Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients.

BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.

DSP p.(Thr2104Glnfs*12) variant presents variably with early onset severe arrhythmias and left ventricular cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a condition characterized by dilatation and systolic dysfunction of the left ventricle in the absence of severe coronary artery disease or abnormal loading conditions. Mutations in the titin (TTN) and lamin A/C (LMNA) genes are the two most significant contributors in familial DCM. Previously mutations in the desmoplakin (DSP) gene have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) and more recently with DCM. METHODS: We describe the cardiac phenotype related to a DSP mutation which was identified in ten unrelated Finnish index patients using next-generation sequencing. Sanger sequencing was used to verify the presence of this DSP variant in the probands' relatives. Medical records were obtained, and clinical evaluation was performed. RESULTS: We identified DSP c.6310delA, p.(Thr2104Glnfs*12) variant in 17 individuals of which 11 (65%) fulfilled the DCM diagnostic criteria. This pathogenic variant presented with left ventricular dilatation, dysfunction and major ventricular arrhythmias. Two patients showed late gadolinium enhancement (LGE) and myocardial edema on cardiac magnetic resonance imaging (MRI) that may suggest inflammatory process at myocardium. CONCLUSIONS: The patients diagnosed with DCM showed an arrhythmogenic phenotype as well as SCD at young age supporting the recently proposed concept of arrhythmogenic cardiomyopathy. This study also demonstrates relatively low penetrance of truncating DSP variant in the probands' family members by the age of 40. Further studies are needed to elucidate the possible relations between myocardial inflammation and pathogenic DSP variants.

Diagnostic utility of next-generation sequencing-based panel testing in 543 patients with suspected skeletal dysplasia.

BACKGROUND: Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. The molecular diagnostic utility of multi-gene panel testing using next-generation sequencing (NGS) has not yet been characterized for an unselected population of individuals with suspected skeletal dysplasia. In this study, we retrospectively reviewed patient reports to assess the diagnostic yield, reported variant characteristics, impact of copy number variation, and performance in prenatal diagnostics of panel tests for variants in genes associated with skeletal dysplasia and growth disorders. RESULTS: Clinical reports of consecutive patients with a clinical indication of suspected skeletal dysplasia who underwent panel testing were examined. The 543 patients included in the study submitted samples for diagnostic genetic testing with an indication of suspected skeletal dysplasia or growth disorder and received one of three nested panel tests. A molecular diagnosis was established in 42.0% of patients (n = 228/543). Diagnostic variants were identified in 71 genes, nearly half of which (n = 35, 49.3%) contributed uniquely to a molecular diagnosis for a single patient in this cohort. Diagnostic yield was significantly higher among fetal samples (58.0%, n = 51/88) than postnatal samples (38.9%, n = 177/455; z = 3.32, p < 0.0009). Diagnostic variants in fetal cases were identified across 18 genes. Thirteen diagnostic CNVs were reported, representing 5.7% of diagnostic findings and ranging in size from 241-bp to whole chromosome aneuploidy. Additionally, 11.4% (36/315) of non-diagnostic patient reports had suspicious variants of unknown significance (VUS), in which additional family studies that provide segregation data and/or functional characterization may result in reclassification to likely pathogenic. CONCLUSIONS: These findings demonstrate the utility of panel testing for individuals with a suspected skeletal dysplasia or growth disorder, with a particularly high diagnostic yield seen in prenatal cases. Pursuing comprehensive panel testing with high-resolution CNV analysis can provide a diagnostic benefit, given the considerable phenotype overlap amongst skeletal dysplasia conditions.

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy.

BACKGROUND: Familial dilated cardiomyopathy (DCM) is typically a monogenic disorder with dominant inheritance. Although over 40 genes have been linked to DCM, more than half of the patients undergoing comprehensive genetic testing are left without molecular diagnosis. Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) were identified in a few patients with sporadic DCM. METHODS AND RESULTS: We determined the frequency of rare NRAP variants in a cohort of DCM patients and control patients to further evaluate role of this gene in cardiomyopathies. A retrospective analysis of our internal variant database consisting of 31,639 individuals who underwent genetic testing (either panel or direct exome sequencing) was performed. The DCM group included 577 patients with either a confirmed or suspected DCM diagnosis. A control cohort of 31,062 individuals, including 25,912 individuals with non-cardiac (control group) and 5,150 with non-DCM cardiac indications (Non-DCM cardiac group). Biallelic (n = 6) or two (n = 5) NRAP variants (two PTVs or PTV+missense) were identified in 11 unrelated probands with DCM (1.9%) but none of the controls. None of the 11 probands had an alternative molecular diagnosis. Family member testing supports co-segregation. Biallelic or potentially biallelic NRAP variants were enriched in DCM vs. controls (OR 1052, p<0.0001). Based on the frequency of NRAP PTVs in the gnomAD reference population, and predicting full penetrance, biallelic NRAP variants could explain 0.25%-2.46% of all DCM cases. CONCLUSION: Loss-of-function in NRAP is a cause for autosomal recessive dilated cardiomyopathy, supporting its inclusion in comprehensive genetic testing.

