A nationwide genetic analysis of inherited retinal diseases in Israel as assessed by the Israeli inherited retinal disease consortium (IIRDC)

Inherited retinal diseases (IRDs) cause visual loss due to dysfunction or progressive degeneration of photoreceptors. These diseases show marked phenotypic and genetic heterogeneity. The Israeli IRD consortium (IIRDC) was established in 2013 with the goal of performing clinical and genetic mapping of the majority of Israeli IRD patients. To date, we recruited 2,420 families including 3,413 individuals with IRDs. On the basis of our estimation, these patients represent approximately 40% of Israeli IRD patients. To the best of our knowledge, this is, by far, the largest reported IRD cohort, and one of the first studies addressing the genetic analysis of IRD patients on a nationwide scale. The most common inheritance pattern in our cohort is autosomal recessive (60% of families). The most common retinal phenotype is retinitis pigmentosa (43%), followed by Stargardt disease and cone/cone–rod dystrophy. We identified the cause of disease in 56% of the families. Overall, 605 distinct mutations were identified, of which 12% represent prevalent founder mutations. The most frequently mutated genes were ABCA4, USH2A, FAM161A, CNGA3, and EYS. The results of this study have important implications for molecular diagnosis, genetic screening, and counseling, as well as for the development of new therapeutic strategies for retinal diseases.     

Health status in patients at risk of inherited arrhythmias and sudden unexpected death compared to the general population

Background  

The possibilities in the molecular genetics of long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM) has made family screening, with diagnostic and predictive genetic testing part of the health care offer in genetic counselling of inherited arrhythmias, potentially affecting the subjective health among these individuals. The study compared health status among patients at risk of arrhythmia because of family history or clinical diagnosis of LQTS and HCM with reference health status scores of the general population.     

Navigating the nuances of clinical sequence variant interpretation in Mendelian disease

Understanding clinical genetic test results in the era of next-generation sequencing has become increasingly complex, necessitating clear and thorough guidelines for sequence variant interpretation. To meet this need the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for a systematic approach for sequence variant interpretation in 2015. This framework is intended to be adaptable to any Mendelian condition, promoting transparency and consistency in variant interpretation, yet its comprehensive nature yields important challenges and caveats that end users must understand. In this review, we address some of these nuances and discuss the evolving efforts to refine and adapt this framework. We also consider the added complexity of distinguishing between variant-level interpretations and case-level conclusions, particularly in the context of the large gene panel approach to clinical diagnostics.     

A quantitative trait rare variant nonparametric linkage method with application to age-at-onset of Alzheimer’s disease

To analyze pedigrees with quantitative trait (QT) and sequence data, we developed a rare variant (RV) quantitative nonparametric linkage (QNPL) method, which evaluates sharing of minor alleles. RV-QNPL has greater power than the traditional QNPL that tests for excess sharing of minor and major alleles. RV-QNPL is robust to population substructure and admixture, locus heterogeneity, and inclusion of nonpathogenic variants and can be readily applied outside of coding regions. When QNPL was used to analyze common variants, it often led to loci mapping to large intervals, e.g., >40 Mb. In contrast, when RVs are analyzed, regions are well defined, e.g., a gene. Using simulation studies, we demonstrate that RV-QNPL is substantially more powerful than applying traditional QNPL methods to analyze RVs. RV-QNPL was also applied to analyze age-at-onset (AAO) data for 107 late-onset Alzheimer’s disease (LOAD) pedigrees of Caribbean Hispanic and European ancestry with whole-genome sequence data. When AAO of AD was analyzed regardless of APOE ε4 status, suggestive linkage (LOD = 2.4) was observed with RVs in KNDC1 and nominally significant linkage (p < 0.05) was observed with RVs in LOAD genes ABCA7 and IQCK. When AAO of AD was analyzed for APOE ε4 positive family members, nominally significant linkage was observed with RVs in APOE, while when AAO of AD was analyzed for APOE ε4 negative family members, nominal significance was observed for IQCK and ADAMTS1. RV-QNPL provides a powerful resource to analyze QTs in families to elucidate their genetic etiology.Subject terms: Genetic linkage study, Heritable quantitative trait     

