The Clinical Variant Analysis Tool: Analyzing the evidence supporting reported genomic variation in clinical practice.

PURPOSE: Genomic test results, regardless of laboratory variant classification, require clinical practitioners to judge the applicability of a variant for medical decisions. Teaching and standardizing clinical interpretation of genomic variation calls for a methodology or tool. METHODS: To generate such a tool, we distilled the Clinical Genome Resource framework of causality and the American College of Medical Genetics/Association of Molecular Pathology and Quest Diagnostic Laboratory scoring of variant deleteriousness into the Clinical Variant Analysis Tool (CVAT). Applying this to 289 clinical exome reports, we compared the performance of junior practitioners with that of experienced medical geneticists and assessed the utility of reported variants. RESULTS: CVAT enabled performance comparable to that of experienced medical geneticists. In total, 124 of 289 (42.9%) exome reports and 146 of 382 (38.2%) reported variants supported a diagnosis. Overall, 10.5% (1 pathogenic [P] or likely pathogenic [LP] variant and 39 variants of uncertain significance [VUS]) of variants were reported in genes without established disease association; 20.2% (23 P/LP and 54 VUS) were in genes without sufficient phenotypic concordance; 7.3% (15 P/LP and 13 VUS) conflicted with the known molecular disease mechanism; and 24% (91 VUS) had insufficient evidence for deleteriousness. CONCLUSION: Implementation of CVAT standardized clinical interpretation of genomic variation and emphasized the need for collaborative and transparent reporting of genomic variation.

Mesenteric cysts, lymphatic leak, and cerebral cavernous malformation in a proband with KRIT1-related disease.

Cerebral cavernous malformations (CCMs) of the central nervous system arise sporadically or secondary to genomic variation. Established genetic etiologies include deleterious variants in KRIT1 (CCM1), malcavernin (CCM2), and PDCD10 (CCM3). KRIT1-related disease has not been described in conjunction with lymphatic defects, although lymphatic defects with abnormal endothelial cell junctions have been observed in mice deficient in HEG1-KRIT1 signaling. We report a proband with CCMs, multiple chylous mesenteric cysts, and chylous ascites with leaky lymphatic vasculature. Clinical short-read exome sequencing detected a disease-associated KRIT1 variant (NM_194456.1:c.[1927C>T];[=], p.(Gln643*)). We postulate an expansion of KRIT1-related disease to include lymphatic malformations and lymphatic endothelial dysfunction.

Long-read genome sequencing resolves a complex 13q structural variant associated with syndromic anophthalmia.

Microphthalmia, anophthalmia, and coloboma (MAC) are a heterogeneous spectrum of anomalous eye development and degeneration with genetic and environmental etiologies. Structural and copy number variants of chromosome 13 have been implicated in MAC; however, the specific loci involved in disease pathogenesis have not been well-defined. Herein we report a newborn with syndromic degenerative anophthalmia and a complex de novo rearrangement of chromosome 13q. Long-read genome sequencing improved the resolution and clinical interpretation of a duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) and terminal deletion. Sequence features at the breakpoint junctions suggested microhomology-mediated break-induced replication (MMBIR) of the maternal chromosome as the origin. Comparing this rearrangement to previously reported copy number alterations in 13q, we refine a putative dosage-sensitive critical region for MAC that might provide new insights into its molecular etiology.

An infant with congenital respiratory insufficiency and diaphragmatic paralysis: A novel BICD2 phenotype?

Monoallelic pathogenic variants in BICD2 are associated with autosomal dominant Spinal Muscular Atrophy Lower Extremity Predominant 2A and 2B (SMALED2A, SMALED2B). As part of the cellular vesicular transport, complex BICD2 facilitates the flow of constitutive secretory cargoes from the trans-Golgi network, and its dysfunction results in motor neuron loss. The reported phenotypes among patients with SMALED2A and SMALED2B range from a congenital onset disorder of respiratory insufficiency, arthrogryposis, and proximal or distal limb weakness to an adult-onset disorder of limb weakness and contractures. We report an infant with congenital respiratory insufficiency requiring mechanical ventilation, congenital diaphragmatic paralysis, decreased lung volume, and single finger camptodactyly. The infant displayed appropriate antigravity limb movements but had radiological, electrophysiological, and histopathological evidence of myopathy. Exome sequencing and long-read whole-genome sequencing detected a novel de novo BICD2 variant (NM_001003800.1:c.[1543G>A];[=]). This is predicted to encode p.(Glu515Lys); p.Glu515 is located in the coiled-coil 2 mutation hotspot. We hypothesize that this novel phenotype of diaphragmatic paralysis without clear appendicular muscle weakness and contractures of large joints is a presentation of BICD2-related disease.

