Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

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LIF-induced STAT3 signaling in murine versus human embryonal carcinoma (EC) cells

Self-renewal and the maintenance of pluripotency of mouse embryonal stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF). Mouse ES cells can be cultured and kept undifferentiated in the absence of embryonal feeder-cell layers when exogenous LIF concentrations are maintained above a threshold concentration. An important downstream target of LIF signal transduction in mouse ES cells is the transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast to mouse ES cells, human ES cells are unresponsive to LIF and depend on feeder cells for undifferentiated growth. Here, we investigated the activation patterns of LIF-downstream effectors in mouse and human embryonal carcinoma (EC) cells. We report that LIF induces both ERK-1 as well as STAT3 activation in mouse P19 EC cells. LIF enhances the proliferation rate of P19 EC cells, which depends on ERK activity but does not require activation of STAT3. In contrast, LIF does not activate STAT3, ERK, or the gp130 receptor in human N tera-2/D1 EC cells, although all receptor components are expressed. The negative feedback protein suppressor of cytokine signaling 1 (SOCS-1) is constitutively expressed in N tera-2/D1 EC cells, suggesting that LIF signal transduction is inhibited by elevated levels of SOCS-1 expression. (C) 2002 Elsevier Science (USA)

Regulation of nodal and BMP signaling by tomoregulin-1 (X7365) through novel mechanisms

During early vertebrate development, members of the transforming growth factor beta (TGFbeta) family play important roles in a variety of processes, including germ layer specification, patterning, cell differentiation, migration, and organogenesis. The activities of TGFbetas need to be tightly controlled to ensure their function at the right time and place. Despite identification of multiple regulators of Bone Morphogenetic Protein (BMP) subfamily ligands, modulators of the activin/nodal class of TGFbeta ligands are limited, and include follistatin, Cerberus, and Lefty. Recently, a membrane protein, tomoregulin-1 (TMEFF1, originally named X7365), was isolated and found to contain two follistatin modules in addition to an Epidermal Growth Factor (EGF) domain, suggesting that TMEFF1 may participate in regulation of TGFbeta function. Here, we show that, unlike follistatin and follistatin-related gene (FLRG), TMEFF1 inhibits nodal but not activin in Xenopus. Interestingly, both the follistatin modules and the EGF motif contribute to nodal inhibition. A soluble protein containing the follistatin and the EGF domains, however, is not sufficient for nodal inhibition; the location of TMEFF1 at the membrane is essential for its function. These results suggest that TMEFF1 inhibits nodal through a novel mechanism. TMEFF1 also blocks mesodermal, but not epidermal induction by BMP2. Unlike nodal inhibition, regulation of BMP activities by TMEFF1 requires the latter's cytoplasmic tail, while deletion of either the follistatin modules or the EGF motif does not interfere with the BMP inhibitory function of TMEFF1. These results imply that TMEFF1 may employ different mechanisms in the regulation of nodal and BMP signals. In Xenopus, TMEFF1 is expressed from midgastrula stages onward and is enriched in neural tissue derivatives. This expression pattern suggests that TMEFF1 may modulate nodal and BMP activities during neural patterning. In summary, our data demonstrate that tomoregulin-1 is a novel regulator of nodal and BMP signaling during early vertebrate embryogenesis. (C) 2003 Elsevier Science (USA). All rights reserved

Patterns and rates of exonic de novo mutations in sporadic Hirschsprung disease patients

WH Gui was selected as one of the candidates for the Young Investigator AwardsD - Concurrent Sessions C16. Developmental syndromes: no. C16.1Hirschsprung disease (HSCR, aganglionic megacolon; 1 in 5000 live births) is a disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. HSCR most commonly presents sporadically, although it can be familial (5-20% of the patients). The sporadic form of the disorder is believed to be a genetically complex disease with both de novo and/or inherited genetic lesions. To assess the role of de novo mutations (DNM) in sporadic HSCR, we performed exome sequencing on 16 HSCR patients and their unaffected parents. Standard BWA/GATK pipeline was used to map the sequence reads to human reference genome 19 and call genomic variants for all 48 samples simultaneously. Exonic DNM mutations were identified using KGGSeq. DNM candidates were scrutinized using Integrative Genomics Viewer (IGV) and those variants deemed plausible were validated by Sanger sequencing. In total we confirmed 20 DNM mutations (17 SNVs, 3 Indels) in 16 genes. Five DNM were identified in RET (major HSCR gene). CCR2, COL6A3, MED26, NUP98, HMCN1 and DENND3 had DNM mutations and were found mutated in independent HSCR patients. Importantly, some of these genes are members of pathways involved in the development of the ENS and the encoded proteins interact with known key signaling molecules. The overall exonic DNM mutation rate is 1.25 per HSCR trio, with 10 out of 16 (62.5%) patients harboring ≥ 1 DNM mutations. Therefore, DNM mutations, as inherited mutations, contribute to the development of sporadic HSCR.link_to_OA_fulltex

Exonic de novo mutations in sporadic Hirschsprung disease

Poster PresentationHirschsprung disease (HSCR) is a disorder of the enteric nervous system (ENS) and is characterized by the absence of enteric neurons along a variable length of the intestine. HSCR most commonly presents sporadically, although it is familial in 5-20% of the patients. The sporadic form of the disorder is believed to be a genetically complex disease. To assess the role of de novo mutations in sporadic HSCR, we performed exome sequencing on 20 HSCR patients, predominantly females with long segment HSCR and their unaffected parents. We identified and confirmed 24 de novo mutations (18 SNVs, 6 Indels) in 17 different genes (1.2 per trio). Non-synonymous de novo mutations were identified in RET in 8 out of 20 patients, corroborating previous findings that RET is the major genetic contributor in long-segment HSCR. A replication study in independent HSCR patients, gene burden tests and functional analysis in both cell lines and zebra fish are currently being conducted. Interestingly, some of the genes harboring de novo mutations are members of pathways involved in the development of the ENS and the encoded proteins interact with known key signaling molecules. We will present all data which will enable us to make conclusion on whether, de novo mutations in genes other that RET also contribute to the development of sporadic HSCR