Original Article

Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders

List of authors.
  • Yaping Yang, Ph.D.,
  • Donna K. Muzny, Thousand.Sc.,
  • Jeffrey M. Reid, Ph.D.,
  • Matthew Due north. Bainbridge, Ph.D.,
  • Alecia Willis, Ph.D.,
  • Patricia A. Ward, M.Due south.,
  • Alicia Braxton, Thousand.S.,
  • Joke Beuten, Ph.D.,
  • Fan Xia, Ph.D.,
  • Zhiyv Niu, Ph.D.,
  • Matthew Hardison, Ph.D.,
  • Richard Person, Ph.D.,
  • Mir Reza Bekheirnia, G.D.,
  • Magalie S. Leduc, Ph.D.,
  • Amelia Kirby, G.D.,
  • Peter Pham, Thousand.Sc.,
  • Jennifer Scull, Ph.D.,
  • Min Wang, Ph.D.,
  • Yan Ding, M.D.,
  • Sharon E. Plon, M.D., Ph.D.,
  • James R. Lupski, G.D., Ph.D.,
  • Arthur Fifty. Beaudet, M.D.,
  • Richard A. Gibbs, Ph.D.,
  • and Christine K. Eng, M.D.

Abstract

Background

Whole-exome sequencing is a diagnostic approach for the identification of molecular defects in patients with suspected genetic disorders.

Methods

We adult technical, bioinformatic, interpretive, and validation pipelines for whole-exome sequencing in a certified clinical laboratory to identify sequence variants underlying illness phenotypes in patients.

Results

Nosotros present data on the first 250 probands for whom referring physicians ordered whole-exome sequencing. Patients presented with a range of phenotypes suggesting potential genetic causes. Approximately eighty% were children with neurologic phenotypes. Insurance coverage was like to that for established genetic tests. Nosotros identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients, achieving a 25% molecular diagnostic rate (95% conviction interval, xx to 31). Among the 62 patients, 33 had autosomal dominant illness, 16 had autosomal recessive disease, and 9 had Ten-linked disease. A total of 4 probands received two nonoverlapping molecular diagnoses, which potentially challenged the clinical diagnosis that had been made on the basis of history and physical examination. A total of 83% of the autosomal dominant mutant alleles and twoscore% of the X-linked mutant alleles occurred de novo. Recurrent clinical phenotypes occurred in patients with mutations that were highly likely to be causative in the aforementioned genes and in unlike genes responsible for genetically heterogeneous disorders.

Conclusions

Whole-exome sequencing identified the underlying genetic defect in 25% of consecutive patients referred for evaluation of a possible genetic condition. (Funded by the National Man Genome Inquiry Institute.)

Introduction

Mendelian diseases are considered to be rare, notwithstanding genetic disorders are estimated to occur at a rate of 40 to 82 per 1000 live births.1 Epidemiologic studies bear witness that if all built anomalies are considered as part of the genetic load, then approximately 8% of persons are identified as having a genetic disorder before reaching adulthood.2 Collectively, rare genetic disorders bear upon substantial numbers of persons.

Many patients with genetic diseases are not given a specific diagnosis. The standard of exercise involves the recognition of specific phenotypic or radiographic features or biopsy findings in add-on to the analysis of metabolites, genomic tests such every bit karyotyping or array-based comparative genomic hybridization,3,4 or the pick of candidate-gene tests, including single-factor analyses and gene-panel tests. The majority of patients remain without a diagnosis.5 The lack of a diagnosis can have considerable adverse effects for patients and their families, including failure to place potential treatments, failure to recognize the run a risk of recurrence in subsequent pregnancies, and failure to provide anticipatory guidance and prognosis. A long-term search for a genetic diagnosis, referred to as the "diagnostic odyssey," also has implications for societal medical expenditures, with unsuccessful attempts consuming limited resources.

Genomic sequencing with the apply of massively parallel next-generation sequencing technologies has proven to be an effective alternative to locus-specific and gene-panel tests in a inquiry setting for establishing a new genetic basis of disease.six-12 The initial application of next-generation sequencing approaches to clinical diagnosis raises challenges. Beyond the technical challenges of the genomic assay and bioinformatic analyses of massive amounts of data, the diagnostic yield in a clinical laboratory setting for unselected patients with a broad range of phenotypes is unknown. Moreover, interrogation of the exome may uncover secondary findings, complicating reporting.13 We analyzed 250 unselected, consecutive cases with the use of clinical whole-exome sequencing in a laboratory certified by the College of American Pathologists (CAP) and the Clinical Laboratory Improvement Amendments (CLIA) program.

