Narasimhan Nagan

Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72 400 specimens

Spinal muscular atrophy (SMA) is a leading inherited cause of infant death with a reported incidence of ∼1 in 10 000 live births and is second to cystic fibrosis as a common, life-shortening autosomal recessive disorder. The American College of Medical Genetics has recommended population carrier screening for SMA, regardless of race or ethnicity, to facilitate informed reproductive options, although other organizations have cited the need for additional large-scale studies before widespread implementation. We report our data from carrier testing (n=72 453) and prenatal diagnosis (n=121) for this condition. Our analysis of large-scale population carrier screening data (n=68 471) demonstrates the technical feasibility of high throughput testing and provides mutation carrier and allele frequencies at a level of accuracy afforded by large data sets. In our United States pan-ethnic population, the calculated a priori carrier frequency of SMA is 1/54 with a detection rate of 91.2%, and the pan-ethnic disease incidence is calculated to be 1/11 000. Carrier frequency and detection rates provided for six major ethnic groups in the United States range from 1/47 and 94.8% in the Caucasian population to 1/72 and 70.5% in the African American population, respectively. This collective experience can be utilized to facilitate accurate pre- and post-test counseling in the settings of carrier screening and prenatal diagnosis for SMA.

Cystic Fibrosis Carrier Testing in an Ethnically Diverse US Population

BACKGROUND The incidence of cystic fibrosis (CF) and the frequency of specific disease-causing mutations vary among populations. Affected individuals experience a range of serious clinical consequences, notably lung and pancreatic disease, which are only partially dependent on genotype. METHODS An allele-specific primer-extension reaction, liquid-phase hybridization to a bead array, and subsequent fluorescence detection were used in testing for carriers of 98 CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations among 364 890 referred individuals with no family history of CF. RESULTS One in 38 individuals carried one of the 98 CFTR mutations included in this panel. Of the 87 different mutations detected, 18 were limited to a single ethnic group. African American, Hispanic, and Asian individuals accounted for 33% of the individuals tested. The mutation frequency distribution of Caucasians was significantly different from that of each of these ethnic groups (P < 1 × 10⁻¹⁰). CONCLUSIONS Carrier testing using a broad mutation panel detects differences in the distribution of mutations among ethnic groups in the US.

Exploring the landscape of pathogenic genetic variation in the ExAC population database: insights of relevance to variant classification.

Purpose:We evaluated the Exome Aggregation Consortium (ExAC) database as a control cohort to classify variants across a diverse set of genes spanning dominant and recessively inherited disorders.Methods:The frequency of pathogenic variants in ExAC was compared with the estimated maximal pathogenic allele frequency (MPAF), based on the disease prevalence, penetrance, inheritance, allelic and locus heterogeneity of each gene. Additionally, the observed carrier frequency and the ethnicity-specific variant distribution were compared between ExAC and the published literature. Results:The carrier frequency and ethnic distribution of pathogenic variants in ExAC were concordant with reported estimates. Of 871 pathogenic/likely pathogenic variants across 19 genes, only 3 exceeded the estimated MPAF. Eighty-four percent of variants with ExAC frequencies above the estimated MPAF were classified as “benign.” Additionally, 20% of the cardiac and 19% of the Lynch syndrome gene variants originally classified as “VUS” occurred with ExAC frequencies above the estimated MPAF, making these suitable for reassessment.Conclusions:The ExAC database is a useful source for variant classification and is not overrepresented for pathogenic variants in the genes evaluated. However, the mutational spectrum, pseudogenes, genetic heterogeneity, and paucity of literature should be considered in deriving meaningful classifications using ExAC.Genet Med 18 8, 850–854.

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 novel duplication/insertion mutation of NEFL in a patient with Charcot-Marie-Tooth disease†

Charcot-Marie-Tooth (CMT) disease is the most common hereditary motor and sensory peripheral neuropathy, affecting approximately 1 in 2,500 individuals. Based on electrophysiological and pathological criteria, CMT is divided into the demyelinating form (CMT1) and the axonal (CMT2) form. CMT type 1 is characterized by severe reduction in motor nerve conduction velocities (NCVs) and segmental demyelination and remyelination of nerve fibers with supernumerary Schwann cells. CMT type 2 is characterized by normal to mildly reduced NCVs and axonal degeneration. Genetic studies have confirmed the heterogeneity of CMT and a number of CMT genes have been identified, including PMP22, MPZ, EGR2, LITAF, GJB1, PRX, KIF1B, RAB7, GARS, HSPB1, SBF2, MTMR2, and NEFL. A large-scale study on CMT patients revealed that mutations of NEFL accounted for about 2% of all CMT cases [Jordanova et al., 2003]. The NEFL gene encodes neurofilament light chain (NEFL or commonly known as NFL), a major component of the intermediate filament network in nerve cells. We evaluated a 71-year-old man with a chief complaint of bilateral, progressive, lower extremity weakness and easy fatigue. Neurological examination reflected no abnormality of formal mental status, cranial nerve or upper extremity motor and sensory function with the exception of mildly depressed reflexes at the brachioradialis, tricep and the bicep. There was a bilateral tendency for foot drop with a tendency to be stooped forward in a propulsive posture. The patient was unable to walk a line heel to toe. Intrinsic foot muscle atrophy was marked with pes cavus. Nerve conduction study demonstrated absent sural sensory responses. Motor responses showed a right peroneal distal latency of 6.3 m/sec (normal <6.0) and velocity of 35 m/sec (normal >40); comparable left peroneal responses measured 8.2 and 32 m/sec, respectively. The F wave latencies measured 57 and 75 m/sec on the right and left, respectively (normal <54m/sec). Electromyography reflected distal more than proximal acute and chronic partial denervation with moderate loss of interference pattern in the most distal muscles and with no involvement of paraspinus muscles. His clinical diagnosis was CMT2 with distal symmetric axonal motor and sensory polyneuropathy. We sequenced a number of CMT genes of the patient, including PMP22 (CMT1A), MPZ (CMT1B and CMT2J), EGR2 (CMT1D), GJB1 (CMTX1), PRX (CMT4F), and NEFL (CMT1F and CMT2E) and tested for deletion/duplication of PMP22. A heterozygous 13 bp duplication/insertion was found in NEFL gene at position þ61 of exon 1 (c.48_60dupGCGCTACGTGGAG; Fig. 1A), which predicts p.T21Xfs (Fig. 1B). The wild-type NEFL (wt NEFL) protein is 543 amino acids and therefore, this mutation either markedly truncates the protein (DNEFL) or the mutation may trigger nonsense mediated decay. The proband is an adopted child and information regarding his parents, siblings and children was unavailable. We could not determine whether this is a spontaneous or an inherited mutation.

