Analysis of 51 proposed hypertrophic cardiomyopathy genes from genome sequencing data in sarcomere negative cases has negligible diagnostic yield.

PURPOSE: Increasing numbers of genes are being implicated in Mendelian disorders and incorporated into clinical test panels. However, lack of evidence supporting the gene-disease relationship can hinder interpretation. We explored the utility of testing 51 additional genes for hypertrophic cardiomyopathy (HCM), one of the most commonly tested Mendelian disorders. METHODS: Using genome sequencing data from 240 sarcomere gene negative HCM cases and 6229 controls, we undertook case-control and individual variant analyses to assess 51 genes that have been proposed for HCM testing. RESULTS: We found no evidence to suggest that rare variants in these genes are prevalent causes of HCM. One variant, in a single case, was categorized as likely to be pathogenic. Over 99% of variants were classified as a variant of uncertain significance (VUS) and 54% of cases had one or more VUS. CONCLUSION: For almost all genes, the gene-disease relationship could not be validated and lack of evidence precluded variant interpretation. Thus, the incremental diagnostic yield of extending testing was negligible, and would, we propose, be outweighed by problems that arise with a high rate of uninterpretable findings. These findings highlight the need for rigorous, evidence-based selection of genes for clinical test panels.

A genome-wide association study of congenital cardiovascular left-sided lesions shows association with a locus on chromosome 20.

Congenital heart defects involving left-sided lesions (LSLs) are relatively common birth defects with substantial morbidity and mortality. Previous studies have suggested a high heritability with a complex genetic architecture, such that only a few LSL loci have been identified. We performed a genome-wide case-control association study to address the role of common variants using a discovery cohort of 778 cases and 2756 controls. We identified a genome-wide significant association mapping to a 200 kb region on chromosome 20q11 [P= 1.72 × 10-8 for rs3746446; imputed Single Nucleotide Polymorphism (SNP) rs6088703 P= 3.01 × 10-9, odds ratio (OR)= 1.6 for both]. This result was supported by transmission disequilibrium analyses using a subset of 541 case families (lowest P in region= 4.51 × 10-5, OR= 1.5). Replication in a cohort of 367 LSL cases and 5159 controls showed nominal association (P= 0.03 for rs3746446) resulting in P= 9.49 × 10-9 for rs3746446 upon meta-analysis of the combined cohorts. In addition, a group of seven SNPs on chromosome 1q21.3 met threshold for suggestive association (lowest P= 9.35 × 10-7 for rs12045807). Both regions include genes involved in cardiac development-MYH7B/miR499A on chromosome 20 and CTSK, CTSS and ARNT on chromosome 1. Genome-wide heritability analysis using case-control genotyped SNPs suggested that the mean heritability of LSLs attributable to common variants is moderately high ([Formula: see text] range= 0.26-0.34) and consistent with previous assertions. These results provide evidence for the role of common variation in LSLs, proffer new genes as potential biological candidates, and give further insight to the complex genetic architecture of congenital heart disease.

Increased Power for Detection of Parent-of-Origin Effects via the Use of Haplotype Estimation.

Parent-of-origin (or imprinting) effects relate to the situation in which traits are influenced by the allele inherited from only one parent and the allele from the other parent has little or no effect. Given SNP genotype data from case-parent trios, the parent of origin of each allele in the offspring can often be deduced unambiguously; however, this is not true when all three individuals are heterozygous. Most existing methods for investigating parent-of-origin effects operate on a SNP-by-SNP basis and either perform some sort of averaging over the possible parental transmissions or else discard ambiguous trios. If the correct parent of origin at a SNP could be determined, this would provide extra information and increase the power for detecting the effects of imprinting. We propose making use of the surrounding SNP information, via haplotype estimation, to improve estimation of parent of origin at a test SNP for case-parent trios, case-mother duos, and case-father duos. This extra information is then used in a multinomial modeling approach for estimating parent-of-origin effects at the test SNP. We show through computer simulations that our approach has increased power over previous approaches, particularly when the data consist only of duos. We apply our method to two real datasets and find a decrease in significance of p values in genomic regions previously thought to possibly harbor imprinting effects, thus weakening the evidence that such effects actually exist in these regions, although some regions retain evidence of significant effects.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium.

Most patients with Ellis-van Creveld syndrome (EvC) are identified with pathogenic changes in EVC or EVC2, however further genetic heterogeneity has been suggested. In this report we describe pathogenic splicing variants in WDR35, encoding retrograde intraflagellar transport protein 121 (IFT121), in three families with a clinical diagnosis of EvC but having a distinctive phenotype. To understand why WDR35 variants result in EvC, we analysed EVC, EVC2 and Smoothened (SMO) in IFT-A deficient cells. We found that the three proteins failed to localize to Wdr35(-/-) cilia, but not to the cilium of the IFT retrograde motor mutant Dync2h1(-/-), indicating that IFT121 is specifically required for their entry into the ciliary compartment. Furthermore expression of Wdr35 disease cDNAs in Wdr35(-/-) fibroblasts revealed that the newly identified variants lead to Hedgehog signalling defects resembling those of Evc(-/-) and Evc2(-/-) mutants. Together our data indicate that splicing variants in WDR35, and possibly in other IFT-A components, underlie a number of EvC cases by disrupting targeting of both the EvC complex and SMO to cilia.

