The PRSS3P2 and TRY7 deletion copy number variant modifies risk for chronic pancreatitis.

BACKGROUND: PRSS1 and PRSS2 constitute the only functional copies of a tandemly-arranged five-trypsinogen-gene cluster (i.e., PRSS1, PRSS3P1, PRSS3P2, TRY7 and PRSS2) on chromosome 7q35. Variants in PRSS1 and PRSS2, including missense and copy number variants (CNVs), have been reported to predispose to or protect against chronic pancreatitis (CP). We wondered whether a common trypsinogen pseudogene deletion CNV (that removes two of the three trypsinogen pseudogenes, PRSS3P2 and TRY7) might be associated with CP causation/predisposition. METHODS: We analyzed the common PRSS3P2 and TRY7 deletion CNV in a total of 1536 CP patients and 3506 controls from France, Germany, India and Japan by means of quantitative fluorescent multiplex polymerase chain reaction. RESULTS: We demonstrated that the deletion CNV variant was associated with a protective effect against CP in the French, German and Japanese cohorts whilst a trend toward the same association was noted in the Indian cohort. Meta-analysis under a dominant model yielded a pooled odds ratio (OR) of 0.68 (95% confidence interval (CI) 0.52-0.89; p = 0.005) whereas an allele-based meta-analysis yielded a pooled OR of 0.84 (95% CI 0.77-0.92; p = 0.0001). This protective effect is explicable by reference to the recent finding that the still functional PRSS3P2/TRY7 pseudogene enhancers upregulate pancreatic PRSS2 expression. CONCLUSIONS: The common PRSS3P2 and TRY7 deletion CNV was associated with a reduced risk for CP. This finding provides additional support for the emerging view that dysregulated PRSS2 expression represents a discrete mechanism underlying CP predisposition or protection.

Do pregnancies reduce iron overload in HFE hemochromatosis women? results from an observational prospective study.

BACKGROUND: HFE hemochromatosis is an inborn error of iron metabolism linked to a defect in the regulation of hepcidin synthesis. This autosomal recessive disease typically manifests later in women than men. Although it is commonly stated that pregnancy is, with menses, one of the factors that offsets iron accumulation in women, no epidemiological study has yet supported this hypothesis. The aim of our study was to evaluate the influence of pregnancy on expression of the predominant HFE p.[Cys282Tyr];[Cys282Tyr] genotype. METHODS: One hundred and forty p.Cys282Tyr homozygous women enrolled in a phlebotomy program between 2004 and 2011 at a blood centre in western Brittany (France) were included in the study. After checking whether the disease expression was delayed in women than in men in our study, the association between pregnancy and iron overload was assessed using multivariable regression analysis. RESULTS: Our study confirms that women with HFE hemochromatosis were diagnosed later than men cared for during the same period (52.6 vs. 47.4 y., P < 0.001). Compared to no pregnancy, having at least one pregnancy was not associated with lower iron markers. In contrast, the amount of iron removed by phlebotomies appeared significantly higher in women who had at least one pregnancy (eß = 1.50, P = 0.047). This relationship disappeared after adjustment for confounding factors (eß = 1.35, P = 0.088). CONCLUSIONS: Our study shows that pregnancy status has no impact on iron markers level, and is not in favour of pregnancy being a protective factor in progressive iron accumulation. Our results are consistent with recent experimental data suggesting that the difference in disease expression observed between men and women may be explained by other factors such as hormones.

Measures to improve microbial quality surveillance of gastrointestinal endoscopes.

BACKGROUND AND STUDY AIM: Infectious outbreaks associated with the use of gastrointestinal endoscopes have increased in line with the spread of highly resistant bacteria. The aim of this study was to determine the measures required to improve microbial quality surveillance of gastrointestinal endoscopes. METHODS: We reviewed the results of all microbiological surveillance testing of gastrointestinal endoscopes and automatic endoscope reprocessors (AERs) performed at Brest Teaching Hospital from 1 January 2008 to 1 June 2015. We analyzed the influence of the time of incubation on the rate of positive results using the Kaplan - Meier method. We also studied risk factors for gastrointestinal endoscope contamination using a multivariable logistic regression model. RESULTS: Over the study period, 1100 microbiological tests of gastrointestinal endoscopes (n = 762) and AERs (n = 338) were performed. A total of 264 endoscope tests (34.6 %) showed a level of contamination higher than the target. After 2 days of incubation, contamination was apparent in only 55.5 % of the endoscopes that were later shown to be contaminated (95 % confidence interval [CI] 49.2 - 61.8). Multivariable analysis showed that the use of storage cabinets for heat-sensitive endoscopes significantly reduced the risk of endoscope contamination (odds ratio [OR] 0.23, 95 %CI 0.09 - 0.54; P  < 0.001) and that the use of endoscopes older than 4 years significantly increased this risk (OR ≥ 6 vs. < 2 years 2.92, 95 %CI 1.63 - 5.24; P < 0.001). CONCLUSIONS: Microbiological culture technique, mainly incubation duration, strongly influenced the results of endoscope sampling. Samples should be cultured for more than 2 days to improve the detection of contaminated endoscopes. Particular attention should be paid to endoscopes older than 2 years and to those that are not stored in storage cabinets for heat-sensitive endoscopes.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

