EMQN: Recommendations for genetic testing in inherited cardiomyopathies and arrhythmias.

Inherited cardiomyopathies and arrhythmias (ICAs) are a prevalent and clinically heterogeneous group of genetic disorders that are associated with increased risk of sudden cardiac death and heart failure. Making a genetic diagnosis can inform the management of patients and their at-risk relatives and, as such, molecular genetic testing is now considered an integral component of the clinical care pathway. However, ICAs are characterised by high genetic and allelic heterogeneity, incomplete / age-related penetrance, and variable expressivity. Therefore, despite our improved understanding of the genetic basis of these conditions, and significant technological advances over the past two decades, identifying and recognising the causative genotype remains challenging. As clinical genetic testing for ICAs becomes more widely available, it is increasingly important for clinical laboratories to consolidate existing knowledge and experience to inform and improve future practice. These recommendations have been compiled to help clinical laboratories navigate the challenges of ICAs and thereby facilitate best practice and consistency in genetic test provision for this group of disorders. General recommendations on internal and external quality control, referral, analysis, result interpretation, and reporting are described. Also included are appendices that provide specific information pertinent to genetic testing for hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies, long QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia.

FMR1 allele size distribution in 35,000 males and females: a comparison of developmental delay and general population cohorts.

PURPOSE: Developmental delay phenotypes have been associated with FMR1 premutation (PM: 55-200 CGG repeats) and "gray zone" (GZ: 45-54 CGG repeats) alleles. However, these associations have not been confirmed by larger studies to be useful in pediatric diagnostic or screening settings. METHODS: This study determined the prevalence of PM and GZ alleles in two independent cohorts of 19,076 pediatric referrals to developmental delay diagnostic testing through Victorian Clinical Genetics Service (cohort 1: N = 10,235; cohort 2: N = 8841), compared with two independent general population cohorts (newborn screening N = 1997; carrier screening by the Victorian Clinical Genetics Service prepair program N = 14,249). RESULTS: PM and GZ prevalence rates were not significantly increased (p > 0.05) in either developmental delay cohort (male PM: 0.12-0.22%; female PM: 0.26-0.33%; male GZ: 0.68-0.69%; female GZ: 1.59-2.13-%) compared with general population cohorts (male PM: 0.20%; female PM: 0.27-0.82%; male GZ: 0.79%; female GZ: 1.43-2.51%). Furthermore, CGG size distributions were comparable across datasets, with each having a modal value of 29 or 30 and ~1/3 females and ~1/5 males having at least one allele with ≤26 CGG repeats. CONCLUSION: These data do not support the causative link between PM and GZ expansions and developmental-delay phenotypes in pediatric settings.

Correction: Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests.

Zoe McDonald, BSc, was omitted from the list of article coauthors. Her name should have been included as the seventh author, following Clare Elizabeth Hunt. Her affiliation is Victorian Clinical Genetics Services, Parkville, Victoria, Australia. The authors regret the error.

Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests.

PurposeTo describe our experience of offering simultaneous genetic carrier screening for cystic fibrosis (CF), fragile X syndrome (FXS), and spinal muscular atrophy (SMA).MethodsCarrier screening is offered through general practice, obstetrics, fertility, and genetics settings before or in early pregnancy. Carriers are offered genetic counseling with prenatal/preimplantation genetic diagnosis available to those at increased risk.ResultsScreening of 12,000 individuals revealed 610 carriers (5.08%; 1 in 20): 342 CF, 35 FXS, 241 SMA (8 carriers of 2 conditions), approximately 88% of whom had no family history. At least 94% of CF and SMA carriers' partners were tested. Fifty couples (0.42%; 1 in 240) were at increased risk of having a child with one of the conditions (14 CF, 35 FXS, and 1 SMA) with 32 pregnant at the time of testing. Of these, 26 opted for prenatal diagnosis revealing 7 pregnancies affected (4 CF, 2 FXS, 1 SMA).ConclusionThe combined affected pregnancy rate is comparable to the population risk for Down syndrome, emphasizing the need to routinely offer carrier screening. The availability of appropriate genetic counseling support and a collaborative approach between laboratory teams, genetics services, health professionals offering screening, and support organizations is essential.

