This page has been archived and is no longer updated
Whole genome sequencing and Charcot-marie-Tooth
Author: Lupski et al
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1181 original article Whole-Genome Sequencing in a Patient with Charcot?Marie?Tooth Neuropathy James R. Lupski, M.D., Ph.D., Jeffrey G. Reid, Ph.D., Claudia Gonzaga-Jauregui, B.S., David Rio Deiros, B.S., David C.Y. Chen, M.Sc., Lynne Nazareth, Ph.D., Matthew Bainbridge, M.Sc., Huyen Dinh, B.S., Chyn Jing, M.Sc., David A. Wheeler, Ph.D., Amy L. McGuire, J.D., Ph.D., Feng Zhang, Ph.D., Pawel Stankiewicz, M.D., Ph.D., John J. Halperin, M.D., Chengyong Yang, Ph.D., Curtis Gehman, Ph.D., Danwei Guo, M.Sc., Rola K. Irikat, B.S., Warren Tom, B.S., Nick J. Fantin, B.S., Donna M. Muzny, M.Sc., and Richard A. Gibbs, Ph.D. From the Department of Molecular and Human Genetics (J.R.L., J.G.R., C.G.-J., M.B., F.Z., P.S., D.M.M., R.A.G.), the Hu- man Genome Sequencing Center (J.G.R., D.R.D., D.C.Y.C., L.N., M.B., H.D., C.J., D.A.W., D.M.M., R.A.G.), the Center for Medical Ethics and Health Policy (A.L.M.), the Department of Pediatrics (J.R.L.), and Texas Children?s Hospital (J.R.L.) ? all at Baylor College of Medicine, Houston; Atlantic Neuroscience Institute, Summit, NJ, and Mount Sinai School of Medicine, New York (J.J.H.); and Life Technologies, Carlsbad, CA (C.Y., C.G., D.G., R.K.I., W.T., N.J.F.). Address reprint requests to Dr. Gibbs at 1 Baylor Plaza, Mail Stop BCM226, Houston, TX 77030, or at agibbs@bcm .edu. This article (10.1056/NEJMoa0908094) was published on March 10, 2010, at NEJM.org. N Engl J Med 2010;362:1181-91. Copyright � 2010 Massachusetts Medical Society. ABSTRACT BACKGROUND Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple pat- terns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. Compre- hensive diagnostic assays must therefore identify all possible DNA changes in each haplotype and determine which are responsible for the underlying disorder. The high number of rare, heterogeneous mutations present in all humans and the pau- city of known functional variants in more than 90% of annotated genes make this challenge particularly difficult. Thus, the identification of the molecular basis of a genetic disease by means of whole-genome sequencing has remained elusive. We therefore aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot?Marie?Tooth disease. METHODS We identified a family with a recessive form of Charcot?Marie?Tooth disease for which the genetic basis had not been identified. We sequenced the whole genome of the proband, identified all potential functional variants in genes likely to be related to the disease, and genotyped these variants in the affected family members. RESULTS We identified and validated compound, heterozygous, causative alleles in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), involving two mutations, in the proband and in family members affected by Charcot?Marie?Tooth disease. Sepa- rate subclinical phenotypes segregated independently with each of the two muta- tions; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome. CONCLUSIONS As shown in this study of a family with Charcot?Marie?Tooth disease, whole-genome sequencing can identify clinically relevant variants and provide diagnostic informa- tion to inform the care of patients. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1182 T he practice of medical genetics re- quires gene-specific analyses of DNA se- quences and mutations to definitively diag- nose disease, provide prognostic information, and guide genetic counseling regarding the risk of re- currence. Studies of autosomal recessive traits such as cystic fibrosis 1 and some dominant traits such as neurofibromatosis type 1 2 revealed the role of single ?disease genes? in conveying traits. However, many phenotypes of mendelian diseases (see the Glossary) are genetically heterogeneous: causative mutations have been identified in more than 100 genes for deafness and retinitis pig- mentosa, for instance. Moreover, specific muta- tions may confer phenotypes that segregate as dominant, recessive, or even digenic 3 or triallel- ic 4 traits. There is also ample evidence of modi- fying loci in mendelian disorders. 5,6 Thus, even when there are simple patterns of inheritance in syndromes with a well-characterized patho- logic course, the underlying mutational events, which need to be resolved for precise molecular diagnosis, within individual families may be complex. Charcot?Marie?Tooth disease is an inherited peripheral neuropathy with two forms: a demyeli- nating form (type 1) affecting the glia-derived myelin and an axonal form (type 2) affecting the nerve axon. The two forms can be distinguished by means of electrophysiological or neuropatho- logical studies. Charcot?Marie?Tooth disease has been used as a model disease to describe genetic heterogeneity, posit the relation of hereditary pattern to clinical severity, and investigate the relative importance of principal and modifying genes in determining human diseases. 