BACKGROUND & AIMS: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. METHODS: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. RESULTS: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). CONCLUSIONS: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy.

PURPOSE: Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test [corrected] was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity. METHODS: During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Française pour le Dépistage et la Prévention des Handicaps de l'Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed. RESULTS: Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity. CONCLUSION: The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.

Insights into RHCE Molecular Analysis in Samples with Partial D Variants: the Experience of Western France.

BACKGROUND: Although systematic blood group genotyping of patients/donors is virtually possible, serological studies remain the gold standard to identify samples of clinical interest that may be further genotyped. In this context, we sought to identify variant D alleles that are likely to be clinically relevant in terms of other Rh antigens in a subset of population genotyped in Western France. METHODS: Samples presenting with the RHD*weak D type 4.2.2 allele (n = 47) were selected for the study. RHCE exons 1-7 were directly sequenced, and expression of Rh antigens was predicted on the basis of the molecular data. RESULTS: Of the 47 samples tested, 19 (40.4%) were predicted to be of potential clinical interest. Moreover, we could show that selecting the samples to be genotyped by the nature of their variant D allele (i.e., RHD*weak D type 4.2.2 allele) rather than by their Duffy-null status appears to increase significantly the likelihood of identifying clinically relevant individuals for Rh status. CONCLUSION: On the basis of our findings we suggest that all individuals genotyped as weak D type 4.2.2 should be systematically screened for RHCE variants by molecular analysis on a routine basis.

Nonvisualization of fetal gallbladder increases the risk of cystic fibrosis.

OBJECTIVE: The aim of our study is to evaluate the prevalence of cystic fibrosis (CF) in fetuses referred for genetic testing because of ultrasonographic sign (nonvisualized fetal gallbladder--NVFGB). METHOD: We reviewed the results of CFTR gene analysis over the period 2002 to 2009 in all consecutive cases referred because of NVFGB in Western France. We correlated these data with the presence of a more classical ultrasonographic finding (fetal echogenic bowel - FEB). RESULTS: Cystic fibrosis was diagnosed in 5 of the 37 fetuses with NVFGB (13.5%, 95% confidence interval (CI): [2.5%; 24.5%]) and in only 9 of the 229 other cases referred because of FEB (3.9%, 95% CI: [3.2%; 14.7%]). In our series, all CF-affected fetuses with NVFGB also had FEB. The risk of CF was 11.6-fold higher in fetuses with both indications (NVFGB + FEB) than in fetuses with isolated FEB (45.5% vs 3.9%, RR = 11.6, 95% CI: [4.7%; 28.8%], p = 0.0001). We also estimated that the residual risk of CF was less than 1 in 68 (1.5%) when a single mutation was identified in the fetus by our molecular protocol. CONCLUSION: Ultrasonographic evidence of NVFGB is an additional risk factor for CF in cases with FEB.

Prevalence of HFE-related haemochromatosis and secondary causes of hyperferritinaemia and their association with iron overload in 1059 French patients treated by venesection.