Vascular endothelial growth factor gene variation and the response to photodynamic therapy in age-related macular degeneration.

PURPOSE: To evaluate the role of vascular endothelial growth factor (VEGF) gene polymorphisms in exudative age-related macular degeneration (AMD). DESIGN: Retrospective, comparative case series. PARTICIPANTS: Patients with recent exudative AMD (n = 162) and age-matched subjects without AMD (n = 85). METHODS: Fluorescein angiography (FA), clinical examination, and single nucleotide polymorphism (SNP) genotyping. MAIN OUTCOME MEASURES: The frequencies of 3 VEGF gene SNPs were analyzed, 1 at the promoter site (rs699947, A-->C) and 2 intronic SNPs (rs2146323, A-->C, and rs3025033, A-->G), in relation to the risk of AMD, to choroidal neovascular (CNV) lesion size and configuration, and to the anatomic response to photodynamic therapy (PDT). These SNPs were chosen to cover all the haploblocks of the VEGF gene. The 86 patients who had undergone PDT were classified as either PDT responders or PDT nonresponders based on the outcome of PDT after the last treatment session. For the PDT responders, the treating physician had deemed the lesion to be clinically dry and without leakage from CNV in FA at a visit scheduled at least 12 weeks after the last PDT treatment. For the PDT nonresponders, the PDT sessions had been discontinued by the treating retina specialist because of an apparently poor response and a still exudative lesion after several PDT sessions. RESULTS: The presence of exudative AMD or lesion size or configuration was not associated with the SNPs studied here. The frequencies of the rs699947 were significantly different in PDT nonresponders and PDT responders. The AA, AC, and CC genotypes were 14%, 39%, and 46%, respectively, in PDT nonresponders, compared with 40%, 48%, and 12%, respectively, in the PDT responders (P = 0.0008). The corresponding frequencies for the rs2146323 AA, AC, and CC genotypes were 4%, 32%, and 64%, respectively, in nonresponders and 24%, 38%, and 38%, respectively, in responders (P = 0.0369). The genotypes of the rs3025033 SNP were distributed evenly between the responders and nonresponders. CONCLUSIONS: The VEGF gene polymorphic SNPs at rs699947 and rs2146323 are strong determinants of the anatomic outcome after PDT, but the SNPs studied were not associated with the presence of exudative AMD or with the CNV lesion size or configuration. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Variation in the vascular endothelial growth factor gene, carotid intima-media thickness and the risk of acute myocardial infarction.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor, but its role in atherogenesis is still unclear. Our goal was to study whether three variants of the VEGF gene, previously associated with VEGF production, are linked to atherosclerosis defined as carotid intima-media thickness (IMT) and as the risk of acute myocardial infarction (AMI). MATERIAL AND METHODS: Three VEGF gene single nucleotide polymorphisms (SNPs) (-2578A>C rs699947, -634C>G rs2010963 and +936C>T rs3025039) were genotyped in 516 control subjects of the OPERA (Oulu Project Elucidating Risk of Atherosclerosis) cohort and in 251 survivors of AMI. In the OPERA cohort, the genotyped SNPs were analysed for their association with IMT. The SNPs were also analysed for their association with the risk of AMI, a complication of advanced atherosclerosis. In addition, haplotype frequencies and their associated effects on IMT and on the risk of AMI were estimated. RESULTS: None of the single genotyped polymorphisms was significantly associated with overall IMT or with the risk of AMI. However, the haplotype CCC was associated with higher overall IMT without plaques in women (p = 0.01, haplotypic effect +0.03 mm), the haplotype CCT with higher IMT without plaques in the internal carotid artery in men (p = 0.001, +0.11), while the haplotype AGT was associated with reduced AMI risk (p = 0.015, OR = 0.46). CONCLUSIONS: Variation in the VEGF gene is weakly associated with IMT and the risk of AMI, but the effect can only be observed when the information of the SNPs is combined by constructing haplotypes.