Methods

Clinical Samples

Nosotros initiated clinical testing with whole-exome sequencing in October 2011. The exam was ordered by the patient's physician, later the physician had explained the risks and benefits of testing to the patient and had obtained written informed consent. Each patient (and their parents or guardians, as appropriate) was advised of the potential disclosure of medically actionable incidental findings, defined as conditions unrelated to the indication for testing that might warrant treatment or additional medical surveillance for the patient and possibly other family members.

Peripheral-blood samples were provided in nigh cases, although other sources of Deoxyribonucleic acid were accepted and samples from both parents were ordinarily provided. Clinical data, provided past the referring md on the requisition form, included findings according to organ arrangement, neurologic status, growth, and development. Nosotros too requested a recent clinic note summarizing the case and the prior workup. Laboratory coordinators monitored the submission of these forms and ensured receipt before interpretation of the data from whole-exome sequencing.

Table i. Table 1. Clinical Description of Patients for Whom Whole-Exome Sequencing Was Ordered.

A brusque clinical synopsis was constructed by the laboratory clinical geneticist and was included in the final report for review by the referring physician. The testing and assay were performed at the Baylor College of Medicine in clinical diagnostic laboratories certified past CAP and CLIA. Here, nosotros describe information from the first 250 sequent probands received between October 2011 and June 2012 for whom whole-exome sequencing was ordered (Table i). The aggregate, deidentified reporting of these data was approved by the local institutional review lath without the need for farther informed consent.

Whole-Exome Sequencing and Variant Confirmation

Whole-exome sequencing and analysis protocols developed by the Human Genome Sequencing Middle at the Baylor College of Medicine were adapted for the clinical exam of whole-exome sequencing. Briefly, genomic Dna samples from probands were fragmented with the employ of sonication, ligated to Illumina multiplexing paired-terminate adapters, amplified by means of a polymerase-chain-reaction assay with the utilise of primers with sequencing barcodes (indexes), and hybridized to biotin-labeled VCRome, version ii.1,14 a solution-based exome capture reagent that was designed in-firm and is commercially available (Roche NimbleGen). Hybridization was performed at 47°C for 64 to 72 hours, and paired-stop sequencing (100 bp) was performed on either the Illumina Genome Analyzer IIx platform (24 cases) or the Illumina HiSeq 2000 platform (226 cases) to provide a mean sequence coverage of more than 130×, with more than 95% of the target bases having at least twenty× coverage (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).

Variants that were deemed clinically significant were confirmed by means of Sanger sequencing. Parental samples, if bachelor, were likewise analyzed past means of Sanger sequencing to determine whether the mutated allele had been transmitted and, if so, by whom. For each example, several rare variants (typically, five to eight) were studied in the proband and family members. Nonpaternity could thus be discovered.

Data Analysis and Annotation

Before clinical interpretation, the data were analyzed and annotated by means of a pipeline that was adult in-house (www.tinyurl.com/HGSC-Mercury; see the Supplementary Appendix). Briefly, the output data from the Illumina Genome Analyzer IIx or HiSeq 2000 were converted from a bcl file to a FastQ file by means of Illumina Consensus Cess of Sequence and Variation software, version 1.eight, and mapped to the reference haploid human-genome sequence (Genome Reference Consortium human genome build 37, human being genome 19) with the utilize of the BWA plan.15 Variant calls, which differed from the reference sequence, were obtained with the use of Atlas-SNP and Atlas-indel.16 Another in-house software program, CASSANDRA, was used for variant filtering and annotation (see the Supplementary Appendix).

Figure 1. Figure 1. Classification, Confirmation, and Reporting of Variants in Samples for Whole-Exome Sequencing.

Variants were identified with the use of Atlas-SNP and Atlas-Indel.xvi ESP denotes ESP5400 information from the National Eye, Lung, and Blood Establish Go Exome Sequencing Project, HGMD Human Gene Mutation Database, MAF pocket-sized allele frequency, TG the 1000 Genomes Project, and VUS variants of unknown clinical significance.

