Adaptation and validation of the ACMG/AMP variant classification framework for MYH7-associated inherited cardiomyopathies: recommendations by ClinGen's Inherited Cardiomyopathy Expert Panel.

PurposeIntegrating genomic sequencing in clinical care requires standardization of variant interpretation practices. The Clinical Genome Resource has established expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification framework for specific genes and diseases. The Cardiomyopathy Expert Panel selected MYH7, a key contributor to inherited cardiomyopathies, as a pilot gene to develop a broadly applicable approach.MethodsExpert revisions were tested with 60 variants using a structured double review by pairs of clinical and diagnostic laboratory experts. Final consensus rules were established via iterative discussions.ResultsAdjustments represented disease-/gene-informed specifications (12) or strength adjustments of existing rules (5). Nine rules were deemed not applicable. Key specifications included quantitative frameworks for minor allele frequency thresholds, the use of segregation data, and a semiquantitative approach to counting multiple independent variant occurrences where fully controlled case-control studies are lacking. Initial inter-expert classification concordance was 93%. Internal data from participating diagnostic laboratories changed the classification of 20% of the variants (n = 12), highlighting the critical importance of data sharing.ConclusionThese adapted rules provide increased specificity for use in MYH7-associated disorders in combination with expert review and clinical judgment and serve as a stepping stone for genes and disorders with similar genetic and clinical characteristics.

Genome-wide association studies in dogs and humans identify ADAMTS20 as a risk variant for cleft lip and palate.

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10(-13); adjusted p= 2.2 x 10(-3)). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 - 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.

A LINE-1 insertion in DLX6 is responsible for cleft palate and mandibular abnormalities in a canine model of Pierre Robin sequence.

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 (24.2 Mb-29.3 Mb; p(raw )= 4.64 × 10(-15)). Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans.

Genome-wide association mapping in dogs enables identification of the homeobox gene, NKX2-8, as a genetic component of neural tube defects in humans.

Neural tube defects (NTDs) is a general term for central nervous system malformations secondary to a failure of closure or development of the neural tube. The resulting pathologies may involve the brain, spinal cord and/or vertebral column, in addition to associated structures such as soft tissue or skin. The condition is reported among the more common birth defects in humans, leading to significant infant morbidity and mortality. The etiology remains poorly understood but genetic, nutritional, environmental factors, or a combination of these, are known to play a role in the development of NTDs. The variable conditions associated with NTDs occur naturally in dogs, and have been previously reported in the Weimaraner breed. Taking advantage of the strong linkage-disequilibrium within dog breeds we performed genome-wide association analysis and mapped a genomic region for spinal dysraphism, a presumed NTD, using 4 affected and 96 unaffected Weimaraners. The associated region on canine chromosome 8 (pgenome  =3.0 × 10(-5)), after 100,000 permutations, encodes 18 genes, including NKX2-8, a homeobox gene which is expressed in the developing neural tube. Sequencing NKX2-8 in affected Weimaraners revealed a G to AA frameshift mutation within exon 2 of the gene, resulting in a premature stop codon that is predicted to produce a truncated protein. The exons of NKX2-8 were sequenced in human patients with spina bifida and rare variants (rs61755040 and rs10135525) were found to be significantly over-represented (p=0.036). This is the first documentation of a potential role for NKX2-8 in the etiology of NTDs, made possible by investigating the molecular basis of naturally occurring mutations in dogs.

Prenatal Testing for Variants in Genes Associated with Hereditary Cancer Risk: Laboratory Experience and Considerations.

Prenatal molecular genetic testing for familial variants that cause inherited disorders has been performed for decades and is accepted as standard of care. However, the spectrum of genes considered for prenatal testing is expanding because of genetic testing for hereditary cancer risk (HCR) and inclusion of conditions with associated cancer risk in carrier screening panels. A few of these disorders, such as ataxia telangiectasia and Bloom syndrome, include increased cancer risk as part of the phenotype, already meet professional guidelines for prenatal testing, and may be associated with increased cancer risk in heterozygous carriers. In addition, recent studies implicate heterozygosity for variants in lysosomal storage disease genes in HCR etiology. Currently, there is no specific professional guidance regarding prenatal testing for HCR. To determine the prevalence of such testing, we reviewed 1345 consecutive prenatal specimens received in our laboratory for familial variant-specific testing and identified 65 (4.8%) with a known or likely HCR component, plus 210 (15.6%) for lysosomal storage disease. These specimens were classified into five distinct categories for clarity and to enable evaluation. Our experience assessing prenatal specimens for variants associated with HCR, with or without a constitutional phenotype, provides metrics for and contributes to the points to consider in prenatal testing for HCR.

Double-Hit and Triple-Hit Follicular Lymphoma.

