5710 − 59 A > G#
Exon 31



5710 − 78 G > A#
Exon 31



5862 + 74 del TT*
Exon 32



5863 − 12 del AT*
Exon 33



5863 − 30 del AT*
Exon 33



5863 − 52 del T#
Exon 33



5874 C > T; S1958S*
Exon 33



5971 A > G; N1994S#
Exon 34



6109 − 54 ins A
Exon 35



6109 − 3 T > C #
Exon 35



6498 − 94 T > C#
Exon 38



6499 − 80 A > G#
Exon 39



6764 − 35 C > G*
Exon 40



6954 + 62 A > G#
Exon 40



6955 − 9 del T*
Exon 41



7861 + 39 G > A
Exon 45



8698 − 8701 del ACAA
Exon 47




(region of 3′ UTR)







*indicates found in patient samples, family members and/or normal controls.



#indicates found in patient samples and family members.



All other polymorphisms were identified in control samples only.






FISH Analysis

Twenty-eight probands (4 familial and 24 sporadic) in whom a NIPBL mutation was not identified were analyzed by FISH with a NIPBL-containing BAC probe (RP 11-14121) to evaluate the possibility of a submicroscopic deletion encompassing the NIPBL gene. RP11-14121 (AC018853.3) encompasses approximately 16 kb of sequence 5′ of NIPBL gene through approximately exon 10 of NIPBL. No deletion of RP1 1-14121 was detected in any of the probands analyzed.
Genotype-Phenotype Correlation
Because of the clinical heterogeneity observed in CdLS (FIG. 1), we evaluated for possible associations between NIPBL genotype and the severity of the phenotype (severity of limb, growth and developmental involvement). The distribution of several major clinical features in our study cohort is shown in Table 6. The results of genotype-phenotype correlation analysis performed for each phenotypic parameter versus presence or absence of a mutation in NIPBL, and versus missense or other types of mutations, are also summarized in Table 6. Statistically significant (P<0.05) differences were observed in the distribution of severity of growth retardation and developmental delay between the mutation-positive and mutation-negative groups with the mutation-positive group displaying a more severe phenotype for these parameters. A similar trend was also observed in the severity of limb defects, although in this case the difference was not statistically significant. When comparing individuals with missense mutations versus all other mutation types, it appeared that the latter were associated with more severe phenotypes in all categories except possibly in growth retardation, although the number of missense mutations was small.







TABLE 6







Distribution of Clinical Severity and Results of Genotype-Phenotype Correlation Analysis.















Missense



MUTATION-POSITIVE

Mutation
versus all



(n = 56)

Positive versus
other
















Frameshift,
MUTATION-
Mutation
Mutations





Splice Site,
NEGATIVE
Negative
P value




Missense
Nonsense
(n = 64)
P value
(Fisher's


PHENOTYPE
SCORE
(%)
(%)
(%)
(Chi Square test)
Exact Test)





Limb Reduction
1
12 (0.92) 
25 (0.60)
47 (0.75)
0.085
0.029



2
1 (0.08)
 3 (0.07)
 9 (0.14)



3
0
14 (0.33)
 7 (0.11)



N/A
0
1
1


Developmental
1
3 (0.23)
 1 (0.03)
12 (0.20)
0.014
0.008


Delay
2
6 (0.46)
10 (0.25)
27 (0.44)



3
4 (0.31)
29 (0.73)
22 (0.36)



N/A
0
3
3


Growth
1
3 (0.33)
 2 (0.07)
17 (0.47)
0.002
0.057


Retardation
2
4 (0.44)
11 (0.38)
13 (0.36)



3
2 (0.22)
16 (0.55)
 6 (0.17)



N/A
4
14 
28 





n = 56 for mutation positive individuals, and n = 64 for mutation-negative individuals


NA = Not assessed.






