Next-generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor-associated variants. It is increasingly recognized that post-zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging because of low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA-related overgrowth spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400× unique read depth for VAF of 10%, and approaching 100% at 1000×. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.

Disorders of somatic mosaicism (DoSM) are a diverse group of syndromic and non-syndromic conditions caused by mosaic variants in genes that regulate cell survival and proliferation. Despite overlap in gene space and technical requirements, few clinical labs specialize in DoSM compared to oncology. We adapted a high-sensitivity next-generation sequencing cancer assay for DoSM in 2014. Some 343 individuals have been tested over the past 5 years, 58% of which had pathogenic and likely pathogenic (P/LP) findings, for a total of 206 P/LP variants in 22 genes. Parameters associated with the high diagnostic yield were: (1) deep sequencing (2,0003 coverage), (2) a broad gene set, and (3) testing affected tissues. Fresh and formalin-fixed paraffin embedded tissues performed equivalently for identification of P/LP variants (62% and 71% of individuals, respectively). Comparing cultured fibroblasts to skin biopsies suggested that culturing might boost the allelic fraction of variants that confer a growth advantage, specifically gain-of-function variants in PIK3CA. Buccal swabs showed high diagnostic sensitivity in case subjects where disease phenotypes manifested in the head or brain. Peripheral blood was useful as an unaffected comparator tissue to determine somatic versus constitutional origin but had poor diagnostic sensitivity. Descriptions of all tested individuals, specimens, and P/LP variants included in this cohort are available to further the study of the DoSM population.

Purpose Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1. Methods Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity. Results The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone. Conclusion Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

The American Board of Genetic Counseling (ABGC) performed a genetic counseling practice analysis (PA) to determine the content of the certification examination. The ABGC-appointed PA Advisory Committee worked with psychometricians to develop a survey which was distributed to 2,038 genetic counselors in the United States and Canada. The survey was also accessible on the ABGC website. Multiple criteria were used to establish the significance of the tasks included in the survey. A total of 677 responses were used in the analysis, representing a 37.1% corrected response rate. Five major content domains with 143 tasks were identified in the PA. New certification test specifications were developed on the basis of PA results and will be used in developing future examination forms. In keeping with credentialing standards, ABGC plans to conduct a PA on a regular basis so that the content of the examination reflects current practice.

Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and utilized targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3, CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines.We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. 9 patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1 was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach.. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.

BackgroundNext-generation sequencing (NGS) is a useful tool for evaluating patients with suspected genetic kidney disease. Clinical practice relies on the use of targeted gene panels that are ordered based on patient presentation. We compare the diagnostic yield of clinical panel-based testing to exome analysis.MethodsIn total, 324 consecutive patients underwent physician-ordered, panel-based NGS testing between December 2014 and October 2018. Gene panels were available for four clinical phenotypes, including atypical hemolytic uremic syndrome (n=224), nephrotic syndrome (n=56), cystic kidney disease (n=26), and Alport syndrome (n=13). Variants were analyzed and clinical reports were signed out by a pathologist or clinical geneticist at the time of testing. Subsequently, all patients underwent retrospective exome analysis to detect additional clinically significant variants in kidney disease genes that were not analyzed as part of the initial clinical gene panel. Resulting variants were classified according to the American College of Medical Genetics and Genomics 2015 guidelines.ResultsIn the initial physician-ordered gene panels, we identified clinically significant pathogenic or likely pathogenic variants in 13% of patients (n=42/324). CFHR3-CFHR1 homozygous deletion was detected in an additional 13 patients with aHUS without a pathogenic or likely pathogenic variant. Diagnostic yield of the initial physician-ordered gene panel was 20% and varied between groups. Retrospective exome analysis identified 18 patients with a previously unknown pathogenic or likely pathogenic variant in a kidney disease gene and eight patients with a high-risk APOL1 genotype. Overall, retrospective exome analysis increased the diagnostic yield of panel-based testing from 20% to 30%.ConclusionsThese results highlight the importance of a broad and collaborative approach between the clinical laboratory and their physician clients that employs additional analysis when a targeted panel of kidney disease–causing genes does not return a clinically meaningful result.

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