# Patent

## Title
Methods for identifying subjects susceptible to charcot-marie-tooth neuropathy type 1C

## Abstract

In one aspect, the invention provides methods of identifying genetic mutations that are associated with peripheral neurological disease. The methods comprise identifying a difference between a nucleic acid sequence of a small integral protein of the lysosome/late endosome (“SIMPLE”) gene from a mammalian subject exhibiting peripheral neuropathy and a nucleic acid sequence of a SIMPLE gene from a subject which is not exhibiting peripheral neuropathy, wherein the difference is a genetic mutation associated with peripheral neurological disease. In another aspect, isolated nucleic acid molecules encoding SIMPLE missense mutations are provided. In another aspect, a method of screening a subject to determine if the subject has a genetic predisposition to develop Charcot-Marie-Tooth type 1C neuropathy is provided. In another aspect, the invention provides kits for determining susceptibility or presence of Charcot-Marie-Tooth type 1C neuropathy in a mammalian subject.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a divisional of co-pending application Ser. No. 12/245,591, filed Oct. 3, 2008, which is a continuation of patent application Ser. No. 10/756,194, filed Jan. 13, 2004, now U.S. Pat. No. 7,449,291, which claims the benefit of Provisional Application No. 60/440,399, filed Jan. 13, 2003. The benefit of the priority of the filing dates of each application is hereby claimed under 35 U.S.C. §§ 120 and 119, respectively. Each application is incorporated herein by reference in its entirety.


STATEMENT OF GOVERNMENT RIGHTS
This invention was made with government support under Grant No. NS38181 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND
The present invention relates to methods and kits for identifying subjects susceptible to Charcot-Marie-Tooth Neuropathy. Charcot-Marie-Tooth (CMT) neuropathy, also called Hereditary Motor and Sensory Neuropathy (HMSN), is a clinically and genetically heterogeneous group of inherited peripheral neuropathies leading to progressive distal muscle weakness and sensory loss. CMT is frequently transmitted in an autosomal dominant manner. An estimated 1 in 2,500 persons has a form of CMT, making it a major diagnostic category within neurogenetic diseases (Skre, H., Clin. Genet. 6:98-118 (1974)). A current classification system divides CMT neuropathy into CMT type 1 (CMT1), which is characterized by demyelination and reduced nerve conduction velocities (NCVs)(typically <40 meters/sec), and CMT type 2 (CMT2), which denotes patients with axonal neuropathy, lack of myelin abnormalities in pathologic specimens, and nearly normal nerve conduction velocities (Dyck and Lambert, Arch. Neurol. 18:603-618 (1968)). Onset of symptoms associated with CMT typically occurs in adolescence or early adulthood; however, presentation may be delayed until mid-adulthood. The severity of symptoms is variable, even among members of the same family, with gradual progression of symptoms. Typical CMT symptoms include pes cavus, distal muscle weakness and atrophy, absent or diminished deep tendon reflexes, and mild sensory loss.
CMT1 has been divided into five subtypes (CMT1A-D, X), based on genetic linkage analysis; however, the CMT1 subtypes are clinically indistinguishable. CMT1A is associated with a 1.4 megabase (Mb) duplication on chromosome 17p11.2-p12 and a gene dosage effect for peripheral myelin protein (PMP22) (Matsumami et al., Nat. Gen.:176-179 (1992)). CMT1B is associated with mutations in the myelin protein zero gene (MPZ) (Hayasaka et al., Nat. Genet. 5:31-34 (1993). CMT1D is associated with mutations in the early growth response 2 element gene (EGR2) (Warner et al., Nat. Gen. 18:382-384 (1998) and CMTX is associated with mutations in the connexin 32 (Cx32) gene (Bergoffen et al., Science 262:2039-2042 (1993). Recently, mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) have been associated with autosomal recessive demyelinating CMT as well as autosomal recessive axonal CMT with vocal cord paralysis (Nelis, E., et al., Neurology 59(12):1835-6 (2002). Patients that exhibit symptoms associated with CMT1, but that lack mutations in these known genes, have been assigned to subtype CMT1C.
Given the prevalence of CMT1 cases not linked to any known genetic loci, there is a need to identify genetic mutations associated with the CMT1 syndrome that can be used in a genetic screen to identify subjects susceptible to CMT1 neuropathy. The present inventors have discovered individuals with mutations in the small integral membrane protein of the lysosome/late endosome (“SIMPLE”) gene and have established a molecular linkage for CMT1C.
SUMMARY
In accordance with the foregoing, in one aspect the present invention provides methods of identifying genetic mutations that are associated with peripheral neurological disease in a mammalian subject, the methods comprising identifying a difference between a nucleic acid sequence of a small integral membrane protein of the lysosome/late endosome (“SIMPLE”) gene from a first mammalian subject exhibiting peripheral neuropathy and a nucleic acid sequence of a SIMPLE gene from a second mammalian subject which is not exhibiting peripheral neuropathy, wherein the first and second mammalian subjects are members of the same species, and wherein the difference between the nucleic acid sequences is a genetic mutation that is associated with peripheral neurological disease. In some embodiments of this aspect of the invention, the method further comprises determining whether the identified mutations co-segregate with peripheral neuropathy.
In another aspect, the present invention provides isolated nucleic acid molecules encoding a SIMPLE protein comprising a missense mutation, the isolated nucleic acid molecules comprising a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
In another aspect, the present invention provides isolated SIMPLE polypeptides comprising a missense mutation, the isolated SIMPLE polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.
In another aspect, the present invention provides a nucleic acid probe for detecting a SIMPLE gene consisting of a nucleic acid sequence selected from the group consisting of a nucleic acid sequence spanning nucleotide 91 to nucleotide 140 of SEQ ID NO: 4, or the complement thereof, and SEQ ID NO: 6, or the complement thereof.
In another aspect, the present invention provides nucleic acid primer molecules consisting of SEQ ID NO: 18 and SEQ ID NO: 19 which are useful for amplifying exon 3 of a SIMPLE gene.
In another aspect, the present invention provides methods of screening a mammalian subject to determine if said subject has a genetic predisposition to develop, or is suffering from Charcot-Marie-Tooth neuropathy type 1C (CMT1C). The method of this aspect of the invention comprises analyzing the nucleic acid sequence of a SIMPLE gene in a mammalian subject to determine whether a genetic mutation that is associated with CMT1C is present in the nucleic acid sequence, wherein the presence of an identified genetic mutation in the SIMPLE gene that co-segregates with CMT1C indicates that the mammalian subject has a genetic predisposition to develop CMT1C or is suffering from CMT1C.
In another aspect, the invention provides a kit for determining susceptibility or presence of CMT1C in a mammalian subject based on the detection of a mutation in a SIMPLE gene, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of a SIMPLE gene and (ii) written indicia indicating a correlation between the presence of said mutation and risk of developing CMT1C. In some embodiments, the kit detects the presence or absence of a mutation in the SIMPLE gene selected from the group consisting of G112S, T115N and W116G.
The invention thus provides methods, reagents and kits for identifying genetic mutations in a SIMPLE gene and thereby facilitates diagnosis of Charcot-Marie-Tooth neuropathy and identification of carriers of the genetic defect. The nucleic acid molecules of the invention are useful as probes to identify genetic mutations in the SIMPLE gene and have therapeutic utility for identifying compounds that can be used to treat Charcot-Marie-Tooth neuropathy.