Variants with suboptimal quality scores were removed from consideration. Remaining variants were compared computationally with the list of reported mutations from the Human Factor Mutation Database.17 Variants in this database with a small allele frequency of less than 5% co-ordinate to either the 1000 Genomes Projecteighteen or the ESP5400 data of the National Heart, Lung, and Blood Institute GO Exome Sequencing Project (http://evs.gs.washington.edu/EVS) were retained. For changes that are not in the Human Gene Mutation Database, synonymous variants, intronic variants that were more than five bp from exon boundaries (which are unlikely to bear upon messenger RNA splicing), and common variants (minor allele frequency, >1%) were as well discarded (Figure 1).

Data Interpretation

Whole-exome sequencing variants (i.e., DNA sequence mutations) that remained after the steps described above were classified as deleterious mutations (potentially pathogenic variants), variants of unknown clinical significance, or benign variants, in accordance with the interpretation guidelines of the American Higher of Medical Genetics and Genomics (ACMG).nineteen Deleterious mutations and variants of unknown clinical significance were further classified as related or unrelated to the patient's phenotype and as potentially medically actionable mutations, recessive mutations in carriers, or mutations with no known disease associations.

Diagnostic Criteria

We applied stringent criteria for determining causative alleles. Confirmed variants were required to take occurred in genes in which mutations had been previously reported to cause disease with a presentation consistent with that observed in the patient. Recurring alleles scored most highly. All alleles were examined to determine their consistency with deleterious mutations of ACMG category i (previously reported to exist deleterious) or category 2 (predicted to exist deleterious).19 Assessment of the deleterious condition of novel or rare changes was aided by a battery of in silico prediction programs,20 which were used simply every bit a guide and were non solely relied on. Patterns of familial segregation were tested to place expected modes of inheritance, and the similarity of identified phenotypes with those described in previous reports was considered (Figure 1).

All putative causative alleles were subjected to all-encompassing literature and database searches, and the results were discussed in roundtable sessions by laboratory directors and physicians with appropriate clinical expertise. This review sometimes resulted in reclassification of the variant status, attributable to ambiguous records in databases or the literature. For each of the 62 cases, a claim of causality depended on the referring physician'due south agreement with the molecular diagnosis.

Data Reporting

The estimation of clinical whole-exome sequencing data at our heart was performed past a team of persons representing several areas of expertise. Scientists with doctorates and expertise in genetics or genomics, clinical molecular geneticists and medical geneticists certified by the American Lath of Medical Genetics, medical directors, and genetic counselors performed several contained levels of review.

Table ii. Table 2. Variant Categories in Clinical Reports of Whole-Exome Sequencing.

The results of whole-exome sequencing were sent in a two-tiered study to the referring physician within approximately xv weeks after the test was requested (Table 2). Tier ane was focused on the disease phenotype and included deleterious mutations and variants of unknown clinical significance related to the phenotype. Medically actionable incidental findings, autosomal recessive carrier status for genes from the ACMG-recommended population-screening console,21 and a limited number of variants that influence the metabolism of the drugs clopidogrel and warfarin were as well reported (Table ii). The expanded set of variants in tier two were provided if they were requested past the physician and if additional consent for tier-ii reporting of results had been obtained from the patient. The expanded report included mutations and variants of unknown clinical significance in genes unrelated to the phenotype, as well as deleterious mutations in genes with no known association with affliction. Mutations in this latter category were monitored every 6 months for the establishment of additional molecular diagnoses by checking the mutations against newly discovered disease genes; if a lucifer was found, the mutation was reported to the referring doctor in an addendum.

Results

Characteristics of the Patients

Of the 250 patients, approximately 80% were children with phenotypes related to neurologic conditions (Table ane). Most patients were younger than 18 years of age; iv specimens from fetuses from terminated pregnancies were also included. All patients had undergone prior genetic testing, which consisted of chromosomal microarray analysis,three,4 metabolic screening, DNA sequencing studies, or a combination of these tests. The prior diagnostic workup of all 15 positive cases from local referrals is shown in Table S2 in the Supplementary Appendix. The office settings of the ordering physicians were equally follows: genetics (61% of offices), pediatrics (24%), and neurology (12%). The remaining 3% were cardiology, endocrinology, slumber medicine, and pathology offices. Samples were bachelor from both parents for 75% of the patients. The costs of testing were billed to the insurance visitor by the Baylor College of Medicine laboratory for 129 cases (52%), 3 of which were denied coverage; 119 (48%) were billed to the referring establishment, and two (ane%) were nonbilled cases. Insurance coverage was like to that of established genetic tests.