7,8 Mutant alleles underlying Charcot?Marie?Tooth disease can segregate in an autosomal dominant, reces- sive, or X-linked manner (Fig. 1). Both single- base variants (single-nucleotide polymorphisms [SNPs]) and copy-number variants, 10 at 39 sepa- rate loci, confer susceptibility to Charcot?Marie? Tooth disease. Most of these susceptibility variants cause dominant forms of the disease, although mutations in genes at 14 of the loci cause reces- sive disease. Adult-onset Charcot?Marie?Tooth disease is highly variable in presentation but is character- ized by distal symmetric polyneuropathy, 9 with slowly progressive distal muscle weakness and atrophy (particularly peroneal muscular atrophy) resulting in foot dorsiflexor weakness, foot drop, and secondary steppage gait. Pes cavus (highly arched feet) or pes planus (flat feet) occurs in most patients. We applied next-generation-sequencing meth- ods to identify the cause of disease in a family with inherited neuropathy that had been previous- ly screened, with negative results, for alterations of some common Charcot?Marie?Tooth genes, including PMP22, 11 MPZ, PRX, GDAP1, and EGR2. Methods Study Participants The study family consisted of four affected sib- lings, four unaffected siblings, and an unaffected mother and father, all of whom provided written informed consent for participation in the study. The study was approved by the institutional re- view board at Baylor College of Medicine. The di- agnosis of Charcot?Marie?Tooth type 1 disease in the proband and the three affected siblings was based on the results of physical examination (dis- tal muscle weakness and wasting, pes cavus, and absence of deep-tendon reflexes) and electrophys- iological studies. Neurophysiological Assessments Neurophysiological studies consisted of a stan- dard battery of nerve-conduction studies, includ- ing motor responses of the median, ulnar, tibial, and peroneal nerves with F-wave latencies; ortho- dromic median-, ulnar-, and sural-nerve sensory potentials; and bilateral tibial H-reflexes. When these studies revealed demyelinating features, tests of blink reflexes were generally performed. Limbs were warmed to a temperature of at least 32�C in all instances. Demyelination was judged to be present if conduction velocities were signif- icantly slowed and the late-response latencies were substantially delayed. Median-nerve mono- neuropathy at the wrist was judged to be present when there was prolonged motor terminal latency or slowed median-nerve sensory velocity with dis- proportionate slowing in the palm-to-wrist seg- ment, or both. The four affected subjects, all of whom had diffuse slowing of conduction, were also thought to have a median-nerve mononeuropathy at the wrist, since the median-nerve motor termi- nal latency was much more prolonged than the ulnar-nerve motor terminal latency (14.9 vs. 8.1, Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . Whole-Genome Sequencing and SH3TC2 Mutations n engl j med 362;13 nejm.org april 1, 2010 1183 10.2 vs. 7.5, 11.6 vs. 6.2, and 9.2 vs. 6.2 msec) (Table 1). DNA Sequence Analysis DNA sequencing was performed with the use of the SOLiD (Sequencing by Oligonucleotide Liga- tion and Detection) system (Applied Biosystems), a next-generation-sequencing platform that involves ligation-based sequencing and a two-base encod- ing method in which four fluorescent dyes are used to tag various combinations of dinucleo- tides. Its accuracy in sequencing 50-base reads is estimated at approximately 99.94%. 12 Multiple se- quences can be read simultaneously, and when the sequence reads overlap, the overall accuracy increases further, reducing the risk of false posi- tive determinations and the need for additional data validation. We determined bases from the primary sequencing data, using the standard SOLiD analysis software. (For details, see the Sup- plementary Appendix, available with the full text of this article at NEJM.org.) Glossary Array-baseduni0020comparativeuni0020genomicuni0020hybridization: A hybridization method for detecting copy-number variations in DNA samples from a patient as compared with a control sample. The method provides higher resolution than cytogenetic methods but lower resolution than sequencing methods. Averageuni0020depthuni0020ofuni0020coverage:uni0020The average number of times each base in the genome was sequenced, as a function of the distribution and number of sequence reads that map to the reference genome. Codinguni0020single-nucleotideuni0020polymorphisms: Single DNA-base changes that occur in the coding regions of genes. Copy-numberuni0020variation: DNA changes that involve sequences of more than 100 bp, larger than single-nucleotide changes or microsatellites, and that vary in their number of copies among individual persons. These variants can be benign and polymorphic, but some can cause disease. DNAuni0020template: An individual fragment of DNA that is available for sequencing. Exonuni0020capture: Methods for isolating and sequencing gene exons, to the exclusion of the remainder of the genome. The DNA templates from exons are ?captured? with the use of probes complementary to the targeted exon sequences. After capture, the targeted DNA is eluted and sequenced. The cost of exon capture can be 10 to 50% lower than that of whole-genome sequencing, although the method is insensitive to copy-number variations and mutations that are out- side the targeted regions. Fragment-sequenceuni0020read: The contiguous nucleotide sequence from one end of a DNA template (as opposed to a mate- pair read). Haploinsufficiency: The state that occurs when a diploid organism has only a single functional copy of a gene, which does not produce enough protein to support normal function. Mapping: The computational process of identifying the specific region of a reference genome from which an individual sequenced DNA template originated. Mappableuni0020yield: The number of bases generated by a DNA-sequencing instrument that can be mapped to the reference genome. Mate-pairuni0020sequencing: A sequencing strategy that permits the inference of structural changes in a genome by sequenc- ing at both the 5prime and 3prime ends of each DNA template (as opposed to the fragment-sequencing approach). Mendelianuni0020disease: Human disease caused by mutations in a single gene. Missenseuni0020mutations: Single DNA-base changes that occur in the coding regions of genes and alter the resulting encoded amino acid sequence. Next-generationuni0020sequencing: DNA-sequencing methods that involve chemical assays other than the traditional Sanger dideoxy-chain-termination method. Next-generation-sequencing methods