OBJECTIVES: To better characterize the clinicopathologic presentation and outcomes of follicular lymphoma with MYC and BCL2 and/or BCL6 rearrangements (double-hit and triple-hit follicular lymphoma), we present three cases from our institution and perform a literature review of 37 published cases. METHODS: Cases were identified using institutional SoftPath software and the MEDLINE database via the PubMed search engine. Clinical and pathologic data were collected with subsequent stratification by histologic grade and treatment for comparison. RESULTS: Similar to classic follicular lymphoma, patients presented most often with low-grade (1-2) but high-stage (III-IV) disease with absence of B symptoms; however, overall survival was worse than that of traditional follicular lymphoma. In a small sample size, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R) achieved better outcomes than a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Specific pathologic features that might prompt testing for MYC rearrangement include elevated proliferation index out of proportion to cytology and aggressive features such as angioinvasion. CONCLUSIONS: Double-hit and triple-hit follicular lymphoma may be better classified as a distinct entity from classical follicular lymphoma with a worse prognosis. Aggressive therapy with a treatment regimen used for high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements might be beneficial, but more evidence is needed to justify aggressive treatment as standard of care.

Multiple Whole Chromosomal Gains Define Angiomatous Meningiomas and Are Absent From the Tumor Vasculature.

Angiomatous meningioma is a variant with prominent vascularity that can mimic other highly vascularized tumors and present diagnostic challenges. Unlike most meningioma variants, where NF2 gene loss on chromosome 22 is the most common genetic abnormality, angiomatous meningiomas are unique in having multiple whole chromosome gains (polysomies). We analyzed 38 meningiomas, 9 angiomatous (including 2 atypical and 1 anaplastic), and 29 nonangiomatous meningiomas, using array comparative genomic hybridization (aCGH). Angiomatous meningiomas showed multiple chromosomal alterations including polysomies and copy neutral loss of heterozygosity in comparison to nonangiomatous variants. The most frequent gains were of chromosomes 5 and 20 (100% and 89% of cases, respectively); none showed chromosome 22 loss. Furthermore, using fluorescence in situ hybridization we show that the vasculature lacked chromosomal polysomy. While generally benign, we present 2 grade II and the first cytogenetically confirmed grade III angiomatous meningioma, demonstrating their potentially aggressive behavior. Thus, multiple polysomies define angiomatous meningioma and aCGH can distinguish this variant from nonangiomatous meningiomas and other histological mimics in diagnostically challenging cases. Furthermore, the prominent vasculature is not neoplastic and likely induced by angiogenic factors. Together, these findings suggest a distinct tumorigenic pathway in angiomatous meningiomas.

Association of early onset myasthenia gravis in Newfoundland dogs with the canine major histocompatibility complex class I.

Acquired Myasthenia Gravis (MG) is an autoimmune neuromuscular disorder whose development in humans has been associated with the Major Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA). There is a form of early onset MG (EOMG) in Newfoundland dogs that mimics the clinical presentation in humans and appears to have familial inheritance. Genotyping of three classical Dog Leukocyte Antigen (DLA) class II genes, DLA-DRB1, DLA-DQA1 and DLA-DQB1, in 16 Newfoundlands with EOMG and 46 unaffected Newfoundlands, identified DLA-DQB1 *00301 (p-value = 0.0051 OR: 7.41) as a risk locus for the development of EOMG in this breed. In order to further investigate the extent of the association to the entire MHC region, 208 additional SNPs were genotyped in two phases. Both a risk locus for EOMG to the DLA class I (chr12: 458483-506460) and a protective locus for EOMG susceptibility that extends outside of the DLA class I (chr12: 89701-475348) were identified. Four additional dog breeds with an elevated risk for the development of MG were SNP genotyped, but no shared or significant associations were found. MHC involvement in canine MG disease manifestation overlaps with loci identified in human studies and highlights the value of dogs as a model for genetic studies of naturally occurring diseases.

Clinical manifestations, response to treatment, and clinical outcome for Weimaraners with hypertrophic osteodystrophy: 53 cases (2009-2011).

OBJECTIVE: To evaluate clinical manifestations, response to treatment, and outcome for Weimaraners with hypertrophic osteodystrophy (HOD). DESIGN: Retrospective case series. ANIMALS: 53 dogs. PROCEDURES: Medical records were reviewed for signalment, vaccination history, clinical signs, laboratory test results, response to treatment, and relapses. Radiographs were reviewed. RESULTS: Clinical signs included pyrexia, lethargy, and ostealgia; signs involving the gastrointestinal, ocular, or cutaneous systems were detected. Of the 53 dogs, 28 (52.8%) had HOD-affected littermates. Dogs with HOD-affected littermates were more likely to relapse, compared with the likelihood of relapse for dogs with no HOD-affected littermates. All 53 dogs had been vaccinated 1 to 30 days before HOD onset; no difference was found between the number of dogs with a history of vaccination with a recombinant vaccine (n … 21) versus a nonrecombinant vaccine (32). Fifty (94.3%) dogs had radiographic lesions compatible with HOD at disease onset, and the other 3 (5.7%) had HOD lesions 48 to 72 hours after the onset of clinical signs. Twelve of 22 (54.5%) dogs treated with NSAIDs did not achieve remission by 7 days after initiation of treatment. All dogs treated initially with corticosteroids achieved remission within 8 to 48 hours. Of the 33 dogs that reached adulthood, 28 (84.8%) were healthy and 5 (15.2%) had episodes of pyrexia and malaise. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with corticosteroids was superior to treatment with NSAIDs in Weimaraners with HOD. It may be necessary to evaluate repeated radiographs to establish a diagnosis of HOD. Most HOD-affected Weimaraners had resolution of the condition with physeal closure.