Common FLG Mutation K4671X Not Associated with Atopic Dermatitis in Han Chinese in a Family Association Study

Background

Filaggrin gene (FLG) mutations have been identified as the cause of ichthyosis vulgaris (IV) and major predisposing factors for atopic dermatitis (AD). The relationship among AD, IV and FLG mutations has not been clarified yet. Mutations 3321delA and K4671X, two of the most common mutations in Chinese patients, were both statistically associated with AD in case-control studies.

Materials and Methods

A group of 100 family trios (a total of 300 members with one affected AD proband and both parents) were recruited and screened for three filaggrin null mutations (3222del4, 3321delA and K4671X). The subjects’ manifestations of AD and IV were assessed by two experienced dermatologists and recorded in detail. The relationship of common mutations to AD were assessed using both case-control and family-based tests of association. Filaggrin expression was measured in skin of 3 subjects with K4671X heterozygote and the normal control using quantitative real-time RT-PCR and immunohistochemistry.

Results

Of 100 probands for AD, 22 were carriers for common FLG mutations and only 2 of them were from 40 none-IV family trios (5.00%), consistent with that of the healthy control group (3.99%, P>0.05). Significant statistical associations were revealed between AD and 3321delA (P<0.001, odds ratio 12.28, 95% confidence interval 3.35–44.98) as well as K4671X (P = 0.002, odds ratio 4.53, 95% confidence interval 1.77–11.60). The family-based approach revealed that 3321delA was over-transmitted to AD offspring from parents (T:U = 12∶1, P = 0.003) but failed to demonstrate transmission disequilibrium between K4671X and AD (T:U = 10∶8, P = 0.815). Moreover, compared to the normal control, filaggrin expression at both mRNA and protein levels in epidermis of subjects with K4671X^heter was not reduced.

Conclusions

AD patients from none-IV family trios have low probability of carrying FLG mutations. The present family samples confirmed the susceptibility of mutation 3321delA to AD in Han Chinese. K4671X was not a pathogenic mutation.     

No Association between Loss-of-Function Mutations in filaggrin and Diabetes,Cardiovascular Disease,and All-Cause Mortality

Background

Common loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing risk factor for atopic disease due to reduced epidermal filaggrin protein levels. We previously observed an association between these mutations and type 2 diabetes and hypothesized that an inherited impairment of skin barrier functions could facilitate low-grade inflammation and hence increase the risk of diabetes and cardiovascular disease. We examined the association between loss-of-function mutations in FLG and diabetes, stroke, ischemic heart disease (IHD), and all-cause mortality in the general population.

Methods

The R501X and 2282del4 loss-of function mutations in FLG were genotyped in four Danish study populations including a total of 13373 adults aged 15-77 years. Two of the studies also genotyped the R2447X mutation. By linkage to Danish national central registers we obtained information for all participants on dates of diagnoses of diabetes, stroke, and IHD, as well as all-cause mortality. Data were analyzed by Cox proportional hazard models and combined by fixed effect meta-analyses.

Results

In meta-analyses combining the results from the four individual studies, carriage of loss-of-function mutations in FLG was not associated with incident diabetes (hazard ratio (HR) (95% confidence intervals (CI)) = 0.95 (0.73, 1.23), stroke (HR (95% CI) = 1.27 (0.97, 1.65), ischemic heart disease (HR (95%CI) = 0.92 (0.71, 1.19), and all-cause mortality (HR (95%CI) = 1.02 (0.83, 1.25)). Similar results were obtained when including prevalent cases in logistic regression models.

Conclusion

Our results suggest that loss-of-function mutations in FLG are not associated with type 2 diabetes, cardiovascular disease, and all-cause mortality. However, larger studies with longer follow-up are needed to exclude any associations.     

Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a complex pathogenesis. Although regulatory T cells (Tregs) have previously been studied in AD, their role remains controversial, likely owing to patient heterogeneity. Thus, we recruited adult AD patients and age‐matched healthy controls, and assessed their filaggrin (FLG) genotype, serum IgE level, and eczema area and severity index (EASI). We found increased proportions of all circulating Treg subpopulations in AD patients. Moreover, we show positive correlations between circulating Tregs and serum IgE FLG null mutations limited the expansion of both memory and effector Tregs and enhanced that of recently thymus‐emigrated Tregs. Furthermore, proportions of circulating Th2‐ or Th17‐Tregs but not Th1‐Tregs were increased in AD patients, and accentuated by FLG null mutations, thereby mimicking the immune deviation observed in Th cell populations. Moreover, ICOS⁺ Tregs showed reduced production of interleukin‐10, suggesting impaired immunosuppression in AD. The level of demethylation of FOXP3i1, which reflects the stability of FOXP3 expression, was similar in the blood and skin of AD patients and healthy controls. Overall, these results show that Tregs may participate into AD pathogenesis and that FLG null mutations exert further modifications on specific subpopulations of circulating Tregs.     

Gene-environment interaction in the onset of eczema in infancy: filaggrin loss-of-function mutations enhanced by neonatal cat exposure

Background

Loss-of-function variants in the gene encoding filaggrin (FLG) are major determinants of eczema. We hypothesized that weakening of the physical barrier in FLG-deficient individuals may potentiate the effect of environmental exposures. Therefore, we investigated whether there is an interaction between FLG loss-of-function mutations with environmental exposures (pets and dust mites) in relation to the development of eczema.

Methods and Findings

We used data obtained in early life in a high-risk birth cohort in Denmark and replicated the findings in an unselected birth cohort in the United Kingdom. Primary outcome was age of onset of eczema; environmental exposures included pet ownership and mite and pet allergen levels. In Copenhagen (n = 379), FLG mutation increased the risk of eczema during the first year of life (hazard ratio [HR] 2.26, 95% confidence interval [CI] 1.27–4.00, p = 0.005), with a further increase in risk related to cat exposure at birth amongst children with FLG mutation (HR 11.11, 95% CI 3.79–32.60, p < 0.0001); dog exposure was moderately protective (HR 0.49, 95% CI 0.24–1.01, p = 0.05), but not related to FLG genotype. In Manchester (n = 503) an independent and significant association of the development of eczema by age 12 mo with FLG genotype was confirmed (HR 1.95, 95% CI 1.13–3.36, p = 0.02). In addition, the risk increased because of the interaction of cat ownership at birth and FLG genotype (HR 3.82, 95% CI 1.35–10.81, p = 0.01), with no significant effect of the interaction with dog ownership (HR 0.59, 95% CI 0.16–2.20, p = 0.43). Mite-allergen had no effects in either cohort. The observed effects were independent of sensitisation.

Conclusions

We have demonstrated a significant interaction between FLG loss-of-function main mutations (501x and 2282del4) and cat ownership at birth on the development of early-life eczema in two independent birth cohorts. Our data suggest that cat but not dog ownership substantially increases the risk of eczema within the first year of life in children with FLG loss-of-function variants, but not amongst those without. FLG-deficient individuals may need to avoid cats but not dogs in early life.     

Carrier Status for the Common R501X and 2282del4 Filaggrin Mutations Is Not Associated with Hearing Phenotypes in 5377 Children from the ALSPAC Cohort

Background

Filaggrin is a major protein in the epidermis. Several mutations in the filaggrin gene (FLG) have been associated with a number of conditions. Filaggrin is expressed in the tympanic membrane and could alter its mechanical properties, but the relationship between genetic variation in FLG and hearing has not yet been tested.

Methodology/Principal Findings

We examined whether loss-of function mutations R501X and 2282del4 in the FLG gene affected hearing in children. Twenty eight hearing variables representing five different aspects of hearing at age nine years in 5,377 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort were tested for association with these mutations. No evidence of association was found between R501X or 2282del4 (or overall FLG mutation carrier status) and any of the hearing phenotypes analysed.

Conclusions/Significance

In conclusion, carrier status for common filaggrin mutations does not affect hearing in children.     

Filaggrin genotype determines functional and molecular alterations in skin of patients with atopic dermatitis and ichthyosis vulgaris

Background

Several common genetic and environmental disease mechanisms are important for the pathophysiology behind atopic dermatitis (AD). Filaggrin (FLG) loss-of-function is of great significance for barrier impairment in AD and ichthyosis vulgaris (IV), which is commonly associated with AD. The molecular background is, however, complex and various clusters of genes are altered, including inflammatory and epidermal-differentiation genes.