produce much larger quantities of data at less expense, but the individual raw sequence reads that are generated from individual amplified DNA-template sequences are shorter and have lower quality. Nonsenseuni0020mutations: DNA-base changes that introduce termination codons in the coding sequences of genes, result- ing in truncated proteins. Sequenceuni0020read:uni0020The sequence generated from a single DNA template. Single-baseuni0020erroruni0020rate: The total number of mismatched bases found in mapped sequence reads from a sequencing run, divided by the mappable yield. This rate estimates the probability that any given mappable base is an error. Two-baseuni0020encoding: A method used in the SOLiD (Sequencing by Oligonucleotide Ligation and Detection) DNA-sequencing platform that represents a DNA sequence as a chain of overlapping dimers encoded as single-base ?colors.? This allows for sequencing of the 16 unique sequence dimers with the use of only four unique dye colors and provides a method for improving the overall accuracy of the sequence reads (reducing the single-base error rate). Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1184 Array-Based Comparative Genomic Hybridization For array-based comparative genomic hybridiza- tion and analysis of the copy-number variants in the proband as compared with those in a male control, we used a 1-million-probe high-resolution oligonucleotide whole-genome array (Agilent), a 2.1-million-oligonucleotide whole-genome array (NimbleGen), and a 44,000-oligonucleotide array (Agilent) that was custom-designed to assay genes previously implicated in inherited neuropathy. Analysis of the copy-number variants was per- formed according to the manufacturer?s instruc- tions and software. Bioinformatic Analysis of SNP Variants Analysis of SNP variants and cross-referencing of them with the Human Gene Mutation Database (www.hgmd.cf.ac.uk), the Online Mendelian In- heritance in Man database (www.ncbi.nlm.nih .gov/omim), and the PolyPhen database (http:// genetics.bwh.harvard.edu/pph/data, based on the 6 col 33p9 CMT phenotype Nerve-conduction studies and electromyography Primary myelinopathy (CMT type 1) Dominant Recessive X-linked Dominant intermediate Primary axonopathy (CMT type 2) Recessive Dominant AUTHOR: FIGURE: RETAKE: SIZE 4-C H/TLine Combo Revised AUTHOR,uni0020PLEASEuni0020NOTE: Figureuni0020hasuni0020beenuni0020redrawnuni0020anduni0020typeuni0020hasuni0020beenuni0020reset. Pleaseuni0020checkuni0020carefully. 1st 2nd 3rd Gibbs (Lupski) 1 of 2 ARTIST: TYPE: ts 04-01-10:EUSSI31263:BOJ MPZ EGR2 LITAF PMP22 SOX10 1q22 10q21 16p13 17p12 22q13 SH3TC2 FIG4 GDAP1 NDRG1 Unknown SBF2 MTMR2 FGD4 CTDP1 PRX 5q32 6q21 8q21 8q24 10q23 11p15 11q22 12p11 18q23 19q13 YARS Unknown ARHGEF10 Unknown DNM2 1p35 3q13 8p23 10q24 ? q25 19p13 MFN2 and KIF1B RAB7A GARS HSPB1 NEFL Unknown HSPB8 and TRPV4 AARS 1p36 3q21 7p14 7q11 8p21 12q12 12q24 16q22 LMNA SLC12A6 GAN Unknown 1q21 15q13 16q24 19q13 GJB1 PRPS1 Unknown Unknown Unknown Xq13 Xq21 Xp22 Xq24 ? q26 Xq26 ? q28 Figureuni00201.uni0020Charcot?Marie?Toothuni0020(CMT)uni0020Diseaseuni0020Phenotypes,uni0020Theiruni0020Geneticuni0020Formsuni0020ofuni0020Inheritance,uni0020anduni0020Theiruni0020Mappeduni0020 Genesuni0020anduni0020Loci. CMT is divided in two major phenotypic types ? glial myelinopathy (CMT type 1) and neuronal axonopathy (CMT type 2) ? according to electrophysiological, clinical, and nerve-biopsy evaluations. Each type can be inherited in a dominant, recessive, or X-linked fashion. There are also autosomal dominant intermediate forms of CMT that can have features of both axonal and demyelinating neuropathies. Several genes have been associated with CMT disease to date, and other loci have been associated and mapped but their genes not yet identified. MPZ, GDAP1, and GJB1 are known to be associated with CMT type 1, but select mutations in these genes can also cause CMT type 2; NEFL is known to be associated with CMT type 2, but select mutations convey a CMT type 1 phenotype. Dominant inter- mediate forms of CMT have been reported to be associated with MPZ mutations. Specific recessive alleles related to CMT have also been reported for EGR2 and PMP22. Of the 31 genes in 39 known CMT loci, only 15 genes are cur- rently available for clinical testing. Current evidence-based clinical guidelines for distal symmetric polyneuropathy recommend genetic testing consisting of screening for common mutations, including the CMT1A duplication copy- number variant and point mutations of the X-linked GJB1 gene. 9 Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . Whole-Genome Sequencing and SH3TC2 Mutations n engl j med 362;13 nejm.org april 1, 2010 1185 National Center for Biotechnology Information [NCBI] dbSNP, build 126) were performed with the use of Perl scripts. Alignment of the orthologous SH3TC2 (SH3 domain and tetratricopeptide re- peats 2) proteins was performed with the use of the ClustalW program and reference SH3TC2 proteins from the following organisms: human (accession number, NP_078853), chimpanzee (XP_527069), macaque (XP_001104761), dog (XP_546315), horse (XP_001501607), cow (XP_616288), mouse (NP_766216), rat (XP_225887), opossum (XP_ 001380773), and chicken (XP_424256). Segregation Analysis Exons 5 and 11 of the SH3TC2 gene were ampli- fied by means of a polymerase-chain-reaction (PCR) assay and directly sequenced in all mem- bers of the study family. To verify the Arg954ter amino acid mutation (R954X), corresponding to a G?A mutation in the genomic DNA in exon 11 of SH3TC2 on chromosome 5 at nucleotide 148,386,628, we also generated a 312-bp PCR fragment and incubated it with the restriction enzyme TaqI; the nucleotide mutation results in elimination of the restriction site for TaqI. Results Nerve-Conduction Studies In addition to the Charcot?Marie?Tooth type 1 phenotype that segregates as a recessive trait, we identified through electrophysiological means an axonal neuropathy in one parent and one grand- parent of the proband. Further evidence of a sub- Tableuni00201.uni0020Neurophysiologicaluni0020Findingsuni0020inuni0020theuni0020Studyuni0020Family.