DETAILED DESCRIPTION
Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Press, Plainsview, N.Y., and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1999) for definitions and terms of art.
The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.
As used herein, the term “peripheral neuropathy” refers to peripheral nerve damage, including sensory and motor nerve damage, producing a variety of symptoms including for example, muscle weakness, numbness, paresthesia and pain in the arms, hands, legs and/or feet, pes cavus and reduced nerve conduction velocity.
As used herein, the term “peripheral neurological disease” refers to the clinical manifestation of a typically slowly progressive peripheral neuropathy.
As used herein, the term “paresthesia” refers to abnormal sensations such as burning, tickling, pricking or tingling.
As used herein, the term “pes cavus” refers to a deformity of the foot producing a high arch that does not flatten with weightbearing. The deformity can be located in the forefoot, midfoot, or hindfoot or in a combination of these sites.
As used herein, the term “proband” refers to the family member through whom a family's medical history comes to light.
As used herein, the term “Charcot-Marie-Tooth neuropathy” or (“CMT”) refers to a peripheral (motor and sensory) neuropathy without an established acquired (non-genetic) cause as described in Sherer et al., (2003) The Molecular and Genetic Basis of Neurological Diseases, 3d Ed., Oxford Butterworth Hiemen Press, pp. 435-453. Unexplained chronic progressive neuropathy in individuals with a negative family history for peripheral neuropathy may represent an instance of inherited CMT on the basis of a new dominant mutation, or a single occurrence of an autosomal recessive or X-linked disorder in a family. CMT patients typically experience slowly progressive symptoms ranging from muscle weakness in the arms, legs, hands and feet, decreased muscle bulk, reduced tendon reflexes and sensory loss. Individuals with CMT may also have foot deformities, such as pes cavus, high arches, hammertoes, inverted heel, flat feet and other orthopedic problems such as mild scoliosis or hip dysplasia.
As used herein, the term “Charcot-Marie-Tooth Neuropathy type 1” or (“CMT1”) includes a large group of inherited autosomal dominant disorders characterized by peripheral nerve demyelination affecting peripheral (motor and/or sensory) nerves. Hallmarks of CMT1 include reduced nerve conduction velocities (typically less than 40 meters/sec), and nerve biopsies that display “onion bulb” formation. Studies have determined that reduction in nerve conduction velocity can be accurately determined in most cases by the age of five years (Nicholson, G. A. Neurology 41:547-552 (1991).
As used herein, the term “Charcot-Marie Tooth Neuropathy type 1C” or (“CMT1C”) refers to one of five clinically indistinguishable subtypes of Charcot-Marie-Tooth Neuropathy type 1 that is designated as type C based on the lack of molecular linkage to genetic loci associated with subtypes CMT1A, CMT1B, CMT1D, and CMT1X and/or based on the presence of mutation(s) in the SIMPLE gene.
As used herein, the term small integral membrane protein of the lysosome/late endosome “SIMPLE,” also known as the lipopolysaccharide-induced TNF-alpha factor “LITAF,” or p53-induced gene “PIG7” refers to any gene that encodes the SIMPLE/LITAF/PIG7 protein. Some SIMPLE genes useful in the practice of this invention are at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 3. Some SIMPLE genes useful in the practice of this invention are at least 95%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 3.
As used herein, the term “primer” means a polynucleotide which can serve to initiate a nucleic acid chain extension reaction. Typically, primers have a length of 5 to about 50 nucleotides, although primers can be longer than 50 nucleotides.
As used herein, the term “sequence identity” or “percent identical” as applied to nucleic acid molecules is the percentage of nucleic acid residues in a candidate nucleic acid molecule sequence that are identical with a subject nucleic acid molecule sequence (such as the nucleic acid molecule sequence set forth in SEQ ID NO: 3), after aligning the sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the sequence identity. No gaps are introduced into the candidate nucleic acid sequence in order to achieve the best alignment. Nucleic acid sequence identity can be determined in the following manner. The subject polynucleotide molecule sequence is used to search a nucleic acid sequence database, such as the Genbank database, using the program BLASTN version 2.1 (based on Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)). The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity as defined in Wootton, J. C., and S. Federhen, Methods in Enzymology 266:554-571 (1996). The default parameters of BLASTN are utilized.
As used herein, the term “genetic mutation” is an alteration of the wild-type SIMPLE gene sequence deposited in GenBank, provided as SEQ ID NO: 3 that is not a recognized polymorphism. A polypmorphism typically has a population frequency of greater than 1% in mammalian control subjects of the same species that do not exhibit peripheral neuropathy.
In one aspect, the present invention provides methods of identifying genetic mutations that are associated with peripheral neurological disease in a mammalian subject. The methods of this aspect of the invention comprise the step of identifying a difference between a nucleic acid sequence of a small integral membrane protein of the lysosome/late endosome (“SIMPLE”) gene from a first mammalian subject exhibiting peripheral neuropathy and a nucleic acid sequence of a SIMPLE gene from a second mammalian subject which is not exhibiting peripheral neuropathy, wherein the first and second mammalian subjects are members of the same species, and wherein the difference between the nucleic acid sequences is a genetic mutation that is associated with peripheral neurological disease. In some embodiments, the method further comprises the step of determining whether the identified genetic mutation co-segregates with peripheral neuropathy.
The methods of this aspect of the invention are useful to identify genetic mutations associated with hereditary peripheral neurological disease in any mammalian subject, particularly human subjects. For example, the methods of the invention may be used to identify genetic mutations in the SIMPLE gene that are associated (i.e., where the mutation is found to occur in subjects predisposed to develop hereditary peripheral neurological disease and the mutation is not found in subjects not predisposed to develop hereditary peripheral neurological disease) with the occurrence of hereditary peripheral neurological disease in individuals at risk for developing this disease.
The present inventors have discovered that mutations in the SIMPLE gene locus are responsible for a portion of cases of the peripheral neurological disease Charcot-Marie-Tooth Neuropathy type 1C (CMT1C). Previously, patients were designated as subtype CMT1C based on the absence of mutations in genetic loci known to be associated with CMT1, such as the peripheral myelin protein 22 (PMP22) gene (associated with CMT1A), the myelin protein zero gene (MPZ) (associated with CMT1B), the early growth response 2 element gene (EGR2) (associated with CMT1D) or the connexin 32 (Cx32) (associated with CMTX). SIMPLE was identified as a candidate gene for CMT1C based in part on chromosomal mapping to a 9 cM region on chromosome 16p as described in Example 1.
The SIMPLE gene appears to be almost ubiquitously expressed (Moriwaki et al., J. Biol Chem 276:23065-23076 (2001), however the biological function of the SIMPLE protein is currently unknown. The protein encoded by the SIMPLE gene possesses a putative membrane association domain flanked by two putative CXXC motifs, known as high affinity zinc binding motifs (Collet et al., J. Biol. Chem 2:2, (2003). In addition, the N-terminus of the SIMPLE protein contains two PPXY motifs (WW domain binding motif) that have been shown to interact with Nedd4, an E3 ubiquitin ligase that plays a role in ubiquitinating membrane proteins (Jolliffe et al., Biochem J 351:557-565 (2000)). The SIMPLE protein is identical to a protein previously described as lipopolysaccharide-induced TNF-alpha factor (“LITAF”). Moriwaki et al., J. Biol Chem 276:23065-23076 (2001). LITAF was originally cloned as a putative nuclear transcription factor involved in binding to a critical region of the TNF-alpha promoter (Myokai et al., Proc Natl Acad Sci 96:4518-4523 (1999)).
The human SIMPLE gene consists of four exons spanning a genomic interval of 37.75 kilobases, with a start codon located in exon 2. The SIMPLE cDNA coding sequence is provided herein as SEQ ID NO: 1 which corresponds to nucleotides 234-719 of GenBank accession number AB034747. Disclosed herein are nucleic acid mutations numbered sequentially with respect to the first nucleotide of SEQ ID NO: 1. The SIMPLE protein encoded by SEQ ID NO: 1 is provided herein as SEQ ID NO: 2. Disclosed herein are amino acid mutations numbered sequentially with respect to the first amino acid residue of SEQ ID NO: 2. The entire 37.75 kilobase genomic locus that encompasses the SIMPLE gene is provided herein as SEQ ID NO: 3. With respect to the first nucleotide in SEQ ID NO: 3, the four exons are as follows: exon 1: nucleotides 1 to 228; exon 2: nucleotides 29,639 to 29,863; exon 3: nucleotides 32,685 to 32,840; and exon 4: nucleotides 36,629 to 37,775. The start codon is in exon 2 at nucleotide 29,644. The nucleic acid sequence encoding exon 3 (nucleotides 29,639 to 29,863 of SEQ ID NO: 3) is provided herein as SEQ ID NO: 4, which encodes the amino acid sequence provided as SEQ ID NO: 5.
The present inventors have identified several missense mutations (for example, G112S, T115N and W116G) in SIMPLE that cause a portion of CMT1C cases. The patients with these mutations in SIMPLE (as further described in Examples 1 and 2) exhibited peripheral neuropathy and met widely accepted criteria for CMT1 including distal muscle weakness and atrophy, depressed deep tendon reflexes and sensory impairment (see Dyck, P. J., and E. H. Lambert, Arch. Neurol. 18:619-625 (1968)). The three missense mutations G112S, T115N, and W116G are clustered in a conserved region of exon 3 encompassing seven amino acids as shown in Table 1. The amino acid sequence of the wild-type human SIMPLE protein AGALTWL (SEQ ID NO: 7) is identically conserved in human, mouse, rat and chicken (Street et al., Neurology 60:22-27). As shown in Table 1, the tight clustering of mutations within exon 3 of the SIMPLE gene suggest this domain, which is immediately adjacent to a membrane association domain, is critical to peripheral nerve function. The first column of Table 1 provides the CMT1 pedigree from which each mutant was identified. The second column provides the altered (underlined) amino acid residue in each CMT1 pedigree.