Objective

The objective was to study whether the functional and molecular alterations in AD and IV skin depend directly on FLG loss-of-function, and whether FLG genotype determines the type of downstream molecular pathway affected.

Methods and Findings

Patients with AD/IV (n = 43) and controls (n = 15) were recruited from two Swedish outpatient clinics and a Swedish AD family material with known FLG genotype. They were clinically examined and their medical history recorded using a standardized questionnaire. Blood samples and punch biopsies were taken and trans-epidermal water loss (TEWL) and skin pH was assessed with standard techniques. In addition to FLG genotyping, the STS gene was analyzed to exclude X-linked recessive ichthyosis (XLI). Microarrays and quantitative real-time PCR were used to compare differences in gene expression depending on FLG genotype. Several different signalling pathways were altered depending on FLG genotype in patients suffering from AD or AD/IV. Disease severity, TEWL and pH follow FLG deficiency in the skin; and the number of altered genes and pathways are correlated to FLG mRNA expression.

Conclusions

We emphasize further the role of FLG in skin-barrier integrity and the complex compensatory activation of signalling pathways. This involves inflammation, epidermal differentiation, lipid metabolism, cell signalling and adhesion in response to FLG-dependent skin-barrier dysfunction.     

Filaggrin gene defects are independent risk factors for atopic asthma in a Polish population: a study in ECAP cohort

Background

FLG null variants of which 2282del4 and R501X are the most frequent in Caucasians are established risk factors for atopic dermatitis (AD) with an effect probably mediated through impairment of epidermal barrier. Among subjects with AD FLG defects are also consistently associated with asthma and allergic rhinitis (AR) but it is less clear to what extent these associations are also present independently from skin disease. The aim of the present study was to evaluate the role of 2282del4 and R501X in predisposing to these allergic phenotypes in a Polish population.

Methodology

2282del4 and R501X were typed among 3,802 participants of the Epidemiology of Allergic Diseases in Poland (ECAP) survey, a cross-sectional population-based study using ECRHS II and ISAAC questionnaires, and ambulatory examination.

Principal Findings

The FLG null variants were associated with AD (OR = 2.01, CI: 1.20–3.36, P = 0.007), allergic rhinitis (in particular persistent form, OR = 1.69, CI:1.12–2.54, P = 0.011), and asthma (in particular atopic asthma, OR = 2.22, CI:1.24–3.96, P = 0.006). Association with atopic asthma (but not persistent allergic rhinitis) was also present in the absence of AD, (OR = 2.02, CI: 1.07–3.81, P = 0.027) as well as in the absence of AD and history of broadly defined inflammatory skin disease (OR = 2.30, CI: 1.07–4.93, P = 0.03). Association to atopic asthma would have not been found if diagnosis was made by questionnaire only (OR = 1.15, CI: 0.58–2.32, P = 0.8). We did not observe an association between FLG variants and allergic sensitizations (P = 0.8) or total IgE. (P = 0.6).

Conclusions/Significance

In a Polish population FLG 2282del4 and R501X carriage increases risk for development of AD and atopic asthma (also in the absence of AD or history thereof). This suggests that interventions aimed at restoring epidermal barrier may have a general role in asthma prophylaxis/treatment.     

Association analysis of single nucleotide polymorphisms at five loci: comparison between atopic dermatitis and asthma in the Chinese Han population

Atopic diseases, such as atopic dermatitis (AD) and asthma, are closely related to clinical phenotypes with hypersensitivity, and often share some similar genetic and pathogenic bases. Our recent GWAS identified three susceptibility gene/loci FLG (rs11204971 and rs3126085), 5q22.1 (rs10067777, rs7701890, rs13360927 and rs13361382) and 20q13.33 (rs6010620) to AD. The effect of these AD associated polymorphisms in asthma is so far unknown. To investigate whether AD relevant genetic variants is identical to asthma and reveal the differences in genetic factors between AD and asthma in Chinese Han population, seven AD associated single nucleotide polymorphisms (SNPs) as well as 3 other SNPs (rs7936562 and rs7124842 at 11q13.5 and rs4982958 at 14q11.2) from our previous AD GWAS were genotyped in 463 asthma patients and 985 controls using Sequenom MassArray system. We found rs4982958 at 14q11.2 was significantly associated with asthma (P = 3.04×10⁻⁴, OR = 0.73). We also detected one significant risk haplotype GGGA from the 4 SNPs (rs10067777, rs7701890, rs13360927 and rs13361382) at 5q22.1 in AD cases (P _correction = 3.60×10⁻¹⁰, OR = 1.26), and the haplotype was suggestive of risk in asthma cases in this study (P = 0.014, P _correction = 0.084, OR = 1.38). These SNPs (rs11204971, rs3126085, rs7936562, rs712484 and rs6010620) at AD susceptibility genes/loci FLG, 11q13.5 and 20q13.33 were not associated with asthma in this study. Our results further comfirmed that 14q11.2 was an important candidate locus for asthma and demonstrated that 5q22.1 might be shared by AD and asthma in Chinese Han population.     

