Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment.

The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy number variants (CNVs) in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). We also demonstrated the screen's high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: results from an ethnically diverse clinical sample of 23,453 individuals.

PURPOSE: Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern. METHODS: A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study. RESULTS: Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease. CONCLUSION: To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of "racial" categories by the physician, as recommended by recent guidelines.

Alternative polyadenylation mediates microRNA regulation of muscle stem cell function.

Pax3, a key myogenic regulator, is transiently expressed during activation of adult muscle stem cells, or satellite cells (SCs), and is also expressed in a subset of quiescent SCs (QSCs), but only in specific muscles. The mechanisms regulating these variations in expression are not well understood. Here we show that Pax3 levels are regulated by miR-206, a miRNA with a previously demonstrated role in myogenic differentiation. In most QSCs and activated SCs, miR-206 expression suppresses Pax3 expression. Paradoxically, QSCs that express high levels of Pax3 also express high levels of miR-206. In these QSCs, Pax3 transcripts are subject to alternative polyadenylation, resulting in transcripts with shorter 3' untranslated regions (3'UTRs) that render them resistant to regulation by miR-206. Similar alternate polyadenylation of the Pax3 transcript also occurs in myogenic progenitors during development. Our findings may reflect a general role of alternative polyadenylation in circumventing miRNA-mediated regulation of stem cell function.

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex.

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics.

Taf1 regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors.

Pax3 plays critical roles during developmental and postnatal myogenesis. We have previously shown that levels of Pax3 protein are regulated by monoubiquitination and proteasomal degradation during postnatal myogenesis, but none of the key regulators of the monoubiquitination process were known. Here we show that Pax3 monoubiquitination is mediated by the ubiquitin-activating/conjugating activity of Taf1, a component of the core transcriptional machinery that was recently reported to be downregulated during myogenic differentiation. We show that Taf1 binds directly to Pax3 and overexpression of Taf1 increases the level of monoubiquitinated Pax3 and its degradation by the proteasome. A decrease of Taf1 results in a decrease in Pax3 monoubiquitination, an increase in the levels of Pax3 protein, and a concomitant increase in Pax3-mediated inhibition of myogenic differentiation and myoblast migration. These results suggest that Taf1 regulates Pax3 protein levels through its ability to mediate monoubiquitination, revealing a critical interaction between two proteins that are involved in distinct aspects of myogenic differentiation. Finally, these results suggest that the components of the core transcriptional are integrally involved in the process of myogenic differentiation, acting as nodal regulators of the differentiation program.

Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors.

Pax3 and Pax7 play distinct but overlapping roles in developmental and postnatal myogenesis. The mechanisms involved in the differential regulation of these highly homologous proteins are unknown. We present evidence that Pax3, but not Pax7, is regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation. Intriguingly, only monoubiquitinated forms of Pax3 could be detected. Mutation of two specific lysine residues in the C-terminal region of Pax3 reduced the extent of its monoubiquitination and susceptibility to proteasomal degradation, whereas introduction of a key lysine into the C-terminal region of Pax7 rendered that protein susceptible to monoubiquitination and proteasomal degradation. Monoubiquitinated Pax3 was shuttled to the intrinsic proteasomal protein S5a by interacting specifically with the ubiquitin-binding protein Rad23B. Functionally, sustained expression of Pax3 proteins inhibited myogenic differentiation, demonstrating that Pax3 degradation is an essential step for the progression of the myogenic program. These results reveal an important mechanism of Pax3 regulation in muscle progenitors and an unrecognized role of protein monoubiquitination in mediating proteasomal degradation.