* Subjectuni0020No. Age Sex Demyelination Axonopathy Median-Nerveuni0020Entrapment Diagnosis yr I-1 80 M No Motor: peroneal, 2.2 mV Yes I-2 77 F No Sural: absent; motor: pero neal, 0.5 mV; tibial, 2.8 mV Yes Axonal neuropathy Maternal grandmother of proband 90 F No Normal Yes; SCV, 43 m/sec MMM II-I 58 F No Normal Yes; SCV, 46 m/sec MMM II-2 57 M No Sural: absent; motor: peroneal, 0.2 mV; tibial, 1.4 mV; H-reflexes: 38 msec Yes Axonal neuropathy III-1 37 M No Normal No III-2 35 M Yes No Yes; median-nerve motor TL, 14.9 msec; ulnar-nerve motor TL, 8.1 msec CMT III-3 34 F No No Yes; SCV, 42 m/sec; median-nerve motor TL, 4.4 msec MMM III-4 (proband) 32 M Yes No Probably; median-nerve motor TL, 10.2 msec; ulnar-nerve motor TL, 7.5 msec CMT III-5 31 F No No No III-6 29 F Yes No Yes; median-nerve motor TL, 11.6 msec; ulnar-nerve motor TL, 6.2 msec CMT III-7 26 F No Motor: peroneal, 36 m/sec; H-reflexes: 35 msec Yes; SCV, 36 m/sec; median-nerve motor TL, 4.8 msec MMM III-8 25 M Yes No Yes; median-nerve motor TL, 9.2 msec; ulnar-nerve motor TL, 6.2 msec CMT * Subject I-1 was a carpenter for more than 50 years. The maternal grandmother of the proband is not included in the pedigree. The ages listed are the ages at the time at which the subjects were evaluated. The normal value for median-nerve motor terminal latency (TL) is less than 4.0 msec and for sensory conduction velocity (SCV) is 48 m/sec or more. The diagnosis column lists the conclusion based on the aggregate findings: severe, widespread slowing of conduction was interpreted as evidence of demyelination, and low-potential amplitudes in multiple nerves with relative preservation of conduction velocities were interpreted as evidence of axonal damage. CMT denotes Charcot?Marie? Tooth disease, CTS the carpal tunnel syndrome, and MMM mild mononeuropathy of the median nerve. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1186 tle phenotype evidenced by, at a minimum, medi- an-nerve mononeuropathy at the wrist was also observed among all the proband?s grandparents and both parents but had an unclear pattern of inheritance. Its variable presentation (Table 1) in- cluded three neurophysiologically defined pheno- types: a normal phenotype with superimposed se- vere median-nerve mononeuropathy at the wrist, thought to be an incidental finding in an 80-year- old man who had been a carpenter for more than 50 years (Subject I-1), a mild median-nerve mononeuropathy at the wrist (the proband?s ma- ternal grandmother and mother [Subject II-1]), and a more severe median-nerve mononeuropathy at the wrist associated with evidence of a more wide- spread axonal polyneuropathy (Subjects I-2 and II-2). The latter phenotype is similar to that of patients with hereditary neuropathy with liability to pressure palsies (Online Mendelian Inheri- tance in Man number, 162500), a disorder patho- logically characterized by patchy myelin abnor- malities and attributed to haploinsufficiency of PMP22 (as a consequence of genomic deletion) 13 ; duplication of PMP22 causes Charcot?Marie?Tooth type 1A disease, the most common form. 14 Genome Variation The sequencing of DNA samples obtained from the proband produced a mappable yield of 89.6 Gb of sequence data, representing an average depth of coverage of approximately 30 times per base. The data from sequential machine runs consisted of 8.3 Gb of 35-bp fragment sequence reads (one run), 30.3 Gb of 25-bp mate-pair sequence reads (two runs), and 51.0 Gb of 50-bp mate-pair se- quence reads (one run). We identified the differences between the con- sensus sequence of the proband and the human genome reference sequence. These were used to produce a list of putative single-base DNA substi- tutions, small insertions, and deletions and po- tential changes in DNA copy number. This list of variants included 3,420,306 SNPs. A total of 2,255,102 of the SNPs were in extragenic regions and 1,165,204 SNPs were within gene regions, including introns, promoters, 3prime and 5prime untrans- lated regions, and splice sites (Table 2). Of the intragenic SNPs, 9069 were nonredundant SNPs predicted to result in nonsynonymous codon changes, and 121 of the 9069 were nonsense mutations. The approximately 3.4 million SNPs identified represent about 0.1% of the reference haploid human genome, 15 and both the total number of SNPs and the number of novel SNPs are similar to those discovered in other diploid genome sequences for individual subjects (Table 3). 12,16-21 Of the more than 3.4 million SNPs, 2,858,587 were present in public databases and 561,719 were novel (Table 3). Data on the se- quence reads, quality, and mapping have been deposited in the NCBI Sequence Read Archive (www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?) (acces- sion number, SRP001734); variant data have been deposited in the dbSNP database. We used two approaches to identifying copy- number variation: array-based comparative ge- nomic hybridization and mate-pair sequencing. We identified 234 copy-number variants ranging in size from 1690 bp to 1,627,813 bp. Of these 234 variants, 132 were confirmed by at least one other method (Table 1 in the Supplementary Ap- pendix); 220 of the 234 (94%) overlap with re- ported regions of copy-number variants in the Database of Genomic Variants (http://projects.tcag .ca/variation). We found no copy-number variants affecting genes known to be involved in Char- cot?Marie?Tooth disease or other neuropathies. Tableuni00202.uni0020SNPsuni0020Identifieduni0020throughuni0020Whole-Genomeuni0020 Sequencinguni0020ofuni0020DNAuni0020fromuni0020theuni0020Proband.