An approach to rapid characterization of DMD copy number variants for prenatal risk assessment.

Prenatal detection of structural variants of uncertain significance, including copy number variants (CNV), challenges genetic counseling, and creates ambiguity for expectant parents. In Duchenne muscular dystrophy, variant classification and phenotypic severity of CNVs are currently assessed by familial segregation, prediction of the effect on the reading frame, and precedent data. Delineation of pathogenicity by familial segregation is limited by time and suitable family members, whereas analytical tools can rapidly delineate potential consequences of variants. We identified a duplication of uncertain significance encompassing a portion of the dystrophin gene (DMD) in an unaffected mother and her male fetus. Using long-read whole genome sequencing and alignment of short reads, we rapidly defined the precise breakpoints of this variant in DMD and could provide timely counseling. The benign nature of the variant was substantiated, more slowly, by familial segregation to a healthy maternal uncle. We find long-read whole genome sequencing of clinical utility in a prenatal setting for accurate and rapid characterization of structural variants, specifically a duplication involving DMD.

Subclinical Cardiomyopathy in Miyoshi Myopathy Detected by Late Gadolinium Enhancement Cardiac Magnetic Resonance Imaging.

Dysferlin is a sarcolemmal protein present in muscle cells. It is responsible for muscle membrane repair. Dysferlin gene (DYSF) mutation, resulting in deficiency in this protein, is termed dysferlinopathy. Clinically, it manifests as early adulthood onset of muscle weakness with markedly elevated creatine kinase levels. The main phenotypes are limb-girdle muscular dystrophy type 2B (LGMD2B), affecting proximal muscles, and Miyoshi myopathy (MM), affecting distal muscles. Dysferlin is also present in cardiomyocytes, and case reports have emerged of cardiac abnormalities in dysferlinopathy. While routine methods of cardiac screening, namely, electrocardiography or echocardiography, are convenient and noninvasive, they often exhibit insufficient diagnostic sensitivity for detecting subclinical cardiac remodeling during early stages of cardiomyopathy. Cardiac magnetic resonance imaging though can provide accurate assessment of cardiac chamber sizes and function. With gadolinium administration, it can also detect areas of myocardial scarring and fibrosis. Early diagnosis of neuromuscular disease-related cardiomyopathy is of clinical significance, as appropriate treatment can retard myocardial fibrosis, delaying cardiomyopathy progression. We present a case of a patient with MM incidentally diagnosed with concomitant cardiomyopathy.

Fetal-onset Congenital Dyserythropoietic Anemia Type 1 due to a Novel Mutation With Severe Iron Overload and Severe Cholestatic Liver Disease.

We report a rare case of severe congenital dyserythropoietic anemia type 1 with fetal onset. Our patient presented with fetal hydrops from 19 weeks of gestation, requiring multiple intrauterine transfusions. At birth, she had severe hemolytic anemia with severe jaundice, and was subsequently transfusion dependent. She eventually developed severe iron overload and fulminant liver failure before her demise at 5 months of age. Genetic testing revealed a novel mutation in CDAN1.

The genetic variation rs6903956 in the novel androgen-dependent tissue factor pathway inhibitor regulating protein (ADTRP) gene is not associated with levels of plasma coagulation factors in the Singaporean Chinese.