BACKGROUND: Venesection is the key therapy in haemochromatosis, but it remains controversial in hyperferritinaemia with moderate iron accumulation. There is substantial evidence that the results of HFE genotyping are routinely misinterpreted, while elevated serum ferritin has become more frequent in recent years in white adult populations following the increase of obesity and metabolic traits. AIMS: To examine the reasons for prescribing venesection in 1,059 French patients during the period 2012-2015, determine the true prevalence of HFE-related haemochromatosis, and compare iron overload profiles between haemochromatosis and non-haemochromatosis patients. RESULTS: Only 258 of the 488 patients referred for haemochromatosis had the p.[Cys282Tyr];[Cys282Tyr] disease causative genotype (adjusted prevalence: 24.4%). Of the 801 remaining patients, 112 (14.0%) had the debated p.[Cys282Tyr];[His63Asp] compound heterozygote genotype, 643 (80.3%) had central obesity, 475 (59.3%) had metabolic syndrome (MetS) and 93 (11.6%) were heavy drinkers. The non-haemochromatosis patients started therapeutic venesection 9 years later than haemochromatosis patients (P < 0.001). Despite similar serum ferritin values, they had lower transferrin saturation (41.1% vs 74.3%; P < 0.001), lower amounts of iron removed by venesection (1.7 vs 3.2 g; P < 0.001) and lower hepatic iron concentrations (107 vs 237 µmol/g; P < 0.001). CONCLUSIONS: Haemochromatosis is over-diagnosed and is no longer the main reason for therapeutic venesection in France. Obesity and other metabolic abnormalities are frequently associated with mild elevation of serum ferritin, the MetS is confirmed in ~50% of treated patients. There is a minimal relationship between serum ferritin and iron overload in non-p.Cys282Tyr homozygotes. Our observations raise questions about venesection indications in non-haemochromatosis patients.

A common SNP near BMP2 is associated with severity of the iron burden in HFE p.C282Y homozygous patients: a follow-up study.

BACKGROUND AND OBJECTIVES: It is now generally admitted that penetrance of the common HFE p.C282Y/p.C282Y genotype is incomplete, and identification of modifier genes is the concern of a growing number of research projects. We recently identified a significant association between pretherapeutic serum ferritin level and the common rs235756 single nucleotide polymorphism (SNP) of the BMP2 gene region. Our results further suggested an interactive effect between the BMP2 rs235756 SNP and the rs16827043 SNP in HJV, with a small additive effect of the rs4901474 SNP in BMP4. DESIGN AND METHODS: The present study has been designed as a replication study in an independent cohort of 450 HFE p.C282Y homozygous patients from a nearby French region (Brittany). Information on individual alcohol consumption and amount of iron removed by phlebotomy being available for a substantial part of this cohort, additional analyses were conducted. RESULTS: Only the use of the Amount of Iron Removed by phlebotomy (AIR) as marker of iron burden has provided positive results. Indeed, a significant association was detected between rs235756 and AIR adjusted for sex and age, with a mean AIR increasing with the number of BMP2 T alleles in the genotype groups. The effect of rs235657 was not strong enough to detect effects of gene combinations. Still, the trend in two-locus genotype risks involving BMP2 and HJV for AIR was concordant with the specific interactive effect described in the initial study. INTERPRETATION AND CONCLUSIONS: Although we failed to replicate results of the initial study, we argue that, altogether, our results help to consider genes involved in the regulation of hepcidin synthesis as potential modifiers of the p.C282Y/pC282Y genotype expression and especially BMP2.

Focus on cystic fibrosis and other disorders evidenced in fetuses with sonographic finding of echogenic bowel: 16-year report from Brittany, France.

OBJECTIVE: Pregnancies medical follow-up and ultrasonography development have enabled detection of fetal echogenic bowel, a sign associated with various pathologies, including cystic fibrosis. Based on the long experience of a region where cystic fibrosis is frequent (Brittany, France), we describe disorders diagnosed in fetal echogenic bowel fetuses and assess ultrasonography ability in detecting cystic fibrosis in utero. STUDY DESIGN: We reviewed the cases of fetal echogenic bowel diagnosed in pregnant women living in Brittany and referred for CFTR gene analysis over the 1992-2007 period (n = 289). RESULTS: A disorder was diagnosed in 32.2% of the fetuses, cystic fibrosis being the most commonly identified (7.6%). We also found digestive malformations (7.0%), chromosomal abnormalities (3.7%), and maternofetal infections (3.7%). Combining these data with our ongoing newborn screening program since 1989 showed that ultrasonography enabled diagnosis of 10.7% of the cystic fibrosis cases. CONCLUSION: This study highlights the importance of pregnancy ultrasound examinations and their efficiency in detecting cystic fibrosis.

Newborn Screening for CF across the Globe-Where Is It Worthwhile?