Discussion
Through the combined use of genome-wide linkage exclusion analysis and mapping of a chromosomal rearrangement on chromosome 5p13, NIPBL was identified as a CdLS disease gene (Krantz et al. 2004; Tonkin et al. 2004). We have identified mutations in 47% of a well-characterized cohort of 120 unrelated probands with both sporadic and familial CdLS. Mutation detection rates were comparable between the sporadic and familial cases: 49 of 106 (46%) sporadic cases and 7 of 14 (50%) of familial cases had identifiable mutations. We expected to detect NIPBL mutations in all of the familial cases previously shown to be positively linked to the 5p13 region (Krantz et al. 2004); however, in our present analysis, we have identified mutations in only 6 of the 11 families available for mutational analysis. This indicates that either the methods used for screening are not identifying all mutations in individuals with CdLS or that potentially additional genes in 5p13 may be responsible for the phenotype.
If NIPBL is the only CdLS disease gene then our mutation detection rate of only 47% may be partly due to the large size of the NIPBL gene and the use of CSGE for mutational analysis. Factors that may account for missed mutations in the gene include: 1) variations in sequence beyond the immediate intron/exon boundaries (such as regulatory regions or intronic sequence); 2) large intragenic deletions; 3) subtle sequence variations (such as point mutations); and 4) difficulty in amplifying and sequencing several NIPBL exons (e.g. exon 33) due to numerous polymorphisms. The multiple splice variants of this gene have made it difficult to screen cDNA accurately for mutations at this time, although this testing is currently being optimized and will allow for improved detection of intronic variations that lead to splice mutations as well as complete exonic deletions. Large-scale deletions of NIPBL were assayed for in those individuals in whom an NIPBL mutation was not identified, and in the 28 mutation-negative individuals studied by FISH no deletion of the region was seen.
Alternatively, it is possible that the linkage established in some of these small families was coincidental and that a second CdLS gene may yet be identified elsewhere in the genome to account for the phenotype in those individuals in whom mutations in NIPBL were not identified. In our initial genome-wide linkage exclusion analysis, 3 other regions were not excluded: chromosome 2q37; chromosome 10p13; and chromosome 14q24. These additional loci may contain a second CdLS gene (Krantz et al. 2004). Likewise several individuals with CdLS have been found to carry an apparently balanced de novo translocation suggesting possible additional loci for a CdLS disease gene. The child with the t(5;13)(p13.1;q12.1) was critical in the identification of NIPBL on chromosome 5p13 as the cause of CdLS when mutated (Hulinsky et al. 2003; Krantz et al. 2004; Tonkin et al. 2004). Two other de novo balanced translocations have been reported. A de novo t(3;17)(q26.3;q23.1) (Ireland et al. 1991) has been extensively evaluated in a child with sporadic CdLS, and to date, no CdLS disease genes have been identified (Tonkin et al. 2001; Tonkin et al. 2004). In this report, a child with a previously described de novo t(14q;21q)(q32;q11) (Wilson et al. 1983) has been found to carry a de novo S1459X NIPBL mutation in exon 20. This may indicate that this translocation is an unrelated event. While not all breakpoints in these rare translocation cases have been completely evaluated, they do not appear to lend additional support to a potential second locus at this time.
Fifty-one different mutations in NIPBL have been identified in 56 patients (7 familial; 49 sporadic) and comprise frameshifts, missense, nonsense, and splice site mutations. Mutations were identified throughout the coding region of the gene (Krantz et al. 2004; Tonkin et al. 2004). To date, no mutations have been identified in exons 4-6, 8, 11-14, 16, 19, 23-25, 30-34, 36, 37 41 and 47. Several exons have been found to have multiple mutations including exons 2, 3, 7, 9, 10, 17, 22, 28, 29, 40, 42, 43 and 45. There is a preponderance of mutations identified in exon 10; however, this exon, at 1625 base pairs, is approximately 8 times the size of the average exon ( ̃200 bp) in the NIPBL gene. Exon 42, at 200 base pairs, was found to contain 4 different mutations in this cohort. The majority of mutations identified are frameshift (22/56 ̃39%) (16 deletions, 5 insertions, 1 complex), followed by missense mutations (13/56 ̃23%), nonsense mutations (12/56 ̃21%), and splice site mutations (9/56 ̃16%). The frameshift, nonsense, and splice site mutations are likely to result in a prematurely truncated protein resulting in haploinsufficiency of NIPBL (a disease mechanism that has been documented in the report of a child with CdLS and a large cytogenetically visible deletion of chromosome 5p13.1-14.2 encompassing the NIPBL gene (Hulinsky et al. 2003)).