TABLE 1







CONSERVED AMINO ACID REGION OF THE SIMPLE


PROTEIN CONTAINING MISSENSE MUTATIONS









CMT1 Pedigree/
Nucleic Acid
Amino Acid


Mutation
Sequence
Sequence





Wild-type
GCCGGTGCTCTGACCT
AGALTWL


human
GGCTG SEQ ID NO: 6
SEQ ID NO: 7


 


K1551, K1552,
GCCAGTGCTCTGACCT
ASALTWL


PN282 G112S
GGCTG SEQ ID NO: 8
SEQ ID NO: 9


 


K1550
GCCGGTGCTCTGAACTG
AGALNWL


T115N
GCTG SEQ ID NO: 10
SEQ ID NO: 11


 


K2900, K1910
GCCGGTGCTCTGACCGG
AGALTGL


W116G
GCTG SEQ ID NO: 12
SEQ ID NO: 13









The practice of this aspect of the invention is therefore useful to identify additional mutations in the SIMPLE gene that are associated with peripheral neurological diseases such as, for example, CMT (type 1 or type 2), Dejerine-Sottas disease, congenital hypomyelination neuropathy, and hereditary neuropathy with liability to pressure palsies. By way of illustrative example, Dejerine-Sottas disease (DSD) is a peripheral neurological disease with clinical features that overlap with those of severe CMT1. Molecular studies indicate that DSD, like CMT, shows genetic heterogeneity and shares several genetic loci in common with those implicated for CMT1, including PMP22, MPZ and EGR2, as well as periaxin (PRX) (Boerkoel et al., Am J Hum Genet 68: 325-33 (2001). Therefore, the methods of this aspect of the invention may be used to identify genetic mutations in SIMPLE that are associated with Dejerine-Sottas disease or other peripheral neurological diseases. In some patients with peripheral neuropathy, mutations in SIMPLE may occur in combination with a mutation in another gene known to be associated with peripheral neurological disease, such as, for example, PMP22, Cx32, MPZ, EGR2, GDAP, and PRX as described herein. For example, mutations in the SIMPLE gene may act to increase or decrease the clinical severity of a peripheral neurological disease that has previously been associated with a mutation in a gene other than SIMPLE.
In the practice of this aspect of the method of the invention, any method of obtaining reliable nucleic acid sequence data from a mammalian subject exhibiting peripheral neuropathy may be utilized. For example, reliable sequence data may be obtained from existing databases of sequence data, or alternatively, a reliable nucleic acid assay that will identify a genetic mutation in the SIMPLE gene may be utilized.
In one embodiment of the method of the invention, a genetic mutation is detected by amplification of all or part of the SIMPLE gene from genomic DNA followed by sequencing of the amplified DNA. For example, each of the four exons of the SIMPLE gene may be amplified individually or in combination using as template genomic DNA from a test subject exhibiting peripheral neuropathy. A method of amplification which is well known by those skilled in the art is the polymerase chain reaction (PCR) (see Current Protocols in Molecular Biology, Ausubel, F. M. et al., John Wiley & Sons; 1995). Alternative amplification techniques may also be used in the method of this aspect of the invention, such as the ligase chain reaction (LCR) (Wu and Wallace, Genomics 4:560-569 (1989)), strand displacement amplification (SDA) (Walker et al., Proc. Nat'l. Acad. Sci. USA 89:392-396 (1992)), self-sustained sequence replication (3SR) (Fahy et al., PCR Methods Appl. 1:25-33 (1992)), and branched chain amplification which are known and available to persons skilled in the art.
The PCR process involves the use of pairs of primers, one for each complementary strand of the duplex DNA (wherein the coding strand is referred to as the “sense strand” and its complementary strand is referred to as the “anti-sense strand”), that will hybridize at sites located on either side of a region of interest in a gene. Chain extension polymerization is then carried out in repetitive cycles to increase the number of copies of the region of interest exponentially. Primers useful in the practice of the method of the invention comprise polynucleotides that hybridize to a region of a SIMPLE gene, which can serve to initiate a chain extension reaction. A “primer pair” is a pair of primers which specifically hybridize to sense (coding) and antisense (non-coding) strands of a duplex polynucleotide to permit amplification of the region lying between the primers of the pair. Primers useful in the practice of this aspect of the invention comprise a polynucleotide of any size that is capable of hybridizing to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 under conditions suitable for PCR amplification and/or sequencing. In a preferred embodiment, primers useful in the practice of this aspect of the invention range from about 5 to 50 bp or longer of continuous sequence chosen from SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4. Table 2 describes sets of primers useful for PCR amplifying and sequencing of each of the four exons of the SIMPLE gene from genomic DNA. The first column of Table 2 describes the exon to be amplified and the second column and third columns provide the nucleotide sequence of the forward and reverse primers used to amplify the exon and their corresponding SEQ ID NOs. Tm refers to the melting temperature of the oligonucleotide pair. The expected PCR product size in base pairs (bp) for each PCR amplification is provided in the fifth column. Example 1 provides a non-limiting example of this embodiment of the method of the invention.







TABLE 2







PRIMERS FOR EXON FRAGMENT AMPLIFICATION AND


SEQUENCING OF THE SIMPLE GENE















Size


Exon
Forward Primer
Reverse Primer
Tm
bp














1
1F 5′ TCAGAAACAAAACCAAAACAAACA 3′
1R 5′ GTCCCACCAGCACCTACCC 3′
59.7
337



(SEQ ID NO: 14)
(SEQ ID NO: 15)




 


2
2F 5' CAACTGAATTTCTTATCTGG 3′
2R 5′ GTAAAACTGGAACGTACTGG 3′
55
387



(SEQ ID NO: 16)
(SEQ ID NO: 17)




 


3
3F 5' ATAGCCAGACGATGAACG 3′
3R 5′ ATGGTGCAGTTGAGAACC 3′
53
385



(SEQ ID NO: 18)
(SEQ ID NO: 19)




 


4
4F 5' GAACATTTTGGCAGC 3′
4R 5′ TAATGGTAGGCACTAAAGG 3′
59
636



(SEQ ID NO: 20)
(SEQ ID NO: 21)