Exome Sequencing

Approximately 200,000 to 400,000 unmarried-nucleotide variants and small insertion and deletion changes were identified in each patient'south personal genome past comparison with the current reference haploid human genome sequence (human genome 19). Multistep filtering retained approximately 400 to 700 variants of potential clinical usefulness per sample (Effigy 1 and Table 2). More than 86% of the variants elected for potential reporting were confirmed past means of Sanger sequencing of the probands. The remaining xiv% were plant to be false positive results; these calls usually had diff allele fractions, poor mapping scores, or sequence information indicating suboptimal alignment to the reference sequence.

Diagnoses Based on Whole-Exome Sequencing

Table 3. Table 3. Molecular Diagnoses in Mendelian Diseases in 62 Positive Cases. Table 4. Table iv. Four Patients with Dual Molecular Diagnoses. Table 5. Table 5. Inheritance Blueprint and Medical Presentation of Patients with Established Molecular Diagnosis.

Of the 250 probands, 62 carried 86 mutated alleles that satisfied criteria for a molecular diagnosis (Tabular array 3, and Table S3 in the Supplementary Appendix). The overall rate of a positive molecular diagnosis was 25%. This grouping included 33 patients with autosomal dominant disease, 16 with autosomal recessive affliction, and 9 with Ten-linked affliction. In improver, 4 patients received molecular diagnoses of two nonoverlapping genetic disorders: 3 with both an autosomal dominant disorder and an autosomal recessive disorder and 1 with an autosomal recessive disorder and an 10-linked disorder (Table iv). There was a tendency toward an association betwixt the charge per unit of a positive diagnosis and the clinical phenotype observed (Table 5), with the highest rate of a positive diagnosis in the grouping of patients with a nonspecific neurologic disorder (33%), followed by the group of patients with a specific neurologic disorder (31%). The 86 mutations included a full range of mutation types: 20 modest frameshift, two in-frame, nine nonsense, 9 splice, and 46 missense mutations.

All positive cases (Table 3 and Tabular array 4, and Tabular array S3 in the Supplementary Appendix) met each of the diagnostic criteria regarding mutation severity, advisable inheritance patterns (when parental data were available), and illness–phenotype cyclopedia. A full of 36 patients had autosomal dominant disorders (including 3 of the patients with ii nonoverlapping genetic disorders); 6 (17%) of these patients, for whom parental data were not available, carried truncating mutations or missense mutations that had previously been reported in afflicted persons, 5 (xiv%) had inherited mutations from symptomatic parents, and 25 (69%; 83% of the 30 patients for whom parental data were available) had de novo mutations, including 1 de novo mutation in the mosaic country. Of the 36 dominant alleles, 24 (67%) were novel variants at the time of diagnosis.

For the 20 patients with autosomal recessive disease (including the iv patients with two nonoverlapping genetic disorders), parental studies indicated that 19 had inherited mutant alleles from each carrier parent. The remaining patient, for whom parental samples were not available, carried an apparently homozygous, mutual, disease-causing mutation.

Among the ten patients with X-linked disorders, 4 (2 boys and 2 girls) carried de novo mutations, 5 (all boys) had maternally inherited mutations, and 1 boy (for whom a maternal sample was not bachelor) carried a previously reported frameshift mutation. Of the 29 full de novo mutations, 23 were single-nucleotide substitutions, including 3 (13%) that occurred at CpG dinucleotides, and vi were small deletions or duplications.

Of the 62 patients with a positive diagnosis, 39 had rare genetic disorders seen simply once in this study, and 23 had recurrent clinical phenotypes (Table 3, and Tabular array S3 in the Supplementary Appendix). The 23 patients with recurrent phenotypes included 4 patients with a Noonan-spectrum disorder involving iii genes (PTPN11, KRAS, and CBL) encoding proteins in the mitogen-activated protein kinase and extracellular signal-regulated kinase pathways22; four patients with intellectual disability or the Coffin–Siris syndrome involving three different SWI–SNF chromatin remodeling genes (ARID1A, ARID1B, and SMARCB1)23-25; 3 patients with the Cornelia de Lange syndrome caused by mutations in genes NIBPL, SMC1A, or HDAC8, whose protein products are involved in sister-chromatid cohesion26; and 12 patients with causative mutations in six genes, each of which was mutated in 2 unrelated patients.