The missense mutations are important in that they may indicate residues of the NIPBL protein that are functionally important. Of the 12 unique missense mutations identified 8 are in amino acid residues that are evolutionarily conserved back to Drosophila (including the M1K change in the initiation codon), and 3 are evolutionarily conserved back to the mouse. One missense mutation, Y2440H, is present in an amino acid that is in a stretch of the human NIPBL protein not seen in rat, mouse or Drosophila. 
Four mutations were identified in unrelated individuals: 2479delAG in exon 10 in 2 individuals; R1536X in exon 22 in 3 individuals; 6109-3T>C in the intronic sequence upstream of exon35 in 2 individuals; and R2298H in exon 40 in 2 individuals. An additional missense mutation in amino acid residue 2298 was also seen in another individual (R2298C). Even among individuals with the same mutation, the phenotype demonstrated some variability. The 3 individuals with the R1536X mutation are all severely affected in growth and development; however, 2 of the 3 have severe limb reduction defects while the third did not have reduction defects. The 2 children with the 2479delAG mutation are also similarly severely affected for growth and development, however one has significant limb reduction defects while the other does not have a reduction defect. The 2 children with the 6109-3T>C and the 2 children with the R2298H missense mutation are all moderately affected for growth and development and none have limb reduction defects. This variability in severity of the phenotypes associated with identical mutations indicates that mutations in NIPBL are not the sole determinants of phenotype and that other factors (genetic and/or environmental) can modify the clinical picture.
In 6 of the 7 familial cases in which an NIPBL mutation has been identified germ line mosaicism is the most likely mechanism. In 5 of these families, DNA from lymphocytes was available for testing from both parents and none carried the mutations identified in the affected siblings and paternity was confirmed in all cases. In one family with a 7151 delAGAC, the father was not available for testing, however he reportedly has no clinical features of CdLS and the mutation was not seen in the mother. Autosomal dominant transmission was demonstrated in the seventh family; a 7321+4 A>G mutation in exon 43 was identified in the mildly affected mother and in 2 of her 4 affected daughters in whom samples were available.
In the 25 sporadic cases in which both parents were available for screening, all mutations were found to have arisen de novo, and in the 17 sporadic cases in which only one parent was available for screening, none of these parents were found to carry the change seen in their child. This would indicate that the vast majority of mutations in individuals with CdLS arise as new events, and in the rare cases of familial recurrence where neither parent is affected germ line mosaicism is the likely explanation. In the family where two male first cousins have CdLS and their mothers, who are sisters, are unaffected, the 2 affected male cousins were each found to carry a different de novo mutation (neither mutation was seen in either set of parents and direct sequencing of the 2 cousins confirmed that they did not share the same mutation). None of the mutations identified in the individuals described here were seen in 300 ethnically matched normal control chromosomes.
A large number of polymorphisms also have been identified in NIPBL (Table 5). There were 3 polymorphisms (N674S, I1206V, and N1994S) that resulted in an amino acid substitution. All 3 of these were identified in probands who had mutations in other exons, and in the case of the N1994S it was present in one of the unaffected parents as well (both parents were not available for screening for the other 2 variants). One polymorphism, N674S, was seen in 25 unrelated probands, 11 of whom had identifiable NIPBL mutations, and in several controls. This amino acid residue is conserved back to the mouse, but is not conserved in Drosophila. If the polymorphism was on the non-mutant allele and had a mild functional effect on the protein, it is possible that it could be a modifier of the phenotype. In the cohort of 11 probands with a mutation and this polymorphism there did not appear to be a marked effect on phenotype. It is of interest that the one individual with a missense (A2390T) mutation who also had this polymorphism was the only one amongst the probands with missense mutations to have limb reduction defects. Further work is needed to evaluate this, and other polymorphisms, as potential modifiers of the phenotype through determining allelic localization of these changes in relation to the mutation as well as functional studies to assess their effects.