In one embodiment of the method of the invention, after amplification, genetic mutations are detected in the amplified DNA by sequence analysis. Methods of DNA sequence analysis are well known in the art. A well known method of sequencing is the “chain termination” method first described by Sanger et al., PNAS (USA) 74(12):5463-5467 (1977) and detailed in SEQUENASETM 2.0 product literature (Amersham Life Sciences, Cleveland). Sequencing can be performed using a single primer or a primer pair. Primers are chosen for sequencing based on their proximity to the region of interest. Non-limiting examples of suitable sequencing primers for each exon are described in Table 2.
Once the nucleic acid sequence from the test subject is obtained, the sequence is compared to the nucleic acid sequence of one or more subjects not exhibiting peripheral neuropathy in order to identify genetic mutations in SIMPLE that are associated with peripheral neurological disease. For example, resulting sequences can be aligned with the known exon sequence using a multiple sequence alignment tool, Sequencher (Gene Codes Corporation, Ann Arbor, Mich.), in order to identify any nucleotide changes as described in Example 5. In one embodiment, the information and analysis can be recorded on a database and the comparisons can be performed by a computer system accessing said database. In this manner, the amplified sequences of SIMPLE from a subject exhibiting peripheral neurological disease are sequenced until a mutation in SIMPLE associated with peripheral neurological disease is identified.
A mutation associated with peripheral neurological disease encompasses any alteration of the wild-type small integral membrane protein of the lysosome/late endosome (“SIMPLE”) sequence deposited in GenBank, provided as SEQ ID NO: 3, that is not a recognized polymorphism. A polymorphism typically has a population frequency of greater than 1% in mammalian control subjects of the same species that do not exhibit peripheral neuropathy, and is not associated with peripheral neurological disease. In contrast, a mutation that is positively associated with peripheral neurological disease typically co-segregates with family members exhibiting peripheral neuropathy. The following characteristics are supportive, but are not required for a genetic mutation to be a causative mutation for peripheral neurological disease: (1) the change results in an amino acid substitution in a highly evolutionarily conserved residue of the SIMPLE protein (such as in exon 3 (SEQ ID NO: 5) or in the conserved region of exon 3 (SEQ ID NO: 7); (2) the change occurs in a functional domain of SIMPLE; (3) the change is predicted to affect splicing; or (4) the change co-segregates with disease in a family in an autosomal dominant manner.
A genetic mutation may be any form of sequence alteration including a deletion, insertion, point mutation or DNA rearrangement in the coding or noncoding regions. Deletions may be small or large and may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Point mutations may also occur in regulatory regions, such as in the promoter of the SIMPLE gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the SIMPLE gene product, or to a decrease in mRNA stability or translation efficiency. DNA rearrangements include a simple inversion of a single segment of DNA, a reciprocal or nonreciprocal translocation disrupting any portion of the gene, or a more complex rearrangement.
In one embodiment of this aspect of the method of the invention, once a mutation is identified in a subject exhibiting peripheral neuropathy, co-segregation analysis is carried out to determine if the particular mutation in the SIMPLE gene co-segregates with the presence of peripheral neuropathy in the subjects tested. The standard test for genetic linkage is described in J. Ott (1999), Analysis of Human Genetic Linkage, 3d ed., The Johns Hopkins University Press. Co-segregation analysis can be done in several ways. In one embodiment, co-segregation analysis is done by sequencing DNA amplified from the corresponding exon in subjects exhibiting peripheral neuropathy utilizing the previously described methods. For example, DNA sequence variations can be identified using DNA sequencing, as described in Example 1. Alternatively, there are several other methods that can be used to detect and confirm DNA sequence variation including, for example, (1) restriction fragment length polymorphism (RFLP) analysis as described in Example 3; (2) single stranded conformation analysis (SSCA) (Orita et al., Proc. Nat'l. Acad. Sci. USA 86:2776-2770 (1989)); (3) denaturing gradient gel electrophoresis (DGGE) based on the detection of mismatches between the two complementary DNA strands (Wartell et al., Nucl. Acids Res. 18:2699-2705 (1990)); (4) RNase protection assays (Finkelstein et al., Genomics 7:167-172 (1990)); (5) hybridization with allele-specific oligonucleotides (ASOs) (Conner et al., Proc. Nat'l. Acad. Sci. USA 80:278-282 (1983)); and (6) allele-specific PCR (Rano & Kidd, Nucl. Acids Res. 17:8392 (1989)). In the SSCA, DGGE and RNase protection assay, a new electrophoretic band appears when a mutation is present. SSCA detects a band which migrates differently because the sequence change causes a difference in single-strand, intramolecular base pairing. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences using a denaturing gradient gel. For allele-specific PCR, primers are used which hybridize at their 3′ ends to a particular SIMPLE mutation. If the particular SIMPLE mutation is not present, an amplification product is not observed. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
In another embodiment, genetic mutations are identified by hybridization of amplified regions of the SIMPLE gene with allele-specific oligonucleotides. For example, a hybridization assay may be carried out by isolating genomic DNA from a mammalian subject exhibiting peripheral neuropathy, contacting the isolated DNA with a hybridization probe specific for a SIMPLE gene mutation under conditions suitable for hybridization of the probe with the isolated genomic DNA, said DNA probe spanning said mutation in said gene, wherein said DNA probe is capable of detecting said mutation; and determining the presence or absence of said hybridized DNA probe as an indication of the presence or absence of said genetic mutation. Desirable probes useful in such a DNA hybridization assay comprise a nucleic acid sequence that is unique to the genetic mutation. Examples of useful DNA probes include SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12 as provided in Table 1. Analysis can involve denaturing gradient gel electrophoresis or denaturing HPLC methods, for example. For guidance regarding probe design and denaturing gel electrophoresis or denaturing HPLC methods, see, e.g., Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.
In another embodiment of this aspect of the method of the invention, restriction fragment length polymorphism (RFLP) for the SIMPLE gene can be used to score for a genetic mutation in a co-segregation analysis. RFLP has been described in U.S. Pat. Nos. 4,965,188 and 4,800,159, incorporated herein by reference. In this technique, restriction enzymes are used which provide a characteristic pattern of restriction fragments, wherein a restriction site is either missing or an additional restriction site is introduced in the mutant allele. Thus, DNA from an individual and from control DNA sequences are isolated and subjected to cleavage by restriction enzymes which are known to provide restriction fragments which differentiate between normal and mutant alleles, and the restriction patterns are identified. Example 3 and Table 4 further illustrate RFLP methods that are useful in the practice of the method of the invention.
Several genetic mutations in SIMPLE that are associated with the peripheral neurological disease CMT1 have been identified by practicing the methods of this aspect of the invention as described in Examples 1 and 2 and shown in Tables 1 and 3. Table 3 provides a list of mutations identified in a SIMPLE gene using the methods of this aspect of the invention. The first column of Table 3 describes the exon each mutation resides in, the second column describes the nucleotide change in the cDNA (numbered sequentially with reference to SEQ ID NO: 1) for each mutant, the third column describes the type of mutation that is present, the fourth column describes primer pairs useful to PCR amplify the exon containing the mutation, and the fifth column describes primers useful for sequencing across the region containing the mutation.







TABLE 3







SUMMARY OF MUTATIONS IDENTIFIED IN THE SIMPLE GENE


THAT CO-SEGREGATE WITH PERIPHERAL NEUROPATHY














Predicted 






Nucleotide
amino acid






change in
change in
Type of
Primers used 
Primers used to


Exon
cDNA
protein
Mutation
to PCR amplify
sequence















3
334G to A
G112S
missense
3F (SEQ ID NO: 18)
3F (SEQ ID NO: 18)






3R (SEQ ID NO: 19)



3
344C to A
T115N
missense
3F (SEQ ID NO: 18)
3F (SEQ ID NO: 18)






3R (SEQ ID NO: 19)



3
346T to G
W116G
missense
3F (SEQ ID NO: 18)
3F (SEQ ID NO: 18)






3R (SEQ ID NO: 19)