Erratum: Hwang Y.T. et al. Molecular Inconsistencies in a Fragile X Male with Early Onset Ataxia. Genes 2016, 7, 68.

n/a.

Molecular Inconsistencies in a Fragile X Male with Early Onset Ataxia.

Mosaicism for FMR1 premutation (PM: 55-199 CGG)/full mutation (FM: >200 CGG) alleles or the presence of unmethylated FM (UFM) have been associated with a less severe fragile X syndrome (FXS) phenotype and fragile X associated tremor/ataxia syndrome (FXTAS)-a late onset neurodegenerative disorder. We describe a 38 year old male carrying a 100% methylated FM detected with Southern blot (SB), which is consistent with complete silencing of FMR1 and a diagnosis of fragile X syndrome. However, his formal cognitive scores were not at the most severe end of the FXS phenotype and he displayed tremor and ataxic gait. With the association of UFM with FXTAS, we speculated that his ataxia might be related to an undetected proportion of UFM alleles. Such UFM alleles were confirmed by more sensitive PCR based methylation testing showing FM methylation between 60% and 70% in blood, buccal, and saliva samples and real-time PCR analysis showing incomplete silencing of FMR1. While he did not meet diagnostic criteria for FXTAS based on MRI findings, the underlying cause of his ataxia may be related to UFM alleles not detected by SB, and follow-up clinical and molecular assessment are justified if his symptoms worsen.

A Prospective Study of Sudden Cardiac Death among Children and Young Adults.

BACKGROUND: Sudden cardiac death among children and young adults is a devastating event. We performed a prospective, population-based, clinical and genetic study of sudden cardiac death among children and young adults. METHODS: We prospectively collected clinical, demographic, and autopsy information on all cases of sudden cardiac death among children and young adults 1 to 35 years of age in Australia and New Zealand from 2010 through 2012. In cases that had no cause identified after a comprehensive autopsy that included toxicologic and histologic studies (unexplained sudden cardiac death), at least 59 cardiac genes were analyzed for a clinically relevant cardiac gene mutation. RESULTS: A total of 490 cases of sudden cardiac death were identified. The annual incidence was 1.3 cases per 100,000 persons 1 to 35 years of age; 72% of the cases involved boys or young men. Persons 31 to 35 years of age had the highest incidence of sudden cardiac death (3.2 cases per 100,000 persons per year), and persons 16 to 20 years of age had the highest incidence of unexplained sudden cardiac death (0.8 cases per 100,000 persons per year). The most common explained causes of sudden cardiac death were coronary artery disease (24% of cases) and inherited cardiomyopathies (16% of cases). Unexplained sudden cardiac death (40% of cases) was the predominant finding among persons in all age groups, except for those 31 to 35 years of age, for whom coronary artery disease was the most common finding. Younger age and death at night were independently associated with unexplained sudden cardiac death as compared with explained sudden cardiac death. A clinically relevant cardiac gene mutation was identified in 31 of 113 cases (27%) of unexplained sudden cardiac death in which genetic testing was performed. During follow-up, a clinical diagnosis of an inherited cardiovascular disease was identified in 13% of the families in which an unexplained sudden cardiac death occurred. CONCLUSIONS: The addition of genetic testing to autopsy investigation substantially increased the identification of a possible cause of sudden cardiac death among children and young adults. (Funded by the National Health and Medical Research Council of Australia and others.).

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis.