Genotype-phenotype correlations between mutation positive and mutation negative individuals as well as between those individuals with different types of mutations were investigated. Severity of limb defects, growth and cognitive development were evaluated (outlined in Tables 1 and 6). Mutations in NIPBL were found in mildly and severely affected individuals with CdLS. Similarly in the group of CdLS probands without identifiable mutations there are also severely and mildly affected individuals. In order to formally evaluate for genotype-phenotype associations between mutation-positive and mutation-negative individuals we performed a contingency table analysis for the three categories of each phenotype tested (limb, growth, and development) versus presence or absence of mutation (Table 6). This analysis demonstrated a trend towards a more severe phenotype in mutation-positive versus mutation-negative individuals. This may indicate that a subset of individuals with “mild” CdLS may either have a different genetic etiology causing their phenotypic findings or mutations in NIPBL that have not yet been detected using CSGE.
We hypothesized that the missense mutations identified in NIPBL may result in either a milder phenotype as a result of a less severe structural effect on the protein, or conversely, a more severe phenotype if they occurred in critical domains of the protein, causing a dominant-negative effect. For these reasons a similar analysis was performed to evaluate genotype-phenotype correlations between missense and all other types of mutations. This analysis suggests that individuals with missense mutations may have a milder phenotype; however, the numbers of individuals with missense mutations are too small to reach definite conclusions at this time.
The role of NIPBL in mammals has yet to be elucidated and what is known about its function has come from Drosophila studies. The Drosophila homolog of NIPBL, Nipped-B, was identified through a screen for mutations that reduce activation by the wing margin enhancer in the presence of a gypsy insertion (Rollins et al. 1999). Gypsy insertions in the cut gene in Drosophila are known to block a remote wing margin enhancer located 85 kb upstream of the promoter. This long range effect on transcription as well as its homology to chromosomal adherins (proteins that have a role in chromosome compaction and sister chromatid cohesion) suggest that the Nipped-B protein performs an architectural role in enhancer-promoter communication (Rollins et al. 1999). These interactions have been demonstrated to be involved in the regulation of multiple developmental pathways in Drosophila including the Notch signaling pathway (Rollins et al. 1999). Recently a role for the Drosophila Nipped-B protein in sister chromatid cohesion has also been demonstrated, and a model of how Nipped-B interacts with the cohesin protein complex to effect gene expression was proposed (Rollins et al. 2004). The ability of distal enhancers to activate promoters and initiate transcription relies on the coordinated interaction of multiple proteins and protein complexes. The large number of additional proteins that interact in these complexes suggest multiple possibilities for modifiers of NIPBL and/or potential additional CdLS disease genes.
We have shown that mutations in NIPBL are detected at present in 47% of individuals with both familial and sporadic CdLS. The mutations are spread throughout the gene, and frameshift, nonsense, splice site and missense mutations have been identified. The majority of mutations are protein truncating, likely leading to haploinsufficiency of the protein product. The 12 unique missense mutations identified in this screening will be important in characterizing functionally important domains of this novel protein. Furthermore, there appears to be a genotype-phenotype correlation between mutation positive and mutation-negative individuals as well as possibly between individuals with missense mutations when compared to all other mutation types.
EXAMPLE 3
Precocious Sister Chromatid Separation (PSCS) in Cornelia de Lange Syndrome
Mutations in NIPBL were identified in sporadic and familial CdLS cases. See Examples 1 and 2. NIPBL is the human homolog of the Drosophila Nipped-B gene. Although its function in mammalian systems has not been elucidated, Nipped-B has been shown to be an essential regulator of cut, Ultrabithorax, and Notch receptor signaling in Drosophila. Sequence homologs of Nipped-B in yeast (Scc2 and Mis4) are required for sister chromatid cohesion during mitosis, and a similar role was recently demonstrated for Nipped-B in Drosophila (Rollins et al., 2004). In order to evaluate NIPBL's role in sister chromatid cohesion in humans, metaphase spreads on a large cohort of mutation positive and mutation negative probands with CdLS were evaluated for evidence of precocious sister chromatid separation (PSCS). PSCS was seen in a significant number of CdLS probands when compared to unaffected matched controls. These studies indicate that NIPBL may play a role in sister chromatid cohesion in humans as has been reported for its homologs in Drosophila and yeast. The identification of PSCS in individuals with CdLS provides an additional diagnostic aid as NIPBL mutational analysis can be labor intensive and to date, mutations are identified in only 45% of affected probands.