* SNPuni0020Type No.uni0020ofuni0020SNPs Nongene 2,255,102 Gene 1,165,204 Intron 1,064,655 Promoter 60,075 3prime UTR 16,350 5prime UTR 3,517 Splice regulatory site 2,089 Splice site 112 Synonymous 9,337 Stop?stop 17 Nonsynonymous 9,069 Stop?gain 121 Stop?loss 27 Total 3,420,306 * Stop?stop refers to synonymous substitutions within a stop codon that maintain the stop codon, stop?gain refers to nonsense mutations, and stop?loss refers to nonsynon- ymous substitutions that change a stop codon to any oth- er codon. SNP denotes single-nucleotide polymorphism, and UTR untranslated region. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . Whole-Genome Sequencing and SH3TC2 Mutations n engl j med 362;13 nejm.org april 1, 2010 1187 We cross-referenced the nonsynonymous SNPs that we detected by using whole-genome sequenc- ing with a database of previously observed muta- tions implicated in human disease (the Human Gene Mutation database) (Table 4, and Table 2 in the Supplementary Appendix). Of the 174 non- synonymous database SNPs identified in the proband, 159 had a clear association with a heritable trait (i.e., the database entry was not annotated with a question mark). Of these, 21 (13%) were described as causing mendelian dis- ease; 16 were heterozygous in the proband, a finding that is consistent with the expected load of autosomal recessive mutations. The other five SNPs might have been erroneously assigned as disease mutations, which would explain why four of them were homozygous in the proband and have been found to be homozygous in unaffected persons. It would also explain why the sequence for the proband, who did not have adrenoleuko- dystrophy, contained a SNP in ABCD1 previously described as a dominant mutation that causes the X-linked disorder adrenoleukodystrophy. 22 An alternative to the interpretation that the five SNPs might have been erroneously assigned as disease mutations is that these alleles might have reduced penetrance. We examined the putative mutations in 40 genes known to cause or be linked to neuro- pathic or related conditions (Table 3 in the Sup- plementary Appendix). This exercise led to closer examination of 3148 putative SNPs, including 54 coding SNPs. Of these 54, 2 were at the SH3TC2 locus ? 1 missense mutation (identified at 7.7 average depth coverage) and 1 nonsense muta- tion (identified at 29.9 average depth coverage) (Fig. 1 in the Supplementary Appendix). Muta- tions in this locus have previously been found to be associated with Charcot?Marie?Tooth type 4C disease, described in families of eastern Euro- pean, Turkish or Spanish Gypsy origin. 23-25 The R954X nonsense mutation has previously been implicated in Charcot?Marie?Tooth disease; the missense mutation (A?G, occurring on chromo- some 5 at nucleotide 148,402,474 and corre- sponding to the amino acid mutation Tyr169His [Y169H]) is novel. Correlation between Genotype and Phenotype Segregation analyses verified independent mater- nal and paternal origins of the mutations (Fig. 2). The nonsense mutation (R954X) appeared in one parent of the proband and in two siblings who did not have Charcot?Marie?Tooth type 1 disease. The missense mutation (Y169H) was found in one parent and one grandparent, neither of whom had Charcot?Marie?Tooth disease. Only the pro- band (Subject III-4) and three of his siblings (Sub- jects III-2, III-6, and III-8) who had inherited both mutant alleles had the Charcot?Marie?Tooth type 1 phenotype (Fig. 2). Tableuni00203.uni0020Individualuni0020Humanuni0020Genomesuni0020Sequenceduni0020touni0020Date.* Genome? Technologyuni0020Used Averageuni0020Depthuni0020uni0020 ofuni0020Coverage SNPs Total Known Novel �10 ?6 Venter Sanger method 7.5 3.21 2.80 0.74 Watson 454 Sequencing System (Roche) 7.4 3.32 2.71 0.61 Chinese (YH) Genome Analyzer (Illumina) 36 3.07 2.67 0.39 African (NA18507) Genome Analyzer (Illumina) 40.6 3.61 2.72 0.88 African (NA18507) SOLiD system (Applied Biosystems) 17.9 3.86 3.13 0.73 Korean (SJK) Genome Analyzer (Illumina) 28.95 3.43 3.01 0.42 Korean (AK1) Genome Analyzer (Illumina) 27.8 3.45 2.88 0.57 Proband in this study SOLiD system (Applied Biosystems) 29.9 3.42 2.85 0.56 * All genomes listed have a ploidy of 2n. SNP denotes single-nucleotide polymorphism. ? The surname of the individual person or the ethnic group (and HapMap sample name, in parentheses) is given. The same African (Yoruban) sample NA18507 was sequenced twice, once with the use of the Genome Analyzer and once with the use of the SOLiD (Sequencing by Oligonucleotide Ligation and Detection) system. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1188 The subjects with the heterozygous missense mutation (Y169H) (Subjects I-2 and II-2) (Fig. 2) also had the apparently dominant axonal neu- ropathy phenotype, as detected by electrophysio- logical studies. These findings of axonal neuropa- thy (Table 1) suggest a gain of function (i.e., a toxic effect) of this mutation. In contrast, the pre- sumed loss-of-function nonsense variant (R954X) was associated with electrophysiological evidence of the carpal tunnel syndrome, regardless of whether it was the sole mutation present (i.e., heterozygous genotype) or was accompanied by the missense variant (Y169H) (i.e., compound heterozygous genotype) (Table 1 and Fig. 2). Discussion We ascertained the molecular basis of an inher- ited disease by using next-generation-sequencing methods. We chose whole-genome sequencing over targeted, exon-capture approaches 26,27 be- cause we did not know whether the ?causative? mutations would reside in known coding ele- ments, and targeted approaches are ill suited to capturing copy-number variants. The heteroge- neity of our sequence data is emblematic of the current rapid progress of sequencing technology: over the 6-month course of this study, sequence read lengths