BACKGROUND: FMR1 full mutations (FMs) (CGG expansion >200) in males mosaic for a normal (<45 CGG) or gray-zone (GZ) (45-54 CGG) allele can be missed with the standard 2-step fragile X syndrome (FXS) testing protocols, largely because the first-line PCR tests showing a normal or GZ allele are not reflexed to the second-line test that can detect FM. METHODS: We used methylation-specific quantitative melt analysis (MS-QMA) to determine the prevalence of cryptic FM alleles in 2 independent cohorts of male patients (994 from Chile and 2392 from Australia) referred for FXS testing from 2006 to 2013. All MS-QMA-positive cases were retested with commercial triplet primed PCR, methylation-sensitive Southern blot, and a methylation-specific EpiTYPER-based test. RESULTS: All 38 FMs detected with the standard 2-step protocol were detected with MS-QMA. However, MS-QMA identified methylation mosaicism in an additional 15% and 11% of patients in the Chilean and Australian cohorts, respectively, suggesting the presence of a cryptic FM. Of these additional patients, 57% were confirmed to carry cryptic expanded alleles in blood, buccal mucosa, or saliva samples. Further confirmation was provided by identifying premutation (CGG 55-199) alleles in mothers of probands with methylation-sensitive Southern blot. Neurocognitive assessments showed that low-level mosaicism for cryptic FM alleles was associated with cognitive impairment or autism. CONCLUSIONS: A substantial number of mosaic FM males who have cognitive impairment or autism are not diagnosed with the currently recommended 2-step testing protocol and can be identified with MS-QMA as a first-line test.

A comprehensive evaluation of myocardial fibrosis in hypertrophic cardiomyopathy with cardiac magnetic resonance imaging: linking genotype with fibrotic phenotype.

AIMS: In hypertrophic cardiomyopathy (HCM), attempts to associate genotype with phenotype have largely been unsuccessful. More recently, cardiac magnetic resonance (CMR) imaging has enhanced myocardial fibrosis characterization, while next-generation sequencing (NGS) can identify pathogenic HCM mutations. We used CMR and NGS to explore the link between genotype and fibrotic phenotype in HCM. METHODS AND RESULTS: One hundred and thirty-nine patients with HCM and 25 healthy controls underwent CMR to quantify regional myocardial fibrosis with late gadolinium enhancement (LGE) and diffuse myocardial fibrosis with post-contrast T1 mapping. Collagen content of myectomy specimens from nine HCM patients was determined. Fifty-six HCM patients underwent NGS for 65 cardiomyopathy genes, including 36 HCM-associated genes. Post-contrast myocardial T1 time correlated histologically with myocardial collagen content (r = -0.70, P = 0.03). Compared with controls, HCM patients had more LGE (4.6 ± 6.1 vs. 0%, P < 0.001) and lower post-contrast T1 time (483 ± 83 vs. 545 ± 49 ms, P < 0.001). LGE negatively correlated with left-ventricular (LV) ejection fraction and outflow tract obstruction, whereas lower post-contrast T1 time, suggestive of more diffuse myocardial fibrosis, was associated with LV diastolic impairment and dyspnoea. Patients with identifiable HCM mutations had more LGE (7.9 ± 8.6 vs. 3.1 ± 4.3%, P = 0.03), but higher post-contrast T1 time (498 ± 81 vs. 451 ± 70 ms, P = 0.03) than patients without. CONCLUSION: In HCM, contrast-enhanced CMR with T1 mapping can non-invasively evaluate regional and diffuse patterns of myocardial fibrosis. These patterns of fibrosis occur independently of each other and exhibit distinct clinical associations. HCM patients with recognized genetic mutations have significantly more regional, but less diffuse myocardial fibrosis than those without.

Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database.

The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch syndrome-associated genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist in variant classification and was recognized through microattribution. The scheme was refined by multidisciplinary expert committee review of the clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants that were not obviously protein truncating from nomenclature. This large-scale endeavor will facilitate the consistent management of families suspected to have Lynch syndrome and demonstrates the value of multidisciplinary collaboration in the curation and classification of variants in public locus-specific databases.

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms.

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease.

Current landscape and new paradigms of proficiency testing and external quality assessment for molecular genetics.

CONTEXT: Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests. OBJECTIVES: To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies. DATA SOURCES: The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers. CONCLUSIONS: Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.

Pathophysiological insights derived by natural history and motor function of spinal muscular atrophy.