Genetic analysis of auditory neuropathy spectrum disorder in the Korean population

Auditory neuropathy spectrum disorder (ANSD) is caused by dys-synchronous auditory neural response as a result of impairment of the functions of the auditory nerve or inner hair cells, or synapses between inner hair cells and the auditory nerve. To identify a causative gene causing ANSD in the Korean population, we conducted gene screening of the OTOF, DIAPH3, and PJVK genes in 19 unrelated Korean patients with ANSD. A novel nonsense mutation (p.Y1064X) and a known pathogenic mutation (p.R1939Q) of the OTOF gene were identified in a patient as compound heterozygote. Pedigree analysis for these mutations showed co-segregation of mutation genotype and the disease in the family, and it supported that the p.Y1064X might be a novel genetic cause of autosomal recessive ANSD. A novel missense variant p.K1017R (c.3050A>G) in the DIAPH3 gene was also identified in the heterozygous state. In contrast, no mutation was detected in the PJVK gene. These results indicate that no major causative gene has been reported to date in the Korean population and that pathogenic mutations in undiscovered candidate genes may have an effect on ANSD.     

Programming social behavior by the maternal fragile X protein

The developing fetus and neonate are highly sensitive to maternal environment. Besides the well‐documented effects of maternal stress, nutrition and infections, maternal mutations, by altering the fetal, perinatal and/or early postnatal environment, can impact the behavior of genetically normal offspring. Mutation/premutation in the X‐linked FMR1 (encoding the translational regulator FMRP) in females, although primarily responsible for causing fragile X syndrome (FXS) in their children, may also elicit such maternal effects. We showed that a deficit in maternal FMRP in mice results in hyperactivity in the genetically normal offspring. To test if maternal FMRP has a broader intergenerational effect, we measured social behavior, a core dimension of neurodevelopmental disorders, in offspring of FMRP‐deficient dams. We found that male offspring of Fmr1^+/? mothers, independent of their own Fmr1 genotype, exhibit increased approach and reduced avoidance toward conspecific strangers, reminiscent of ‘indiscriminate friendliness’ or the lack of stranger anxiety, diagnosed in neglected children and in patients with Asperger's and Williams syndrome. Furthermore, social interaction failed to activate mesolimbic/amygdala regions, encoding social aversion, in these mice, providing a neurobiological basis for the behavioral abnormality. This work identifies a novel role for FMRP that extends its function beyond the well‐established genetic function into intergenerational non‐genetic inheritance/programming of social behavior and the corresponding neuronal circuit. As FXS premutation and some psychiatric conditions that can be associated with reduced FMRP expression are more prevalent in mothers than full FMR1 mutation, our findings potentially broaden the significance of FMRP‐dependent programming of social behavior beyond the FXS population.     

Expectant Mothers Maximizing Opportunities: Maternal Characteristics Moderate Multifactorial Prenatal Stress in the Prediction of Birth Weight in a Sample of Children Adopted at Birth

Background

Mothers’ stress in pregnancy is considered an environmental risk factor in child development. Multiple stressors may combine to increase risk, and maternal personal characteristics may offset the effects of stress. This study aimed to test the effect of 1) multifactorial prenatal stress, integrating objective “stressors” and subjective “distress” and 2) the moderating effects of maternal characteristics (perceived social support, self-esteem and specific personality traits) on infant birthweight.