Clinical and ultrastructural findings in an ataxic variant of Kufor-Rakeb syndrome.

INTRODUCTION: Kufor-Rakeb syndrome (KRS) is a rare autosomal recessive neurodegenerative disorder manifesting as juvenile-onset atypical parkinsonism with pyramidal signs, supranuclear gaze palsy, dementia and characteristic minimyoclonus, with a notable phenotype variability. The responsible gene ATP13A2 was also associated with hereditary spastic paraplegia, uncomplicated early - or late-onset parkinsonism and a form of neuronal ceroid lipofuscinosis. We present clinical and ultrastructural findings in a 28-year-old woman with novel biallelic ATP13A2 mutations. MATERIAL AND METHODS: An ultrastructural study of the skin and muscle sample was carried out. Sequence analysis of all protein coding exons and exon-intron boundaries of genes was performed on patient's genomic DNA. A proprietary oligonucleotide-selective sequencing method was used for capturing genomic targets and sequencing was performed using Illumina sequencing system. RESULTS: The patient presented with juvenile-onset progressive parkinsonian syndrome and cognitive deterioration, accompanied by mild spastic paraplegia, supranuclear gaze palsy, cerebellar syndrome, peripheral neuropathy and fine myoclonus. Plentiful and varied osmiophilic deposits were found in skin and muscle biopsy. Sequence analysis identified two novel heterozygous variants in ATP13A2: a nonsense variant c.2209C>T, p.(Gln737*) and a 2-bp deletion c.2366_2367delTC, p.(Leu789Argfs*15) causing a frameshift leading to a premature stop codon. Oral levodopa treatment was initiated resulting in marked improvement of bradykinesia, rigidity, speech and swallowing. CONCLUSIONS: We report two novel ATP13A2 pathogenic mutations, further expanding the phenotype of Kufor-Rakeb syndrome with the unusual features of ataxia and polyneuropathy. We thoroughly describe ultrastructural findings and document a meaningful response to levodopa.

Impaired HDL2-mediated cholesterol efflux is associated with metabolic syndrome in families with early onset coronary heart disease and low HDL-cholesterol level.

OBJECTIVE: The potential of high-density lipoproteins (HDL) to facilitate cholesterol removal from arterial foam cells is a key function of HDL. We studied whether cholesterol efflux to serum and HDL subfractions is impaired in subjects with early coronary heart disease (CHD) or metabolic syndrome (MetS) in families where a low HDL-cholesterol level (HDL-C) predisposes to early CHD. METHODS: HDL subfractions were isolated from plasma by sequential ultracentrifugation. THP-1 macrophages loaded with acetyl-LDL were used in the assay of cholesterol efflux to total HDL, HDL2, HDL3 or serum. RESULTS: While cholesterol efflux to serum, total HDL and HDL3 was unchanged, the efflux to HDL2 was 14% lower in subjects with MetS than in subjects without MetS (p<0.001). The efflux to HDL2 was associated with components of MetS such as plasma HDL-C (r = 0.76 in men and r = 0.56 in women, p<0.001 for both). The efflux to HDL2 was reduced in men with early CHD (p<0.01) only in conjunction with their low HDL-C. The phospholipid content of HDL2 particles was a major correlate with the efflux to HDL2 (r = 0.70, p<0.001). A low ratio of HDL2 to total HDL was associated with MetS (p<0.001). CONCLUSION: Our results indicate that impaired efflux to HDL2 is a functional feature of the low HDL-C state and MetS in families where these risk factors predispose to early CHD. The efflux to HDL2 related to the phospholipid content of HDL2 particles but the phospholipid content did not account for the impaired efflux in cardiometabolic disease, where a combination of low level and poor quality of HDL2 was observed.

Plasma levels of antibodies against oxidized LDL are inherited but not associated with HDL-cholesterol level in families with early coronary heart disease.