Results
In order to evaluate NIPBL's role in sister chromatid cohesion in humans, metaphase spreads on 90 CdLS probands (40 NIPBL mutation positive and 50 NIPBL mutation negative) were evaluated for evidence of precocious sister chromatid separation (PSCS). We screened a minimum of 50 metaphases from each proband and found evidence of PSCS in 37 of 90 probands (41%) (FIG. 10). Of these, 37 probands with PSCS, 16 (43%) were mutation positive and 21 (57%) mutation negative. Of the 53 probands without evidence of PSCS, 24 (45%) were mutation positive and 29 (54%) were mutation negative. Ninety control slides were screened and 8 (9%) demonstrated evidence of PSCS. Both severe and mild CdLS phenotypes were seen in the PSCS positive and negative groups. Missense, frameshift and nonsense mutations have been seen in both groups. Two of the patients with PSCS have the same missense mutation: an R2298H in exon 40 in a highly conserved amino acid residue. An average of 2.05 metaphases (4%) (Range: 2-10%) were found to have PSCS in the CdLS probands. In the control samples that were positive for PSCS an average of 1.125 metaphases (2%) (Range 0-4%) were found to have PSCS. Additionally, several metaphase spreads from individuals with CdLS demonstrated some evidence of breakage. See FIG. 10-I.
Discussion
In eukaryotic cells, replicated DNA molecules remain physically connected from their synthesis in S phase until they are separated during anaphase. This phenomenon, called sister chromatid cohesion, is essential for the temporal separation of DNA replication and mitosis and for the equal separation of the duplicated genome. Sister chromatids in metaphase chromosomes are physically connected until their separation during anaphase (Nasmyth et al., 2000; Nasmyth et al., 2001). PSCS is a phenomenon whereby separate and splayed chromatids with discernible centromeres are seen and involves all or most chromosomes of a metaphase (Kajii and Asamoto 2004). It involves not only the centromere but also the entire sister chromatids of almost all mitotic chromosomes in a given metaphase (Kajii and Ikeuchi 2004). PSCS has been described in a number of conditions including Roberts Syndrome (German 1979), Fanconi Anemia and Ataxia Teleangiectasia (Mehes and Buhler 1995), Alzheimer disease (Moorhead and Heyman 1983; Spremo-Potparevic et al., 2004) Tuberous Sclerosis (Scappaticci et al., 1988), Variegated Aneuploidy (Kajii et al., 1998; Plajaet al., 2003; Plaja et al., 2001) and in normal individuals after exposure to genotoxic chemicals (Major et al., 1999) as well as in association with cancer such as Wilms tumor (Mehes et al., 2002) and breast cancer (Rao et al., 1996) and has been seen in spontaneous abortions (Keser et al., 1996). Recently mutations in the BUB1B gene were found to be a cause of multiple variegated aneuploidy (Hanks et al., 2004). BUB1B encodes BUBR1, a key protein in the mitotic spindle checkpoint. PSCS has also been reported to be present in a low percentage (less than 2-3%) of normal individuals (Dominguez and Rivera 1992; Kajii and Ikeuchi 2004).
Due to the role played by the yeast homologs of NIPBL (Scc2, Rad21, Mis4) in sister chromatid cohesion and the evidence that a similar cohesion abnormality is seen in Drosophila (Rollins et al., 2004), we hypothesized that a similar phenomenon may be present in individuals with CdLS. In studying a minimum of 50 metaphase spreads in 90 CdLS individuals and 90 matched control subjects we found a prevalence of PSCS of 41% in the CdLS samples and 9% in the control samples. The presence or absence of PSCS in CdLS did not appear to be influenced by the presence or absence of an identified mutation in NIPBL or by the age or sex of the individuals with CdLS.
The finding of several metaphases in some of the CdLS probands demonstrating apparently increased breakage (not observed in any of the controls) (FIG. 10-I) indicates that there may be some predisposition to chromosomal fragility in CdLS probands; however, this finding warrants further investigation.
The identification of PSCS in individuals with CdLS has diagnostic value. Presently mutations in NIPBL are identified in approximately 45% of individuals with a clear diagnosis of CdLS (Gillis et al., 2004; Krantz et al., 2004; Tonkin et al., 2004). There has not been any clear evidence that there are other loci for a CdLS gene at this time and it may be that the low mutation detection rate is complicated by the large size of the NIPBL gene and incomplete characterization of the entire coding region (Gillis et al., 2004). The development of an auxiliary test, such as screening for PSCS, provides another parameter to assess for supporting a diagnosis in individuals with CdLS in whom an NIPBL mutation has not been identified, or in whom testing was not performed.
REFERENCES