doubled (from 25 bp to 50 bp), the density of samples on the sequencing slide in- creased, and mapping technology improved. Over- all, the sequence yield increased by a factor of three, with no appreciable increase in expense. This rapid pace of technological improvement makes it difficult to accurately determine the ex- pense of repeating this experiment, but given that the expense of sequencing reagents for a single run on the SOLiD instrument was $25,000 in April 2009, we estimate that the entire effort would currently cost less than $50,000. The whole-genome sequencing approach used in this proband enabled us to identify the cause of his disease as compound heterozygous muta- tions in the SH3TC2 gene and thus to delineate the specific biologic basis of disease in his fam- ily. The SH3TC2 protein contains both SH3 and TPR motifs; SH3 motifs mediate the assembly of protein complexes binding to proline-rich pro- teins, and TPR motifs are involved in protein? protein interactions. The mouse orthologue of SH3TC2 is specifical- ly expressed in Schwann cells, and the SH3TC2 protein localizes to the plasma membrane and to the perinuclear endocytic recycling compart- ment, which is consistent with a role in myelina- tion or in axon?glia interactions. 28 Mice lacking Sh3tc2 have abnormal organization of the node of Ranvier. 28 Consistent with a role of SH3TC2 in endocytic processes 29 is the finding that SH3TC2 mutations result in disruption of the endocytic and membrane recycling pathways. 30 We observed that both of the SH3TC2 muta- tions, when heterozygous, have phenotypic con- sequences that can be detected by electrophysi- ological means. The Y169H missense variant segregates with an axonal neuropathy, whereas the nonsense R954X mutation is associated with subclinical evidence of the carpal tunnel syn- drome; therefore, haploinsufficiency of SH3TC2 may cause susceptibility to the carpal tunnel syndrome. This susceptibility may also result from mutations in other genes related to Charcot? Marie?Tooth disease in addition to PMP22 and SH3TC2. Whole-genome sequencing of other members of the proband?s family might help clarify whether the additional 69 SNPs at the SH3TC2 locus and 3146 SNPs at the other 39 neuropathy-associated gene loci examined (in- cluding many rare variants, 466 of which have not previously been described [Table 3 in the Supplementary Appendix]) can modify the highly Tableuni00204.uni0020Diseaseuni0020anduni0020Traituni0020Associationsuni0020ofuni0020 Nonsynonymousuni0020SNPsuni0020Identifieduni0020inuni0020theuni0020Proband,uni0020 Accordinguni0020touni0020theuni0020Humanuni0020Geneuni0020Mutationuni0020Database.* Diseaseuni0020oruni0020Traituni0020Associateduni0020withuni0020Mutation SNPs no. (%) Total 159 (100) Behavioral disorder 6 (4) Cancer 33 (21) Association 7 Increased risk 9 Reduced risk 3 Susceptibility 14 Complex disease 48 (30) Mendelian disease 21 (13) Metabolic trait 17 (11) Pharmacogenetic trait 14 (9) Other traits 20 (13) * SNP denotes single-nucleotide polymorphism. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . Whole-Genome Sequencing and SH3TC2 Mutations n engl j med 362;13 nejm.org april 1, 2010 1189 6 col 33p9 Axonal neuropathy CMT1 Normal AUTHOR: FIGURE: RETAKE: SIZE 4-C H/TLine Combo Revised AUTHOR,uni0020PLEASEuni0020NOTE: Figureuni0020hasuni0020beenuni0020redrawnuni0020anduni0020typeuni0020hasuni0020beenuni0020reset. Pleaseuni0020checkuni0020carefully. 1st 2nd 3rd Gibbs (Lupski) 2 of 2 ARTIST: TYPE: ts 04-01-10JOB: 36213 ISSUE: A B C I II III I-1 +/+ I-2 +/Y169H II-1 R954X/+ II-2 +/Y169H III-1 +/+ III-2 R954X/ Y169H III-3 R954X/+ III-4 R954X/ Y169H III-5 +/+ III-6 R954X/ Y169H III-7 R954X/+ III-8 R954X/ Y169H G?Auni0020mutant (R954X) Wilduni0020type Y169 Homo sapiens Pan troglodytes Macaca mulatta Canis familiaris Equus caballus Bos taurus Mus musculus Rattus norvegicus Monodelphis domestica Gallus gallus EHLLFDHKYWLNCILVEDTEIQVSVDDKHLETIYLGLLIQEGHFFCRALCSVTPPAEKEG-ECLTL EHLLFDHKYWLNCILVEDTEIQVSVDDKHLETIYLGLLIQEGHFFCRALCSVTPPAEKEG-ECLTL EHLLFDHKYWLNCILVEDTEIQVSVDDKHLETIYLGLLIQEGHFFCRALCSVIPPAEKEG-ECLTL EHLLFDHKYWLNCRLVEDTEIQVSVDEKHLETIYLALLIQEGHFFCRAMCSVAQPAEKEG-EYLTL EHLLFDHKYWLNSRLVEDTEIQVSVDDKHLESIYLGLLIQEGHFFCRAMCSVAQPAEKEG-EYLTL EHLLFDHKYWLNCRLVEDTEIHVSIDDKHLETIYLGLLIQEGHFFCRAMCSVAQPAEKEG-EYLTL EHLFFDHTYWLNSRLVDDTEIQVSVDDNHLENIYLGLLLQEGHFFCRAVCSVAQPADKEG-EYLTL EHLFFDHTYWLNSRLVDDTEIQVSVDDTHLENIYLGLLLQEGHFFCRAMCSVTQPADKEG-EYLTL EQLLFDHNYWLNFRLVEDTKIQVIVNYEHLEAIYQSLLIQEGH-FCRTVHTVFRSGEKEGGEYLKL EQLLFEQEYWLNCALVEDTEIRVSMDENRLATIYLGLLLQEGHFFSRAVPGVCQPG-GEGQEGLQL SH3TC2 Genotypeuni0020anduni0020Phenotype Resultsuni0020ofuni0020R954Xuni0020Genotyping Sequenceuni0020Alignment Figureuni00202.uni0020Pedigreeuni0020ofuni0020theuni0020Studyuni0020Familyuni0020anduni0020Segregationuni0020anduni0020Conservationuni0020ofuni0020SH3TC2uni0020Mutations. Panel A shows the pedigree of the proband (arrow) and his family and their SH3TC2 genotypes: plus signs indicate the wild-type allele; Y169H indicates the A?G mutation on chromosome 5 at nucleotide 148,402,474 and corre- sponding to the amino acid missense mutation Tyr169His, and R954X indicates the G?A mutation in the genomic DNA in exon 11 of SH3TC2 on chromosome 5 at nucleotide 148,386,628, leading to the amino acid nonsense muta- tion Arg954ter. (Genomic coordinates for the mutations in the proband are based on the human genome reference sequence, build 36.2.) Squares indicate male subjects, and circles female subjects; slashes indicate deceased sub- jects. Subjects in generations I and II had three phenotypes. The paternal grandfather (Subject I-1) was studied 20 years ago, at 80 years of age, and had normal results, with the sole exception of a median-nerve mononeuropathy at the wrist, thought to be caused by his occupation as a carpenter. The paternal grandmother (Subject