High-throughput discovery of rare insertions and deletions in large cohorts

Pooled-DNA sequencing strategies enable fast, accurate, and cost-effect detection of rare variants, but current approaches are not able to accurately identify short insertions and deletions (indels), despite their pivotal role in genetic disease. Furthermore, the sensitivity and specificity of these methods depend on arbitrary, user-selected significance thresholds, whose optimal values change from experiment to experiment. Here, we present a combined experimental and computational strategy that combines a synthetically engineered DNA library inserted in each run and a new computational approach named SPLINTER that detects and quantifies short indels and substitutions in large pools. SPLINTER integrates information from the synthetic library to select the optimal significance thresholds for every experiment. We show that SPLINTER detects indels (up to 4 bp) and substitutions in large pools with high sensitivity and specificity, accurately quantifies variant frequency (r = 0.999), and compares favorably with existing algorithms for the analysis of pooled sequencing data. We applied our approach to analyze a cohort of 1152 individuals, identifying 48 variants and validating 14 of 14 (100%) predictions by individual genotyping. Thus, our strategy provides a novel and sensitive method that will speed the discovery of novel disease-causing rare variants.     

Analysis of rare variant population structure in Europeans explains differential stratification of gene-based tests

There is substantial interest in the role of rare genetic variants in the etiology of complex human diseases. Several gene-based tests have been developed to simultaneously analyze multiple rare variants for association with phenotypic traits. The tests can largely be partitioned into two classes – ‘burden'' tests and ‘joint'' tests – based on how they accumulate evidence of association across sites. We used the empirical joint site frequency spectra of rare, nonsynonymous variation from a large multi-population sequencing study to explore the effect of realistic rare variant population structure on gene-based tests. We observed an important difference between the two test classes: their susceptibility to population stratification. Focusing on European samples, we found that joint tests, which allow variants to have opposite directions of effect, consistently showed higher levels of P-value inflation than burden tests. We determined that the differential stratification was caused by two specific patterns in the interpopulation distribution of rare variants, each correlating with inflation in one of the test classes. The pattern that inflates joint tests is more prevalent in real data, explaining the higher levels of inflation in these tests. Furthermore, we show that the different sources of inflation between tests lead to heterogeneous responses to genomic control correction and the number of variants analyzed. Our results indicate that care must be taken when interpreting joint and burden analyses of the same set of rare variants, in particular, to avoid mistaking inflated P-values in joint tests for stronger signals of true associations.     

DNAJ mutations are rare in Chinese Parkinson's disease patients and controls

Mutations in DNAJC13, DNAJC6 and DNAJC5 have been implicated in Parkinson's disease (PD). To determine if rare coding variants in these genes play a role in PD risk in the Chinese population, we sequenced all coding exons of the three genes in 99 early-onset PD cases and 99 controls, and genotyped 8 missense variants in another 711 PD cases and 539 controls. Besides two common missense variants that did not show association with PD, the remaining missense variants were extremely rare (<0.5%), found in healthy population controls and did not show enrichment in PD cases. Our results suggest that missense mutations in DNAJC13, DNAJC5 and DNAJC6 do not play a major role in PD in the Chinese population.     

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease

PurposeCurrent diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test.MethodsWe performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.ResultsWe found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs.ConclusionRobust identification of CNVs by GS is possible within a clinical testing environment.     

Phylogenetic analysis of the mitochondrial genome indicates significant differences between patients with Alzheimer disease and controls in a French-Canadian founder population

The activity of cytochrome oxidase (CO), the terminal enzyme of the mitochondrial electron transport chain, has been reported to be lower in the brains of Alzheimer disease (AD) patients. This suggests that a modification of mitochondrial DNA (mtDNA) may be responsible for this decrease of CO activity. Many mtDNA variants were found by different studies at a higher frequency in AD patients, suggesting that mtDNA variants could confer a genetic susceptibility to AD. In this study, we sequenced the entire mitochondrial genome region that encompasses the three CO genes and the 22 mitochondrial tRNA in 69 AD patients and 83 age-matched controls. We detected a total of 95 mtDNA variants. The allele frequencies of the majority of these variants were similar in patients and controls. However, a haplotype composed of three different modifications (positions: 5633, 7476, and 15812) was present in three of the 69 late-onset AD patients (4.3%) and also in 1 of 16 early-onset AD patients (6.2%) but not in control individuals. Given that one of these variants (15812) has already been shown to be associated with another neurodegenerative disease and that all three modifications are relatively conserved and their frequencies in the general population is only 0.1%, our data suggest that the presence of this haplotype may represent a risk factor for AD. We also found a significant association (P < 0.05) of two other variants at positions 709 (rRNA 12S) and 15928 (tRNA^Thr). These two mtDNA variants are three times more frequent in control individuals compared with AD patients, suggesting that they may be protective against AD. Am. J. Med. Genet 85:20–30, 1999. © 1999 Wiley-Liss, Inc.     

