Genetic variants within the second intron of the KCNQ1 gene affect CTCF binding and confer a risk of Beckwith-Wiedemann syndrome upon maternal transmission.

BACKGROUND: Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith-Wiedemann (BWS; MIM 130650) and the Silver-Russell (SRS; MIM 180860) syndromes. DNA methylation defects account for 60% of BWS and SRS cases and, in most cases, occur without any identified mutation in a cis-acting regulatory sequence or a trans-acting factor. METHODS: We investigated whether 11p15 cis-acting sequence variants account for primary DNA methylation defects in patients with SRS and BWS with loss of DNA methylation at ICR1 and ICR2, respectively. RESULTS: We identified a 4.5 kb haplotype that, upon maternal transmission, is associated with a risk of ICR2 loss of DNA methylation in patients with BWS. This novel region is located within the second intron of the KCNQ1 gene, 170 kb upstream of the ICR2 imprinting centre and encompasses two CTCF binding sites. We showed that, within the 4.5 kb region, two SNPs (rs11823023 and rs179436) affect CTCF occupancy at DNA motifs flanking the CTCF 20 bp core motif. CONCLUSIONS: This study shows that genetic variants confer a risk of DNA methylation defect with a parent-of-origin effect and highlights the crucial role of CTCF for the regulation of genomic imprinting of the CDKN1C/KCNQ1 domain.

Helicobacter pylori CagA triggers expression of the bactericidal lectin REG3γ via gastric STAT3 activation.

BACKGROUND: Most of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3γ encodes a secreted C-type lectin that exerts direct bactericidal activity against Gram-positive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3γ expression is triggered by CagA in the H. pylori-infected stomach. METHODOLOGY/PRINCIPAL FINDINGS: In human gastric mucosal tissues, REG3γ expression was significantly increased in CagA-positive, compared to CagA-negative H. pylori infected individuals. Using transfected CagA-inducible gastric MKN28 cells, we recapitulated REG3γ induction in vitro, also showing that tyrosine phosphorylated, not unphosphorylated CagA triggers REG3γ transcription. In concert with induced REG3γ, pro-inflammatory signalling downstream of the gp130 cytokine co-receptor via the signal transducer and activator of transcription (STAT)3 and transcription of two cognate ligands, interleukin(IL)-11 and IL-6, were significantly increased. Exogenous IL-11, but not IL-6, directly stimulated STAT3 activation and REG3γ transcription. STAT3 siRNA knockdown or IL-11 receptor blockade respectively abrogated or subdued CagA-dependent REG3γ mRNA induction, thus demonstrating a requirement for uncompromised signalling via the IL-11/STAT3 pathway. Inhibition of the gp130-related SHP2-(Ras)-ERK pathway did not affect CagA-dependent REG3γ induction, but strengthened STAT3 activation as well as augmenting transcription of mucosal innate immune regulators, IL-6, IL-8 and interferon-response factor (IRF)1. CONCLUSIONS/SIGNIFICANCE: Our results support a model of CagA-directed REG3γ expression in gastric epithelial cells via activation of the IL-11/gp130/STAT3 pathway. This response might allow Gram-negative H. pylori to manipulate host immunity to favour its own survival, by reducing the fitness of co-habiting Gram-positive bacteria with which it competes for resources in the gastric mucosal niche.

New insights into the pathogenesis of Beckwith-Wiedemann and Silver-Russell syndromes: contribution of small copy number variations to 11p15 imprinting defects.

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis-duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS.

A novel gastrokine, Gkn3, marks gastric atrophy and shows evidence of adaptive gene loss in humans.

BACKGROUND & AIMS: Gastrokines are stomach mucus cell-secreted proteins; 2 gastrokines are known, GKN1 and GKN2. Gastrokine expression is lost in gastric cancer, indicating a possible function in tumor suppression. We have identified a third gastrokine gene in mammals. METHODS: Gkn3 was characterized by studies of molecular structure, evolutionary conservation, and tissue expression as well as transcriptional/translational outcome in mouse genetic models of gastric pathology. The functional consequences of Gkn3 overexpression were evaluated in transfected cell lines. RESULTS: Gkn3 encodes a secreted (approximately 19 kilodalton) protein that is co-expressed with trefoil factor (Tff)2 in the distal stomach and discriminates a Griffinia simplicifolia lectin (GS)-II-positive mucus neck cell (MNC) subpopulation in the proximal stomach. In humans, widespread homozygosity for a premature stop codon polymorphism, W59X, has likely rendered GKN3 non-functional. Population genetic analysis revealed an ancestral GKN3 read-through allele that predominates in Africans and indicates the rapid expansion of W59X among non-Africans during recent evolution. Mouse Gkn3 expression is strongly up-regulated in (Tff2-deficient) gastric atrophy, a pre-cancerous state that is typically associated with Helicobacter pylori and marks a non-proliferative, GS-II positive lineage with features of spasmolytic polypeptide-expressing metaplasia (SPEM). Gkn3 overexpression inhibits proliferation in gastric epithelial cell lines, independently of incubation with recombinant human TFF2 or apoptosis. CONCLUSIONS: Gkn3 encodes a novel, functionally distinct gastrokine that is overexpressed and might restrain epithelial cell proliferation in gastric atrophy. Spread of the human GKN3 stop allele W59X might have been selected for among non-Africans because of its effects on pre-neoplastic outcomes in the stomach.