In another aspect, the present invention provides isolated nucleic acid molecules encoding a SIMPLE protein comprising a mutation selected from the group consisting of G112 S, T115N, and W116G. The mutations in the SIMPLE protein are numbered sequentially according to the first amino acid of SEQ ID NO: 2. The nucleotide sequences are numbered sequentially according to the first nucleotide of SEQ ID NO: 1. Each mutation is further described as follows:
Mutation G112S results from a nucleotide change of G to A at nucleotide 334, which results in a codon change from GGT to AGT which in turn results in the missense mutation at amino acid G112 to S, substituting a serine for a glycine at amino acid position 112 in the SIMPLE protein. In some embodiments, mutation G112S is encoded by SEQ ID NO: 8 as shown in Table 1.
Mutation T115N results from a nucleotide change of C to A at nucleotide 344, which results in a codon change from ACC to AAC which in turn results in the missense mutation at amino acid T115 to N, substituting an asparagine for a threonine at amino acid position 115 in the SIMPLE protein. In some embodiments, mutation T115N is encoded by SEQ ID NO: 10 as shown in Table 1.
Mutation W116G results from a nucleotide change of T to G at nucleotide 346, which results in a codon change from TGG to GGG which in turn results in the missense mutation at amino acid W116 to G, substituting a glycine for a tryptophan at amino acid position 116 in the SIMPLE protein. In some embodiments, mutation W116G is encoded by SEQ ID NO: 12 as shown in Table 1.
In some embodiments, the isolated nucleic acid molecules described herein comprise a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12. In this regard, in some embodiments, the isolated nucleic acid molecules described herein are at least 90% identical to a portion of SEQ ID NO: 1 or its complement. In some embodiments, the isolated nucleic acid molecules described herein are at least 90% identical to a portion of SEQ ID NO: 3 or its complement. In some embodiments, the isolated nucleic acid molecules described herein are at least 90% identical to a portion of SEQ ID NO: 4 or its complement. In some embodiments, the isolated nucleic acid molecules described herein are at least 90% identical to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12 as described in Table 1. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 1 under conditions of 5×SSC at 50° C. for 1 hr. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 1 under conditions of 5×SSC at 60° C. for 1 hr. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 3 under conditions of 5×SSC at 50° C. for 1 hr. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 3 under conditions of 5×SSC at 60° C. for 1 hr. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 4 under conditions of 5×SSC at 50° C. for 1 hr. In some embodiments, the isolated nucleic acid molecules described herein hybridize to the complement of SEQ ID NO: 4 under conditions of 5×SSC at 60° C. for 1 hr.
Some nucleic acid embodiments, for example, include genomic DNA, RNA, and cDNA encoding the mutant proteins or fragments thereof. In some embodiments, the invention also encompasses DNA vectors such as, for example DNA expression vectors that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the coding sequences the nucleic acids above, and genetically engineered host cells that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. The nucleic acids encoding the SIMPLE protein mutations can be manipulated using conventional techniques in molecular biology so as to create recombinant constructs that express mutant polypeptides.
The nucleic acid sequences described above have diagnostic as well as therapeutic use. The nucleic acid sequences can be used as probes to identify more genetic mutations in the SIMPLE gene and to detect the presence or absence of wild-type or mutant genes in an individual, such as in nucleic acid hybridization assays, southern and northern blot analysis, and as controls for screening assays and the kits described herein. The sequences described herein can also be incorporated into constructs for preparing recombinant mutant proteins or used in methods of searching or identifying agents that modulate SIMPLE levels and/or activity, for example, candidate therapeutic agents. For example, agents that modulate SIMPLE levels may be utilized to treat diseases of the nervous system. Because the mutations of this aspect of the invention are dominant negative or gain of function mutations, they have also have therapeutic utility for use in the identification and development and design of drugs which circumvent or overcome the mutated SIMPLE gene function. The sequences of the nucleic acids and/or proteins described herein can also be incorporated into computer systems and used with modeling software so as to enable rational drug design. Information from genotyping methods provided herein can be used, for example, in computer systems, in pharmacogenomic profiling of therapeutic agents to predict effectiveness of an agent in treating an individual for a neurological disease.
The identification of mutants T115N and G112S is described in Example 1. The identification of mutant W116G is described in Example 2. The co-segregation analysis of these mutations is described in Example 3 and Table 4. Further characterization of these mutations is described in Tables 1 and 3.
In another aspect, the invention provides a nucleic acid probe for detecting a SIMPLE gene, the probe consisting of a nucleic acid sequence selected from the group consisting of nucleotide 91 to nucleotide 140 of SEQ ID NO: 4, or the complement thereof and SEQ ID NO: 6, or the complement thereof. The nucleic acid probes of this aspect of the invention are useful to detect the presence of a wild-type gene in an individual, such as in nucleic acid hybridization assays, southern and northern blot analysis and as controls for the screening assays and kits described herein. The nucleic acid probes of this aspect of the invention may be used in nucleic acid hybridization assays with genomic DNA isolated from a mammalian subject as described herein.
In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO: 18 and SEQ ID NO: 19. The primer molecules of the invention can be used individually as sequencing primers, or together as a primer pair for amplifying exon 3 of the SIMPLE gene, under conditions as disclosed in Table 2. SEQ ID NO: 18 and SEQ ID NO: 19 can be used, for example, to screen for mutations in the SIMPLE gene that are associated with peripheral neuropathy and are useful reagents in the methods and kits described herein.
In another aspect, the invention provides isolated mutant SIMPLE polypeptides and peptide fragments. Mutant SIMPLE polypeptides are SIMPLE proteins encoded by a SIMPLE gene having at least one of the mutations associated with peripheral neuropathy, as described above. In some embodiments, the isolated polypeptide includes mutation G112S, such as, for example, an isolated polypeptide comprising SEQ ID NO: 9. In other embodiments, the isolated polypeptide includes mutation T115N, such as, for example, an isolated polypeptide comprising SEQ ID NO: 11. In further embodiments, the isolated polypeptide includes mutation W116G, such as, for example, an isolated polypeptide comprising SEQ ID NO: 13. The isolated mutant SIMPLE polypeptides and peptide fragments are useful, for example, as antigens for raising antibodies which specifically bind to mutant SIMPLE polypeptides.
In another aspect, the present invention provides methods of screening a mammalian subject to determine if said subject has a genetic predisposition to develop, or is suffering from Charcot-Marie-Tooth type 1C (CMT1C) neuropathy. The methods of this aspect of the invention comprise the step of analyzing the nucleic acid sequence of a SIMPLE gene in a subject to determine whether a genetic mutation that is associated with CMT1C is present in the nucleic acid sequence, wherein the presence of such a mutation indicates that the mammalian subject has a genetic predisposition to develop CMT1C or is diagnosed as suffering from such a disease. In some embodiments, the method further comprises determining whether the mammalian subject is exhibiting peripheral neuropathy. The clinical examination of a mammalian subject for symptoms related to peripheral neuropathy may be done either prior to, or after nucleic acid analysis of the test subject.
The method of this aspect of the invention is useful for screening any mammalian subject, such as for example, a human subject, for the genetic predisposition to develop CMT1C disease. The method is useful for preimplantation, prenatal and postnatal diagnosis of neurological disease caused by mutation in the SIMPLE gene that facilitates genetic counseling and therapeutic intervention. The method is especially useful for screening and diagnosing presymptomatic at-risk family members for the presence or absence of mutations in SIMPLE associated with the disease. The method is also useful for screening subjects exhibiting peripheral neuropathy to determine whether their symptoms are caused by a genetic mutation in the SIMPLE gene.
Any genetic mutation in the SIMPLE gene that co-segregates with CMT1C is useful in the practice of the method of this aspect of the invention. Examples of such mutations are shown in Table 3. In one embodiment, genetic mutations that co-segregate with CMT1C are missense mutations in which a nucleic acid base change results in an amino acid substitution in the SIMPLE protein. Examples of such missense mutations include, for example, G112S, T115N, and W116G as shown in Table 3.
In another embodiment, the method of this aspect of the invention can be practiced using mutations that cause deletions or silent mutations which do not alter the amino acid sequence, but may change splicing or gene regulation.
In some embodiments of the invention, subjects are screened for genetic mutations at one or more of the SIMPLE protein positions: 111, 112, 113, 114, 115, or 116.