Newborn screening (NBS) for cystic fibrosis (CF) has been performed in many countries for as long as four decades and has transformed the routine method for diagnosing this genetic disease and improved the quality and quantity of life for people with this potentially fatal disorder. Each region has typically undertaken CF NBS after analysis of the advantages, costs, and challenges, particularly regarding the relationship of benefits to risks. The very fact that all regions that began screening for CF have continued their programs implies that public health and clinical leaders consider early diagnosis through screening to be worthwhile. Currently, many regions where CF NBS has not yet been introduced are considering options and in some situations negotiating with healthcare authorities as policy and economic factors are being debated. To consider the assigned question (where is it worthwhile?), we have completed a worldwide analysis of data and factors that should be considered when CF NBS is being contemplated. This article describes the lessons learned from the journey toward universal screening wherever CF is prevalent and an analytical framework for application in those undecided regions. In fact, the lessons learned provide insights about what is necessary to make CF NBS worthwhile.

The Changing Epidemiology of Cystic Fibrosis: Incidence, Survival and Impact of the CFTR Gene Discovery.

Significant advances in the management of cystic fibrosis (CF) in recentdecades have dramatically changed the epidemiology and prognosis of this serious disease, which is no longer an exclusively pediatric disease. This paper aims to review the changes in the incidence and survival of CF and to assess the impact of the discovery of the responsible gene (the CFTR gene) on these changes. The incidence of CF appears to be decreasing in most countries andpatient survival, which can be monitored by various indicators, has improved substantially, with an estimated median age of survivalof approximately50 years today. Cloning of the CFTR gene 30 years ago and efforts to identify its many mutations have greatly improved the management of CF. Implementation of genetic screening policies hasenabled earlier diagnosis (via newborn screening), in addition to prevention within families or in the general population in some areas (via prenatal diagnosis, family testing or population carrier screening). In the past decade, in-depth knowledge of the molecular bases of CF has also enabled the emergenceof CFTR modulator therapies which have led to major clinical advances in the treatment of CF. All of these phenomena have contributed to changing the face of CF. The advent of targeted therapies has paved the way for precision medicine and is expected to further improve survival in the coming years.

Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis.

Extensive genetic studies of chronic pancreatitis over the past decade have highlighted the importance of a tightly regulated balance between activation and inactivation of trypsin within the pancreas to disease susceptibility and resistance. The recent identification of chymotrypsin C (CTRC) as enzyme Y, which was proposed to protect the pancreas by degrading prematurely activated trypsinogen within the pancreas 20 years ago, made CTRC an excellent candidate gene for disease-association studies. Here, we analyzed all eight exons of the CTRC gene for conventional genetic variants and copy number variations (CNVs) by direct sequencing and quantitative fluorescent multiplex PCR, respectively, in a total of 287 French white patients (idiopathic x 216; familial x 42; hereditary x 29). While no CNVs were found in any of the 287 subjects, 20 conventional variations including a nonsense mutation (p.W55X), a microdeletion mutation (p.K247_R254del) and nine missense mutations were found in the 216 patients with idiopathic chronic pancreatitis (ICP). Except for two common polymorphisms, all the remaining 18 mutational events represent rare variations, with a minor allele frequency of 0-0.3% in the control population. All these rare variants were always found more frequently in the ICP patients than in the controls, and their combined frequency in the ICP patients (26/216; 12.0%) is significantly different from that in the controls (4/350; 1.1%) (OR = 11.8 [3.9-40.6]), chi (2) = 31.58, P < 10(-6)). This genetic finding, when considered in the perceived role of CTRC in eliminating prematurely activated trypsin, indicated that CTRC is a new pancreatitis susceptibility gene.

Time trends in birth incidence of cystic fibrosis in two European areas: data from newborn screening programs.

OBJECTIVE: To determine the incidence of cystic fibrosis (CF) and its time trends over a 16-year period (1990 to 2005) in 2 European regions with a long history of newborn screening (NBS) for CF, and to investigate the impact of some external factors. STUDY DESIGN: This study focused on data from NBS and prenatal diagnosis (PD) in Brittany (western France) and Veneto/Trentino Alto-Adige (northeastern Italy). RESULTS: Similar birth incidences of CF were observed in the 2 regions (1/3153 vs 1/3540; P = .245). Time trend analysis using Poisson regression revealed that the birth incidence decreased significantly in the Italian area only (average annual percent change [AAPC] = -4.7%; 95% confidence interval [CI] = -7.3 to -2; P = .0008). The use of PD appeared more common in Brittany, and considering the terminations of CF-affected fetuses, the adjusted incidence was 1/2191 in Brittany and 1/3116 in Veneto/Trentino, corresponding to variations of 30.5% (highly significant; P = .0002) and 12% (not significant; P = .16), respectively. Recording the reason for each PD allowed ready assessment of the affect of various public health policies on incidence. The affect of population mixing also appeared to be relevant in the Italian area. CONCLUSIONS: This study highlights how the incidence of CF has evolved in 2 European regions that have different attitudes toward PD and immigration policy.