Method

Hierarchical regression modeling was used to examine cross-sectional data on 403 birth mothers and their newborns from an adoption study.

Results

Distress during pregnancy showed a statistically significant association with birthweight (R² = 0.032, F _{(2, 398)} = 6.782, p = .001). The hierarchical regression model revealed an almost two-fold increase in variance of birthweight predicted by stressors as compared with distress measures (R² Δ = 0.049, F _{(4, 394)} = 5.339, p < .001). Further, maternal characteristics moderated this association (R² Δ = 0.031, F _{(4, 389)} = 3.413, p = .009). Specifically, the expected benefit to birthweight as a function of higher SES was observed only for mothers with lower levels of harm-avoidance and higher levels of perceived social support. Importantly, the results were not better explained by prematurity, pregnancy complications, exposure to drugs, alcohol or environmental toxins.

Conclusions

The findings support multidimensional theoretical models of prenatal stress. Although both objective stressors and subjectively measured distress predict birthweight, they should be considered distinct and cumulative components of stress. This study further highlights that jointly considering risk factors and protective factors in pregnancy improves the ability to predict birthweight.     

Polymorphism C1420T of Serine hydroxymethyltransferase gene on maternal risk for Down syndrome

Recent researches have investigated the factors that determine the maternal risk for Down syndrome (DS) in young woman. In this context, some studies have demonstrated the association between polymorphisms in genes involved on folate metabolism and the maternal risk for DS. These polymorphisms may result in abnormal folate metabolism and methyl deficiency, which is associated with aberrant chromosome segregation leading to trisomy 21. In this study, we analyzed the influence of the polymorphism C1420T in Serine hydroxymethyltransferase (SHMT) gene on maternal risk for DS and on metabolites concentrations of the folate pathway (serum folate and plasma homocysteine and methylmalonic acid). The study group was composed by 105 mothers with DS children (case group) and 185 mothers who had no children with DS (control group). The genotype distribution did not show significant statistical difference between case and control mothers (P?=?0.24) however a protective effect between genotypes CC (P?=?0.0002) and CT (P?<?0.0001) and maternal risk for DS was observed. Furthermore, the SHMT C1420T polymorphism (rs1979277) does not affect the concentration of metabolites of folate pathway in our DS mothers. In conclusion, our data showed a protective role for the genotypes SHMT CC and CT on maternal risk for DS. The concentrations of metabolites of folate pathway did not differ significantly between the genotypes SHMT.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Role of maternal 5‐HT1A receptor in programming offspring emotional and physical development

Serotonin_1A receptor (5‐HT_1AR) deficiency has been associated with anxiety and depression and mice with genetic receptor inactivation exhibit heightened anxiety. We have reported that 5‐HT_1AR is not only a genetic but also a maternal ‘environmental’ factor in the development of anxiety in Swiss‐Webster mice. Here, we tested whether the emergence of maternal genotype‐dependent adult anxiety is preceded by early behavioral abnormalities or whether it is manifested following a normal emotional development. Pups born to null or heterozygote mothers had significantly reduced ultrasonic vocalization (USV) between postnatal day (P) 4 and 12, indicating an influence of the maternal genotype. The offspring's own genotype had an effect limited to P4. Furthermore, we observed reduced weight gain in the null offspring of null but not heterozygote mothers, indicating that a complete maternal receptor deficiency compromises physical development of the offspring. Except a short perinatal deficit during the dark period, heterozygote females displayed normal maternal behavior, which, with the early appearance of USV deficit, suggests a role for 5‐HT_1AR during pre‐/perinatal development. Consistent with this notion, adult anxiety in the offspring is determined during the pre‐/perinatal period. In contrast to heterozygote females, null mothers exhibited impaired pup retrieval and nest building that may explain the reduced weight gain of their offspring. Taken together, our data indicate an important role for the maternal 5‐HT_1AR in regulating emotional and physical development of their offspring. Because reduced receptor binding has been reported in depression, including postpartum depression, reduced 5‐HT_1AR function in mothers may influence the emotional development of their offspring.     

Occult Hepatitis B Virus Infection in Anti-HBs-Positive Infants Born to HBsAg-Positive Mothers in China

Objective

To investigate the prevalence of occult HBV infection (OBI) among children and to characterize virology of occult HBV, we conducted an epidemiological survey.