OBJECTIVE: Oxidized low-density lipoproteins (oxLDL) and antibodies against them (anti-oxLDLs) are thought to play a central role in atherosclerosis. One proposed antiatherosclerotic mechanism for HDL is to prevent oxidation of LDL. This study examined whether plasma HDL-cholesterol (HDL-C) is related to plasma anti-oxLDL levels. METHODS: We collected families based on probands with low HDL-C and premature coronary heart disease (CHD). Antibody levels were determined in samples from 405 subjects. Immunoglobulin G, M and A levels against two in vitro models of oxLDL, malondialdehyde-acetaldehyde-modified LDL (MAA-LDL) and copper oxidized LDL (CuOx-LDL), were measured by ELISA. We carried out heritability estimation of antibody traits and bivariate analyses between HDL-C, LDL-C and antibody traits. RESULTS: All the antibody levels were significantly inherited (p < 0.001), heritability estimates ranging from 0.28 to 0.65. HDL-C exhibited no environmental or genetic cross-correlations with antibody levels. Significant environmental correlations were detected between LDL-C and both IgG levels (ρ(E) = 0.40, p = 0.046 and ρ(E) = 0.39, p < 0.001). There were no differences in antibody levels between subjects with normal and low HDL-C, or between CHD-affected and non-affected subjects. CONCLUSION: In this study, low HDL-C level displayed no significant associations with the anti-oxLDL levels measured. The heritability of the anti-oxLDL levels was a novel and interesting finding.

Genome scan for loci regulating HDL cholesterol levels in Finnish extended pedigrees with early coronary heart disease.

Coronary heart disease (CHD) is the leading cause of mortality in Western societies. Its risk is inversely correlated with plasma high-density lipoprotein cholesterol (HDL-C) levels, and approximately 50% of the variability in these levels is genetically determined. In this study, the aim was to carry out a whole-genome scan for the loci regulating plasma HDL-C levels in 35 well-defined Finnish extended pedigrees (375 members genotyped) with probands having low HDL-C levels and premature CHD. The additive genetic heritability of HDL-C was 43%. A variance component analysis revealed four suggestive quantitative trait loci (QTLs) for HDL-C levels, with the highest LOD score, 3.1, at the chromosomal locus 4p12. Other suggestive LOD scores were 2.1 at 2q33, 2.1 at 6p24 and 2.0 at 17q25. Three suggestive loci for the qualitative low HDL-C trait were found, with a nonparametric multipoint score of 2.6 at the chromosomal locus 10p15.3, 2.5 at 22q11 and 2.1 at 6p12. After correction for statin use, the strongest evidence of linkage was shown on chromosomes 4p12, 6p24, 6p12, 15q22 and 22q11. To search for the underlying gene on chromosome 6, we analyzed two functional and positional candidate genes (peroxisome proliferator-activated receptor-delta (PPARD), and retinoid X receptor beta, (RXRB)), but found no significant evidence of association. In conclusion, we identified seven chromosomal regions for HDL-C regulation exceeding the level for suggestive evidence of linkage.

Genetic and environmental determinants of total and high-molecular weight adiponectin in families with low HDL-cholesterol and early onset coronary heart disease.

OBJECTIVE: Plasma adiponectin and high-density lipoprotein cholesterol (HDL-C) exhibit a well-known positive metabolic correlation. Neither heritability nor genome-wide linkage analysis for the high-molecular weight (HMW) adiponectin is available. This work estimates the genetic and environmental determinants and the heritabilities of the adiponectins and lipid traits in Finnish families with early onset coronary heart disease (CHD) and low HDL-C. METHODS: Heritability and genome-wide univariate linkage analysis was performed for total and HMW adiponectin in extended families from Northern Finland with early onset CHD and low HDL-C using a variance components approach. The genetic and environmental correlations between the plasma adiponectins and various lipid traits were also studied and a bivariate analysis for HDL-C and the adiponectins carried out. RESULTS: In the partial correlation analysis (adjusted for sex, age, BMI and statin use) the adiponectins showed a stronger correlation with HDL-C (total 0.57, p=0.001, HMW 0.51, p<0.005) than with any other lipid trait in unrelated subjects. Our estimates detected strong heritability for total (0.53+/-0.10), HMW (0.51+/-0.10) and the HMW/total adiponectin ratio (0.68+/-0.11). Univariate linkage analysis showed suggestive evidence of linkage on chromosome 11p15 for total adiponectin and on 3q13.2-q24 and 6p21 for the HMW adiponectin. The strongest environmental cross-correlation between the adiponectins and lipids was seen between HDL-C and total adiponectin (rhoe=0.64, p<0.05), whereas the strongest genetic correlation was detected between low-density lipoprotein cholesterol and the HMW adiponectin (rhog=-0.48, p<0.05). CONCLUSION: No significant genetic correlations between HDL-C and the adiponectins were observed. Therefore, the metabolic association between HDL-C and adiponectin is most likely regulated by complex genetic pathways and environmental factors.