Faculty Bibliography

BACKGROUND:Hirschsprung's disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes. METHODS:We genotyped and exome-sequenced samples from 190 patients with Hirschsprung's disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung's disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos. RESULTS:). For individual patients, the estimated risk of Hirschsprung's disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120). CONCLUSIONS:. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.).

Hirschsprung disease (HSCR) is a congenital disorder with a population incidence of ~1/5000 live births, defined by an absence of enteric ganglia along variable lengths of the colon. HSCR genome-wide association studies (GWAS) have found common associated variants at RET, SEMA3, and NRG1, but they still fail to explain all of its heritability. To enhance gene discovery, we performed a GWAS of 170 cases identified from the Danish nationwide pathology registry with 4717 controls, based on 6.2 million variants imputed from the haplotype reference consortium panel. We found a novel low-frequency variant (rs144432435), which, when conditioning on the lead RET single-nucleotide polymorphism (SNP), was of genome-wide significance in the discovery analysis. This conditional association signal was replicated in a Swedish HSCR cohort with discovery plus replication meta-analysis conditional odds ratio of 6.6 (P = 7.7 × 10^-10; 322 cases and 4893 controls). The conditional signal was, however, not replicated in two HSCR cohorts from USA and Finland, leading to the hypothesis that rs144432435 tags a rare haplotype present in Denmark and Sweden. Using the genome-wide complex trait analysis method, we estimated the SNP heritability of HSCR to be 88%, close to estimates based on classical family studies. Moreover, by using Lasso (least absolute shrinkage and selection operator) regression we were able to construct a genetic HSCR predictor with a area under the receiver operator characteristics curve of 76% in an independent validation set. In conclusion, we combined the largest collection of sporadic Hirschsprung cases to date (586 cases) to further elucidate HSCR's genetic architecture.

Gene expression changes, the driving forces for cellular diversity in multicellular organisms, are regulated by a diverse set of gene regulatory elements that direct transcription in specific cells. Mutations in these elements, ranging from chromosomal aberrations to single-nucleotide polymorphisms, are a major cause of human disease. However, we currently have a very limited understanding of how regulatory element genotypes lead to specific phenotypes. In this review, we discuss the various methods of regulatory element identification, the different types of mutations they harbor, and their impact on human disease. We highlight how these variations can affect transcription of multiple genes in gene regulatory networks. In addition, we describe how novel technologies, such as massively parallel reporter assays and CRISPR/Cas9 genome editing, are beginning to provide a better understanding of the functional roles that these elements have and how their alteration can lead to specific phenotypes.

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 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 presence by acetylcholinesterase staining and embryonic day 12.5 (E12.5) intestine transcriptome by RNA-sequencing. Survival rates of Ret wild-type, 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 intestinal innervation. As compared with wild-type mice intestinal gene expression, loss of Ret in null homozygotes led to differential expression of approximately 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 intestine transcriptome. Overall, given the null alleles and phenotypic assays used, we did not find evidence for genetic interaction between Ret and Sema3d affecting survival, presence of myenteric plexus or intestine transcriptome.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.

This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

BACKGROUND:Gastroschisis is a developmental disorder involving the extrusion of fetal intestines through a defect in the abdominal wall. The mechanism is presumed to be a dual vascular/thrombotic pathogenesis, where normal right umbilical vein involution forms a possible site for thrombosis adjacent to the umbilical ring. PURPOSE/OBJECTIVE:The aim of this study was to demonstrate that the 3 common prothrombotic polymorphisms, MTHFR c.677C>T, F2 c.20210G>A, and F5 Leiden, were elevated in frequency in Indonesian gastroschisis patients. MATERIAL AND METHODS/METHODS:Three genetic markers were investigated in 46 patients with gastroschisis and 89 ethnicity-matched controls for association studies using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) or TaqMan Genotyping Assays on genomic DNA. RESULTS:MTHFR c.677C>T showed a significant association with gastroschisis (OR = 2.1, 95% CI = 1.13-3.86; p = .018) but no affected infants had risk alleles for either F2 c.20210G>A or F5 Leiden. Further, the frequency of MTHFR risk allele (T) in patients with maternal age <25 years is marginally significant higher than those in cases with maternal age ≥25 years (p = .069) with an OR of 2.7 (95% CI = 0.90-8.07). CONCLUSIONS:MTHFR is a common susceptibility factor for gastroschisis in Indonesia. The increased gastroschisis risk in offspring of younger maternal age suggests the thrombotic pathogenesis model. A founder effect is the most likely explanation for the rarity of the F2 and F5 Leiden polymorphisms in Indonesian population.

Intestinal neuronal dysplasia type B (IND) denotes an increased proportion of hyperplastic submucosal ganglia, as resolved histochemically in 15-mum-thick frozen sections. IND has been reported proximal to the aganglionic segment in patients with Hirschsprung disease (HSCR) and is putatively associated with a higher rate of postsurgical dysmotility. We developed and validated histological criteria to diagnose IND-like submucosal ganglion cell hyperplasia (IND-SH) in paraffin sections and used the approach to study the incidence and clinical and/or genetic associations of IND-SH at the proximal margins of HSCR pull-through resection specimens. Full-circumference paraffin sections from the proximal margins of 64 HSCR colonic pull-through specimens and 24 autopsy controls were immunostained for neuron-specific Hu antigen, and nucleated ganglion cells in each submucosal ganglion were counted. In controls, an age-related decline in the relative abundance of "giant" ganglia (>/=7 nucleated Hu-positive [Hu+] ganglion cells) was observed. A conservative diagnostic threshold for IND-SH (control mean +/- 3x standard deviation) was derived from 15 controls less than 25 weeks of age. No control exceeded this threshold, whereas in the same age range, IND-SH was observed at the proximal margins in 15% (7 of 46) of HSCR resections, up to 15 cm proximal to the aganglionic segment. No significant correlation was observed between IND-SH and length of or distance from the aganglionic segment, sex, trisomy 21, RET or SEMA3C/D polymorphisms, or clinical outcome, but analysis of more patients, with better long-term follow-up will be required to clarify the significance of this histological phenotype.