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Newborn screening for cystic fibrosis (CF-NBS) using an IRT-DNA algorithm with a 12 CFTR-variant panel and an IRT/IRT failsafe was officially implemented in the French-speaking Community of Belgium in January 2020. This screening protocol was evaluated after 4 years according to the criteria defined by the European Cystic Fibrosis Society's working group on neonatal screening. Immunoreactive trypsinogen concentration (IRT) was measured on dried blood spots collected between the second and the fourth day of life. CFTR variants genotyping was initiated when IRT ≥ 99th percentile (P99). If at least 1 variant was identified, the child was referred to a CF center for a sweat test (ST). If IRT ≥ 99.9th percentile with no variant identified, a failsafe was provided with IRT repeated on day 21 and subsequent ST in case of persistent IRT > P99. Extensive Gene Analysis was initiated if sweat chloride level was ≥ 30 mmol/L. Over a period of 4 years, 212.979 newborns were screened. Forty-two were diagnosed with CF: 34 by CF-NBS, 3 following a meconium ileus, 3 by family history and 2 missed cases. Additionally, 112 healthy carriers and 14 CFSPID were identified. The median age at the first consultation was 23 days. The sensitivity of our CF-NBS is 95% (target ≥ 95%), the positive predictive value 18% (target ≥ 30%) and CF/CFSPID ratio 2.8., Conclusion:  Although follow-up is limited, this first evaluation demonstrates encouraging sensitivity and early management before one month of age., What Is Known: • Newborn screening for cystic fibrosis improves the prognosis of patients., What Is New: • This article presents an initial assessment of our screening after 4 years., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.) more...

Since January 2020, neonatal screening for cystic fibrosis (CF-NBS) has been implemented in the Wallonia-Brussels Federation. It's based on the immunoreactive trypsin (IRT1) assay between day 2 and day 4, associated with a 12 CFTR pathogenic variants analysis and with an IRT control on day 21. The aim of this study is to evaluate the performance of our CF-NBS in Liège according to the quality criteria defined by the European Cystic Fibrosis Society's working group on neonatal screening. After four years, 58.762 newborns have been screened. Nineteen children with cystic fibrosis were diagnosed : 14 by NBS, 3 following a meconium ileus, 1 by family history and 1 false negative diagnosed on clinical basis. Furthermore, 39 healthy carriers and 2 uncertain diagnosis (CFSPID) were identified. The sensitivity of CF NBS is 93,3 % (target ≥ 95 %), the positive predictive value (PPV) 17,7 % (target ≥ 30 %). Increasing the TIR1 threshold by 0,1 in 0,1 from P99 to P99,5, would be associated with a lower sensitivity and a non-significant improvement of PPV. A national assessment of CF NBS needs to be carried out. more...

It is estimated that in highly medicalised countries, median life expectancy for most newborns with cystic fibrosis now exceeds 70 years, approaching that of the general population. However, socio-economic disparities between countries continue to have a devastating impact on the prognosis of patients in Eastern Europe, Africa, India and South America. In Morocco, very limited genetic data suggest that the prevalence of this disease is at least of the same order as in Belgium. But as it is not really recognised by the national health system, patients are denied access even to symptomatic treatment. As a result, their outcome is tragic, similar to what it was 60 years ago in the most medicalised countries. A pilot project for a first paediatric reference centre in Casablanca is currently being set up. If properly resourced, this project can only be a success and should be the first step on the road towards cystic fibrosis care in this country. In a very humble way, several Belgian stakeholders are trying to support this project. more...