I-2, 77 years of age at the time of evaluation) and the father (Subject II-2) had evidence of a patchy axonal polyneuropathy, with def- inite median-nerve mono neuropathy at the wrist. The maternal grandmother (evaluated at 90 years of age; data not shown) and the mother (Subject II-1) had normal findings except for very mild median-nerve mononeuropathy at the wrist. Two of the proband?s sisters (Subjects III-3 and III-7) had this same phenotype. Two members of this generation had completely normal findings (Subjects III-1 and III-5). The other four siblings had diffuse, dispropor- tionate conduction slowing in the distal median nerve, without evidence of conduction block, findings that are sug- gestive of a superimposed median mononeuropathy at the wrist. Subjects III-2, III-4, III-6, and III-8 had Charcot? Marie?Tooth type 1 (CMT1) disease. Panel B shows the results of TaqI restriction digestion of the SH3TC2 exon 11 polymerase-chain-reaction product on which the G?A mutation, corresponding to the R954X allele, occurs. This mutation was present in the proband?s mother and six of the eight siblings, as well as in the maternal grandmother (not shown). The mutation destroys the restriction site for TaqI; the wild type yields two small bands and the heterozygous mutant yields three bands, the upper of which is the uncut DNA. Panel C shows sequence alignment of the SH3TC2 protein among various species. The downward arrowhead indicates the location of the highly con- served Tyr169 amino acid that, in persons with the novel missense mutation Y169H, is changed to His. Sequences were obtained from the National Center for Biotechnology Information. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . The new england journal of medicine n engl j med 362;13 nejm.org april 1, 2010 1190 penetrant Y169H and R954X mutations and there- by influence the neuropathy phenotype. The whole-genome sequencing approach that we describe here contrasts with other diagnostic approaches. A clinical-testing panel that screens for a copy-number variant that commonly causes Charcot?Marie?Tooth disease 14 and nucleotide- sequence variants in 15 of the genes known to be mutated in patients with the disease can cost more than $15,000. 31 Mutations in two or more genes related to Charcot?Marie?Tooth disease have been described as causing a phenotype more severe than that of our proband or other patients affected by the disease. 32-34 Such groups of mu- tations include a combination of two SNPs at the ACBD1 locus and a copy-number variant af- fecting PMP22, as well as the combination of a SNP and a copy-number variant at the same lo- cus. 35,36 There is also a report of mutations in two genes related to Charcot?Marie?Tooth dis- ease segregating in the same family as either a recessive trait or a sporadic trait, the latter of which was attributed to a de novo copy-number variant. 37 Given this locus heterogeneity, with evidence of a mutational load that has clinical consequences, as well as the ease of use and accuracy of the whole-genome sequencing meth- ods we applied, clinical and genetics experts struggling to explain poorly understood high- penetrance genetic diseases can now seriously consider this approach for illuminating the mo- lecular causes of these diseases. The approach may ultimately contribute to the care of patients and families living with such diseases. Our results suggest that haploinsufficiency of SH3TC2 confers predisposition to a mild polyneu- ropathy with particular susceptibility to the carpal tunnel syndrome. More generally, they demon- strate the diagnostic power of whole-genome se- quencing in the context of genetically heteroge- neous mendelian disease and inform efforts to decipher the genetic bases of complex traits. As new, rare alleles at other gene loci are implicated in conditions such as diabetes, obesity, heart dis- ease, and cancer and as the patterns of interac- tion of the alleles with a patient?s phenotype are delineated, genetic susceptibility to such dis- eases may become clearer. As a practical matter, the identification of rare, heterogeneous alleles by means of whole-genome sequencing may be the only way to definitively determine genetic contri- butions to the associated clinical phenotypes. Supported in part by grants from the National Human Ge- nome Research Institute (5 U54 HG003273, to Dr. Gibbs) and the National Institute of Neurological Disorders and Stroke (R01 NS058529, to Dr. Lupski). Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank Kevin McKernan, Michael Rhodes, Francisco de la Vega, Quynh Doan, and Fiona Hyland for extensive discussion and support and Cristian Coarfa for structural-variation analysis and insights. References Rommens JM, Iannuzzi MC, Kerem B, 1.uni0020 et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245:1059-65. Wallace MR, Marchuk DA, Andersen 2.uni0020 LB, et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupt- ed in three NF1 patients. Science 1990; 249:181-6. Kajiwara K, Berson EL, Dryja TP. Di-3.uni0020 genic retinitis pigmentosa due to muta- tions at the unlinked peripherin/RDS and ROM1 loci. Science 1994;264:1604-8. Katsanis N, Ansley SJ, Badano JL, et al. 4.uni0020 Triallelic inheritance in Bardet-Biedl syn- drome, a Mendelian recessive disorder. Science 2001;293:2256-9. Dipple KM, McCabe ERB. Phenotypes 5.uni0020 of patients with ?simple? Mendelian dis- orders are complex traits: thresholds, mod- ifiers, and systems dynamics. Am J Hum Genet 2000;66:1729-35. Badano JL, Katsanis N. Beyond Men-6.uni0020 del: an evolving view of human genetic disease transmission. Nat Rev Genet 2002;3:779-89. Allan W. Relation of hereditary pat-7.uni0020 tern to clinical severity as illustrated by peroneal atrophy. Arch Intern Med 1939; 63:1123-31. Haldane JBS. The relative importance 8.uni0020 of principal and modifying