Randomized feeding intervention in infants at high risk for celiac disease.

BACKGROUND: A window of opportunity has been suggested for reducing the risk of celiac disease by introducing gluten to infants at 4 to 6 months of age. METHODS: We performed a multicenter, randomized, double-blind, placebo-controlled dietary-intervention study involving 944 children who were positive for HLA-DQ2 or HLA-DQ8 and had at least one first-degree relative with celiac disease. From 16 to 24 weeks of age, 475 participants received 100 mg of immunologically active gluten daily, and 469 received placebo. Anti-transglutaminase type 2 and antigliadin antibodies were periodically measured. The primary outcome was the frequency of biopsy-confirmed celiac disease at 3 years of age. RESULTS: Celiac disease was confirmed by means of biopsies in 77 children. To avoid underestimation of the frequency of celiac disease, 3 additional children who received a diagnosis of celiac disease according to the 2012 European Society for Pediatric Gastroenterology, Hepatology, and Nutrition diagnostic criteria (without having undergone biopsies) were included in the analyses (80 children; median age, 2.8 years; 59% were girls). The cumulative incidence of celiac disease among patients 3 years of age was 5.2% (95% confidence interval [CI], 3.6 to 6.8), with similar rates in the gluten group and the placebo group (5.9% [95% CI, 3.7 to 8.1] and 4.5% [95% CI, 2.5 to 6.5], respectively; hazard ratio in the gluten group, 1.23; 95% CI, 0.79 to 1.91). Rates of elevated levels of anti-transglutaminase type 2 and antigliadin antibodies were also similar in the two study groups (7.0% [95% CI, 4.7 to 9.4] in the gluten group and 5.7% [95% CI, 3.5 to 7.9] in the placebo group; hazard ratio, 1.14; 95% CI, 0.76 to 1.73). Breast-feeding, regardless of whether it was exclusive or whether it was ongoing during gluten introduction, did not significantly influence the development of celiac disease or the effect of the intervention. CONCLUSIONS: As compared with placebo, the introduction of small quantities of gluten at 16 to 24 weeks of age did not reduce the risk of celiac disease by 3 years of age in this group of high-risk children. (Funded by the European Commission and others; PreventCD Current Controlled Trials number, ISRCTN74582487.).

Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior.

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration-response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50 (ß = -0.67, r2 = 0.017, P = 2 × 10(-4)) or by response to low nicotine (ß = -0.29, r2 = 0.02, P = 6 × 10(-5)) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (ß = -0.04, r2 = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.

Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3ß4α5 nicotinic acetylcholine receptors.

The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3ß4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3ß4* receptor did not affect total [(125)I]-epibatidine binding or surface expression of the receptor. However, addition of α5(D398) into α3ß4* receptor decreased the maximal response to agonist without significantly affecting EC(50) in aequorin intracellular calcium assay. α3ß4α5(N398) nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3ß4α5 nAChRs led to significantly greater intracellular calcium release from IP(3) stores relative to α3ß4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC(50) for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3ß4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of α5 into an α3ß4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling.

Screening for unrecognized coeliac disease in subfertile couples.

OBJECTIVE: Subfertility has been reported as a long-term complication of unrecognized and/or untreated coeliac disease (CD); however, the results from studies on this topic are ambiguous. We aimed to determine the prevalence of unrecognized CD in subfertile male-female couples visiting a fertility clinic compared with the general population. METHODS: Subjects included 1038 male-female couples (n = 2076) who visited the fertility clinic of the Leiden University Medical Center in the Netherlands between 2003 and 2009. All consecutive patients were routinely, serologically screened, and those with positive test results for antibodies against IgA anti-tissue transglutaminase type 2 and IgA endomysial antibodies were considered to have unrecognized CD. Clinical data on gender, age, height, weight, diagnosis of subfertility, and previously diagnosed CD were collected from the clinical files. Subsequently, after serological screening, all patients were anonymized. The prevalence of unrecognized CD was compared with the one in the general adult population in the Netherlands (0.35%). RESULTS: The prevalence of unrecognized CD in subfertile male-female couples was 0.48% (10/2076; 6 females and 4 males) and was not significantly more frequent compared with the general population. Compared with the control group, similar CD prevalences were found within the different subfertility categories separately: unexplained subfertility, anovulation, tubal pathology, and male factor (p = NS). CONCLUSION: In our large study cohort of subfertile male-female couples, the prevalence of unrecognized CD is comparable to the general population in the Netherlands. No association was observed between CD and subfertility in the different subfertility categories and genders.

Specific celiac disease antibodies in children on a gluten-free diet.