In some embodiments of this aspect of the method of the invention, subjects are screened for the presence of a genetic mutation that is associated with CMT1C disease in exon 3 of a SIMPLE gene, such as, for example, nucleotides 32,685 to 32,840 of SEQ ID NO: 3. Exon 3 encodes a region of highly conserved amino acid residues as shown in Table 1, provided as SEQ ID NO: 7. Examples of mutations found in exon 3 within the highly conserved region of SEQ ID NO: 7 that co-segregate with CMT1C are shown in Table 1 and include G112S, T115N, and W116G.
Individuals carrying particular mutations in the SIMPLE gene may be identified using a variety of techniques of analyzing nucleic acid sequence that are well known in the art such as, for example, direct sequencing, PCR amplification and sequencing, restriction fragment length polymorphism (RFLP), nucleic acid hybridization, and single strand conformation polymorphism (SSCP). For each of these techniques, the test subject provides a biological sample containing genomic DNA to be analyzed. The test sample may be obtained from body cells, such as those present in peripheral blood, cheek cells, urine, saliva, surgical specimen, and autopsy specimens. The test sample can be processed to inactivate interfering compounds, and to purify or partially purify the nucleic acids in the sample. Any suitable purification method can be employed to obtain purified or partially purified nucleic acids from the test sample. A lysing reagent optionally can be added to the sample, particularly when the nucleic acids in the sample are sequestered or enveloped, for example, by cellular or nuclear membranes. Additionally, any combination of additives, such as buffering reagents, suitable proteases, protease inhibitors, nucleases, nuclease inhibitors and detergents can be added to the sample to improve the amplification and/or detection of the nucleic acids in the sample. Additionally, when the nucleic acids in the sample are purified or partially purified, the use of precipitation can be used, or solid support binding reagents can be added to or contacted to the sample, or other methods and/or reagents can be used. One of ordinary skill in the art can routinely select and use additives for, and methods for preparation of a nucleic acid sample for amplification.
In one embodiment of the method of the invention, the nucleic acid sequence is analyzed by direct sequencing for differences in nucleic acid sequence from the wild-type SIMPLE gene by sequencing of the subject's SIMPLE gene using primers specific for the region of interest, such as, for example, the sequencing primers described in Table 2 and Table 3.
In another embodiment, prior to sequencing the DNA is amplified enzymatically in vitro through use of PCR (Saiki et al., Science 239:487-491 (1988)) or other in vitro amplification methods as previously described herein. In a further embodiment, the DNA from an individual can be evaluated using RFLP techniques are described in Example 3 and elsewhere herein. The previously described methods useful for determining co-segregation analysis are also useful in this aspect of the method of the invention, such as, for example, nucleic acid hybridization techniques and single strand conformation polymorphism (SSCP). SSCP is a rapid and sensitive assay for nucleotide alterations, including point mutations (see Orita, M., et al., Genomics 5:874-879 (1989)). DNA segments ranging in length from approximately 100 bp to approximately 400 bp are amplified by PCR, heat denatured and electrophoresed on high resolution-non-denaturing gels. Under these conditions, each single-stranded DNA fragment assumes a secondary structure determined in part by its nucleotide sequence. Even single base changes can significantly affect the electrophoretic mobility of the PCR product.
In another aspect, the present invention provides kits for determining susceptibility or presence of CMT1C in a subject. The kits of the invention include (i) one or more nucleic acid primer molecules for amplification of a portion of the SIMPLE gene; and (ii) written indicia indicating a correlation between the presence of said mutation and risk of developing CMT1C. In one embodiment, the kits of the invention further comprise means for determining whether a mutation associated with CMT1C is present. In some embodiments, the kits of the invention comprise detection components specific for one or more of the particular genetic mutations described herein.
Primer molecules for amplification of a portion of the SIMPLE gene can be of any suitable length and composition and are selected to facilitate amplification of at least one or more regions (in the case of duplexed or multiplexed amplification) of the SIMPLE gene as shown in SEQ ID NO: 3 that potentially contains a genetic mutation. For example, oligonucleotide primers can be in the range of 5 bp to 50 bp or longer, and are chosen as primer pairs so that primers hybridize to sequences flanking the putative mutation. Primer pairs typically have an annealing temperature within about 20° C. of each other. Computer programs are useful in the design of primers with the required specificity and optimal amplification properties. See, e.g., Oligo version 5.0 (available from National Biosciences Inc., 3001 Harbor Lane, Suite 156, Plymouth, Minn.). Examples of primer pairs suitable for inclusion in the kit of the invention are provided in Table 2.
Similarly, a kit of the invention can also provide reagents for a duplexed amplification reaction (with two pairs of primers) a multiplexed amplification reaction (with three or more pair of primers) so as to amplify multiple sites of SIMPLE nucleotide mutations in one reaction.
Also included in the kit of the invention are written indicia indicating a correlation (typically a positive correlation) between the presence of a particular mutation in the SIMPLE gene and the risk of developing CMT1C disease.
The kit optionally also comprises one or more enzymes useful in the amplification or detection of nucleic acids and/or nucleotide sequences. Suitable enzymes include DNA polymerases, RNA polymerases, ligases, and phage replicases. Additional suitable enzymes include kinases, phosphatases, endonucleases, exonucleases, RNAses specific for particular forms of nucleic acids (including, but not limited to, RNAse H), and ribozymes. Other suitable enzymes can also be included in the kit.
The kit optionally comprises amplification reaction reagents suitable for use in nucleic acid amplification. Such reagents are well known and include, but are not limited to: enzyme cofactors such as magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD), and deoxynucleoside triphosphates (dNTPs). The kit optionally can also comprise detection reaction reagents, such as light or fluorescence generating substrates for enzymes linked to probes.
The kit optionally includes control DNA, such as positive and negative control samples. Negative control samples may comprise for example, genomic DNA or SIMPLE cDNA from a mammalian subject with no predisposition to CMT1C, or portions thereof. Positive control samples may comprise, for example, nucleic acid molecules containing an identified mutation in the SIMPLE gene as described herein.
The kit optionally includes instructions for using the kit in the detection of mutations in SIMPLE associated with CMT1C disease. The kit also preferably includes instructions on the appropriate parameters for the amplification reaction. Any suitable set of amplification parameters can be employed. For example, the precise temperature at which double-stranded nucleic acid sequences dissociate, primers hybridize or dissociate, and polymerase is active, are dependent on the length and composition of the sequences involved, the salt content of the reaction, the oligonucleotide concentration, the viscosity of the reaction and the type of polymerase. One of ordinary skill in the art can easily determine appropriate temperatures for the amplification reaction (see, e.g., Wetmur, J. Critical Reviews in Biochemistry and Molecular Biology 26:227-59 (1991)). For example, temperatures above about 90° C., such as between about 92° C., and about 100° C., are typically suitable for the dissociation of double-stranded nucleic acid sequences. Temperatures for forming primer hybrids are preferably between about 45° C. and about 65° C. Temperatures for the polymerization/extension phase are typically between about 60° C. and about 90° C., depending on the polymerase utilized in the reaction.
A multiplicity of suitable methods may be used to analyze the amplified nucleic acid product to determine whether a mutation associated with CMT1C disease is present. Suitable means include DNA sequencing, northern blotting, southern blotting, Southwestern blotting, probe shift assays (see, e.g., Kumar et al., AIDS Res. Hum. Retroviruses 5:345-54 (1989), T4 Endonuclease VII-mediated mismatch-cleavage detection (see, e.g., Youil et al., Proc. Nat'l. Acad. Sci. USA 92:87-91 (1995), Fluorescence Polarization Extension (FPE), Single Strand Length Polymorphism (SSLP), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), Immobilized Mismatch Binding Protein Mediated (MutS-mediated) Mismatch detection (see, e.g., Wagner et al., Nucleic Acids Research 23:3944-48 (1995), reverse dot blotting, (see, e.g., European Patent Application No. 0 511 559), hybridization-mediated enzyme recognition (see, e.g., Kwiatkowski et al., Mol. Diagn. 4(4):353-64 (1999)), describing the InvaderTM embodiment of this technology by Third-Wave Technologies, Inc.), detection, single-strand conformation polymorphism (SSCP) and gradient denaturing gel electrophoresis to detect probe-target mismatches (e.g., “DGGE”, see, e.g., Abrams et al., Genomics 7:463-75 (1990), Ganguly et al., Proc. Nat'l. Acad. Sci. USA 90:10325-29 (1993), and Myers et al., Methods Enzymology 155:501-27 (1987)).