Incidental Findings

In addition to diagnostic findings, thirty of the 250 patients had medically actionable incidental findings in a total of 16 genes (Table S4 in the Supplementary Appendix). Of the 16 genes, 9 were amongst the medically actionable genes recently recommended for reporting by the ACMG.27 Carrier-status mutations in genes from the ACMG-recommended population-screening panel21 were too detected in 13 of the 250 patients (Table S5 in the Supplementary Appendix).

Discussion

On applying whole-exome sequencing to the diagnoses of 250 unselected, sequent patients, we observed a molecular diagnostic yield of 25%, which is higher than the positive rates of other genetic tests, such equally karyotype analysis (5 to xv%),28,29 chromosomal microarray analysis (15 to 20%),30 and Sanger sequencing for unmarried genes. In our laboratory, the positive charge per unit for single-factor tests by means of Sanger sequencing ranges from 3 to 15% for genes such equally FOXG1 and MECP2, which are associated with relatively nonspecific phenotypes, to a loftier of 47% for CHD7, which is associated with the more specific, readily identifiable phenotype of the Accuse syndrome (coloboma of the eye, heart bibelot, atresia of the choanae, retarded growth and development, and genital and ear anomalies) (Fig. S1 in the Supplementary Appendix). Amid the 500 additional clinical exomes completed during the review procedure for this commodity, we obtained a similar diagnostic yield, at 26% (data non shown).

Previous studies accept shown that 31% of patients with nonsyndromic, sporadic cases of intellectual disability (16 of 51 patients) and 13% of those with severe intellectual disability (13 of 100) can be provided with a specific molecular diagnosis by means of adjacent-generation sequencing approaches.11,12 The 25% diagnostic charge per unit in our clinical study may be the result of different categories of presentation; 200 of 250 patients had intellectual disability as 1 of the clinical features, and the diagnosis was determined in 51 of these patients (26%) by means of whole-exome sequencing. Overall, amid patients who had nonsyndromic disorders with a neurologic phenotype (intellectual disability or developmental delay), the diagnostic charge per unit was 33%. Whole-exome sequencing provided a diagnosis in 31% of persons with a specific neurologic finding, such equally a movement disorder. These results advise that these two groups of patients in particular are practiced candidates for testing with whole-exome sequencing.

Before ordering whole-exome sequencing, physicians had carried out extensive clinical diagnostic workups, some of which exceeded the time and cost of the clinical whole-exome sequencing. For case, one patient (Patient fourteen in Table S3 in the Supplementary Appendix) had whole-exome sequencing ordered at 26 months of age. He had previously been evaluated by ways of chromosomal microarray analysis, Deoxyribonucleic acid methylation, eight unmarried-gene sequencing tests, mitochondrial genome sequencing by side by side-generation sequencing, respiratory-chain enzyme analysis, and multiple biochemical analyte studies. On the basis of the charges listed for these tests, we found that the cost of this patient's previous genetic testing was three times as high equally the current price of whole-exome sequencing. This patient carried a mutation in SYNGAP1, 31 which is associated with a newly recognized nonsyndromic mental retardation that may not have been identified past conventional genetic testing. He also had an incidental, medically actionable mutation in FBN1 that would have escaped detection without whole-exome sequencing.

The 25% diagnostic rate that nosotros observed volition probably increase in future example series. Gains will be made through improved detection of copy-number variation; such genomic changes contribute substantively to disease brunt,32 but not all are detected past electric current array-comparative genomic hybridization testing. The diagnoses in approximately 25% of our 62 patients with positive cases were based on disease-gene discoveries made within the past ii years, which suggests that most of the genes that underlie mendelian diseases have yet to be discovered. For example, vii patients, including those with mutations in ARID1A, ARID1B (in 2 patients), KANSL1, SMARCB1, SRCAP, and C5orf42, would not take received a diagnosis if this report had been conducted before 2012, when certain written report reports became available. Periodic monitoring of the literature and databases is therefore likely to help diagnose numerous additional cases.33

Additional data from family studies or further feedback from referring physicians may also found more than diagnoses among the cases in our report that take not even so been identified through whole-exome sequencing. Clinical confirmation is often the only ways of establishing the veracity of the diagnosis. Often, a 2d laboratory assay is not available to independently confirm the diagnosis. The possibility of simulated positive results exists just is small and similar to that for other laboratory diagnoses that demand to be considered in the context of the clinical presentation. There is also the possibility of an evolving phenotype that might at some point alter or add to the diagnosis in some patients.

