Clinical validity assessment of genes frequently tested on intellectual disability/autism sequencing panels.

PURPOSE: Neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and autism spectrum disorder (ASD), exhibit genetic and phenotypic heterogeneity, making them difficult to differentiate without a molecular diagnosis. The Clinical Genome Resource Intellectual Disability/Autism Gene Curation Expert Panel (GCEP) uses systematic curation to distinguish ID/ASD genes that are appropriate for clinical testing (ie, with substantial evidence supporting their relationship to disease) from those that are not. METHODS: Using the Clinical Genome Resource gene-disease validity curation framework, the ID/Autism GCEP classified genes frequently included on clinical ID/ASD testing panels as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. RESULTS: As of September 2021, 156 gene-disease pairs have been evaluated. Although most (75%) were determined to have definitive roles in NDDs, 22 (14%) genes evaluated had either Limited or Disputed evidence. Such genes are currently not recommended for use in clinical testing owing to the limited ability to assess the effect of identified variants. CONCLUSION: Our understanding of gene-disease relationships evolves over time; new relationships are discovered and previously-held conclusions may be questioned. Without periodic re-examination, inaccurate gene-disease claims may be perpetuated. The ID/Autism GCEP will continue to evaluate these claims to improve diagnosis and clinical care for NDDs.

A single NGS-based assay covering the entire genomic sequence of the DMD gene facilitates diagnostic and newborn screening confirmatory testing.

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two-tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time-consuming and expensive. We have developed a comprehensive next-generation sequencing (NGS)-based single-step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single-exon or multi-exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full-sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD.

Laboratory testing for fragile X, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Pathogenic variants in the FMR1 gene are associated with fragile X syndrome, fragile X-associated tremor ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (FXPOI). This document provides updated information regarding FMR1 pathogenic variants, including prevalence, genotype-phenotype correlations, and variant nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction (PCR) amplification of FMR1, including triplet repeat-primed and methylation-specific PCR.The American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document ACMG Technical Standards for Clinical Genetics Laboratories, which is now maintained online ( http://www.acmg.net ). This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document is the fourth update to the original standards and guidelines for fragile X testing that were published in 2001, with revisions in 2005 and 2013, respectively.This versionClarifies the clinical features associated with different FMRI variants (Section 2.3)Discusses important reporting considerations (Section 3.3.1.3)Provides updates on technology (Section 4.1).

Whole genome sequencing identifies a cryptic SOX9 regulatory element duplication underlying a case of 46,XX ovotesticular difference of sexual development.

Ovotesticular differences of sexual development (OT-DSD) are rare genetic variances defined by the coexistence of both testicular and ovarian tissues. Various molecular etiologies including SRY translocation or SOX9 pathogenic variants with different modes of inheritance have been associated with 46,XX OT-DSD. Here we describe a child diagnosed with SRY-negative 46,XX OT-DSD after completing a series of complex clinical genetic analyses, including chromosomal microarray, DSD gene panel (sequencing and deletion/duplication analysis), whole exome sequencing, and whole genome sequencing. Of these, only whole genome sequencing reported a pathogenic duplication in a non-coding region that contains the RevSex regulatory element, which modifies SOX9 expression and is associated with 46,XX OT-DSD and complete sex reversal. This is the first clinical RevSex duplication detected by clinical whole genome sequencing. We highlight the utility of whole genome sequencing in shortening the diagnostic odyssey and the importance of optimal counseling through a team-based multi-specialty approach for patients with DSDs.

Diagnostic capabilities of nanopore long-read sequencing in muscular dystrophy.

Many individuals with muscular dystrophies remain genetically undiagnosed despite clinical diagnostic testing, including exome sequencing. Some may harbor previously undetected structural variants (SVs) or cryptic splice sites. We enrolled 10 unrelated families: nine had muscular dystrophy but lacked complete genetic diagnoses and one had an asymptomatic DMD duplication. Nanopore genomic long-read sequencing identified previously undetected pathogenic variants in four individuals: an SV in DMD, an SV in LAMA2, and two single nucleotide variants in DMD that alter splicing. The DMD duplication in the asymptomatic individual was in tandem. Nanopore sequencing may help streamline genetic diagnostic approaches for muscular dystrophy.

A Hemizygous Deletion Within the PGK1 Gene in Males with PGK1 Deficiency.

Phosphoglycerate kinase-1 (PGK1) deficiency is a rare X-linked disorder caused by pathogenic variants in the PGK1 gene. Complete loss-of-function variants have not been reported in this gene, indicating that residual enzyme function is critical for viability in males. Therefore, copy number variants (CNVs) that include single exon or multiple exon deletions or duplications are generally not expected in individuals with PGK1 deficiency. Here we describe a 64-year-old male presenting with a family history (three additional affected males) and a personal history of childhood-onset metabolic myopathy that involves episodes of muscle pain, stiffness after activity, exercise intolerance, and myoglobinuria after exertion. Biochemical analysis on a muscle biopsy indicated significantly reduced activity (15% compared to normal) for phosphoglycerate kinase (PGK1), a glycolytic enzyme encoded by PGK1. A diagnosis of PGK1 deficiency was established by molecular analysis which detected an approximately 886 kb deletion involving the polyadenylation site in the 3'UTR of the PGK1 gene. RNA analysis showed significantly reduced PGK1 transcript levels (30% compared to normal). This is the first deletion reported in the PGK1 gene and is the first pathogenic variant involving the 3'UTR polyadenylation site of this gene. Our report emphasizes the role of 3'UTR variants in human disorders and underscores the need for exploring noncoding regions of disease-associated genes when seeking a molecular diagnosis.