Corey Braastad

The allelic spectrum of Charcot–Marie–Tooth disease in over 17,000 individuals with neuropathy

We report the frequency, positive rate, and type of mutations in 14 genes (PMP22, GJB1, MPZ, MFN2, SH3TC2, GDAP1, NEFL, LITAF, GARS, HSPB1, FIG4, EGR2, PRX, and RAB7A) associated with Charcot–Marie–Tooth disease (CMT) in a cohort of 17,880 individuals referred to a commercial genetic testing laboratory. Deidentified results from sequencing assays and multiplex ligation‐dependent probe amplification (MLPA) were analyzed including 100,102 Sanger sequencing, 2338 next‐generation sequencing (NGS), and 21,990 MLPA assays. Genetic abnormalities were identified in 18.5% (n = 3312) of all individuals. Testing by Sanger and MLPA (n = 3216) showed that duplications (dup) (56.7%) or deletions (del) (21.9%) in the PMP22 gene accounted for the majority of positive findings followed by mutations in the GJB1 (6.7%), MPZ (5.3%), and MFN2 (4.3%) genes. GJB1 del and mutations in the remaining genes explained 5.3% of the abnormalities. Pathogenic mutations were distributed as follows: missense (70.6%), nonsense (14.3%), frameshift (8.7%), splicing (3.3%), in‐frame deletions/insertions (1.8%), initiator methionine mutations (0.8%), and nonstop changes (0.5%). Mutation frequencies, positive rates, and the types of mutations were similar between tests performed by either Sanger (n = 17,377) or NGS (n = 503). Among patients with a positive genetic finding in a CMT‐related gene, 94.9% were positive in one of four genes (PMP22, GJB1, MPZ, or MFN2).

Technical standards and guidelines for spinal muscular atrophy testing

Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron (SMN1) gene, affecting approximately 1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brainstem nuclei. The disease is classified on the basis of age of onset and clinical course. Two almost identical SMN genes are present on 5q13: the SMN1 gene, which is the spinal muscular atrophy-determining gene, and the SMN2 gene. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being used as a reliable and sensitive spinal muscular atrophy diagnostic test. Although SMN2 produces less full-length transcript than SMN1, the number of SMN2 copies has been shown to modulate the clinical phenotype. Carrier detection relies on the accurate determination of the SMN1 gene copies. This document follows the outline format of the general Standards and Guidelines for Clinical Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and methods of analysis.

Ovarian failure in ataxia with oculomotor apraxia type 2

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disorder associated with mutations in the Senataxin (SETX) gene. Clinical manifestations (ataxia, peripheral neuropathy, oculomotor apraxia) of this disease have previously been limited to the nervous system. We describe a patient homozygous for a novel mutation of SETX who manifested not only ataxia but also ovarian failure.

A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.

We developed a rules‐based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co‐occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re‐evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting.

BRCA Share: A Collection of Clinical BRCA Gene Variants

As next‐generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Severe Dilated Cardiomyopathy in a Patient With Myotonic Dystrophy Type 2 and Homozygous Repeat Expansion in ZNF9

, in the firstintron of the gene Zinc Finger Protein 9, ZNF9 (CNBP) causes DM2. The normal allele sizeof the complex repeat is between 104 and 176 bp. RNA transcribed from the expanded alleleresults in toxic effects within the cell. Specifically in DM1, RNA containing the abnormalrepeat interacts with RNA-binding proteins that then lead to downstream effects that candisrupt regulation of alternative splicing, mRNA translation, or mRNA stability.

Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS)

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.