    Bankier A, Haan E, Birrell R (1986) Familial occurrence of Brachmann-de Lange syndrome. Am J Med Genet 25:163-5
    Beratis N G, Hsu L Y, Hirschhorn K (1971) Familial de Lange syndrome. Report of three cases in a sibship. Clin Genet 2:170-6
    Brachmann W (1916) Ein fall von symmetrischer monodactylie durch ulnadefekt, mit symmetrischer flughautbildung in den ellenbeugen, sowie anderen abnormalitaten (zwerghaftigkeit, halsrippen, behaarung). Jahrbuch Kinderheilkd 84:225-235
    Caksen H, Kurtoglu S, Cesur Y, Ozturk A (2001) An analysis of seven infants with Brachmann-de Lange syndrome, of whom two identical twin sisters. Genet Couns 12:373-7
    Chodirker B N, Chudley A E (1994) Male-to-male transmission of mild Brachmann-de Lange syndrome. Am J Med Genet 52:331-3
    de Lange C (1933) Sur un type nouveau de degeneratio (typus Amstelodamensis). Arch Med Enfants 36:713-719
    Feingold M, Lin A E (1993) Familial Brachmann-de Lange syndrome: further evidence for autosomal dominant inheritance and review of the literature. Am J Med Genet 47:1064-7
    Fryns J P, Dereymaeker A M, Hoefnagels M, D'Hondt F, Mertens G, van den Berghe H (1987) The Brachmann-de Lange syndrome in two siblings of normal parents. Clin Genet 31:413-5
    Ganguly A, Rock M J, Prockop D J (1993) Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 90:10325-9
    Halal F, Silver K (1992) Syndrome of microcephaly, Brachmann-de Lange-like facial changes, severe metatarsus adductus, and developmental delay: mild Brachmann-de Lange syndrome. Am J Med Genet 42:381-6
    Hulinsky R, Winesette H, Dent K, Silver R, King J, Lowichik A, Chen Z, Viskochil D (2003) Prenatal diagnosis dilemma: fetus with del(5)(p13.1p14.2) diagnosed postnatally with Cornelia de Lange syndrome. Am J Hum Genet suppl. 73:602
    Ireland M, Donnai D, Burn J (1993) Brachmann-de Lange syndrome. Delineation of the clinical phenotype. Am J Med Genet 47:959-64
    Ireland M, English C, Cross I, Houlsby W T, Burn J (1991) A de novo translocation t(3;17)(q26.3;q23.1) in a child with Cornelia de Lange syndrome. J Med Genet 28:639-40
    Jackson L, Kline A D, Barr M A, Koch S (1993) de Lange syndrome: a clinical review of 310 individuals. Am J Med Genet 47:940-6
    Kline A D, Barr M, Jackson L G (1993a) Growth manifestations in the Brachmann-de Lange syndrome. Am J Med Genet 47:1042-9
    Kline A D, Stanley C, Belevich J, Brodsky K, Barr M, Jackson L G (1993b) Developmental data on individuals with the Brachmann-de Lange syndrome. Am J Med Genet 47:1053-8
    Kozma C (1996) Autosomal dominant inheritance of Brachmann-de Lange syndrome. Am J Med Genet 66:445-8
    Krajewska-Walasek M, Chrzanowska K, Tylki-Szymanska A, Bialecka M (1995) A further report of Brachmann-de Lange syndrome in two sibs with normal parents. Clin Genet 47:324-7
    Krantz I D, McCallum J, DeScipio C, Kaur M, Gillis L A, Yaeger D, Jukofsky L, Wasserman N, Bottani A, Morris C A, Nowaczyk M J, Toriello H, Bamshad M J, Carey J C, Rappaport E, Kawauchi S, Lander A D, Calof A L, Li H H, Devoto M, Jackson L G (2004) Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat Genet 36:631-5
    Krantz I D, Tonkin E, Smith M, Devoto M, Bottani A, Simpson C, Hofreiter M, Abraham V, Jukofsky L, Conti B P, Strachan T, Jackson L (2001) Exclusion of linkage to the CDL1 gene region on chromosome 3q26.3 in some familial cases of Cornelia de Lange syndrome. Am J Med Genet 101:120-9
    Krantz I D, Rand E B, Genin A, Hunt P, Jones M, Louis A A, Graham J M, Jr., Bhatt S, Piccoli D A, Spinner N B (1997) Deletions of 20p12 in Alagille syndrome: frequency and molecular characterization. Am J Med Genet 70:80-6
    Lieber E, Glaser J H, Jhaveri R (1973) Brachmann-de Lange syndrome. Report of two cases in a sibship. Am J Dis Child 125:717-8
    McConnell V, Brown T, Morrison PJ (2003) An Irish three-generation family of Cornelia de Lange syndrome displaying autosomal dominant inheritance. Clin Dysmorphol 12:241-4
    Naguib K K, Teebi A S, Al-Awadi S A, Marafie M J (1987) Brachmann-de Lange syndrome in sibs. J Med Genet 24:627-9
    Opitz J M (1985) The Brachmann-de Lange syndrome. Am J Med Genet 22:89-102
    Robinson L K, Wolfsberg E, Jones K L (1985) Brachmann-de Lange syndrome: evidence for autosomal dominant inheritance. Am J Med Genet 22:109-15
    Rollins R A, Korom M, Aulner N, Martens A, Dorsett D (2004) Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene. Mol Cell Biol 24:3100-11
    Rollins R A, Morcillo P, Dorsett D (1999) Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152:577-93
    Russell K L, Ming J E, Patel K, Jukofsky L, Magnusson M, Krantz I D (2001) Dominant paternal transmission of Cornelia de Lange syndrome: a new case and review of 25 previously reported familial recurrences. Am J Med Genet 104:267-76
    Saul R A, Rogers R C, Phelan M C, Stevenson R E (1993) Brachmann-de Lange syndrome: diagnostic difficulties posed by the mild phenotype. Am J Med Genet 47:999-1002
    Selicorni A, Lalatta F, Livini E, Briscioli V, Piguzzi T, Bagozzi D C, Mastroiacovo P, Zampino G, Gaeta G, Pugliese A, Cerutti-Mainaroli P, Guala A, Zelante L, Stabile M, Belli S, Franceschini P, Gianotti A, Scarano G (1993) Variability of the Brachmann-de Lange syndrome. Am J Med Genet 47:977-82
