Copy number variants in candidate genes are genetic modifiers of Hirschsprung disease
ABSTRACT: Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. The HSCR phenotype is highly variable with respect to gender, segment length of aganglionosis, familiality and the presence of additional anomalies. By molecular genetic analysis, a minimum of 11 neuro-developmental genes (RET, GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, KIAA1279, TCF4) are known to harbor rare high-penetrance mutations that confer a large risk to the bearer. In addition, two other genes (RET, NRG1) harbor common low-penetrance polymorphisms that contribute only partially to risk and act as genetic modifiers. To broaden this search, we examined whether a set of 67 proven and candidate HSCR genes harbored additional modifier alleles. In this pilot study, we utilized a custom-designed array CGH with ~33,000 test probes at an average resolution of ~185bp to detect gene-sized or smaller copy number variants (CNVs) within these 67 genes in 18 heterogeneous HSCR patients. Using stringent criteria, we identified CNVs at three loci (MAPK10, ZFHX1B, SOX2) that are novel, involve regulatory and coding sequences of these neuro-developmental genes and show association with HSCR in combination with other congenital anomalies. Two-condition experiment: Patient vs. Control. Sex-matched controls. Technical replicates: 4 were examined twice and 3 were studied in triplicate. Technical replicates: 408.3.1, 408.3.2 Technical replicates: 300.3.1, 300.3.2 Technical replicates: 354.3.1, 354.3.2 Technical replicates: 355.3.1, 355.3.2 Technical replicates: 63.3.1, 63.3.2, 63.3.3 Technical replicates: 122.7.1, 122.7.2, 122.7.3 Technical replicates: 413.3.1, 413.3.2, 413.3.3
Project description:Analysis of Retinoblastoma protein (Rb) (Hs.408528) dependent transcriptional changes following siRNA mediated ablation of the RET finger protein/ Tripartite protein (RFP/TRIM27) (Hs.440382). Common reference design, two biological replicates with two technical replicates each.
Project description:For most multigenic disorders, clinical manifestation (penetrance) and presentation (expressivity) are likely to be an outcome of genetic interaction between multiple susceptibility genes. Here, using gene knockouts in mice we evaluated genetic interaction between loss of Ret and loss of Sema3d, two Hirschsprung disease (HSCR) susceptibility genes. We intercrossed Ret and Sema3d double null heterozygotes to generate mice with the nine possible genotypes and assessed survival by counting various genotypes, myenteric plexus development by acetylcholinesterase (AchE) staining and embryonic day 12.5 (E12.5) gut transcriptome by RNA-sequencing. Survival rates of Ret wildtype, null heterozygote and null homozygote mice at E12.5, birth and weaning were not influenced by the genotypes at Sema3d locus and vice-versa. Loss of myenteric plexus was observed only in all Ret null homozygotes, irrespective of the genotypes at Sema3d locus, and Sema3d null heterozygote and homozygote mice had normal gut innervation. As compared to wildtype mice gut gene expression, loss of Ret in null homozygotes led to differential expression of ~300 genes, whereas loss of Sema3d in null homozygotes had no major consequence and there was no evidence supporting major interaction between the two genes influencing gut transcriptome. Overall, given the null alleles and phenotypic assays used, we did not find evidence for genetic interaction between Ret and Sema3d affecting survival, myenteric plexus formation or gut transcriptome. Overall design: poly-A RNA-seq in embryonic day 12.5 mouse gut from 3 wildtype males, 3 wildtype females, 3 Ret null homozyogote males, 3 Ret null homozyogote females, 3 Sema3d null homozyogote males, 3 Sema3d null homozyogote females, 3 Ret-Sema3d double null homozyogote males, 3 Ret-Sema3d double null homozyogote females
Project description:The cystic leukoencephalopathy without megalencephaly has been defined as distinct autosomal-recessive syndrome of quasi-static encephalopathy with normo- or microcephaly, impaired psychomotor development and characteristic pattern on brain MRI. We identified mutations in the gene encoding the RNASET2 glycoprotein in seven affected individuals as the cause of disease. The results suggest that RNASET2 plays an important role in central nervous system myelination and development. 2-Color dye swap design independent for two different families including technical and biologial replicates for affected family members (disease) and not affected family members (controls).