Phylogenetic analysis of the mitochondrial genome indicates significant differences between patients with Alzheimer disease and controls in a French-Canadian founder population.

The activity of cytochrome oxidase (CO), the terminal enzyme of the mitochondrial electron transport chain, has been reported to be lower in the brains of Alzheimer disease (AD) patients. This suggests that a modification of mitochondrial DNA (mtDNA) may be responsible for this decrease of CO activity. Many mtDNA variants were found by different studies at a higher frequency in AD patients, suggesting that mtDNA variants could confer a genetic susceptibility to AD. In this study, we sequenced the entire mitochondrial genome region that encompasses the three CO genes and the 22 mitochondrial tRNA in 69 AD patients and 83 age-matched controls. We detected a total of 95 mtDNA variants. The allele frequencies of the majority of these variants were similar in patients and controls. However, a haplotype composed of three different modifications (positions: 5633, 7476, and 15812) was present in three of the 69 late-onset AD patients (4.3%) and also in 1 of 16 early-onset AD patients (6.2%) but not in control individuals. Given that one of these variants (15812) has already been shown to be associated with another neurodegenerative disease and that all three modifications are relatively conserved and their frequencies in the general population is only 0.1%, our data suggest that the presence of this haplotype may represent a risk factor for AD. We also found a significant association (P < 0.05) of two other variants at positions 709 (rRNA 12S) and 15928 (tRNA(Thr)). These two mtDNA variants are three times more frequent in control individuals compared with AD patients, suggesting that they may be protective against AD.     

Linkage analysis of anorexia and bulimia nervosa cohorts using selected behavioral phenotypes as quantitative traits or covariates.

To increase the likelihood of finding genetic variation conferring liability to eating disorders, we measured over 100 attributes thought to be related to liability to eating disorders on affected individuals from multiplex families and two cohorts: one recruited through a proband with anorexia nervosa (AN; AN cohort); the other recruited through a proband with bulimia nervosa (BN; BN cohort). By a multilayer decision process based on expert evaluation and statistical analysis, six traits were selected for linkage analysis (1): obsessionality (OBS), age at menarche (MENAR), and anxiety (ANX) for quantitative trait locus (QTL) linkage analysis; and lifetime minimum body mass index (BMI), concern over mistakes (CM), and food-related obsessions (OBF) for covariate-based linkage analysis. The BN cohort produced the largest linkage signals: for QTL linkage analysis, four suggestive signals: (for MENAR, at 10p13; for ANX, at 1q31.1, 4q35.2, and 8q13.1); for covariate-based linkage analyses, both significant and suggestive linkages (for BMI, one significant [4q21.1] and three suggestive [3p23, 10p13, 5p15.3]; for CM, two significant [16p13.3, 14q21.1] and three suggestive [4p15.33, 8q11.23, 10p11.21]; and for OBF, one significant [14q21.1] and five suggestive [4p16.1, 10p13.1, 8q11.23, 16p13.3, 18p11.31]). Results from the AN cohort were far less compelling: for QTL linkage analysis, two suggestive signals (for OBS at 6q21 and for ANX at 9p21.3); for covariate-based linkage analysis, five suggestive signals (for BMI at 4q13.1, for CM at 11p11.2 and 17q25.1, and for OBF at 17q25.1 and 15q26.2). Overlap between the two cohorts was minimal for substantial linkage signals.     