OBJECTIVE: To examine the natural history of spinal muscular atrophy (SMA) to gain further insight into the clinical course and pathogenesis. STUDY DESIGN: Survival pattern, age of onset, and ambulatory status were retrospectively analyzed in 70 patients with SMA with deletions of the survival motor neuron 1 genes that presented to a specialized neuromuscular clinic. The Kaplan-Meier method was used to obtain survival curves. Hammersmith Functional Motor Scale-Expanded and abductor pollicis brevis compound muscle action potential amplitudes were assessed in 25 of the surviving cohort and correlated with survival motor neuron 2 copy number. RESULTS: Survival probabilities at ages 1, 2, 4, 10, 20, and 40 years were 40%, 25%, 6%, and 0%, respectively, for patients with SMA type 1; 100%, 100%, 97%, 93%, 93%, and 52% for patients with SMA type 2 and all patients with SMA type 3 were alive (age range 7-33 years). There were significant associations between age of onset and long-term outcome, specifically survival in SMA type 1 (P < .01) and Hammersmith Functional Motor Scale-Expanded (P < .0001), and compound muscle action potential (P = .001) in SMA types 2 and 3. Motor function in patients with long-standing SMA reduced over prolonged periods or remained stable. Survival motor neuron 2 copy number related to continuing changes in motor function with age. CONCLUSION: The natural history of SMA suggests considerable early loss of motor neurons, with severity related to differences in the number of remaining motor neurons. As the ensuing chronic course in milder phenotypes suggests relative stability of remaining motor neurons, the maximal therapeutic window presents early.

Dysfunction of axonal membrane conductances in adolescents and young adults with spinal muscular atrophy.

Spinal muscular atrophy is distinct among neurodegenerative conditions of the motor neuron, with onset in developing and maturing patients. Furthermore, the rate of degeneration appears to slow over time, at least in the milder forms. To investigate disease pathophysiology and potential adaptations, the present study utilized axonal excitability studies to provide insights into axonal biophysical properties and explored correlation with clinical severity. Multiple excitability indices (stimulus-response curve, strength-duration time constant, threshold electrotonus, current-threshold relationship and recovery cycle) were investigated in 25 genetically characterized adolescent and adult patients with spinal muscular atrophy, stimulating the median motor nerve at the wrist. Results were compared with 50 age-matched controls. The Medical Research Council sum score and Spinal Muscular Atrophy Functional Rating Scale were used to define the strength and motor functional status of patients with spinal muscular atrophy. In patients with spinal muscular atrophy, there were reductions in compound muscle action potential amplitude (P < 0.0005) associated with reduction in stimulus response slope (P < 0.0005), confirming significant axonal loss. In the patients with mild or ambulatory spinal muscular atrophy, there was reduction of peak amplitude without alteration in axonal excitability; in contrast, in the non-ambulatory or severe spinal muscular atrophy cohort prominent changes in axonal function were apparent. Specifically, there were steep changes in the early phase of hyperpolarization in threshold electrotonus (P < 0.0005) that correlated with clinical severity. Additionally, there were greater changes in depolarizing threshold electrotonus (P < 0.0005) and prolongation of the strength-duration time constant (P = 0.001). Mathematical modelling of the excitability changes obtained in patients with severe spinal muscular atrophy supported a mixed pathology comprising features of axonal degeneration and regeneration. The present study has provided novel insight into the pathophysiology of spinal muscular atrophy, with identification of functional abnormalities involving axonal K(+) and Na(+) conductances and alterations in passive membrane properties, the latter linked to the process of neurodegeneration.

A novel deletion-insertion mutation identified in exon 3 of FXN in two siblings with a severe Friedreich ataxia phenotype.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound heterozygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124-127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron-sulphur cluster assembly factor IscU, would be impaired.

Premature arthritis is a distinct type II collagen phenotype.

Mutations in the gene encoding type II collagen (COL2A1) give rise to a spectrum of phenotypes predominantly affecting cartilage and bone. These chondrodysplasias are typically characterized by disproportionately short stature, eye abnormalities, cleft palate, and hearing loss. It is less recognized that mutations in COL2A1 can also present as degenerative joint disease in the absence of any other phenotypic clues. We report 2 Australian families presenting with an isolated arthritis phenotype, segregating as a dominant trait affecting both large and small joints, prior to age 30 years. Sequencing of COL2A1 in the propositi revealed 2 sequence changes resulting in glycine substitutions in the triple-helical domain of type II collagen. We review the increasing evidence implicating COL2A1 mutations in individuals presenting with isolated degenerative joint disease, aiming to alert physicians who assess these patients to this possibility. The importance of finding a COL2A1 mutation in such patients lies in the subsequent ability to accurately assess recurrence risks, offer early (including prenatal) diagnosis, and provide information regarding the natural history of the condition. Most importantly, it enables at-risk individuals to be identified for implementation of preventative strategies (i.e., weight loss, joint-friendly exercise programs) and early ameliorative management of their condition.