Project description:Parkinson’s disease (PD) has a neuro-developmental component with multiple genetic predispositions. The most prevalent mutation, LRRK2-G2019S is linked to familial and sporadic PD. Based on the multiple origins of PD and the incomplete penetrance of LRRK2-G2019S, we hypothesize that modifiers in the patient genetic background act as susceptibility factors for developing PD. To assess the developmental component of LRRK2-G2019S pathogenesis, we used 19 human iPSC-derived neuroepithelial stem cell lines (NESCs). Isogenic controls distinguish between LRRK2-G2019S dependent and independent cellular phenotypes. LRRK2-G2019S patient and healthy mutagenized lines showed altered NESC self-renewal. Within patients, phenotypes were only partly LRRK2-G2019S dependent, suggesting Parkinson’s disease (PD) has a neuro-developmental component with multiple genetic predispositions. The most prevalent mutation, LRRK2-G2019S is linked to familial and sporadic PD. Based on the multiple origins of PD and the incomplete penetrance of LRRK2-G2019S, we hypothesize that modifiers in the patient genetic background act as susceptibility factors for developing PD. To assess the developmental component of LRRK2-G2019S pathogenesis, we used 19 human iPSC-derived neuroepithelial stem cell lines (NESCs). Isogenic controls distinguish between LRRK2-G2019S dependent and independent cellular phenotypes. LRRK2-G2019S patient and healthy mutagenized lines showed altered NESC self-renewal. Within patients, phenotypes were only partly LRRK2-G2019S dependent, suggesting a significant contribution of the genetic background. We identified Serine racemase (SRR) as a novel patient-specific, developmental, genetic modifier contributing to the abberant phenotypes. Its enzymatic product, D-Serine, rescued altered NESC renewal. Susceptibility factors in the genetic background, such as SRR, could be new targets for early PD diagnosis and treatment. Overall design: 51 samples analyzed, 4 outliers excluded based on QC, 17 coditions, 3 biological replicates, 6 LRRK2G2019S patients, 6 healthy indiviuals, 3 gene corrected lines, 2 inserted mutated lines
Project description:A M. tuberculosis transposon library was used to infect WT and iNOS-/- mice. Surviving mutants were recovered from spleens, genomic DNA was extracted, and labeled probes were synthesized from transposon ends. Probes from each WT mouse were hybridized with probes from a similar iNOS-/- mouse. Two-condition experiment, Growth in WT vs. iNOS-/- mice. Biological replicates: TraSH probe made from 5 wild type and 5 iNOS-/- mice after 3 weeks of infection and from 8 wild type and 8 iNOS-/- mice after 4 weeks of infection. Technical replicates: TraSH probe was synthesized twice from each mouse and dyes were swapped. One array contains probe from one iNOS-/- and one WT mouse. Each biological replicate has two arrays, representing technical replicate dye swaps from one iNOS-/- and one WT mouse.
Project description:We have recently reported an allelic series of dominant, conditional, temperature-sensitive (DTS) mutations in the type IV collagen gene col4a1 in Drosophila and the onset of severe myopathy by massive degradation of striated muscle fibers, degeneration of epithelial and circular smooth muscle cells of the gut. In order to get a closer look at the molecular causes of the observed phenomena we have executed microarray experiments with the aforementioned mutant. Two growth temperatures for both DTS-3 and OreR flies with dye-swap technical control replicates.
Project description:miRNA profiling of undifferentiated and neuro-differentiated adipose-derived stem cells. Adipose-derived stem cells obtained from 4 different donors were divided into 2 groups, undifferentiated "control" group and neurally-differentiated "neuro" group. miRNA of each cells were analyzed using Agilent miRNA microarray.