In the cases that went undiagnosed, the etiologic mutations may exist located in noncoding regions, such as regulatory or deep intronic regions that cannot be detected by means of whole-exome sequencing. Sequencing of all annotated coding exons of the 10 chromosome in 208 families with Ten-linked mental retardation identified causative alleles in only 25% of the families that underwent analysis,34 which is consistent with a bias in mutation type in the Human Factor Mutation Database and suggests that our understanding of the allelic architecture of fifty-fifty mendelizing traits is far from consummate.

Technical limitations may also account for a pocket-size but considerable fraction of cases in which whole-exome sequencing did non place the variation underlying an credible mendelian disorder. The mutant alleles may be located in the coding regions that are not well covered by whole-exome sequencing (virtually 5% of the coding regions) (Table S1 in the Supplementary Appendix). A potential remedy for this problem is whole-genome sequencing, but it is more than expensive than whole-exome sequencing and results in a depth of sequence coverage that is lower than that achieved by whole-exome sequencing. Other technical limitations may result from the presence of multiple pseudogenes or repetitive regions that obscure the specific copy to which the variant maps.35

Although most patients who receive a diagnosis on the basis of whole-exome sequencing are likely to accept rare genetic diseases, it was expected that some of the diagnoses would be relatively mutual syndromes. In fact, 4 patients received a molecular diagnosis of Noonan-spectrum disorder, a common and relatively well-defined group of disorders. The diagnosis in one of these four patients was suspected on the footing of clinical examination, but sequencing analyses of Noonan-console genes failed to identify a causative mutation. Whole-exome sequencing detected a deleterious mutation in CBL, a relatively new Noonan gene that had not been included in the Noonan cistron panel at the time that the patient's DNA was analyzed with the utilise of that panel. The other three patients presented with atypical clinical phenotypes, and Noonan-spectrum disorders were not in the immediate differential diagnosis. Nosotros suggest that as testing with whole-exome sequencing evolves to characterize more patients with atypical presentations of known genetic diseases, the spectrum of phenotypes associated with genetic disorders will aggrandize.

Whole-exome sequencing has likewise proved useful in the characterization of patients with multiple diagnoses. Among the 62 patients for whom whole-exome sequencing provided a positive result, we identified mutations that were responsible for more than one condition with genetic bases in 4 patients (half-dozen%); this was unexpected, given the heuristic paradigm of a singular unifying diagnosis in clinical medicine. It is likely that as whole-exome sequencing and whole-genome sequencing reach more than widespread clinical implementation, multiple "hits" in a patient that explain the superimposed traits or blended phenotypes volition become more commonplace.

In conclusion, the use of whole-exome sequencing to analyze 250 consecutive clinical cases yielded a diagnosis in 25% of these cases, which supports the employ of whole-exome sequencing as a diagnostic exam for patients with nonspecific or unusual disease presentations of possible genetic cause and for patients with clinical diagnoses of heterogeneous genetic weather condition. Questions about price-effectiveness, accurateness, yield, and effective integration of genome-based diagnosis in medical care must exist addressed in future studies and will require prospective study designs.

Funding and Disclosures

Supported in part by grants from the National Human being Genome Enquiry Constitute (U54-HG003273, to Dr. Gibbs; and U01 HG006485-01, to Dr. Plon).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

This commodity was published on October 2, 2013, at NEJM.org.

We thank the patients and their families for participating in this study and their physicians for submitting the clinical samples; Eric Boerwinkle, Ph.D., for expert advice and collaboration; Alicia Hawes, Mark Scheel, Nehad Saada, Wendy Liu, Irene Miloslavskaya, and Wenmiao Zhu for expert technical and bioinformatics development and support; Linda Guynn for patient-chart review; and Jeffrey Mize, Sean Kim, Doreen Ng, and Michelle Rives for administrative program support.

Author Affiliations

From the Departments of Molecular and Human Genetics (Y.Y., A.W., P.A.West., A.B., J.B., F.Ten., Z.North., M.H., R.P., M.R.B., Chiliad.S.Fifty., A.K., J.Due south., South.E.P., J.R.50., A.L.B., C.Thousand.E.) and Pediatrics (S.East.P., J.R.50.) and the Human Genome Sequencing Center (D.One thousand.Thou., J.1000.R., M.N.B., P.P., M.W., Y.D., J.R.L., R.A.G.), Baylor College of Medicine, Houston.

Address reprint requests to Dr. Eng at the Section of Molecular and Man Genetics, NAB 2015, Baylor College of Medicine, Houston, TX 77030, or at [email protected].

Supplementary Cloth

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