The kit is preferably provided in a microbiologically stable form. Microbiological stability can be achieved by any suitable means, such as by (i) freezing, refrigeration, or lyophilization of kit components; (ii) by heat-, chemical-, or filtration-mediated sterilization or partial sterilization; and/or (iii) by the addition of antimicrobial agents such as azide, detergents, and other suitable reagents to other kit components. The kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling by a clinically useful sample analyzer. For example, the kit can optionally comprise multiple liquids, each of which are stored in distinct compartments within the housing. In turn, each compartment can be sealed by a device that can be removed, or easily penetrated, by a mechanical device.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. All literature citations herein are expressly incorporated by reference
EXAMPLE 1
This example describes the identification of the T115N and the G112S missense mutations in the SIMPLE gene and demonstrates that these mutations co-segregate with Charcot-Marie-Tooth neuropathy type 1C.
Mapping an Autosomal Dominant Charcot-Marie-Tooth Neuropathy to Chromosome 16p
Subjects:
A four generation family of Irish descent comprising 37 family members, some of which exhibited unexplained Charcot-Marie-Tooth neuropathy was identified and designated pedigree K1550 (see Street et al., Am J. Hum. Genet. 70:244-250 (2002)). Another four generation family of English descent with 38 family members also comprising some family members exhibiting unexplained Charcot-Marie-Tooth neuropathy was identified and designated pedigree K1551. Id. Affected family members met widely accepted criteria for CMT1 disease including distal muscle weakness and atrophy, depressed deep tendon reflexes and sensory impairment (see Dyck and Lambert, Arch. Neurol. 18:603-618 (1968)). The mean ulnar (16.7 m/s [n=3], 25.3 [n=8]), median (23 m/s [n=5], 25.8 m/s [n=12]) and peroneal (20.4 m/s [n=4, 21 m/s [n=6]) motor nerve conduction velocities of affected K1550 and K1551 patients were consistent with CMT1 (see Street et al., Neurology 60:22-26 (2003)). One affected individual in pedigree K1550 had a sural nerve biopsy taken during reconstructive foot surgery that demonstrated “onion-bulb hypertrophy” typical of demyelinating CMT. Id. 200 unrelated control DNA samples for mutational analysis were taken from a collection of predominantly Caucasians of European descent.
Mapping and Identification of SIMPLE as a Candidate for CMT1:
To identify the locus responsible for the phenotype in these families, a whole genome-wide scan was performed in pedigrees K1550 and K1551, with informative microsatellite markers spaced at 10 cM intervals using the methods as described in Street et al., Am. J. Med. Genet. 70:244-250 (2002). Using two markers, D165764 and D165519 (obtained from Research Genetics), the CMT1 gene was mapped to chromosome 16p within a 9-cM interval. Id. SIMPLE was identified as one of 20 candidate genes that mapped to the critical region on chromosome 16p and was evaluated for DNA sequence alterations in families K1550 and K1551 (Street et al., Neurology 60:22-26 (2003)).
Molecular Analysis:
The following protocol of informed consent was approved by the institutional review board (IRB) of the University of Washington, Seattle. 15 to 20 mL of blood was obtained by venipuncture for high-molecular weight DNA (as described by Neitzel et al., Hum. Genet. 73:320-326 (1986)) and used as a template for PCR amplification. The three coding exons of the SIMPLE gene were PCR amplified from subject genomic DNA utilizing primer pairs listed in Table 2. PCR reactions were carried out in 25 μl containing 1×PCR buffer of 10 mM Tris-HCL (pH 8.3 at 25° C.), 50 mM KCL, 2 mM MgCl2, 0.2 mM each dNTP (dATP, dCTP, dGTP, dTTP), 0.66 μM each oligonucleotide forward and reverse primer, and 0.6 U of 5 U/μ1 Ampli-Taq Polymerase (Sigma, St. Louis, Mo.). 5 μl of PCR product was characterized by gel electrophoresis/ethidium bromide staining for the presence of a single correctly sized band, as shown in Table 2.
Direct DNA Sequencing of the PCR Fragments:
5 μl of PCR product from each sample confirmed to have a single correctly sized band was treated with 1 μl of ExoSAP-IT (US Biochemical, Cleveland, Ohio) at 37° C. for 2 hours followed by heat inactivation at 85° C. for 10 minutes. Direct DNA sequencing of the purified fragments was carried out by using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc., Foster City, Calif.). The primers used for sequencing are the same primers used for PCR amplification as listed in Table 2. For initial mutation screening, either forward or reverse primer was used. The PCR reaction contained 3 μl of treated PCR product ( ̃100 ng), 3 pmol primer, 1 μl sequencing buffer and 2 μl of BigDye reagent in a total volume of 10 μl. The sequencing reaction was carried out in a PTC-100 Programmable Thermal Controller (MJ Research Inc., Waltham, Mass.) with cycle conditions of 96° C. for 2 min, 30 cycles of 96° C. for 15 sec, 50° C. for 10 sec, and 60° C. for 4 min. The sequencing product was purified by ethanol/EDTA precipitation, then electrophoresed on an ABI DNA Sequencer (Applied Biosystems Inc., Foster City, Calif.).
Results:
Analysis of the three SIMPLE coding exons and flanking intron nucleotide sequences in pedigrees K1550 and K1551 revealed mutations in exon 3. In K1550, a C to A transversion at nucleotide 344 (as counted from the cDNA start codon as shown in SEQ ID NO: 1) was detected in exon 3, which predicts substitution of asparagine for threonine at amino acid position 115 (T115N).
In K1551, a G to A transition at nucleotide 344 was detected in exon 3, which predicts substitution of serine for glycine at amino acid position 112 (G112S).
Evaluation of Co-Segregation of CMT1C and Genetic Mutations
The C to A transversion at nucleotide 344 and the G to A transition at nucleotide 334 each introduced a novel BsrI restriction endonuclease site which was utilized to verify that the mutation co-segregates with subjects exhibiting peripheral neuropathy as further described in Example 3 and Table 4.
EXAMPLE 2
This example describes the identification of the W116G missense mutation in the SIMPLE gene.
Subjects Tested:
A family of Dutch descent comprising 17 family members, some of which exhibited unexplained Charcot-Marie-Tooth neuropathy was identified and designated pedigree K2900 (see Street et al., Neurology 60:22-26 (2003)). The proband in this family had decreased nerve conduction velocities ranging from 15 to 30 m/s, consistent with CMT1C. Affected family members had previously been evaluated and shown not to have alterations in the PMP22, MPZ or EGR2 genes. 100 unrelated control chromosomes were also included in the study.
Methods:
The entire coding region of SIMPLE was sequenced in genomic DNA of one affected individual by first PCR amplifying the 3 coding exons (exons 2-4) and sequencing each using the primers shown in Table 2 as described in Example 1.
Results:
In K2900, a T to G transversion at nucleotide 346 (as counted from the cDNA start codon as shown in SEQ ID NO: 1) was detected in exon 3, which predicts substitution of glycine for tryptophan at amino acid position 116 (W116G).
Evaluation of Co-Segregation of CMT1C and Genetic Mutations
The T to G transversion at nucleotide 346 introduced a novel Nci1 restriction endonuclease site which was utilized to verify that the mutation co-segregates with subjects exhibiting peripheral neuropathy as further described in Example 3 and Table 4.
EXAMPLE 3
This example describes the use of restriction fragment length polymorphism (RFLP) analysis to evaluate co-segregation of peripheral neuropathy with genetic mutation in the SIMPLE gene.
Restriction Fragment Length Polymorphism (RFLP) Analysis:
Each of the identified mutations, G112S, T115N and W116G, alter the restriction endonuclease digestion pattern of specific restriction endonucleases as shown in Table 4. The first column of Table 4 describes the mutations amenable to RFLP analysis, the second column provides a useful primer set for amplification of the region encompassing the mutation, the third column provides the relevant restriction endonuclease for use in digestion of the PCR fragment, and the fourth and fifth columns provide the expected restriction enzyme digested fragments for wild-type and mutant genes, respectively. The final two columns of Table 4 provide the reaction conditions appropriate for each restriction enzyme digestion listed.
Results of Segregation Analysis:
G112S Mutation:
RFLP analysis was performed on samples from 33 individuals from the K1551 pedigree (described in Example 1), including 18 individuals exhibiting demyelinating peripheral neuropathy. Bsr1 digestion of the 380 bp exon 3 fragment resulted in the pattern shown in Table 4 for mutant-type samples in all 18 subjects exhibiting peripheral neuropathy. Of the 15 samples from individuals not exhibiting peripheral neuropathy, all Bsr1 restriction patterns corresponded to the expected fragment pattern for wild-type shown in Table 4. The expected wild-type fragment pattern was observed in 200 unrelated samples of control chromosomes.
T115N Mutation:
RFLP analysis was performed on samples from 29 individuals from the K1550 pedigree (described in Example 1), including 21 individuals exhibiting peripheral neuropathy. Bsr1 digestion of the 380 bp exon 3 fragment resulted in the pattern shown in Table 4 for mutant-type samples in all 21 subjects exhibiting peripheral neuropathy. Of the 8 samples from individuals not exhibiting peripheral neuropathy, all Bsr1 restriction patterns corresponded to the expected fragment pattern for wild-type shown in Table 4. The expected wild-type fragment pattern was observed in 200 unrelated samples of control chromosomes.
W116G Mutation:
RFLP analysis was performed on samples from 8 individuals from the K2900 pedigree (described in Example 2), including 4 individuals exhibiting peripheral neuropathy. Nci1 digestion of the 380 bp exon fragment resulted in the pattern shown in Table 4 for mutant-type samples in all 4 individual subjects exhibiting peripheral neuropathy. Of the 4 samples from individuals not exhibiting peripheral neuropathy, all Nci1 restriction patterns corresponded to the expected fragment pattern for wild-type shown in Table 4. The expected wild-type fragment pattern was observed in 100 unrelated samples of control chromosomes.