    Tonkin E, Smith M, Eichhorn P, Hagan D M, Herrell S, Lusher M, Ireland M, Burn J, Strachan T (2001) A novel gene is disrupted by a Cornelia de Lange-associated translocation breakpoint at 3q26.3. Am J Hum Genet 69:A618
    Tonkin E T, Wang T J, Lisgo S, Bamshad M J, Strachan T (2004) NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat Genet 36:636-41
    Van Allen M I, Filippi G, Siegel-Bartelt J, Yong S L, McGillivray B, Zuker R M, Smith C R, Magee J F, Ritchie S, Toi A, Reynolds J F (1993) Clinical variability within Brachmann-de Lange syndrome: a proposed classification system. Am J Med Genet 47:947-58
    Wilson W G, Kennaugh J M, Kugler J P, Wyandt H E (1983) Reciprocal translocation 14q;21q in a patient with the Brachmann-de Lange syndrome. J Med Genet 20:469-71
    Dominguez M G, Rivera H. 1992. C-anaphases: a mitotic variant. Ann Genet 35(3):183-5.
    German J. 1979. Roberts' syndrome. I. Cytological evidence for a disturbance in chromatid pairing. Clin Genet 16(6):441-7.
    Gillis L A, McCallum J, Kaur M, DeScipio C, Yaeger D, Mariani A, Kline A D, Li H H, Devoto M, Jackson L G, Krantz I D. 2004. NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype-phenotype correlations. Am J Hum Genet 75(4):610-23.
    Hanks S, Coleman K, Reid S, Plaja A, Firth H, Fitzpatrick D, Kidd A, Mehes K, Nash R, Robin N, Shannon N, Tolmie J, Swansbury J, Irrthum A, Douglas J, Rahman N. 2004. Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUBIB. Nat Genet 36(11):1159-61.
    Ireland M, Burn J. 1993. Cornelia de Lange syndrome—photo essay. Clin Dysmorphol 2(2):151-60.
    Kajii T, Asamoto A. 2004. Prenatal diagnosis of a heterozygous carrier of premature chromatid separation (PCS) trait. Am J Med Genet A 126(4):432.
    Kajii T, Ikeuchi T. 2004. Premature chromatid separation (PCS) vs. premature centromere division (PCD). Am J Med Genet A 126(4):433-4.
    Kajii T, Kawai T, Takumi T, Misu H, Mabuchi O, Takahashi Y, Tachino M, Nihei F, Ikeuchi T. 1998. Mosaic variegated aneuploidy with multiple congenital abnormalities: homozygosity for total premature chromatid separation trait. Am J Med Genet 78(3):245-9.
    Keser I, Luleci G, Gunduz G. 1996. Premature centromere division in three unrelated families. Ann Genet 39(2):87-90.
    Major J, Jakab M G, Tompa A. 1999. The frequency of induced premature centromere division in human populations occupationally exposed to genotoxic chemicals. Mutat Res 445(2):241-9.
    Mehes K, Buhler E M. 1995. Premature centromere division: a possible manifestation of chromosome instability. Am J Med Genet 56(1):76-9.
    Mehes K, Kajtar P, Kosztolanyi G. 2002. Association of nonsyndromic Wilms tumor with premature centromere division (PCD). Am J Med Genet 112(2):215-6.
    Moorhead P S, Heyman A. 1983. Chromosome studies of patients with Alzheimer disease. Am J Med Genet 14(3):545-56.
    Nasmyth K, Peters J M, Uhlmann F. 2000. Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288(5470): 1379-85.
    Nasmyth K, Peters J M, Uhlmann F. 2001. Splitting the chromosome: cutting the ties that bind sister chromatids. Novartis Found Symp 237:113-33; discussion 133-8, 158-63.
    Plaja A, Mediano C, Cano L, Vendrell T, Sarret E, Farran I, Sanchez M A. 2003. Prenatal diagnosis of a rare chromosomal instability syndrome: variegated aneuploidy related to premature centromere division (PCD). Am J Med Genet A 117(1):85-6.
    Plaja A, Vendrell T, Smeets D, Sarret E, Gili T, Catala V, Mediano C, Scheres J M. 2001. Variegated aneuploidy related to premature centromere division (PCD) is expressed in vivo and is a cancer-prone disease. Am J Med Genet 98(3):216-23.
    Rao N M, Joshi N N, Shined S R, Adjani S H, Gosh S N. 1996. Premature separation of centromere and aneuploidy: an indicator of high risk in unaffected individuals from familial breast cancer families? Euro J Cancer Prey 5(5):343-50.
    Scappaticci S, Caramel D, Toni M, Vivarelli R, Fois A, Fraccaro M. 1988. Chromosome abnormalities in tuberous sclerosis. Hum Genet 79(2): 151-6.
    Spremo-Potparevic B, Zivkovic L, Djelic N, Bajic V. 2004. Analysis of premature centromere division (PCD) of the X chromosome in Alzheimer patients through the cell cycle. Exp Gerontol 39(5):849-54.
    Kousseff, B. G., Newkirk, P. & Root, A. W. Brachmann-de Lange syndrome. 1994 update. Arch Pediatr Adolesc Med 148, 749-55 (1994).
    Bugge, M. et al. Disease associated balanced chromosome rearrangements: a resource for large scale genotype-phenotype delineation in man. J Med Genet 37, 858-65 (2000).
    Falek, A., Schmidt, R. & Jervis, G. A. Familial de Lange syndrome with chromosome abnormalities. Pediatrics 37, 92-101 (1966).
    Aqua, M. S. et al. Duplication 3q syndrome: molecular delineation of the critical region. Am J Med Genet 55, 33-7 (1995).
    Ireland, M., English, C., Cross, I., Lindsay, S. & Strachan, T. Partial trisomy 3q and the mild Cornelia de Lange syndrome phenotype. J Med Genet 32, 837-8 (1995).
    Rizzu, P. et al. Delineation of a duplication map of chromosome 3q: a new case confirms the exclusion of 3q25-q26.2 from the duplication 3q syndrome critical region. Am J Med Genet 68, 428-32 (1997).
    Kruglyak, L., Daly, M. J., Reeve-Daly, M. P. & Lander, E. S. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 58, 1347-63 (1996).
    Kawauchi, S. et al. Regulation of lens fiber cell differentiation by transcription factor c-Maf. J Biol Chem 274, 19254-60 (1999).


