The new england journal of medicine 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. ABSTRACT BACKGROUND Whole-genome sequencing may revolutionize medical diagnostics through rapid From the Department of Molecular and identification of alleles that cause disease. However, even in cases with simple pat- 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- terns of inheritance and unambiguous diagnoses, the relationship between disease man Genome Sequencing Center (J.G.R., phenotypes and their corresponding genetic changes can be complicated. Compre- D.R.D., D.C.Y.C., L.N., M.B., H.D., C.J., hensive diagnostic assays must therefore identify all possible DNA changes in each D.A.W., D.M.M., R.A.G.), the Center for Medical Ethics and Health Policy (A.L.M.), haplotype and determine which are responsible for the underlying disorder. The the Department of Pediatrics (J.R.L.), and high number of rare, heterogeneous mutations present in all humans and the pau- Texas Children’s Hospital (J.R.L.) — all at city of known functional variants in more than 90% of annotated genes make this Baylor College of Medicine, Houston; Atlantic Neuroscience Institute, Summit, challenge particularly difficult. Thus, the identification of the molecular basis of a NJ, and Mount Sinai School of Medicine, genetic disease by means of whole-genome sequencing has remained elusive. We New York (J.J.H.); and Life Technologies, therefore aimed to assess the usefulness of human whole-genome sequencing for Carlsbad, CA (C.Y., C.G., D.G., R.K.I., W.T., N.J.F.). Address reprint requests to Dr. genetic diagnosis in a patient with Charcot–Marie–Tooth disease. Gibbs at 1 Baylor Plaza, Mail Stop BCM226, Houston, TX 77030, or at agibbs@bcm METHODS .edu. We identified a family with a recessive form of Charcot–Marie–Tooth disease for This article (10.1056/NEJMoa0908094) was which the genetic basis had not been identified. We sequenced the whole genome published on March 10, 2010, at NEJM.org. of the proband, identified all potential functional variants in genes likely to be related N Engl J Med 2010;362:1181-91. to the disease, and genotyped these variants in the affected family members. Copyright © 2010 Massachusetts Medical Society. 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. n engl j med 362;13 nejm.org april 1, 2010 1181 Downloaded from www.nejm.org at HHS LIBRARIES CONSORTIUM on May 12, 2010 . Copyright © 2010 Massachusetts Medical Society. All rights reserved. The new england journal of medicine he practice of medical genetics re- resulting in foot dorsiflexor weakness, foot drop, quires gene-specific analyses of DNA se- and secondary steppage gait. Pes cavus (highly Tquences and mutations to definitively diag- arched feet) or pes planus (flat feet) occurs in nose disease, provide prognostic information, and most patients. guide genetic counseling regarding the risk of re- We applied next-generation-sequencing meth- currence. Studies of autosomal recessive traits ods to identify the cause of disease in a family such as cystic f ibrosis1 and some dominant traits with inherited neuropathy that had been previous- such as neurofibromatosis type 12 revealed the ly screened, with negative results, for alterations role of single “disease genes” in conveying traits. of some common Charcot–Marie–Tooth genes, However, many phenotypes of mendelian diseases including PMP22,11 MPZ, PRX, GDAP1, and EGR2. (see the Glossary) are genetically heterogeneous: causative mutations have been identified in more Methods than 100 genes for deafness and retinitis pig- mentosa, for instance. Moreover, specific muta- Study Participants tions may confer phenotypes that segregate as The study family consisted of four affected sib- dominant, recessive, or even digenic3 or triallel- lings, four unaffected siblings, and an unaffected ic4 traits. There is also ample evidence of modi- mother and father, all of whom provided written fying loci in mendelian disorders.5,6 Thus, even informed consent for participation in the study. when there are simple patterns of inheritance The study was approved by the institutional re- in syndromes with a well-characterized patho- view board at Baylor College of Medicine. The di- logic course, the underlying