Context: Idiopathic central precocious puberty (iCPP) is defined by the premature reactivation of the hypothalamic-pituitary-gonadal axis with normal magnetic resonance imaging scan of the central nervous system, causing the development of secondary sexual characteristics before age 8 years in girls and 9 years in boys. MKRN3 loss of function variants now represent the most common genetic cause of iCPP., Objective: This work aims to document the clinical course of puberty in 8 families harboring pathogenic MKRN3 variants., Methods: This is an observational case series study of patients with CPP due to MKRN3 variants followed in a single center., Results: Genetic analysis of MKRN3 was carried out in 28 unrelated patients with iCPP and a family history of paternal inheritance or no/unavailable maternal inheritance, particularly in case of very early and rapidly evolving CPP. We identified 6 novel and 2 recently described variants in the MKRN3 gene in 9 girls, 1 boy, and their family members. These mutations were all predicted to be deleterious by in silico prediction programs., Conclusion: We have identified 6 novel MKRN3 mutations in children with CPP. An MKRN3 loss of function should be considered after careful history pinpointing paternally inherited CPP. A family segregation study allowed the detection of an MKRN3 variant in 2 young brothers still prepubertal, raising the question of screening and management of asymptomatic prepubertal family members., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.) more...

Background: Two recent studies identified a mutation (p.Asp620Asn) in the vacuolar protein sorting 35 gene as a cause for an autosomal dominant form of Parkinson disease . Although additional missense variants were described, their pathogenic role yet remains inconclusive., Methods and Results: We performed the largest multi-center study to ascertain the frequency and pathogenicity of the reported vacuolar protein sorting 35 gene variants in more than 15,000 individuals worldwide. p.Asp620Asn was detected in 5 familial and 2 sporadic PD cases and not in healthy controls, p.Leu774Met in 6 cases and 1 control, p.Gly51Ser in 3 cases and 2 controls. Overall analyses did not reveal any significant increased risk for p.Leu774Met and p.Gly51Ser in our cohort., Conclusions: Our study apart from identifying the p.Asp620Asn variant in familial cases also identified it in idiopathic Parkinson disease cases, and thus provides genetic evidence for a role of p.Asp620Asn in Parkinson disease in different populations worldwide. more...

Complex diseases usually harbour hereditary factors linked with multiple susceptibility genes. The additive effects of genetic and environmental factors are responsible for the pathology. The impact of heredity has been demonstrated through family studies, but also, and mostly, through the study of adopted people and twins. Recently, genome wide association studies (GWAS) allowed the identification of many susceptibility genes for most complex diseases. However, a large part of the heritability is still missing, probably because of insufficient exploration of rare genetic variants and/or epigenetic factors. The ultimate goal of these genetic studies is the definition of an individual risk leading to specific preventive measures (model "predict and prevent"), but this purpose remains very remote for the majority of complex diseases. more...

Background: Neonatal screening programs for sickle cell disease are common in North America and in some European countries. Isoelectric Focusing or High Performance Liquid Chromatography is the main technique used for hemoglobin variant detection., Methods: Since tandem mass spectrometry is being used for screening of inherited metabolic disorders and allows protein identification, we had developed an application to identify the most relevant hemoglobin mutations with this technology., Results: This approach had been previously validated and has been routinely applied in our laboratory for the last three years. We report here our experience with this new method in the field, applied to our East-Belgian population., Conclusions: To conclude, mass spectrometry provides an efficient alternative approach for laboratories performing neonatal screening of hemoglobin disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.) more...

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 × 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis. more...

Genome-wide association studies (GWAS) have identified dozens of risk loci for many complex disorders, including Crohn's disease. However, common disease-associated SNPs explain at most ∼20% of the genetic variance for Crohn's disease. Several factors may account for this unexplained heritability, including rare risk variants not adequately tagged thus far in GWAS. That rare susceptibility variants indeed contribute to variation in multifactorial phenotypes has been demonstrated for colorectal cancer, plasma high-density lipoprotein cholesterol levels, blood pressure, type 1 diabetes, hypertriglyceridemia and, in the case of Crohn's disease, for NOD2 (refs. 14,15). Here we describe the use of high-throughput resequencing of DNA pools to search for rare coding variants influencing susceptibility to Crohn's disease in 63 GWAS-identified positional candidate genes. We identify low frequency coding variants conferring protection against inflammatory bowel disease in IL23R, but we conclude that rare coding variants in positional candidates do not make a large contribution to inherited predisposition to Crohn's disease. more...

We undertook a meta-analysis of six Crohn's disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 × 10⁻⁸). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohn's disease. more...

Background: A multicenter genome-wide association scan for Crohn's Disease (CD) has recently reported 40 CD susceptibility loci, including 29 novel ones (19 significant and 10 putative). To gain insight into the genetic overlap between CD and ankylosing spondylitis (AS), these markers were tested for association in AS patients., Principal Findings: Two previously established associations, namely with the MHC and IL23R loci, were confirmed. In addition, rs2872507, which maps to a locus associated with asthma and influences the expression of the ORMDL3 gene in lymphoblastoid cells, showed a significant association with AS (p = 0.03). In gut biopsies of AS and CD patients, ORMDL3 expression was not significantly different from controls and no correlation was found with the rs2872507 genotype (Spearman's rho: -0.067). The distribution of p-values for the remaining 36 SNPs was significantly skewed towards low p-values unless the top 5 ranked SNPs (ORMDL3, NKX2-3, PTPN2, ICOSLG and MST1) were excluded from the analysis., Conclusions: Association analysis using risk variants for CD led to the identification of a new risk variant associated with AS (ORMDL3), underscoring a role for ER stress in AS. In addition, two known and five potentially relevant associations were detected, contributing to common susceptibility of CD and AS. more...

The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are common causes of morbidity in children and young adults in the western world. Here we report the results of a genome-wide association study in early-onset IBD involving 3,426 affected individuals and 11,963 genetically matched controls recruited through international collaborations in Europe and North America, thereby extending the results from a previous study of 1,011 individuals with early-onset IBD. We have identified five new regions associated with early-onset IBD susceptibility, including 16p11 near the cytokine gene IL27 (rs8049439, P = 2.41 x 10(-9)), 22q12 (rs2412973, P = 1.55 x 10(-9)), 10q22 (rs1250550, P = 5.63 x 10(-9)), 2q37 (rs4676410, P = 3.64 x 10(-8)) and 19q13.11 (rs10500264, P = 4.26 x 10(-10)). Our scan also detected associations at 23 of 32 loci previously implicated in adult-onset Crohn's disease and at 8 of 17 loci implicated in adult-onset ulcerative colitis, highlighting the close pathogenetic relationship between early- and adult-onset IBD. more...

Background and Aims: The familial Mediterranean fever (FMF) gene (MEFV) encodes pyrin, a major regulator of the inflammasome platform controlling caspase-1 activation and IL-1beta processing. Pyrin has been shown to interact with the gene product of NLRP3, NALP3/cryopyrin, also an important active member of the inflammasome. The NLRP3 region was recently reported to be associated with Crohn's disease (CD) susceptibility. We therefore sought to evaluate MEFV as an inflammatory bowel disease (IBD) susceptibility gene., Methodology and Results: MEFV colonic mucosal gene expression was significantly increased in experimental colitis mice models (TNBS p<0.0003; DSS p<0.006), in biopsies from CD (p<0.02) and severe ulcerative colitis (UC) patients (p<0.008). Comprehensive genetic screening of the MEFV region in the Belgian exploratory sample set (440 CD trios, 137 UC trios, 239 CD cases, 96 UC cases, and 107 healthy controls) identified SNPs located in the MEFV 5' haplotype block that were significantly associated with UC (rs224217; p = 0.003; A allele frequency: 56% cases, 45% controls), while no CD associations were observed. Sequencing and subsequent genotyping of variants located in this associated haplotype block identified three synonymous variants (D102D/rs224225, G138G/rs224224, A165A/rs224223) and one non-synonymous variant (R202Q/rs224222) located in MEFV exon 2 that were significantly associated with UC (rs224222: p = 0.0005; A allele frequency: 32% in cases, 23% in controls). No consistent associations were observed in additional Canadian (256 CD trios, 91 UC trios) and Scottish (495 UC, 370 controls) sample sets. We note that rs224222 showed marginal association (p = 0.012; G allele frequency: 82% in cases, 70% in controls) in the Canadian sample, but with a different risk allele. None of the NLRP3 common variants were associated with UC in the Belgian-Canadian UC samples and no significant interactions were observed between NLRP3 and MEFV that could explain the observed flip-flop of the rs224222 risk allele., Conclusion: The differences in association levels observed between the sample sets may be a consequence of distinct founder effects or of the relative small sample size of the cohorts evaluated in this study. However, the results suggest that common variants in the MEFV region do not contribute to CD and UC susceptibility. more...

Inflammatory Bowel Disease, Crohn's disease and ulcerative colitis, are complex, multifactorial, polygenic diseases. Huge progresses in the knowledge of human genome and genotyping techniques have allowed the identifications of dozens of genes and loci associated with these diseases. These discoveries set the path for a new molecular classification of these diseases and let us see new therapeutic possibilities. The present paper highlights, in the setting of the Synthèse CHU 2009 meeting, the contributions of the team of the CHU and university of Liège in this field. more...

We used a candidate gene approach to identify a set of SNPs, located in a predicted regulatory region on chromosome 1q44 downstream of NLRP3 (previously known as CIAS1 and NALP3) that are associated with Crohn's disease. The associations were consistently replicated in four sample sets from individuals of European descent. In the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these SNPs were strongly associated with risk of Crohn's disease (P(combined) = 3.49 x 10(-9), odds ratio = 1.78, confidence interval = 1.47-2.16 for rs10733113), reaching a level consistent with the stringent significance thresholds imposed by whole-genome association studies. In addition, we observed significant associations between SNPs in the associated regions and NLRP3 expression and IL-1beta production. Mutations in NLRP3 are known to be responsible for three rare autoinflammatory disorders. These results suggest that the NLRP3 region is also implicated in the susceptibility of more common inflammatory diseases such as Crohn's disease. more...

Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease (a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development. more...

The interferon regulatory factor 5 (IRF5) gene encodes a transcription factor that plays an important role in the innate as well as in the cell-mediated immune responses. The IRF5 gene has been shown to be associated with systemic lupus erythematosus and rheumatoid arthritis. We studied whether the IRF5 gene is also associated with inflammatory bowel diseases (IBD), Crohn disease (CD) and ulcerative colitis (UC). Twelve polymorphisms in the IRF5 gene were genotyped in a cohort of 1007 IBD patients (748 CD and 254 UC) and 241 controls from Wallonia, Belgium. The same polymorphisms were genotyped in a confirmatory cohort of 311 controls and 687 IBD patients (488 CD and 192 UC) from Leuven, Belgium. A strong signal of association [P = 1.9 x 10(-5), odds ratio (OR) 1.81 (1.37-2.39)] with IBD was observed for a 5 bp indel (CGGGG) polymorphism in the promoter region of the IRF5 gene. The association was detectable also in CD patients (P = 6.8 x 10(-4)) and was particularly strong among the UC patients [P = 5.3 x 10(-8), OR = 2.42 (1.76-3.34)]. The association of the CGGGG indel was confirmed in the second cohort [P = 3.2 x 10(-5), OR = 1.59 (1.28-1.98)]. The insertion of one CGGGG unit is predicted to create an additional binding site for the transcription factor SP1. Using an electrophoretic mobility shift assay, we show allele-specific differences in protein binding to this repetitive DNA-stretch, which suggest a potential function role for the CGGGG indel. more...

The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15-1.48; P=0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16-1.54; P=0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04-1.30; P=0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway - in this case, TLR4 and its signaling molecule TIRAP - plays a role in susceptibility to IBD. more...

To identify novel susceptibility loci for Crohn disease (CD), we undertook a genome-wide association study with more than 300,000 SNPs characterized in 547 patients and 928 controls. We found three chromosome regions that provided evidence of disease association with p-values between 10(-6) and 10(-9). Two of these (IL23R on Chromosome 1 and CARD15 on Chromosome 16) correspond to genes previously reported to be associated with CD. In addition, a 250-kb region of Chromosome 5p13.1 was found to contain multiple markers with strongly suggestive evidence of disease association (including four markers with p < 10(-7)). We replicated the results for 5p13.1 by studying 1,266 additional CD patients, 559 additional controls, and 428 trios. Significant evidence of association (p < 4 x 10(-4)) was found in case/control comparisons with the replication data, while associated alleles were over-transmitted to affected offspring (p < 0.05), thus confirming that the 5p13.1 locus contributes to CD susceptibility. The CD-associated 250-kb region was saturated with 111 SNP markers. Haplotype analysis supports a complex locus architecture with multiple variants contributing to disease susceptibility. The novel 5p13.1 CD locus is contained within a 1.25-Mb gene desert. We present evidence that disease-associated alleles correlate with quantitative expression levels of the prostaglandin receptor EP4, PTGER4, the gene that resides closest to the associated region. Our results identify a major new susceptibility locus for CD, and suggest that genetic variants associated with disease risk at this locus could modulate cis-acting regulatory elements of PTGER4., Competing Interests: Competing interests. The authors have declared that no competing interests exist. more...

Cultured L-929 cells respond to media-made hyperosmotic (600 mOsmol/kg H2O) by addition of NaCl, sorbitol or proline by adjusting successively their intracellular level in different osmolytes: Na+, K+, amino acids and sorbitol. In the NaCl medium, Na+ and K+ are first to increase. Their concentration is then down-regulated while they are replaced by less disrupting osmolytes: amino acids and sorbitol. The amino-acid level is also adjusted with respect to the increase in sorbitol which starts only after 24 h, depending on the induction of aldose reductase. A similar evolution in the amount of these osmolytes is observed, with different time scales and amplitudes, depending on whether the osmotic shocks are applied abruptly or slowly, in a more physiological way. The interplay between the osmolytes is also different depending on their availability in the external medium. Such complex evolutions indicate that a cascade of interacting signals must be considered to account for the overall regulation process. It can hardly be fitted into a model implicating a single primary signalling event (early increase in ions or decrease in cell volume) as usually postulated. Also, the volume up-regulation is not significantly different in the different conditions, showing that it is not primarily dependent on the adjustment of the intracellular osmolarity which is reached immediately upon cell shrinkage and is maintained all over, independently of the availability and changes in nature of the osmolytes. more...

L-929 cells acclimated to media made hyperosmotic (600 mosmol/kgH2O) by addition of NaCl, sorbitol, or mannitol show, on SDS-polyacrylamide gels, a markedly enhanced protein band at 40 kDa, most likely corresponding to the enzyme aldose reductase. The effect was not observed in cells acclimated to a medium rendered hyperosmotic by addition of proline. The major organic osmolyte accumulated is sorbitol in cells acclimated to high-sorbitol or high-NaCl medium, proline in cells acclimated to high-proline medium. Cells acclimated to any of these hyperosmotic media display unaltered Na+ levels and similarly increased K+ levels and decreased Cl-levels. These results are interpreted in terms of the mechanisms involved in aldose reductase induction and in regulation of the enzyme activity in long-term acclimation to hyperosmotic media. more...

Application of abrupt, large hyperosmotic shocks induces in L929 cultured cells changes similar to those previously described in other cell types, notably a hypercondensation of the nuclear chromatin. This paper shows that; 1) this phenomenon is concomitant with a complete disappearance of deoxyribonucleic acid, as visualized by immunogold labelling, from the nucleoplasmic spaces; 2) acclimation to high osmolarities (600 mOsm) by addition to the culture medium of NaCl, sorbitol or proline protects the cells from these effects, which appear to be largely attenuated-acclimated cells also survive much better to the osmotic shock than do control cells and; 3) the best protection seems to be provided by sorbitol and NaCl. Proline acclimation is less effective. These effects are discussed in terms of increased tolerance to NaCl load induced at the level of different macromolecules by so-called 'compensatory' organic compounds. more...

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures. more...