BACKGROUND: Genome-wide association study (GWAS) has reported that rs6903956 within the first intron of androgen-dependent tissue factor pathway inhibitor (TFPI) regulating protein (ADTRP) gene is associated with coronary artery disease (CAD) risk in the Chinese population. Although ADTRP is believed to be involved in the upregulation of TFPI, the underlying mechanism involved is largely unknown. This study investigated the association of rs6903956 with plasma Factor VII coagulant activity (FVIIc) and fibrinogen levels, which are regulated by TFPI and are independent risk predictors for CAD. METHODS: We conducted the analysis in both Chinese adult (N = 309) and neonatal cohorts (N = 447). The genotypes of the rs6903956 single nucleotide polymorphism (SNP) were determined by the polymerase chain reaction restriction fragment length polymorphism method (PCR-RFLP). FVIIc and fibrinogen level were measured from citrated plasma. The association between rs6903956 and coagulation factors was tested by linear regression with adjustment for possible confounders. Analysis was carried out in adults and neonates separately. RESULTS: No significant association was observed between rs6903956 and plasma FVIIc nor fibrinogen levels with adjustment for age, gender, body mass index (BMI) and cigarette smoking in adults (P for FVIIc = 0.464; P for fibrinogen = 0.349). The SNP was also not associated with these two coagulation factors in the neonates (P for FVIIc = 0.579; P for fibrinogen = 0.359) after adjusting for gestational age, gender and birth weight. CONCLUSIONS: SNP rs6903956 on ADTRP gene was not associated with plasma FVIIc nor fibrinogen levels.

A clinical approach to diagnosis and management of mitochondrial myopathies.

This paper provides an overview of the different types of mitochondrial myopathies (MM), associated phenotypes, genotypes as well as a practical clinical approach towards disease diagnosis, surveillance, and management. nDNA-related MM are more common in pediatric-onset disease whilst mtDNA-related MMs are more frequent in adults. Genotype-phenotype correlation in MM is challenging due to clinical and genetic heterogeneity. The multisystemic nature of many MMs adds to the diagnostic challenge. Diagnostic approaches utilizing genetic sequencing with next generation sequencing approaches such as gene panel, exome and genome sequencing are available. This aids molecular diagnosis, heteroplasmy detection in MM patients and furthers knowledge of known mitochondrial genes. Precise disease diagnosis can end the diagnostic odyssey for patients, avoid unnecessary testing, provide prognosis, facilitate anticipatory management, and enable access to available therapies or clinical trials. Adjunctive tests such as functional and exercise testing could aid surveillance of MM patients. Management requires a multi-disciplinary approach, systemic screening for comorbidities, cofactor supplementation, avoidance of substances that inhibit the respiratory chain and exercise training. This update of the current understanding on MMs provides practical perspectives on current diagnostic and management approaches for this complex group of disorders.

Clinical application of targeted long read sequencing in prenatal beta-thalassemia testing and genetic counseling.

BACKGROUND: Beta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease-associated variant, there is ambiguity on risk to their offspring. METHODS: We describe a case report of a proband with elevated HbA2, no identifiable HBB disease-associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5'UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants. RESULTS: This process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded. CONCLUSION: This finding provided clarity and enabled family planning for the proband and her family.

Targeting heterozygous dominant negative variant of KCNA2 using Gapmer ASO for the treatment of drug-resistant epilepsy.

A missense mutation c.1220C>G of KCN2A gene was recently identified in an infant with epilepsy. KCNA2 encodes KV1.2 subunits that form voltage-gated potassium channels (VGKC) via tetrameric assembly. The mutation results in amino acid change P407R at the highly conserved PVP motif. Functional characterization revealed that mutant KV1.2_P407R subunits formed loss-of-function channels and suppressed both KV1.2 and KV1.1 channel activities. Hetero-tetrameric assembly of the KV1.2_P407R subunits with other neuronal voltage-gated potassium channels of Shaker subfamily could lead to general deficit of repolarizing potassium current and potentially underlie the enhanced seizure susceptibility. Indeed, expression of human KV1.2_P407R in early postnatal rat cortical neurons or genetically engineered hESC-derived neurons disclosed broadening of action potential duration and early afterdepolarization (EAD), associating with reduced potassium current. We hypothesize that Gapmer antisense oligonucleotides (ASOs) targeted to c.1220C>G mutation will selectively degrade the mutant mRNA while allowing the remaining wild-type (WT) subunits to form functional channels. As a proof of principle, delivery of Gapmer packaged in lipid nanoparticle into cortical neurons selectively suppressed KV1.2_P407R over the WT protein expression, reversing the broadening of action potential duration, abrogating the EAD and leading to overall increase in potassium current.

Pitfalls in clinical genetics.

