Amy Knight Johnson

Clinical laboratories collaborate to resolve differences in variant interpretations submitted to ClinVar

Purpose:Data sharing through ClinVar offers a unique opportunity to identify interpretation differences between laboratories. As part of a ClinGen initiative, four clinical laboratories (Ambry, GeneDx, Partners Healthcare Laboratory for Molecular Medicine, and University of Chicago Genetic Services Laboratory) collaborated to identify the basis of interpretation differences and to investigate if data sharing and reassessment resolve interpretation differences by analyzing a subset of variants.Methods:ClinVar variants with submissions from at least two of the four participating laboratories were compared. For a subset of identified differences, laboratories documented the basis for discordance, shared internal data, independently reassessed with the American College of Medical Genetics and Genomics–Association for Molecular Pathology (ACMG-AMP) guidelines, and then compared interpretations.Results:At least two of the participating laboratories interpreted 6,169 variants in ClinVar, of which 88.3% were initially concordant. Laboratories reassessed 242/724 initially discordant variants, of which 87.2% (211) were resolved by reassessment with current criteria and/or internal data sharing; 12.8% (31) of reassessed variants remained discordant owing to differences in the application of the ACMG-AMP guidelines.Conclusion:Participating laboratories increased their overall concordance from 88.3 to 91.7%, indicating that sharing variant interpretations in ClinVar—thereby allowing identification of differences and motivation to resolve those differences—is critical to moving toward more consistent variant interpretations.Genet Med advance online publication 09 March 2017

A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories

Purpose:While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization.Methods:Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews.Results:Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data.Conclusion:This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016

Inflammatory pseudotumor of the heart caused by Listeria monocytogenes infection

Inflammatory pseudotumor (IPT) of the heart is rare and of unknown etiology. We present a case of cardiac IPT caused by Listeria monocytogenes that evolved following gastroenteritis in a previously healthy child. L. monocytogenes, known to cause acute invasive infections, has not been reported previously as a cause of cardiac infection in children or of IPT. The literature concerning infectious IPT is reviewed.

A novel mutation in the promoter of RARS2 causes pontocerebellar hypoplasia in two siblings.

Pontocerebellar hypoplasia (PCH) is characterized by hypoplasia and atrophy of the cerebellum, variable pontine atrophy, microcephaly, severe mental and motor impairments and seizures. Mutations in 11 genes have been reported in 8 out of 10 forms of PCH. Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase gene (RARS2) have been recently associated with PCH type 6, which is characterized by early-onset encephalopathy with signs of oxidative phosphorylation defect. Here we describe the clinical presentation, neuroimaging findings and molecular characterizations of two siblings with a clinical diagnosis of PCH who displayed a novel variant (c.-2A>G) in the 5′-UTR of the RARS2 gene in the homozygous state. This variant was identified through next-generation sequencing testing of a panel of nine genes known to be involved in PCH. Gene expression and functional studies demonstrated that the c.-2A>G sequence change directly leads to a reduced RARS2 messenger RNA expression in the patients by decreasing RARS2 promoter activity, thus providing evidence that mutations in the RARS2 promoter are likely to represent a new causal mechanism of PCH6.

Clinical utility of gene panel-based testing for hereditary myelodysplastic syndrome/acute leukemia predisposition syndromes.

Clinical utility of gene panel-based testing for hereditary myelodysplastic syndrome/acute leukemia predisposition syndromes

Improved molecular diagnosis of patients with neonatal diabetes using a combined next-generation sequencing and MS-MLPA approach.

Abstract Background: We evaluated a methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA) assay for the molecular diagnosis of transient neonatal diabetes mellitus (TNDM) caused by 6q24 abnormalities and assessed the clinical utility of using this assay in combination with next generation sequencing (NGS) analysis for diagnosing patients with neonatal diabetes (NDM). Methods: We performed MS-MLPA in 18 control samples and 42 retrospective NDM cases with normal bi-parental inheritance of chromosome 6. Next, we evaluated 22 prospective patients by combining NGS analysis of 11 NDM genes and the MS-MLPA assay. Results: 6q24 aberrations were identified in all controls and in 19% of patients with normal bi-parental inheritance of chromosome 6. The MS-MLPA/NGS combined approach identified a genetic cause in ~64% of patients with NDM of unknown etiology. Conclusions: MS-MLPA is a reliable method to identify all known 6q24 abnormalities and comprehensive testing of all causes reveals a causal mutation in ~64% of patients.

