BACKGROUND Whole-exome sequencing is a diagnostic approach for the identification of molecular defects in patients with suspected genetic disorders. METHODS We developed technical, bioinformatic, interpretive, and validation pipelines for whole-exome sequencing in a certified clinical laboratory to identify sequence variants underlying disease phenotypes in patients. RESULTS We 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 80% were children with neurologic pheno-types. Insurance coverage was similar to that for established genetic tests. We identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients, achieving a 25% molecular diagnostic rate (95% confidence interval, 20 to 31). Among the 62 patients, 33 had autosomal dominant disease, 16 had auto-somal recessive disease, and 9 had X-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 40% 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 same genes and in different 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 Human Genome Research Institute.)

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.

BACKGROUND Whole-exome sequencing can provide insight into the relationship between observed clinical phenotypes and underlying genotypes. METHODS We conducted a retrospective analysis of data from a series of 7374 consecutive unrelated patients who had been referred to a clinical diagnostic laboratory for whole-exome sequencing; our goal was to determine the frequency and clinical characteristics of patients for whom more than one molecular diagnosis was reported. The phenotypic similarity between molecularly diagnosed pairs of diseases was calculated with the use of terms from the Human Phenotype Ontology. RESULTS A molecular diagnosis was rendered for 2076 of 7374 patients (28.2%); among these patients, 101 (4.9%) had diagnoses that involved two or more disease loci. We also analyzed parental samples, when available, and found that de novo variants accounted for 67.8% (61 of 90) of pathogenic variants in autosomal dominant disease genes and 51.7% (15 of 29) of pathogenic variants in X-linked disease genes; both variants were de novo in 44.7% (17 of 38) of patients with two monoallelic variants. Causal copy-number variants were found in 12 patients (11.9%) with multiple diagnoses. Phenotypic similarity scores were significantly lower among patients in whom the phenotype resulted from two distinct mendelian disorders that affected different organ systems (50 patients) than among patients with disorders that had overlapping phenotypic features (30 patients) (median score, 0.21 vs. 0.36; P = 1.77×10−7). CONCLUSIONS In our study, we found multiple molecular diagnoses in 4.9% of cases in which whole-exome sequencing was informative. Our results show that structured clinical ontologies can be used to determine the degree of overlap between two mendelian diseases in the same patient; the diseases can be distinct or overlapping. Distinct disease phenotypes affect different organ systems, whereas overlapping disease phenotypes are more likely to be caused by two genes encoding proteins that interact within the same pathway. (Funded by the National Institutes of Health and the Ting Tsung and Wei Fong Chao Foundation.)

involves karyotyping, whereas in the Netherlands, patients who undergo amniocentesis have a more limited assessment only for trisomies 13, 18, and 21 and the sex chromosomes; thus, the tradeoff is a bit different.In addition, cfDNA is provided as a screening test for trisomies 13, 18, and 21, not just for Down syndrome. However, the performance characteristics of cfDNA for trisomies 13 and 18 are not as favorable as for T21, with a higher rate of falsenegative and false-positive results. In addition, a percentage of patients-somewhere between 1.5% and 8%-fail to obtain a result, usually because of insufficient fetal DNA. Such "low fetal fraction" is associated with obesity, which is a significant problem affecting a high percentage of reproductive-aged women in the United States. It is estimated that 20% to 50% of cfDNA tests fail to provide adequate fetal DNA in obese women. In addition, low fetal fraction is also associated with aneuploidy; therefore, women with cfDNA test failure should be considered high risk and offered follow-up with diagnostic testing (as well as a second attempt at cfDNA). When these potential outcomes are all considered, the performance characteristics of cfDNA versus traditional screening are not as clearly superior.Like the authors of this abstracted paper, several other authors and experts (Prenat Diagn 2013;33 (7):636-642) have suggested a contingent approach, using multiple marker screening as an initial screening tool, and then offering NIPT to intermediate-risk patients and either cfDNA or invasive testing to the highest-risk patients. Before completely changing the current standard of care, we need to understand this tradeoff. These authors consider comparative costs, but do not really provide incremental cost-effectiveness ratios, which are the best way to compare these strategies. While cfDNA is a better test if we are looking at a very precise test for a single disorder, only in patients in whom the test is successful at providing a result current screening may be preferable if we are looking to screen the entire population for a broad range of birth defects. Cost utility analyses, conducted by independent investigators and considering all important outcomes, are clearly needed before our approach completely changes. -MEN)

IMPORTANCE Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown. OBJECTIVE To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors. DESIGN Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children’s hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record. MAIN OUTCOMES AND MEASURES Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations. RESULTS Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%). CONCLUSIONS AND RELEVANCE Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

Dominant optic atrophy (DOA)1,2 and axonal peripheral neuropathy (Charcot-Marie-Tooth Type 2 or CMT2)3 are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively4. In yeast, homologs of OPA1(Mgm1) and MFN2(Fzo1) work in concert with Ugo15,6, which has no human equivalent to date7. By whole exome sequencing patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein, mitofilin(Fcj1). Loss-of-function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2, while exemplifying a novel class of modified solute transporters linked to mitochondrial dynamics.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.