Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods: WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases. Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56

PurposeIntegrating genomic sequencing in clinical care requires standardization of variant interpretation practices. The Clinical Genome Resource has established expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification framework for specific genes and diseases. The Cardiomyopathy Expert Panel selected MYH7, a key contributor to inherited cardiomyopathies, as a pilot gene to develop a broadly applicable approach.MethodsExpert revisions were tested with 60 variants using a structured double review by pairs of clinical and diagnostic laboratory experts. Final consensus rules were established via iterative discussions.ResultsAdjustments represented disease-/gene-informed specifications (12) or strength adjustments of existing rules (5). Nine rules were deemed not applicable. Key specifications included quantitative frameworks for minor allele frequency thresholds, the use of segregation data, and a semiquantitative approach to counting multiple independent variant occurrences where fully controlled case-control studies are lacking. Initial inter-expert classification concordance was 93%. Internal data from participating diagnostic laboratories changed the classification of 20% of the variants (n = 12), highlighting the critical importance of data sharing.ConclusionThese adapted rules provide increased specificity for use in MYH7-associated disorders in combination with expert review and clinical judgment and serve as a stepping stone for genes and disorders with similar genetic and clinical characteristics.

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

Original research article INTRODUCTIONSignificant advances in copy-number detection have broadened the mutation spectrum for many clinical genetic disorders. 1,2 Intragenic deletion mutations are of considerable frequency in many disease genes, such as PAX6, CDKL5, and STXPB1. Recurrent rearrangements between segmentally duplicated sequences are also associated with a number of syndromic disorders. 3 For these known disorders, targeted gene testing by multiplex ligation-dependent amplification or exon-focused arrays has been useful. With the increasing uptake of exome sequencing into the clinical diagnostic approach, the need for testing previously uncharacterized genes for pathogenic copynumber variation (CNV) is a significant consideration, not only to detect aberrations in genes that may cause disease when haplo-insufficient but also in genes associated with recessive disorders for which the mutation has been identified in only one of the alleles by exome sequencing. 4 Whereas exome sequencing is still gaining popularity as a powerful clinical tool, whole-genome chromosomal microarray analysis (CMA) has become an indispensable screening method that is now routinely used as a first-tier test for children with intellectual disability, developmental delay, or congenital anomalies. 5 In less than 10 years, the CMA designs have evolved from low-resolution arrays containing large bacterial artificial chromosome clones or <100,000 oligonucleotide probes to high-resolution versions with more than 1 million probes. 6 As a result, several groups have identified single-gene pathogenic aberrations that boost the analytical sensitivity of CMA. 7 However, although some of these more recent arrays have higher density at disease genes, they do not all cover every exon in those genes and are therefore not capable of detecting some pathogenic intragenic mutations. Separately, data from exon-focused arrays have shown that up to 40% of intragenic mutations can involve just one or two exons within a gene, and therefore it is essential to cover all exons within targeted genes. 1 Copy-number detection in clinical genetic testing eventually will occur entirely through examination of next-generation data, whereas array comparative genomic hybridization (aCGH) and other assays will serve as complementary and confirmatory methods. 8 To complement whole-exome sequencing (WES) or whole-genome sequencing data in a meaningful way, an array with coverage of virtually all exons is essential. Until the time that WES/whole-genome sequencing can be used routinely and reliably for copy-number detection, a whole-exome array can be used as the ultimate whole-genome CMA platform. Purpose: Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its ut...

Cornelia de Lange Syndrome (CdLS) is a dominantly inherited heterogeneous genetic disorder with multi-system abnormalities. 60% of probands with CdLS have heterozygous mutations in the Nipped-B- like (NIPBL) gene, 5% have mutations in the SMC1A gene, and 1 proband was found to have a mutation in the SMC3 gene. Cohesin is a multi-subunit complex consisting of a SMC1A and SMC3 heterodimer and two non-SMC subunits. SMC1A is located on the human X chromosome and is reported to escape X inactivation. We show that 29 unrelated CdLS probands with 21 unique SMC1A mutations have been identified by our group and others including 7 males. All mutations identified to date are either missense or small deletions with all presumably preserving the protein open reading frame. Both wild type and mutant alleles are expressed. Females quantitatively express twice the amount of SMC1A mRNA as compared to males. The transcriptional profiling of 23 selected genes is different in SMC1A mutant probands, controls and NIPBL mutant probands. These results suggest that mechanistically SMC1A-related CdLS is not due to altered levels of the SMC1A transcript, but rather that the mutant proteins maintain a residual function in males and enact a dominant negative effect in females.