Variant frequencies of KCNQ1, KCNH2, and SCN5A in a Chinese inherited arrhythmia cohort and other disease cohorts undergoing genetic testing

KCNQ1, KCNH2, and SCN5A are the most common genes responsible for long QT syndrome and Brugada syndrome. However, the genetic variant frequencies of the three genes in different Chinese disease cohorts are largely unknown. In this study, we analyzed the genetic variants of KCNQ1, KCNH2, and SCN5A in patients from seven cohorts (total N = 11945, including patients clinically suspected to have inherited arrhythmia [n = 122], other cardiovascular diseases [n = 1045], epilepsy [n = 4797], or other diseases [n = 5841], and healthy controls [n = 140]) who had undergone genetic testing. All of these variants were identified via genetic testing by two Chinese companies using the Hi-Seq 2000 platform. A total of 1018 variants (minor allele frequency <0.01) were identified, with 186 (18%), 374 (37%), and 458 (46%) variants in the coding exons of KCNQ1, KCNH2, and SCN5A, respectively. Of these variants, 84% had unknown or uncertain clinical significance. The frequency of identified ClinVar pathological/likely pathological variants was higher for KCNQ1 (13/186, 7.0%) than for KCNH2 (6/374, 1.6%) or SCN5A (10/458, 2.2%), and KCNH2 held the highest number and proportion of radical mutations (30/374, 8%). The prevalence of variants was highest in the inherited arrhythmia cohort (35%) and lowest in the healthy controls (<4%), as expected. Noticeably, the variant prevalence was relatively high in the epilepsy cohort (27%). Finally, only 22 of the 82 variants (26%) identified by both companies had consistent interpretations of pathogenicity between the two companies. Our study demonstrated a comprehensive spectrum of variants in KCNQ1, KCNH2, and SCN5A in a large number of Chinese individuals, including inherited arrhythmia, cardiovascular diseases, and epilepsy. The detailed variant frequency data of each cohort could serve as a valuable reference to facilitate further variant classification by others. We also found that the interpretations of pathogenicity differed greatly among the companies.     

Diagnosing and preventing inherited disease: An analysis of multinucleated blastomere formation in human embryos

Human embryos were disaggregated into component blastomeres42–72 h after insemination. The blastomeres were scoredfor the number of nuclei present and blastomeres of known nuclearmorphology were returned to individual culture drops for 16–20h, after which they were scored for cleavage and nuclear morphology.In all, 48% of mononucleated blastomeres cleaved during thisperiod, but only 76% of these produced two mononucleated daughterblastomeres; in the remainder, one or more of the blastomereswas abnormally nucleated. During overnight culture, 30% of multinucleatedblastomeres and 30% of anucleate blastomeres cleaved, the majorityproducing abnormally nucleated daughter blastomeres. The majorityof blastomeres which showed no sign of cleavage after overnightculture retained the same nuclear morphology as when originallydisaggregated. However, a small number of mononucleated blastomerescontained two nuclei after culture, indicating that karyokinesismay have taken place in the absence of cytokinesis. Overall,30% of blastomeres with more than one nucleus seemed to ariseby this mechanism, the remainder probably arising by errorsof chromosome segregation and/or packaging at mitosis. In addition,25/111 mononucleated daughter cells arose either after abnormaldivision of mononucleated parent cells or after division ofmultinucleated cells, suggesting that 23% of newly formed mononucleatedcells might be chromosomally abnormal. The results of DNA quantitationindicated that very few (12/131, 9.2%) blastomeres (whetherunior multinucleated) had a DNA content outside the 2–4Crange. The embryos used for these studies had been culturedin one of three commonly used in-vitro fertilization (IVF) media:modified T6, Earle‘s balanced salts or Universal IVF medium(a commercial medium from Medi-Cult). A retrospective analysiswas carried out of the number of embryos containing multinucleatedblastomeres at disaggregation and of the total proportion ofisolated blastomeres which were multinucleated in three groupsof embryos, each of which had been cultured in one of the IVFmedia. Both these parameters were found to vary between cohortsof embryos cultured in the different media. The mechanism(s)by which culture medium composition might affect multinucleationof human blastomeres is discussed, as is the significance ofthese data for reliable preimplantation diagnosis of geneticstatus.     

Primary cutaneous Rosai-Dorfman disease; a case-based review of a diagnostically and therapeutically challenging rare variant

Primary cutaneous Rosai-Dorfman disease is a rare form of Rosai-Dorfman disease limited to the skin. The diagnosis of primary cutaneous disease is based on a combination of clinical presentation, histopathology, and the detection of S100+, CD68+, and CD1a− histiocytic immunophenotyping. However, the diagnosis of primary cutaneous disease is often difficult and significantly delayed due to the non-specific nature of its histologic and clinical features. In this review, we describe four cases in order to familiarize pathologists and dermatopathologists with the clinicopathologic correlation of primary cutaneous Rosai-Dorfman disease and to help facilitate early diagnosis. In addition, we discuss the proposed pathophysiology and molecular etiology of this tumor, and its relationship with IgG4 sclerosing disease.     