TABLE 4







THE CONDITIONS OF RFLP ANALYSIS FOR DETERMINING


COSEGREGATION WITH DEMYELINATING NEUROPATHY














Restriction Fragment






Sizes (bp)
Conditions













Mutation
Primer Set
Enzyme 
Wild-type
Mutant
Temp
Buffer





334G to A
3F (SEQ ID NO: 18)
Bsr1
380
121,259
65° C.
NEBuffer 3


(G112S)
3R (SEQ ID NO: 19)







344C to A
3F (SEQ ID NO: 18)
Bsr1
380
108,272
65° C.
NEBuffer 3


(T115N)
3R (SEQ ID NO: 19)







346T to G
3F (SEQ ID NO: 18)
Nci1
380
104, 276
37° C.
NEBuffer 4


(W116G)
3R (SEQ ID NO: 19)









EXAMPLE 4
This example describes the analysis of SIMPLE gene expression after nerve injury in a rat model.
Methods:
Young adult Sprague-Dawley rats were anesthetized and the sciatic nerves were transected at the sciatic notch, and both cut ends were ligated and pulled apart to prevent axonal regeneration into the distal stump. The entire distal nerve stump (about 4 cm in length) was harvested during the next 1 to 58 days later and divided into 2-cm segments, termed P (the segment immediately adjacent to the crush) and D (the more distal segment). Further description of this experimental method is provided in Scarlato et al., J Neurosci. Res. 66:16-22 (2001).
RNA Expression Analysis:
RNA Isolation:
RNA was isolated by CsCl2 gradient centrifugation as described by Chirgwin et al., Biochem. 18:5294-5299 (1979). For the lesioned adult rat sciatic nerves, total RNA was isolated from distal stumps of sciatic nerves that were transected or crushed and a Northern Blot was probed with the following cDNAs: a 1.4 kb fragment of SIMPLE, a full-length cDNA of rat myelin protein zero; and a full-length cDNA of rat GAPDH.
Results:
Northern blot analysis indicated that the 2.4 kb SIMPLE message was present at moderate levels in rat sciatic nerve, with expression remaining constant during sciatic nerve development. Following axotomy of transected sciatic nerve, SIMPLE expression remained essentially constant for a 48 day time course. Following crush injury, a general increase in SIMPLE expression was observed over a 58 day time course, and was more pronounced in the nerve region proximal to the site of injury. The fact that SIMPLE expression was unchanged as a result of nerve injury stands in distinct contrast to other CMT1 genes such as MPZ, PMP22, connexin-32 and EGR2, all of which have been found to demonstrate altered expression as a result of nerve injury (see Sherer et al., J. Neurosci. 15:8281-8294 (1995); Snipes et al., J. Cell. Biol. 117:225-238 (1992) and Zorick et al., Mol. Cell. Neurosci. 8:129-145 (1996)).
Protein Expression:
Blood samples were cleared of red blood cells by lysis in osmotic buffer (PureGene). Intact lymphocytes remaining in the lysate were then pelleted by centrifugation, washed in phosphate-buffered saline, and lysed in boiling SDS-PAGE loading buffer. 50 μg of each extract was resolved on a SDS-PAGE gel and transferred to PVDF membrane. Blots were then incubated with anti-LITAF monoclonal antibodies (Transduction Labs; 1:5000), followed by horseradish peroxidase-conjugated goat anti-mouse antibodies (Sigma; 1:20,000). Detection was performed with the ECL Plus system (Amersham).
Results:
Western blot analysis of peripheral blood lymphocytes indicated that the T115N and W116G substitutions do not appear to alter the SIMPLE protein level compared to a control individual and an individual carrying the PMP22 duplication. This result is in contrast to the observation that overexpression of the PMP22 gene in CMT1A is associated with demyelination and formation of perinuclear protein aggregates (Matsumami et al., Nat. Genet. 1:176-179 (1992)).
EXAMPLE 5
This example describes a kit and method of use for identifying genetic mutations associated with peripheral neurological disease in a mammalian subject, and for determining susceptibility or presence of CMT1C in a test subject.
Methods Utilized:
PCR Amplification:
Carried out as described in Example 1
Direct Sequencing:
Carried out as described in Example 1
Data Analysis:
The resulting sequences are aligned with the known exon sequence using a multiple sequence alignment tool, Sequencher (Gene Codes Corporation, Ann Arbor, Mich.), in order to identify any nucleotide changes. Electrophergrams are also visually examined to detect heterozygous base changes that might be missed by Sequencher.
Confirmation of the Nucleotide Changes:
Once a nucleotide change is detected, the exon fragment encompassing the suspected mutation is subjected to PCR amplification and direct sequencing again, using both forward and reverse primers as shown in Table 2.
For familial cases, when the nucleotide change is confirmed, with consent, the available family members, including affected and at risk unaffected individuals, are tested to confirm that the mutation segregates with the disease. After appropriate consent for clinical testing is obtained, the test may also be used for presymptomatic diagnosis in at-risk individuals.
Contents of the SIMPLE Mutation Kit:

    
    
        1. 10×PCR Buffer (100 mM Tris-HCL (pH 8.3 at 25° C.), 500 mM KCL, 20 mM MgCl2
        2. dNTP mix: dATP, dCTP, dGTP, dTTP at 10 mM each (Sigma, St. Louis, Mo.)
        3. Ampli-Taq DNA Polymerase (Sigma, St. Louis, Mo.)
        4. Primers for amplification of each SIMPLE exon and the adjacent intronic sequences at 0.66 μM each (as shown in Table 2)
        5. Exo-SAP-IT (US Biochemical, Cleveland, Ohio)
        6. BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc., Foster City, Calif.)
        7. Control DNA
        8. Written instructions and indicia indicating a positive correlation between the presence of a particular mutation in the SIMPLE gene and the risk of CMT1C disease.
    
    


While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

## Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence comprising at least 21 contiguous nucleotides of a variant of the nucleotide sequence set forth in SEQ ID NO:1, wherein:
(i) the nucleotide sequence comprises a nucleotide corresponding to position 334 of SEQ ID NO:1 and said variant encodes G112S; or
(ii) the nucleotide sequence comprises a nucleotide corresponding to position 344 of SEQ ID NO:1 and said variant encodes T115N; or
(iii) the nucleotide sequence comprises a nucleotide corresponding to position 346 of SEQ ID NO:1 and said variant encodes W116G;
wherein the isolated nucleic acid molecule comprising the nucleotide sequence (i), (ii) or (iii) is linked to a detectable moiety.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a nucleotide corresponding to position 334 of SEQ ID NO:1 and said variant comprises the nucleic acid substitution G334A.

3. The isolated nucleic acid molecule of claim 2, wherein the nucleic acid sequence comprises SEQ ID NO:8.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a nucleotide corresponding to position 344 of SEQ ID NO:1 and said variant comprises the nucleic acid substitution C344A.

5. The isolated nucleic acid molecule of claim 4, wherein the nucleic acid sequence comprises SEQ ID NO:10.

6. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a nucleotide corresponding to position 346 of SEQ ID NO:1 and said variant comprises the nucleic acid substitution T346G.

7. The isolated nucleic acid molecule of claim 6 wherein the nucleic acid sequence comprises SEQ ID NO:12.

8. An isolated nucleic acid probe for detecting a human small integral membrane protein of the lysosome/late endosome (SIMPLE) gene comprising at least 5 contiguous nucleotides having a nucleic acid sequence identical to, or complementary to, at least a portion of SEQ ID NO:4, wherein the nucleic acid probe comprises a mutation selected from the group consisting of G334A, C344A and T346G, wherein the nucleic; acrid molecule is linked to a detectable moiety.

9. The isolated nucleic acid probe of claim 8, wherein the nucleic acid probe comprises a nucleic acid sequence identical to, or complementary to, a nucleic acid sequence present in the region between nucleotide 91 and nucleotide 140 of SEQ ID NO:4.

10. A kit to determine if a human subject has a genetic predisposition to develop Charcot-Marie-Tooth neuropathy type 1C based on the detection of a mutation in a human small integral membrane protein of the lysosome/late endosome (SIMPLE) gene, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of a SIMPLE gene comprising SEQ ID NO:3 and (ii) a written indicia indicating a correlation between the presence of said mutation and risk of developing Charcot-Marie-Tooth neuropathy type 1C, wherein the nucleic acid primer molecule comprises a mutation selected from the group consisting of G334A, C344A and T346G, and wherein the nucleic acid primer molecule is linked to a detectable moiety.

11. The kit of claim 10, further comprising nucleic acid primer molecules for sequencing across an amplified portion of a SIMPLE gene, wherein each primer molecule is identical to at least 10 contiguous nucleotides occurring in the sequence of the SIMPLE gene disclosed in SEQ ID NO:3, or the complement thereof.

12. The kit of claim 10, wherein said mutation consists of an alteration in the nucleic acid sequence of SIMPLE gene that encodes an amino acid residue selected from the group consisting of amino acid residues 111, 112, 113, 114, 115, 116, and 117.

13. The kit of claim 12, wherein said mutation comprises at least one of G112S, T115N or W116G.