With the increasing availability of genetic tests, more doctors are offering and ordering such tests for their patients. Ordering a genetic test appears to be a simple process of filling in paperwork, drawing 3 mL of blood in an ethylenediaminetetraacetic acid tube and receiving a test report. This is identical to sending off a full blood count. However, it is far more complex than that. There are many potential pitfalls, as shown by the increasing number of complaints and lawsuits filed against doctors and allied health staff. Furthermore, clinical genetics involves more than just ordering tests; in fact, focusing on genetic tests alone is a potential pitfall. In this review, we discuss the common pitfalls in clinical genetics and how doctors can avoid these pitfalls to ensure patient safety and to safeguard their practice.

Challenges associated with the identification of germline variants on myeloid malignancy genomic profiling-a Singaporean experience.

Genomic profiling to identify myeloid-malignancy-related gene mutations is routinely performed for patients with suspected or definite myeloid malignancies. The most common specimen types in our experience are peripheral blood and bone marrow aspirates. Although primarily intended to identify somatic mutations, not infrequently, potentially clinically significant germline variants are also identified. Confirmation of the germline status of these variants is typically performed by hair follicle or skin fibroblast testing. If the germline variant is classified as a pathogenic or likely pathogenic variant and occurs in a gene known to be associated with a disease relevant to the patient's phenotype (for example, the identification of a DDX41 pathogenic variant in an individual with acute myeloid leukemia), the management algorithm is typically quite straightforward. Challenging situations may occur such as when the germline variant is classified as a pathogenic or likely pathogenic variant and occurs in a gene not known to be associated with the patient's phenotype/presenting complaint. We have encountered several such challenging cases in which potentially clinically significant germline variants were identified on the initial genomic profiling of peripheral blood or bone marrow aspirate. In this article, we present these cases and discuss the genetic counseling and management approaches.

Genetics in Ischemic Stroke: Current Perspectives and Future Directions.

Ischemic stroke is a heterogeneous condition influenced by a combination of genetic and environmental factors. Recent advancements have explored genetics in relation to various aspects of ischemic stroke, including the alteration of individual stroke occurrence risk, modulation of treatment response, and effectiveness of post-stroke functional recovery. This article aims to review the recent findings from genetic studies related to various clinical and molecular aspects of ischemic stroke. The potential clinical applications of these genetic insights in stratifying stroke risk, guiding personalized therapy, and identifying new therapeutic targets are discussed herein.

Using gene panels in the diagnosis of neuromuscular disorders: A mini-review.

The diagnosis of inherited neuromuscular disorders is challenging due to their genetic and phenotypic variability. Traditionally, neurophysiology and histopathology were primarily used in the initial diagnostic approach to these conditions. Sanger sequencing for molecular diagnosis was less frequently utilized as its application was a time-consuming and cost-intensive process. The advent and accessibility of next-generation sequencing (NGS) has revolutionized the evaluation process of genetically heterogenous neuromuscular disorders. Current NGS diagnostic testing approaches include gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS). Gene panels are often the most widely used, being more accessible due to availability and affordability. In this mini-review, we describe the benefits and risks of clinical genetic testing. We also discuss the utility, benefits, challenges, and limitations of using gene panels in the evaluation of neuromuscular disorders.

Can leaky splicing and evasion of premature termination codon surveillance contribute to the phenotypic variability in Alkuraya-Kucinskas syndrome?

Disease-associated variants in KIAA1109 associate with autosomal recessive Alkuraya-Kucinskas syndrome, which is typified by cerebral parenchymal underdevelopment, clubfeet, and arthrogryposis. Biallelic truncating variants occur with severe disease resulting in miscarriage or early neonatal death, whereas biallelic missense variants can occur with a milder phenotype of global developmental delay and intracranial malformation. This suggests that hypomorphic alleles in KIAA1109 give rise to a milder phenotype than do amorphic alleles. We describe a consanguineous family with pseudodominant segregation of a homozygous noncanonical splice donor variant (NM_015312.2:c.[13438+3A>G];[13438+3A>G]) in mother and daughter. In peripheral blood, sequencing of cDNA detected skipping of exon 76 (NM_015312.3:c.13281_13438del) and, by qRT-PCR quantification, occurred in 82-95% of peripheral blood KIAA1109 mRNA. Although the deletion of exon 76 is predicted to encode p.(Trp4428Serfs*4), 46-83% of KIAA1109 mRNA in peripheral blood evaded nonsense mediated mRNA decay as measured by qRT-PCR. These observations expand understanding of the genotype-phenotype association in KIAA1109-related disease and suggest hypotheses for milder presentations of Alkuraya-Kucinskas syndrome.