Alu-mediated deletion of PIGL in a Patient with CHIME syndrome

CHIME syndrome is a rare autosomal recessive neuroectodermal disorder associated with biallelic mutations in PIGL. To date, six molecularly confirmed cases of CHIME syndrome have been reported. Here, we report the seventh patient with biallelic PIGL mutations associated with CHIME syndrome and describe the first characterization of an intragenic deletion in PIGL. Our characterization of the deletion breakpoint junction demonstrated that the breakpoints occurred within Alu repeats and the deletion was most likely mediated by a microhomology event. Analysis of PIGL genomic sequences for repetitive elements demonstrated that Alu repeats represent ∼34% of its intronic sequence, suggesting that the genomic architecture may predispose the gene to disease‐causing copynumber changes. Taken together, these findings indicate that patients with a clinical diagnosis of CHIME syndrome and a single identifiable mutation in PIGL warrant further investigation for copynumber changes involving PIGL.

Reinitiation of mRNA translation in a patient with X-linked infantile spasms with a protein-truncating variant in ARX

Mutations in the Aristaless-related homeobox gene (ARX) lead to a range of X-linked intellectual disability phenotypes, with truncating variants generally resulting in severe X-linked lissencephaly with ambiguous genitalia (XLAG), and polyalanine expansions and missense variants resulting in infantile spasms. We report two male patients with early-onset infantile spasms in whom a novel c.34G>T (p.(E12*)) variant was identified in the ARX gene. A similar variant c.81C>G (p.(Y27*)), has previously been described in two affected cousins with early-onset infantile spasms, leading to reinitiation of ARX mRNA translation resulting in an N-terminal truncated protein. We show that the novel c.34G>T (p.(E12*)) variant also reinitiated mRNA translation at the next AUG codon (c.121–123 (p.M41)), producing the same N-terminally truncated protein. The production of both of these truncated proteins was demonstrated to be at markedly reduced levels using in vitro cell assays. Using luciferase reporter assays, we demonstrate that transcriptional repression capacity of ARX was diminished by both the loss of the N-terminal corepressor octapeptide domain, as a consequence of truncation, and the marked reduction in mutant protein expression. Our study indicates that premature termination mutations very early in ARX lead to reinitiation of translation to produce N-terminally truncated protein at markedly reduced levels of expression. We conclude that even low levels of N-terminally truncated ARX is sufficient to improve the patient’s phenotype compared with the severe phenotype of XLAG that includes malformations of the brain and genitalia normally seen in complete loss-of-function mutations in ARX.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

Frequency of germline mutations in cancer susceptibility genes in malignant mesothelioma.

PURPOSE The aim of the current study was to determine the prevalence and clinical predictors of germline cancer susceptibility mutations in patients with malignant mesothelioma (MM). METHODS We performed targeted capture and next-generation sequencing of 85 cancer susceptibility genes on germline DNA from 198 patients with pleural, peritoneal, and tunica vaginalis MM. RESULTS Twenty-four germline mutations were identified in 13 genes in 23 (12%) of 198 patients. BAP1 mutations were the most common (n = 6; 25%). The remaining were in genes involved in DNA damage sensing and repair (n = 14), oxygen sensing (n = 2), endosome trafficking (n = 1), and cell growth (n = 1). Pleural site (odds ratio [OR], 0.23; 95% CI, 0.10 to 0.58; P < .01), asbestos exposure (OR, 0.28; 95% CI, 0.11 to 0.72; P < .01), and older age (OR, 0.95; 95% CI, 0.92 to 0.99; P = .01) were associated with decreased odds of carrying a germline mutation, whereas having a second cancer diagnosis (OR, 3.33; 95% CI, 1.22 to 9.07; P = .02) significantly increased the odds. The odds of carrying a mutation in BAP1 (OR, 1,658; 95% CI, 199 to 76,224; P < .001), BRCA2 (OR, 5; 95% CI, 1.0 to 14.7; P = .03), CDKN2A (OR, 53; 95% CI, 6 to 249; P < .001), TMEM127 (OR, 88; 95% CI, 1.7 to 1,105; P = .01), VHL (OR, 51; 95% CI, 1.1 to 453; P = .02), and WT1 (OR, 20; 95% CI, 0.5 to 135; P = .049) were significantly higher in MM cases than in a noncancer control population. Tumor sequencing identified mutations in a homologous recombination pathway gene in 52% (n = 29 of 54). CONCLUSION A significant proportion of patients with MM carry germline mutations in cancer susceptibility genes, especially those with peritoneal MM, minimal asbestos exposure, young age, and a second cancer diagnosis. These data support clinical germline genetic testing for patients with MM and provide a rationale for additional investigation of the homologous recombination pathway in MM.