Population screening and cascade testing for carriers of SMA.

Spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseases, caused by absence of both copies of the survival motor neuron 1 (SMN1) gene. Identification of SMA carriers has important implications for individuals with a family history and the general population. SMA carriers are completely healthy and most are unaware of their carrier status until they have an affected child. A total of 422 individuals have been studied to identify SMA carriers. This cohort included 117 parents of children homozygously deleted for SMN1 (94% were carriers and 6% had two copies of SMN1; of these individuals, two in seven had the '2+0' genotype, two in seven were normal but had children carrying a de novo deletion and three in seven were unresolved), 158 individuals with a significant family history of SMA (47% had one copy, 49% had two copies and 4% had three copies of SMN1) and 146 individuals with no family history of SMA (90% had two copies, 2% had one copy and 8% had three copies of SMN1). The SMA carrier frequency in the Australian population appears to be 1/49 and the frequency of two-copy SMN1 alleles and de novo deletion mutations are both at least 1.7%. A multimodal approach involving quantitative analysis, linkage analysis and genetic risk assessment (GRA), facilitates the resolution of SMA carrier status in individuals with a family history as well as individuals of the general population, providing couples with better choices in their family planning.

Detection of mutations in genes associated with hearing loss using a microarray-based approach.

Knowing the etiology of hearing loss in a person has implications for counseling and management of the condition. More than 50% of cases of early onset, nonsyndromic sensorineural hearing loss are attributable to genetic factors. However, deafness is a genetically heterogeneous condition and it is therefore currently not economically and practically feasible to screen for mutations in all known deafness genes. We have developed a microarray-based hybridization biochip assay for the detection of known mutations. The current version of the hearing loss biochip detects nine common mutations in the connexin 26 gene, four mutations in the pendrin gene, one mutation in the usherin gene, and one mutation in mitochondrial DNA. The biochip was validated using DNA from 250 people with apparent nonsyndromic, moderate to profound sensorineural hearing loss. The hearing loss biochip detected with 100% accuracy the mutations it was designed for. No false-positives or false-negative results were seen. The biochip can easily be expanded to test for additional mutations in genes associated with hearing impairment or other genetic conditions.

Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH.

BACKGROUND & AIMS: Hyperplastic polyposis syndrome (HPS) is defined phenotypically with multiple, large and/or proximal hyperplastic polyps. There is no known germ-line predisposition. We aimed to characterize the clinicopathologic features of 38 patients with HPS and explore the role of germ-line mutations in the base excision repair genes MBD4 and MYH. METHODS: Utilizing clinical databases of The Royal Melbourne Hospital Bowel Cancer Surveillance Service and the Familial Cancer Clinic, 38 patients with HPS were recruited. The patients were analyzed for age at first diagnosis, features of hyperplastic polyposis, family histories of polyposis and colorectal cancer (CRC), coexisting adenomas, serrated adenomas, incidence of CRC, and microsatellite instability in the tumours. Mutation analysis of MBD4 and MYH were performed. RESULTS: Serrated adenomas were common (26%), and 19 (50%) of the 38 patients had a first-degree relative with CRC. Family history of HPS was uncommon, with only 2 cases found. Ten patients developed CRC, and 3 required surgery for polyposis. No pathogenic mutations in MBD4 were detected in the 27 patients tested, but 6 single nucleotide polymorphisms of uncertain functional significance were identified. Pathogenic biallelic MYH mutations were detected in 1 patient. CONCLUSIONS: Mutations in MBD4 are unlikely to be implicated in HPS; MYH mutations should be studied, especially when adenomas occur in the same patient. The clinical, histopathologic, and molecular findings of this study should contribute to our understanding of HPS and its relationship to the serrated neoplasia pathway.