Project description:Congenital Heart Disease (CHD) accounts for 1% of birth defects, and while large-scale genetic studies have uncovered genes associated with CHDs, identifying causal mutations remains a challenge. We hypothesized that genetic determinants for CHDs could be found in the protein interactomes of GATA4 and TBX5, two cardiac transcription factors (TFs) associated with CHDs. Defining their interactomes in human cardiac progenitors via affinity purification-mass spectrometry and integrating the results with genetic data from the Pediatric Cardiac Genomic Consortium (PCGC) revealed an enrichment of de novo variants among proteins that interact with GATA4 or TBX5. A consolidative score designed to prioritize TF interactome members based on distinctive variant, gene and proband features identified numerous likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied cardiac developmental genes resulting in co-activation and the GLYR1 variant associated with CHD disrupted interaction with GATA4. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating the contribution of genetic variants in CHD and can be extended to other genetic diseases.
Project description:Hypoplastic left heart syndrome (HLHS) is a heterogeneous, lethal combination of congenital malformations that result in a heart unable to sustain systemic circulation. The genetic determinants of this disorder are largely unknown. Evidence of copy number variants (CNVs) contributing to the genetic etiology of HLHS and other congenital heart defects (CHDs) has been mounting. However, the functional effects of such CNVs have not been examined, particularly in cases where the variant of interest is found in only a single patient. Initially whole-genome SNP microarrays were employed to detect CNVs in two patient cohorts (N = 70 total) predominantly diagnosed with some form of nonsyndromic HLHS. We discovered 16 rare variants adjacent to or overlapping 20 genes associated with cardiovascular or premature lethality phenotypes in mouse knockout models. Fifteen of the 16 variants were identified in separate patients, suggestive of a private mutation model of disease. We evaluated the impact of selected variants on the expression of nine of these genes through quantitative PCR on cDNA derived from patient heart tissue. Four genes displayed significantly altered expression in patients with an overlapping or proximal CNV verses patients without such CNVs. Thus, rare and private genomic imbalances perturb transcription of genes affecting cardiogenesis in a subset of nonsyndromic HLHS patients. Some of these genes influence extracellular matrix structure, cardiac neural crest development, and coronary vascularization. A total of 70 samples from CHD patients, mostly with nonsyndromic HLHS, yielded SNP genotypes and probe intensity ratios of sufficient quality for CNV identification. Two classes of CNV detection algorithms (HMM and CBS) were employed. After identification, concordant entries were subjected to selection criteria based on rarity and gene content, which produced putative candidate genes for follow-up experiments.
Project description:Proteome profiles of neutrophil granulocytes from patients with severe congenital neutropenia of the genotypes SRP54, SRP19, SRPRA, ELANE, and HAX1
Project description:Transcriptional profiling of rat liver comparing male rats with congenital hypothyrodism (CH) vs intact at adulhood. Here we studied how CH influences liver gene expression program in adulthood. Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology but its specific influence in liver is less understood. Here we studied how CH influences liver gene expression program in adulthood. Pregnant rats were given anti-thyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as a reduction in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, feed efficiency increased in CH and this was accompanied by significant catch-up growth. On PND80, significant reduction in body mass, tail length, and circulating IGF-I remained in CH rats. On the other hand, mRNA levels of known GH targeted genes were significantly up-regulated. Serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in expression for hepatic genes involved in lipid metabolism with an increased transcription of PPAR and reduced expression of genes involved in fatty acids and cholesterol uptake, cellular sterol efflux, triglycerides assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to hypothyroidism onset in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters, and to T3 replacement with enhanced activation of lipogenic transcriptional program. In summary, we provided in vivo evidence that neonatal hypothyroidism causes long-lasting effects on hepatic transcriptional program and tissue sensitivity to hormone treatment. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood. Two conditions CH vs INTACT male rats. Biological replicates: Four independent hybridizations: 4 controls (age-matched intact rats) vs 4 CH (male rats with congenital hypothyroidism) on postnatal day 80 for a total of four arrays. One replicate per array.
Project description:Although most disease associations detected by GWAS are nongenic, very few have been mapped to causal regulatory variants. Here, we present a method for detecting regulatory QTLs that does not require genotyping or whole-genome sequencing. The method combines deep, long-read ChIP-seq with a new statistical test that simultaneously scores peak height correlation and allelic imbalance: the Genotype-independent Signal Correlation and Imbalance (G-SCI) test. We performed histone acetylation ChIP-seq on 57 human lymphoblastoid cell lines and used the resulting reads to call 500,066 SNPs de novo within regulatory elements. The G-SCI test annotated 8,764 of these as histone acetylation QTLs (haQTLs) - an order of magnitude larger than the set of candidates detected by expression QTL analysis. Lymphoblastoid haQTLs were highly predictive of autoimmune disease mechanisms. Thus, our method facilitates large-scale regulatory variant detection in any moderately-sized cohort for which functional profiling data can be generated, thus simplifying identification of causal variants within GWAS loci. We applied our method, named Regulatory Variant Ascertainment and chromatin Regression by sequencing (RegVAR-seq), to 57 cell lines from a single population group. We used the resulting sequence data for variant calling, and validated calls using an independent platform. We then identified histone acetylation QTLs (haQTLs) using a novel statistical test that requires no prior genotype information and combines peak height and allelic imbalance data across the 57 individuals. Transcription factor binding site analysis was used to independently support the functionality of haQTLs. Finally, we examined the association between haQTLs and SNPs associated with human phenotypes.
Project description:Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal dominant inheritance. Here, we describe a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation affecting the rod domain of the protein. To demonstrate pathogenicity of this homozygous FLNC-mutation described, ultra-morphological, proteomic and functional investigations were performed in addition to immunological studies of known marker proteins for dominant filaminopathies. Our results showed that the mutant protein is expressed to similar quantities as the wildtype variant in control skeletal muscle fibres, alters the proteomic signature of quadriceps muscle, and results in the presence of ultrastructural perturbations. Moreover, comparable findings for filaminopathy marker proteins were found in both, our homozygous and a dominant case. The mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wildtype variant. These combined findings extend the currently recognized clinical, genetic and biochemical spectrum of filaminopathies. The unusual congenital presentation of the disease indicates that homozygosity for a mutation in filamin C severely aggravates the phenotype.