genes in deter- mining some human diseases. J Genet 1941;41:149-57. England JD, Gronseth GS, Franklin G, 9.uni0020 et al. Practice parameter: evaluation of dis- tal symmetric polyneuropathy: role of lab- oratory and genetic testing (an evidence- based review): report of the American Academy of Neurology, American Associ- ation of Neuromuscular and Electrodiag- nostic Medicine, and American Academy of Physical Medicine and Rehabilitation. Neurology 2009;72:185-92. Lupski JR. Structural variation in the 10.uni0020 human genome. N Engl J Med 2007;356: 1169-71. Roa BB, Garcia CA, Suter U, et al. 11.uni0020 Charcot?Marie?Tooth disease type 1A: association with a spontaneous point mu- tation in the PMP22 gene. N Engl J Med 1993;329:96-101. McKernan KJ, Peckham HE, Costa 12.uni0020 GL, et al. Sequence and structural varia- tion in a human genome uncovered by short-read, massively parallel ligation se- quencing using two-base encoding. Ge- nome Res 2009;19:1527-41. Del Colle R, Fabrizi GM, Turazzini M, 13.uni0020 Cavallaro T, Silvestri M, Rizzuto N. Heredi- tary neuropathy with liability to pressure palsies: electrophysiological and genetic study of a family with carpal tunnel syn- drome as only clinical manifestation. Neurol Sci 2003;24:57-60. Lupski JR, de Oca-Luna RM, Slaugen-14.uni0020 haupt S, et al. DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 1991;66:219-32. International Human Genome Sequenc-15.uni0020 ing Consortium. Finishing the euchromat- ic sequence of the human genome. Nature 2004;431:931-45. Levy S, Sutton G, Ng PC, et al. The 16.uni0020 diploid genome sequence of an individual human. PLoS Biol 2007;5(10):e254. Wheeler DA, Srinivasan M, Egholm M, 17.uni0020 et al. The complete genome of an indi- Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . Whole-Genome Sequencing and SH3TC2 Mutations n engl j med 362;13 nejm.org april 1, 2010 1191 vidual by massively parallel DNA sequenc- ing. Nature 2008;452:872-6. Bentley DR, Balasubramanian S, Swerd-18.uni0020 low HP, et al. Accurate whole human ge- nome sequencing using reversible termi- nator chemistry. Nature 2008;456:53-9. Wang J, Wang W, Li R, et al. The dip-19.uni0020 loid genome sequence of an Asian indi- vidual. Nature 2008;456:60-5. Ahn SM, Kim TH, Lee S, et al. The 20.uni0020 first Korean genome sequence and analy- sis: full genome sequencing for a socio- ethnic group. Genome Res 2009;19:1622-9. Kim JI, Ju YS, Park H, et al. A highly 21.uni0020 annotated whole-genome sequence of a Ko- rean individual. Nature 2009;460:1011-5. Dvor�kov� L, Stork�nov� G, Unter-22.uni0020 rainer G, et al. Eight novel ABCD1 gene mutations and three polymorphisms in patients with X-linked adrenoleukodystro- phy: the first polymorphism causing an amino acid exchange. Hum Mutat 2001; 18:52-60. Senderek J, Bergmann C, Stendel C, et 23.uni0020 al. Mutations in a gene encoding a novel SH3/TPR domain protein cause autoso- mal recessive Charcot-Marie-Tooth type 4C neuropathy. Am J Hum Genet 2003;73: 1106-19. Azzedine H, Ravis� N, Verny C, et al. 24.uni0020 Spine deformities in Charcot-Marie-Tooth 4C caused by SH3TC2 gene mutations. Neurology 2006;67:602-6. Gooding R, Colomer J, King R, et al. A 25.uni0020 novel Gypsy founder mutation, p.Arg1109X in the CMT4C gene, causes variable periph- eral neuropathy phenotypes. J Med Genet 2005;42(12):e69. Albert TJ, Molla MN, Muzny DM, et al. 26.uni0020 Direct selection of human genomic loci by microarray hybridization. Nat Methods 2007;4:903-5. Ng SB, Buckingham KJ, Lee C, et al. 27.uni0020 Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 2010;42: 30-5. Arnaud E, Zenker J, de Preux Charles 28.uni0020 A-S, et al. SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the pe- ripheral nervous system. Proc Natl Acad Sci U S A 2009;106:17528-33. Roberts RC, Peden AA, Buss F, et al. 29.uni0020 Mistargeting of SH3TC2 away from the re- cycling endosome causes Charcot-Marie- Tooth disease type 4C. Hum Mol Genet 2010;19:1009-18. Lupo V, Galindo M, Mart�nez-Rubio 30.uni0020 D, et al. Mutations in the SH3TC2 protein causing Charcot-Marie-Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway. Hum Mol Genet 2009;18:4603-14. Athena Diagnostics home page. (Ac-31.uni0020 cessed March 5, 2010, at http://www .athenadiagnostics.com.) Chung KW, Sunwoo IN, Kim SM, et al. 32.uni0020 Two missense mutations of EGR2 R359W and GJB1 V136A in a Charcot-Marie- Tooth disease family. Neurogenetics 2005; 6:159-63. Auer-Grumbach M, Fischer C, Papi? L, 33.uni0020 et al. Two novel mutations in the GDAP1 and PRX genes in early onset Charcot- Marie-Tooth syndrome. Neuropediatrics 2008;39:33-8. Hodapp JA, Carter GT, Lipe HP, Michel-34.uni0020 son SJ, Kraft GH, Bird TD. Double trouble in hereditary neuropathy: concomitant mutations in the PMP-22 gene and another gene produce novel phenotypes. Arch Neurol 2006;63:112-7. Roa BB, Garcia CA, Pentao L, et al. 35.uni0020 Evidence for a recessive PMP22 point mu- tation in Charcot-Marie-Tooth disease type 1A. Nat Genet 1993;5:189-94. Shy ME, Scavina MT, Clark A, et al. 36.uni0020 T118M PMP22 mutation causes partial loss of function and HNPP-like neuropa- thy. Ann Neurol 2006;59:358-64. Verny C, Ravis� N, Leutenegger AL, 37.uni0020 et al. Coincidence of two genetic forms of Charcot-Marie-Tooth disease in a single family. Neurology 2004;63:1527-9. Copyright � 2010 Massachusetts Medical Society. R E C E I V E I M M E D I A T E N O T I F I C A T I O N W H E N A J O U R N A L A R T I C L E I S R E L E A S E D E A R L Y To be notified when an article is released early on the Web and to receive the table of contents of the Journal by e-mail every Wednesday evening, sign up through our Web site at NEJM.org. Copyright � 2010 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at EDWARD G MINER LIBRARY on April 5, 2010 . "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment