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.

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.

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.

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.

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.

Effects of AMPD1 common mutation on the metabolic-chronotropic relationship: Insights from patients with myoadenylate deaminase deficiency.

PURPOSE: Current evidence indicates that the common AMPD1 gene variant is associated with improved survival in patients with advanced heart failure. Whilst adenosine has been recognized to mediate the cardioprotective effect of C34T AMPD1, the precise pathophysiologic mechanism involved remains undefined to date. To address this issue, we used cardio-pulmonary exercise testing data (CPX) from subjects with myoadenylate deaminase (MAD) defects. METHODS: From 2009 to 2013, all the patients referred in our laboratory to perform a metabolic exercise testing, i.e. a CPX with measurements of muscle metabolites in plasma during and after exercise testing, were prospectively enrolled. Subjects that also underwent an open muscle biopsy for diagnosis purpose were finally included. The metabolic-chronotropic response was assessed by calculating the slope of the linear relationship between the percent heart rate reserve and the percent metabolic reserve throughout exercise. MAD activity was measured using the Fishbein's technique in muscle biopsy sample. The common AMPD1 mutation was genotyped and the AMPD1 gene was sequenced to screen rare variants from blood DNA. RESULTS: Sixty-seven patients were included in the study; 5 had complete MAD deficiency, 11 had partial MAD deficiency, and 51 had normal MAD activity. Compared with normal MAD activity subjects, MAD deficient subjects appeared to have a lower-than-expected metabolic-chronotopic response during exercise. The metabolic-chronotropic relationship is more closely correlated with MAD activity in skeletal muscle (Rs = 0.57, p = 5.93E-7, Spearman correlation) than the presence of the common AMPD1 gene variant (Rs = 0.34, p = 0.005). Age-predicted O2 pulse ratio is significantly increased in MAD deficient subjects, indicating a greater efficiency of the cardiovascular system to deliver O2 (p < 0.01, Scheffé's post hoc test). CONCLUSION: The metabolic-chronotropic response is decreased in skeletal muscle MAD deficiency, suggesting a biological mechanism by which AMPD1 gene exerts cardiac effect.

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.

Highlighting the impact of cascade carrier testing in cystic fibrosis families.

BACKGROUND: Cascade carrier testing within cystic fibrosis (CF) affected families offers relatives of CF patients the opportunity to know their status regarding the mutation that segregates within their family, and thus to make informed reproductive choices. As an Australian study has recently shown that this test seemed underused, we searched to assess uptake of this test in a European area where CF is common, and to report its public health implications. METHODS: This study relied on 40 CF-affected families from western Brittany, France. Investigations included drawing of family trees and registration of carrier tests performed in those families. RESULTS: Of the 459 relatives eligible for testing, 185 were tested, leading to an adjusted uptake rate of testing of 40.7% (95% CI: [34.1%; 47.3%]). The main predictors for having testing were being female (p=0.031) and having a high prior risk (p<0.001). Planning a pregnancy or expecting a child (reported in at least 38.4% of tested relatives) also appeared critical in choosing to be tested. Overall, carrier testing allowed to reassure more than 1/4 of the relatives and to detect five new 1-in-4 at-risk couples who then requested prenatal diagnosis. CONCLUSIONS: This observational study assesses, for first time in Europe, uptake of CF cascade carrier testing within CF families, which is a critical tool to reassure non-carriers and to detect early new at-risk couples.

Diagnostic Algorithm for Glycogenoses and Myoadenylate Deaminase Deficiency Based on Exercise Testing Parameters: A Prospective Study.

AIM: Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies. METHODS: From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise), and recovery (2, 5, 10, and 15 min). Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test). Myoadenylate deaminase (MAD) activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC) curve analysis. RESULTS: The study involved 51 patients. Omitting patients with glycogenoses (n = 3), MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067) to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0) for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%. CONCLUSION: The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis.

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.

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.

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.

Evidence for the high importance of co-morbid factors in HFE C282Y/H63D patients cared by phlebotomies: results from an observational prospective study.

Despite type I haemochromatosis (HC) is mainly associated with the HFE C282Y/C282Y genotype, a second genotype -C282Y/H63D- has mostly been described in other patients. Its association with HC, apart from any associated co-morbid factors, remains unclear and complex to interpret for physicians. This study assesses the weight of this genotype and the role of co-morbid factors in the occurrence of iron overload. This prospective study included the C282Y/C282Y (n = 172) and C282Y/H63D (n = 58) patients enrolled in a phlebotomy program between 2004 and 2007 in a blood centre of western Brittany (Brest, France), where HC is frequent. We compared prevalence of these two genotypes, as well as patients' profile regarding degree of iron overload and prevalence of co-morbid factors. First, we confirmed the obvious deficit of C282Y/H63D compound heterozygotes among patients cared by phlebotomies. This genotype was 3.0 times less frequent than the C282Y/C282Y genotype among those patients (18.9% vs. 56.0%) whereas it was 4.9 times more frequent in the general population (4.3% vs. 0.9%; p<0.0001). Despite a similar level of hyperferritinaemia, the C282Y/H63D patients who came to medical attention had a milder plasma iron overload, reflected by a lower transferrin saturation median (52.0% vs. 84.0%; p<0.0001). They also exhibited more frequently co-morbid factors, as heavy drinking (26.0% vs. 13.9%; p = 0.0454), overweight (66.7% vs. 39.4%; p = 0.0005) or both (21.3% vs. 2.6%; p<0.0001). Ultimately, they required a lower amount of iron removed to reach depletion (2.1 vs. 3.4 g; p<0.0001), clearly reflecting their lower tissue iron. This study confirms that H63D is a discrete genetic susceptibility factor whose expression is most visible in association with other co-factors. It highlights the importance of searching for co-morbidities in these diagnostic situations and of providing lifestyle and dietary advice.

Cascade testing in families of carriers identified through newborn screening in Western Brittany (France).

BACKGROUND: Newborn screening (NBS) for cystic fibrosis (CF) can lead to the detection of healthy carriers. We report a unique assessment of family testing following the identification of carriers by NBS for over 20 years, in an area where CF is frequent. METHODS: We reviewed all of the carriers identified by NBS between 1991 and 2010 and registered the tests done in those families. RESULTS: NBS identified 0.1% of the newborns as carriers, which correspond only to 2.6% of the expected carriers born within the period, and 1/3 of those with an increased IRT level. Of the 195 families, 75.9% requested testing (2.5 tests per family). We identified 183 carriers and five 1-in-4 risk couples. Reassurance about genetic status was provided to 96% of the couples. CONCLUSIONS: Carriers detected by NBS appeared to be well managed in our area, and cascade testing that informs on genetic status seems relatively active.

Evidence for decline in the incidence of cystic fibrosis: a 35-year observational study in Brittany, France.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disorder whose incidence has long been estimated as 1/2500 live births in Caucasians. Expanding implementation of newborn screening (NBS) programs now allows a better monitoring of the disease incidence, what is essential to make reliable predictions for disease management. This study assessed time trends in the birth incidence of CF over a long period (35 years: 1975-2009) in an area where CF is frequent (Brittany, France) and where NBS has been implemented for more than 20 years. METHODS: This study enrolled CF patients born in Brittany between January 1st 1975 and December 31st 2009 (n = 483). Time trends in incidence were examined using Poisson regression and mainly expressed using the average percent change (APC). RESULTS: The average number of patients born each year declined from 18.6 in the late 1970's (period 1975-79) to 11.6 nowadays (period 2005-09). The corresponding incidence rates dropped from 1/1983 to 1/3268, which represented a decline close to 40% between these two periods (APC = -39.3%, 95% CI = -55.8% to -16.7%, p = 0.0020). A clear breakpoint in incidence rate was observed at the end of the 1980's (p < 0.0001). However, the incidence rate has remained quite stable since that time (annual APC = -1.0%, 95% CI = -3.0% to 1.1%, p = 0.3516). CONCLUSIONS: This study provides an accurate picture of the evolution of the incidence of a genetic disease over a long period and highlights how it is influenced by the health policies implemented. We observed a 40% drop in incidence in our area which seems consecutive to the availability of prenatal diagnosis.