Genotype-phenotype correlations in Cornelia de Lange syndrome: Behavioral characteristics and changes with age.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder associated with unusual facial features, limb abnormalities, a wide range of health conditions, and intellectual disability. Mutations in five genes that encode (SMC1A, SMC3, RAD21) or regulate (NIPBL, HDAC8) the cohesin complex have been identified in up to 70% of individuals. Genetic cause remains unknown for a proportion of individuals. There is substantial heterogeneity in all aspects of CdLS but very little is known about what predicts phenotypic heterogeneity. In this study, we evaluated genotype-phenotype associations in 34 individuals with CdLS. Participants with NIPBL mutations had significantly lower self help skills and were less likely to have verbal skills relative to those who were negative for the NIPBL mutation. No significant differences were identified between the groups in relation to repetitive behavior, mood, interest and pleasure, challenging behavior, activity, impulsivity, and characteristics of autism spectrum disorder whilst controlling differences in self help skills. Significant correlations indicating lower mood, interest and pleasure, and increased insistence on sameness with older age were identified for those who were NIPBL mutation positive. The findings suggest similarities in the behavioral phenotype between those with and without the NIPBL mutation once differences in self help skills are controlled for. However, there may be subtle differences in the developmental trajectory of these behaviors according to genetic mutation status in CdLS.

Using population data for assessing next-generation sequencing performance.

MOTIVATION: During the past 4 years, whole-exome sequencing has become a standard tool for finding rare variants causing Mendelian disorders. In that time, there has also been a proliferation of both sequencing platforms and approaches to analyse their output. This requires approaches to assess the performance of different methods. Traditionally, criteria such as comparison with microarray data or a number of known polymorphic sites have been used. Here we expand such approaches, developing a maximum likelihood framework and using it to estimate the sensitivity and specificity of whole-exome sequencing data. RESULTS: Using whole-exome sequencing data for a panel of 19 individuals, we show that estimated sensitivity and specificity are similar to those calculated using microarray data as a reference. We explore the effect of frequency misspecification arising from using an inappropriately selected population and find that, although the estimates are affected, the rankings across procedures remain the same. AVAILABILITY AND IMPLEMENTATION: An implementation using Perl and R can be found at busso.ncl.ac.uk (Username: igm101; Password: Z1z1nts).

The Cockayne Syndrome Natural History (CoSyNH) study: clinical findings in 102 individuals and recommendations for care.

PURPOSE: Cockayne syndrome (CS) is a rare, autosomal-recessive disorder characterized by microcephaly, impaired postnatal growth, and premature pathological aging. It has historically been considered a DNA repair disorder; fibroblasts from classic patients often exhibit impaired transcription-coupled nucleotide excision repair. Previous studies have largely been restricted to case reports and small series, and no guidelines for care have been established. METHODS: One hundred two study participants were identified through a network of collaborating clinicians and the Amy and Friends CS support groups. Families with a diagnosis of CS could also self-recruit. Comprehensive clinical information for analysis was obtained directly from families and their clinicians. RESULTS AND CONCLUSION: We present the most complete evaluation of Cockayne syndrome to date, including detailed information on the prevalence and onset of clinical features, achievement of neurodevelopmental milestones, and patient management. We confirm that the most valuable prognostic factor in CS is the presence of early cataracts. Using this evidence, we have created simple guidelines for the care of individuals with CS. We aim to assist clinicians in the recognition, diagnosis, and management of this condition and to enable families to understand what problems they may encounter as CS progresses.Genet Med 18 5, 483-493.

Positional mapping of PRKD1, NRP1 and PRDM1 as novel candidate disease genes in truncus arteriosus.

BACKGROUND: Truncus arteriosus (TA) is characterised by failure of septation of the outflow tract into aortic and pulmonary trunks and is associated with high morbidity and mortality. Although ranked among the least common congenital heart defects, TA provides an excellent model for the role of individual genes in cardiac morphogenesis as exemplified by TBX1 deficiency caused by point mutations or, more commonly, hemizygosity as part of the 22q11.2 deletion syndrome. The latter genetic lesion, however, is only observed in a proportion of patients with TA, which suggests the presence of additional disease genes. OBJECTIVE: To identify novel genes that cause Mendelian forms of TA. METHODS AND RESULTS: We exploited the occurrence of monogenic forms of TA in the Saudi population, which is characterised by high consanguinity, a feature conducive to the occurrence of Mendelian phenocopies of complex phenotypes as we and others have shown. Indeed, we demonstrate in two multiplex consanguineous families that we are able to map TA to regions of autozygosity in which whole-exome sequencing revealed homozygous truncating mutations in PRKD1 (encoding a kinase derepressor of MAF2) and NRP1 (encoding a coreceptor of vascular endothelial growth factor (VEGFA)). Previous work has demonstrated that Prkd1(-/-) is embryonic lethal and that its tissue-specific deletion results in abnormal heart remodelling, whereas Nrp1(-/-) develops TA. Surprisingly, molecular karyotyping to exclude 22q11.2 deletion syndrome in the replication cohort of 17 simplex TA cases revealed a de novo hemizygous deletion that encompasses PRDM1, deficiency of which also results in TA phenotype in mouse. CONCLUSIONS: Our results expand the repertoire of molecular lesions in chromatin remodelling and transcription factors that are implicated in the pathogenesis of congenital heart disease in humans and attest to the power of monogenic forms of congenital heart diseases as a complementary approach to dissect the genetics of these complex phenotypes.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.