Integrated analysis of metabolomic profiling and exome data supplements sequence variant interpretation,classification, and diagnosis

PurposeA primary barrier to improving exome sequencing diagnostic rates is the interpretation of variants of uncertain clinical significance. We aimed to determine the contribution of integrated untargeted metabolomics in the analysis of exome sequencing data by retrospective analysis of patients evaluated by both exome sequencing and untargeted metabolomics within the same clinical laboratory.MethodsExome sequencing and untargeted metabolomic data were collected and analyzed for 170 patients. Pathogenic variants, likely pathogenic variants, and variants of uncertain significance in genes associated with a biochemical phenotype were extracted. Metabolomic data were evaluated to determine if these variants resulted in biochemical abnormalities that could be used to support their interpretation using current American College of Genetics and Genomics (ACMG) guidelines.ResultsMetabolomic data contributed to the interpretation of variants in 74 individuals (43.5%) over 73 different genes. The data allowed for the reclassification of 9 variants as likely benign, 15 variants as likely pathogenic, and 3 variants as pathogenic. Metabolomic data confirmed a clinical diagnosis in 21 cases, for a diagnostic rate of 12.3% in this population.ConclusionUntargeted metabolomics can serve as a useful adjunct to exome sequencing by providing valuable functional data that may not otherwise be clinically available, resulting in improved variant classification.     

Lipid screening in an elderly population: difficulty in interpretation and in detection of occult metabolic disease.

AIMS: To determine lipid profiles and associations with other metabolic disease in a representative British elderly population. METHODS: Part of a prevalence survey of dementia in all 75+ year olds conducted from the large general practice serving the town and surrounding area of Melton Mowbray, Leicestershire (the M-old study). Patients (n = 224) aged from 75 to 98 years, and representative of the overall population, also provided pre-prandial blood samples on which various age and nutrition related analytes were determined. These included documented medical history, thyroid stimulating hormone (TSH), glucose, immunoglobulins, and lipid profile in plasma. RESULTS: Cholesterol and lipid variables showed wide scatter, with some negative trends but no significant associations with age for total cholesterol, high density lipoprotein (HDL) cholesterol, the ratio of total to HDL cholesterol or triglycerides. Women had significantly higher concentrations of total and HDL cholesterol at all ages. Serum TSH was above 6.0 mU/1 in 10/205 patients, random glucose was above 11.2 mmol/l in nine of 207 patients, borderline dysglobulinaemia was present in four of 210 patients, all without correlation with cholesterol concentrations. CONCLUSION: This British data is consistent with an inverse correlation between survival and cholesterol, but wide scatter restricts reliance on single result lipid data in individual patient management. Random lipid screening is also unhelpful, inefficient and without added value in revealing other age related and unrecognised occult metabolic disease.     

De novo and rare inherited copy-number variations in the hemiplegic form of cerebral palsy

PurposeHemiplegia is a subtype of cerebral palsy (CP) in which one side of the body is affected. Our earlier study of unselected children with CP demonstrated de novo and clinically relevant rare inherited genomic copy-number variations (CNVs) in 9.6% of participants. Here, we examined the prevalence and types of CNVs specifically in hemiplegic CP.MethodsWe genotyped 97 unrelated probands with hemiplegic CP and their parents. We compared their CNVs to those of 10,851 population controls, in order to identify rare CNVs (<0.1% frequency) that might be relevant to CP. We also sequenced exomes of “CNV-positive” trios.ResultsWe detected de novo CNVs and/or sex chromosome abnormalities in 7/97 (7.2%) of probands, impacting important developmental genes such as GRIK2, LAMA1, DMD, PTPRM, and DIP2C. In 18/97 individuals (18.6%), rare inherited CNVs were found, affecting loci associated with known genomic disorders (17p12, 22q11.21) or involving genes linked to neurodevelopmental disorders.ConclusionWe found an increased rate of de novo CNVs in the hemiplegic CP subtype (7.2%) compared to controls (1%). This result is similar to that for an unselected CP group. Combined with rare inherited CNVs, the genomic data impacts the understanding of the potential etiology of hemiplegic CP in 23/97 (23.7%) of participants.