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. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

## Claims

1. A method for diagnosing a human as having an increased risk of developing Cornelia de Lange Syndrome (CdLS) comprising:
a) providing a biological sample from said human comprising a nucleic acid molecule comprising an NIPBL (Nipped-B like) gene wherein said NIPBL gene encodes an NIPBL protein; and
b) detecting in the NIPBL gene of step a) a genetic alteration which alters the length of the NIPBL protein encoded by the NTPBL gene of step a) such that the NIPBL protein encoded by the NIPBL gene of step a) is shorter than a protein consisting of SEQ ID NO: 3;
wherein said genetic alteration in the NIPBL gene of step a) corresponds to an alteration in SEQ ID NO: 2 selected from the group consisting of: deletion of G at position 276, replacement of the nucleotides at positions 325-334 with SEQ ID NO: 121, deletion of CT at positions 868-869, deletion of A at position 1087, insertion of G at position 1672, insertion of C at position 1795, insertion of A at position 2028, deletion of AG at positions 2605-2606, deletion of T at position 2646, deletion of G at position 3095, deletion of TGTCT at positions 3149-3153, deletion of TAGA at positions 3183-3186, deletion of AGAG at positions 3186-3189, insertion of G at position 4095, deletion of AAAAA at positions 4691-4695, deletion of C at position 4693, deletion of AAGAC at positions 7277-7281, deletion of C at position 7336, deletion of G at position 7557, deletion of C at position 7906, and insertion of G at position 7951;
or wherein said genetic alteration in the NIPBL gene of step a) is a nonsense mutation creating a stop codon at a position corresponding to the codon encoding an amino acid in the NIPBL protein of SEQ ID NO: 3 selected from the group consisting of: Arg at position 479, Arg at position 797, Arg at position 832, Glu at position 977, Ser at position 1024, Ser at position 1398, Ser at position 1459, Arg at position 1536, Arg at position 1723, and Arg at position 1758; and
wherein said genetic alteration in the NIPBL gene of step a) is correlated with an increased risk of developing CdLS in said human.

2. The method according to claim 1, wherein said biological sample is selected from the group consisting of blood, saliva, amniotic fluid, embryonic tissue and tissue.

3. The method according to claim 2, wherein said biological sample is blood.

4. The method of claim 1, wherein the genetic alteration detected in step b) is in exon 10 of the NIPBL gene.

5. The method according to claim 1, wherein step b) is performed by a method selected from the group consisting of direct sequencing, conformation sensitive gel electrophoresis, single strand polymorphism assay, restriction fragment length polymorphism assay, ligase chain reaction, enzymatic cleavage and southern hybridization.

6. The method according to claim 5, wherein step b) is performed by direct sequencing.

7. The method according to claim 5, wherein step b) is performed by conformation sensitive gel electrophoresis.

8. The method according to claim 1, wherein said nucleic acid molecule comprising the NIPBL gene is a deoxyribonucleic acid.

9. The method according to claim 1, wherein said nucleic acid molecule comprising the NIPBL gene is a messenger ribonucleic acid.

10. The method according to claim 1, further comprising isolating the protein product encoded by said NIPBL gene of step a).

11. The method of claim 1, further comprising isolating cells from said human and assessing said cells for the presence or absence of precocious sister chromatid separation (PSOS).

12. The method as claimed in claim 1, wherein said genetic alteration has been previously identified in a patient having CdLS.

13. The method as claimed in claim 1, wherein said human is an infant, and said method further comprises performance of genotype-phenotype correlation analysis.

14. The method of claim 13, wherein the phenotype for the genotype-phenotype correlation is selected from the group consisting of limb reduction, developmental delay, and growth retardation.