Estimating the age of CFTR mutations predominantly found in Brittany (Western France).

BACKGROUND: Disparities in the spectrum of mutations within the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are commonly observed in populations from different ethnical and/or geographical origins. The occurrence of CF in Brittany (western France) is one of the highest in populations from Caucasian origin (<1/2000 in specific areas). The W846X(2), 1078delT and G551D mutations, as well as the I1027T polymorphism in cis with the DeltaF508 mutation (currently referred to as p.F508del) are particularly frequent in this area. We investigated the age of the respective variants in the region of interest. METHODS: Several polymorphic markers surrounding the CFTR gene were genotyped. Allele frequencies as well as mutation rates and other parameters were used to calculate the respective age of the most recent common ancestors in the region of interest by a previously employed, simple likelihood-based method. RESULTS: Following haplotype reconstruction and simulation, the ages were estimated to be approximately 600, 1000, 1200 and 600 years, respectively (with a 95% confidence interval). CONCLUSIONS: These datings thus provide historical insights in the context of understanding population migrations. They also underline the usefulness of this method for estimating the age of rare mutations with a limited number of carriers.

Primary sclerosing cholangitis is associated with abnormalities in CFTR.

BACKGROUND: The etiology of primary sclerosing cholangitis (PSC) is unknown. PSC and Cystic Fibrosis related liver disease have common features: chronic inflammation, biliary damage and similar cholangiographic findings. It is unknown whether or not PSC is related to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We hypothesize that a sub-group of PSC patients may be a "single-organ" presentation of CF. METHODS: Patients with PSC underwent nasal potential difference (NPD) measurement, sweat chloride measurement and complete CFTR sequencing by new generation sequencing. RESULTS: 6/32 patients aged 46 ±â€¯13 yrs. had CFTR causing mutations on one allele and 19 had CFTR polymorphisms; 6/23 tested had abnormal and 21 had intermediate sweat tests; 4/32 patients had abnormal NPD. One patient had chronic pancreatitis and was infertile. CONCLUSIONS: 19% of PSC patients had features of CFTR related disorder, 19% carry CFTR mutations and 50% had CFTR polymorphisms. In some patients, PSC may be a single organ presentation of CF or a CFTR-related disorder.

Polymorphic length of FOXE1 alanine stretch: evidence for genetic susceptibility to thyroid dysgenesis.

Familial cases of congenital hypothyroidism from thyroid dysgenesis (TD) (OMIM 218700) occur with a frequency 15-fold higher than by chance, FOXE1 is one of the candidate genes for this genetic predisposition and contains an alanine tract. Our purpose is to assess the influence of length of the alanine tract of FOXE1 on genetic susceptibility to TD. A case-control association study (based on 115 patients affected by TD and 129 controls genotyped by direct sequencing) and transmission disequilibrium testing (TDT) analyses were performed. The transcriptional activities of FOXE1 constructs containing 14 or 16 alanines were also studied. In the case-control association study, the 16/16 and 16/14 genotypes were inversely associated with TD (OR = 0.39, 95%CI = 0.22-0.68, P = 0.0005), strongly suggesting that the presence of 16 alanines in the tract protect against the occurrence of TD. This association was stronger in the subgroup of patients with ectopic thyroid (OR = 0.28, 95%CI = 0.13-0.58, P = 0.00015). The protection was confirmed by the TDT analysis performed in 39 trios (chi(2) = 4.3, P = 0.0374). Alternatively, the presence of the 14/14 genotype is associated with an increase risk of TD (OR = 2.59, 95%CI = 1.56-4.62, P = 0.0005). The expression studies showed that the transcriptional activities of FOXE1 with 16 alanines were significantly higher (1.55-fold) than FOXE1 containing 14 alanines (P < 0.003), while the nuclear localisation of the proteins was not affected. We conclude that FOXE1 through its alanine containing stretch modulates significantly the risk of TD occurrence, enhancing a mechanism linking an alanine containing transcription factor to disease.