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.

Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and the most common genetic cause of infant mortality, affecting ∼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 brain stem nuclei. The disease is classified on the basis of age of onset and clinical course. SMA is caused by mutations in the telomeric copy of the survival motor neuron 1 (SMN1) gene, but all patients retain a centromeric copy of the gene, SMN2. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being utilized as a reliable and sensitive SMA diagnostic test. In the majority of cases, the disease severity correlates inversely with an increased SMN2 gene copy number. Carrier detection, in the deletion cases, relies on the accurate determination of the SMN1 gene copies. Since SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1 in 40 to 1 in 60, direct carrier dosage testing has been beneficial to many families. This unit attempts to highlight the molecular genetics of SMA with a focus on the advantages and limitations of the current molecular technologies.

Scaling resolution of variant classification differences in ClinVar between 41 clinical laboratories through an outlier approach

ClinVar provides open access to variant classifications shared from many clinical laboratories. Although most classifications are consistent across laboratories, classification differences exist. To facilitate resolution of classification differences on a large scale, clinical laboratories were encouraged to reassess outlier classifications of variants with medically significant differences (MSDs). Outliers were identified by first comparing ClinVar submissions from 41 clinical laboratories to detect variants with MSDs between the laboratories (650 variants). Next, MSDs were filtered for variants with ≥3 classifications (244 variants), of which 87.6% (213 variants) had a majority consensus in ClinVar, thus allowing for identification of outlier classifications in need of reassessment. Laboratories with outlier classifications were sent a custom report and encouraged to reassess variants. Results were returned for 204 (96%) variants, of which 62.3% (127) were resolved. Of those 127, 64.6% (82) were resolved due to reassessment prompted by this study and 35.4% (45) resolved by a previously completed reassessment. This study demonstrates a scalable approach to classification resolution and capitalizes on the value of data sharing within ClinVar. These activities will help the community move toward more consistent variant classifications, which will improve the care of patients with, or at risk for, genetic disorders.

Evaluation of a 27-gene inherited cancer panel across 630 consecutive patients referred for testing in a clinical diagnostic laboratory

BackgroundExtensive clinical and genetic heterogeneity of inherited cancers has allowed multi-gene panel testing to become an efficient means for identification of patients with an inherited predisposition to a broad spectrum of syndromic and nonsyndromic forms of cancer. This study reports our experience with a 27-gene inherited cancer panel on a cohort of 630 consecutive individuals referred for testing at our laboratory with the following objectives: 1. Determine the rates for positive cases and those with variants of uncertain clinical significance (VUS) relative to data published in the recent literature, 2. Examine heterogeneity among the constituent genes on the panel, and 3. Review test uptake in the cohort relative to other reports describing outcomes for expanded panel testing.MethodsClinical and genomic data were reviewed on 630 individuals tested on a panel of 27 genes selected on the basis of high (≥ 40%) or moderate to low (≤ 40%) lifetime risk of hereditary cancer. These patients were not enriched for adherence to the National Comprehensive Cancer Network (NCCN) criteria for Hereditary Breast and Ovarian Cancer (HBOC) or Lynch Syndrome (LS) and constitute a referral laboratory cohort.ResultsSixty-five individuals with variants classified as pathogenic or likely pathogenic across 14 genes were identified for an overall positive rate of 10.3%. Although a family history of cancer constituted a major reason for referral, accounting for 84% of our cohort, excluding patients with a known familial variant did not have a significant impact on the observed positive rate (9% vs 10.3%). More than half (58%) of the pathogenic or likely pathogenic variants were observed in high or moderate to low risk genes on the panel, while only 42% occurred in classic HBOC or LS-associated genes.ConclusionThese results provide the actual percentage of family or personal history of cancer that can be attributed to pathogenic or likely pathogenic variants in one or more of the genes on our panel and corroborate the utility of multi-gene panels over sequential testing to identify individuals with an inherited predisposition to cancer.