OBJECTIVE: Celiac disease (CD) is characterized by histologic alterations in small bowel biopsies. Circulating specific CD antibodies at the time of diagnosis and their disappearance after a gluten-free diet support the diagnosis of CD. We aimed to determine the behavior of the CD antibodies immunoglobulin A anti-tissue transglutaminase (anti-TG2) and immunoglobulin A endomysium (EMA) in children with CD after starting a gluten-free diet. METHODS: This was a retrospective multicenter study in the Netherlands between 2001 and 2009. Inclusion criteria were all newly diagnosed patients with CD younger than 19 years who had at least 1 anti-TG2 and/or EMA measurement before and after starting a gluten-free diet. Eight different anti-TG2 kits were used with substrates of guinea pig TG2 in 1 (Sigma) and 7 human-recombinant TG2: Varelisa and EliA Celikey Phadia-GmbH; Orgentec Diagnostica-GmbH; Diarect AG; Roboscreen GmbH; Aeskulisa Diagnostics; Binding Site Ltd. EMA was analyzed with indirect immunofluorescence tests. Statistical analyses were performed by using mixed-model repeated measurements and survival analysis. RESULTS: There were 129 children with CD included (mean age: 5.6 years; SD ± 4.2). The mean concentration of anti-TG2 decreased significantly within 3 months after starting a gluten-free diet (P < .0001). The cumulative percentage of children who became negative for EMA after ½, 1, 1½, and 2 years was 31%, 60%, 74%, and 87%, respectively. For anti-TG2, a comparable trend was shown: 35%, 55%, 64%, and 78%, respectively. CONCLUSIONS: Doctors taking care of children with CD should be aware that the mean concentration of anti-TG2 will show a 74% decrease (95% confidence interval: 69%-79%) after 3 months of gluten-free diet, and ∼80% of the children will be sero-negative for EMA and anti-TG2 after 2 years of the diet.

Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site.

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA(A) receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the α subunits of the receptor, whereas GABA binds to pockets in the interfaces between ß and α subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ß-α pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ß-α pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the α subunit from the same or the other ß-α pair. We conclude that steroids potentiate the opening of the GABA(A) receptor induced by either agonist binding site.

The PreventCD Study design: towards new strategies for the prevention of coeliac disease.

BACKGROUND: PreventCD (www.preventcd.com) is a European multicentre study, which studies the influence of infant nutrition, and that of genetic, immunologic and environmental factors, on the risk of developing coeliac disease (CD). The hypothesis is that it is possible to induce tolerance to gluten by introducing small quantities of gluten to infants, preferably while they are still being breast-fed, and that this might also reduce the risk for related autoimmune disorders. AIM: To describe the design of this ongoing European CD research project. METHODS: PreventCD encompasses two study designs and two study populations: (i) a European multicentre study: a prospective, double-blind, randomized dietary-intervention study among infants from families with high risk of CD, and (ii) a Swedish population-based CD screening study among 12-year-olds from the general population, divided into two birth cohorts that differ with respect to infant feeding practices. DISCUSSION: PreventCD is expected to elucidate some of the genetic and immunological mechanisms involved in the process of immune intolerance.

Phenotypic variance in childhood coeliac disease and the HLA-DQ/DR dose effect.

OBJECTIVE: Coeliac disease (CD) is associated with HLA-DQ2 and DQ8. The clinical picture is variable and certain human leucocyte antigen (HLA) DQ/DR combinations have a higher relative risk (RR) for CD than others. Moreover, the HLA-DQ gene-dose effect has an impact on the strength of the gluten-specific T-cell response and thus may correlate with clinical presentation and severity of CD. The aim of this study was to determine the correlation between HLA-DQ/DR-based genotypes and the variation in phenotypes of the disease. MATERIAL AND METHODS: A total of 113 non-related Caucasian children clinically diagnosed with CD during the period 1980-2003 with a known HLA type were included in the study. Patients were divided into four categories according to amount of disease expression predisposing to HLA-DQ2 or HLA-DQ8 molecules and the known RR of their HLA-DR/DQ type for CD: high (DR3DQ2 homozygous and DR3DQ2/DR7DQ2), substantial (DR3DQ2/DR5DQ7 and DR5DQ7/DR7DQ2), moderate (DR3DQ2-DR4DQ8 and DR3DQ2/DR*DQ*) and low (DR7DQ2/DR*DQ*, DR4DQ8- DR*DQ* and DR*DQ*- DR*DQ*). The clinical data and HLA genotypes of these patients were compared. RESULTS: The 113 children were diagnosed with CD at a mean age of 4.6 years and boys were significantly older than girls when diagnosed (p=0.01). RR for having CD was highest for the high HLA-risk group (RR 8.1). With the exception of a greater frequency of abdominal distension and fewer non-gastrointestinal symptoms in the substantial HLA-risk group, there were no significant differences in clinical characteristics or degree of severity of the small-bowel histological findings between the children in the different HLA-risk groups. CONCLUSION: No correlation was found between disease severity and a double HLA-DQ2 gene dose.

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4.

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.