Methods

186 HB-vaccinated infants born to HBsAg-positive mothers were included in the study. Serological tests for HBV markers were performed using commercial ELISA kits. Real-time quantitative PCR and nested PCR were used to detect HBV DNA. PCR products of the C and pre-S/S regions were sequenced and analyzed.

Results

1.61% (3/186) infants were HBsAg positive, and 4.92% (9/183) infants were considered as occult infection. The viral load of mothers was associated with occult infection (P = 0.020). Incomplete three-dose injections of HB vaccine was associated with HBV infection (P = 0.022). Six OBI infants were positive for anti-HBs, but their titers were not greater than 100 mIU/mL. Seven isolated HBV pre-S/S sequences were obtained from nine OBI infants. Three of the sequences were genotype C, and four of the sequences were genotype C/D. Escape mutation S143L was found in the four sequences of genotype C/D. All seven sequences lacked G145R and other escape mutation in S region.

Conclusions

Occult HBV infection was detected in anti-HBs positive infants born to HBsAg-positive mothers in China. Occult infection was associated with absent anti-HBs or with low anti-HBs level, high maternal viral loads and escape mutations in the S gene.     

Association of single nucleotide polymorphism rs2065955 of the <Emphasis Type="Italic">filaggrin</Emphasis> gene with susceptibility to Epstein-Barr virus-associated gastric carcinoma and EBV-negative gastric carcinoma

The relationship between the Filaggrin gene (FLG) rs2065955 polymorphism and susceptibility to Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC) was investigated in Shandong Province, China. We detected the FLG rs2065955 genotype and allele distribution by using PCR and restriction fragment length polymorphism (RFLP) in 64 EBVaGC, 82 EBVnGC, and 111 normal control samples. Immunohistochemistry was used to detect the level of FLG protein in 35 EBVaGC and 51 EBVnGC tumor tissues. Compared with normal controls, the genotype CC and allele C of FLG rs2065955 showed higher frequency in EBVaGC and EBVnGC. There was no significant difference between EBVaGC and EBVnGC in allele distribution of FLG rs2065955, but the genotype CC was found more frequently in EBVaGC than in EBVnGC. The risk of developing either EBVaGC or EBVnGC in genotype CC was higher than in other genotypes. Furthermore, genotype CC of FLG rs2065955 may contribute more to the risk of developing EBVaGC than EBVnGC. There was no significant difference in the expression level of FLG protein between EBVaGC and EBVnGC. In conclusion, the FLG rs2065955 polymorphism was significantly related to gastric carcinoma. Allele C of FLG rs2065955 could be a risk factor for EBVaGC or EBVnGC, while genotype CC of FLG rs2065955 was especially associated with EBVaGC.

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Altered LINE-1 Methylation in Mothers of Children with Down Syndrome

Down syndrome (DS, also known as trisomy 21) most often results from chromosomal nondisjunction during oogenesis. Numerous studies sustain a causal link between global DNA hypomethylation and genetic instability. It has been suggested that DNA hypomethylation might affect the structure and dynamics of chromatin regions that are critical for chromosome stability and segregation, thus favouring chromosomal nondisjunction during meiosis. Maternal global DNA hypomethylation has not yet been analyzed as a potential risk factor for chromosome 21 nondisjunction. This study aimed to asses the risk for DS in association with maternal global DNA methylation and the impact of endogenous and exogenous factors that reportedly influence DNA methylation status. Global DNA methylation was analyzed in peripheral blood lymphocytes by quantifying LINE-1 methylation using the MethyLight method. Levels of global DNA methylation were significantly lower among mothers of children with maternally derived trisomy 21 than among control mothers (P = 0.000). The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation (R² = 4.5%, P = 0.046). The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate. Although our findings revealed an association between maternal global DNA hypomethylation and trisomy 21 of maternal origin, further progress and final conclusions regarding the role of global DNA methylation and the occurrence of trisomy 21 are facing major challenges.     

Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V₂ receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication.     

Suppression of Conformation-Compromised Mutants of Salmonella enterica Serovar Typhimurium MelB

The crystal structure of the Na⁺-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelB_St, with little effect on binding affinities for both sugar and Na⁺. Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelB_St mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelB_St, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.