mutational events, agnosis of Charcot–Marie–Tooth type 1 disease which need to be resolved for precise molecular in the proband and the three affected siblings was diagnosis, within individual families may be based on the results of physical examination (dis- complex. tal muscle weakness and wasting, pes cavus, and Charcot–Marie–Tooth disease is an inherited absence of deep-tendon reflexes) and electrophys- peripheral neuropathy with two forms: a demyeli- iological studies. nating form (type 1) affecting the glia-derived myelin and an axonal form (type 2) affecting the Neurophysiological Assessments nerve axon. The two forms can be distinguished Neurophysiological studies consisted of a stan- by means of electrophysiological or neuropatho- dard battery of nerve-conduction studies, includ- logical studies. Charcot–Marie–Tooth disease has ing motor responses of the median, ulnar, tibial, been used as a model disease to describe genetic and peroneal nerves with F-wave latencies; ortho- heterogeneity, posit the relation of hereditary dromic median-, ulnar-, and sural-nerve sensory pattern to clinical severity, and investigate the potentials; and bilateral tibial H-reflexes. When relative importance of principal and modifying these studies revealed demyelinating features, genes in determining human diseases.7,8 Mutant tests of blink reflexes were generally performed. alleles underlying Charcot–Marie–Tooth disease Limbs were warmed to a temperature of at least can segregate in an autosomal dominant, reces- 32°C in all instances. Demyelination was judged sive, or X-linked manner (Fig. 1). Both single- to be present if conduction velocities were signif- base variants (single-nucleotide polymorphisms icantly slowed and the late-response latencies [SNPs]) and copy-number variants,10 at 39 sepa- were substantially delayed. Median-ner ve mono- rate loci, confer susceptibility to Charcot–Marie– neuropathy at the wrist was judged to be present Tooth disease. Most of these susceptibility variants when there was prolonged motor terminal latency cause dominant forms of the disease, although or slowed median-nerve sensory velocity with dis- mutations in genes at 14 of the loci cause reces- proportionate slowing in the palm-to-wrist seg- sive disease. ment, or both. The four affected subjects, all of Adult-onset Charcot–Marie–Tooth disease is whom had diffuse slowing of conduction, were also highly variable in presentation but is character- thought to have a median-nerve mononeuropathy ized by distal symmetric polyneuropathy,9 with at the wrist, since the median-nerve motor termi- slowly progressive distal muscle weakness and nal latency was much more prolonged than the atrophy (particularly peroneal muscular atrophy) ulnar-nerve motor terminal latency (14.9 vs. 8.1, 1182 n engl j med 362;13 nejm.org april 1, 2010 Downloaded from www.nejm.org at HHS LIBRARIES CONSORTIUM on May 12, 2010 . Copyright © 2010 Massachusetts Medical Society. All rights reserved. Whole-Genome Sequencing and SH3TC2 Mutations 10.2 vs. 7.5, 11.6 vs. 6.2, and 9.2 vs. 6.2 msec) tides. Its accuracy in sequencing 50-base reads is (Table 1). estimated at approximately 99.94%.12 Multiple se- quences can be read simultaneously, and when DNA Sequence Analysis the sequence reads overlap, the overall accuracy DNA sequencing was performed with the use of increases further, reducing the risk of false posi- the SOLiD (Sequencing by Oligonucleotide Liga- tive determinations and the need for additional tion and Detection) system (Applied Biosystems), a data validation. We determined bases from the next-generation-sequencing platform that involves primary sequencing data, using the standard ligation-based sequencing and a two-base encod- SOLiD analysis software. (For details, see the Sup- ing method in which four fluorescent dyes are plementary Appendix, available with the full text used to tag various combinations of dinucleo- of this article at NEJM.org.) Glossary Array-based comparative genomic hybridization: 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. Average depth of coverage: The 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. Coding single-nucleotide polymorphisms: Single DNA-base changes that occur in the coding regions of genes.