Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis.

Mutations in the serine protease inhibitor Kazal type 1 gene (SPINK1) encoding pancreatic secretory trypsin inhibitor (PSTI) have recently been found to be associated with chronic pancreatitis. Nevertheless, knowledge of severe mutations is particularly scarce, both in terms of number and in the extent of clinical information. The aim of this study was to expand the known spectrum of such mutations. 46 unrelated families, each including at least two pancreatitis patients and carrying neither cationic trypsinogen (PRSS1) mutations nor the frequent SPINK1 N34S mutation, participated in this study. The four exons and their flanking sequences of the SPINK1 gene were screened by denaturing high performance liquid chromatography analysis (DHPLC); and mutations were identified by direct sequencing. A heterozygous microdeletion mutation (c.27delC), which occurs within a symmetric element, was identified in two families. In one family, c.27delC showed segregation with the disease across two generations, with a penetrance of up to 75%. But in the other family, however, the same mutation manifested as a low-penetrance susceptibility factor. In addition, a novel heterozygous splicing mutation, c.87+1G>A (G>A substitution at nucleotide +1 of intron 2) was found in one family with familial pancreatitis. Our results also helped to resolve the sharply differing views about PSTI's role in pancreatitis.

Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography.

BACKGROUND: R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365 through 366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. MATERIALS AND METHODS: DNA samples containing either the known c.365G>A or c.365 through 366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay. RESULTS: DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C). CONCLUSIONS: A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.

[The hemochromatosis gene (HFE). Molecular analysis--diagnostic applications]. / Le gène de l'hémochromatose (HFE). Analyse moléculaire--applications diagnostiques.

Hemochromatosis is the most common single gene disorder in Caucasian populations. Regulation of iron balance by intestine is impaired, leading to a widespread deposition of iron, and the disease is associated with an increased risk of hepatocellular carcinoma. Typically the excess of iron treated by phlebotomies is performed in our Blood Center. In 1996 an original paper identifying HFE as a strong candidate gene for hemochromatosis was published and two mutations were described (C282Y and H63D). The former results in a cysteine to tyrosine substitution at amino acid 282 and was found in different patient populations up to 80-90% of patients homozygous for the C282Y mutation. The frequency of the second variant H63D is also increased in hemochromatosis patients but its penetrance is probably not complete. Assessing clinical implications is a new way of identifying patients at risk for this frequent and probably underdiagnosed disease, and important because treatment by venesections is safe with a proven benefit in preventing development of the disease. Four hundred and eighty patients were included in our study and we have shown in this work a correlation between the genotype and the phenotypic presentation of the disorder, with patients homozygous for the C282Y mutation having a greater excess of iron.

HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.

Hereditary hemochromatosis (HH) is a common autosomal recessive genetic disorder of iron metabolism. The HFE candidate gene encoding an HLA class I-like protein involved in HH was identified in 1996. Two missense mutations have been described: C282Y, accounting for 80% to 90% of HH chromosomes, and H63D, which is associated with a milder form of the disease representing 40% to 70% of non-C282Y HH chromosomes. We report here on the analysis of C282Y, H63D, and the 193A-->T substitution leading to the S65C missense substitution in a large series of probands and controls. The results confirm that the C282Y substitution was the main mutation involved in hemochromatosis, accounting for 85% of carrier chromosomes, whereas the H63D substitution represented 39% of the HH chromosomes that did not carry the C282Y mutation. In addition, our screening showed that the S65C substitution was significantly enriched in probands with at least one chromosome without an assigned mutation. This substitution accounted for 7.8% of HH chromosomes that were neither C282Y nor H63D. This enrichment of S65C among HH chromosomes suggests that the S65C substitution is associated with the mild form of hemochromatosis.

HLA class II typing and idiopathic IgA nephropathy (IgAN): DQB1*0301, a possible marker of unfavorable outcome.

Previous studies have reported associations between HLA antigens and Idiopathic IgA Nephropathy (IgAN). Nevertheless most of the studies were performed by serology. Thus we decided to perform the HLA class II typing of 58 patients by molecular biology techniques. We report a small increase of DRB1*04. But the main result of our study is the identification of a strong association between HLA DQB1*0301 and IgAN patients with an unfavorable outcome.

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here. This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.

Identification of a novel pancreatitis-associated missense mutation, R116C, in the human cationic trypsinogen gene (PRSS1).

Over the past 5 years, several gain-of-function missense mutations in the human cationic trypsinogen gene (PRSS1, OMIM 276000) have been associated with hereditary and/or sporadic pancreatitis. This study reports a new pancreatitis-associated mutation--R116C (CGT > TGT: c.346C > T)--in the gene.

Phenotype-genotype correlation in haemochromatosis subjects.

Haemochromatosis is a common autosomal recessive genetic disorder of iron metabolism. A candidate gene was recently identified (HLA-H) and two amino acid substitutions (C282Y and H63D) were characterized. Haemochromatosis probands (n = 478) from Brittany were selected from their iron status markers, primarily serum iron, serum ferritin and transferrin saturation. We investigated the relationships between haemochromatosis phenotype and genotypes at the HLA-H locus and surrounding markers. As already reported, we observed that the C282Y substitution is unambiguously associated with the haemochromatosis phenotype, haemochromatosis patients homozygous for the substitution (Tyr/Tyr) accounting for 81.2% of all haemochromatosis patients. A clear heterogeneity in serum ferritin and transferrin saturation values, and in iron removed by phlebotomy was observed among haemochromatosis patients that is correlated with the presence of two subgroups of individuals homozygous and non-homozygous for the mutant allele C282Y, the latter being characterized by lower phenotypic values. In this subgroup, sequencing did not reveal any other mutation in the HLA-H gene, hence the genotype remained unclear. Thus, an additional non-genetic cause, other mutations or another gene can not be excluded as explanations for the results in these patients.

Linkage disequilibrium between the four most common cystic fibrosis mutations and microsatellite haplotypes in the Celtic population of Brittany.

Microsatellite haplotypes were determined for 117 chromosomes carrying the four most frequent mutations in the cystic fibrosis (CF) gene identified in the Breton population of Celtic origin, as well as for 83 normal chromosomes (noncarriers of a CF mutation). Each of the three non-delta F508 mutations was associated with a single haplotype: 1078deIT with 16-31-13, G55ID with 16-7-17, and W846X with 16-32-13. Although these results suggest identity-by-descent for each mutation, recurrent mutations, although unlikely, could not be completely ruled out. The four most frequent haplotypes on normal chromosomes and the three most frequent haplotypes on delta F508 chromosomes are the same as those found in Ireland, Spain, and Italy. This suggests that some haplotypes, associated or not with the delta F508 mutation, were present in an ancestral population from which all four populations descended.

Prenatal diagnosis of cystic fibrosis in different European populations: application of denaturing gradient gel electrophoresis.

The cystic fibrosis transmembrane regulator gene, one of the most commonly mutated in the European population, was cloned in 1989 and since then has been extensively analysed in patients of various ethnic backgrounds. We have screened the entire coding sequences of the cystic fibrosis transmembrane regulator gene and identified many mutations and polymorphisms. In this paper we propose a general strategy to improve prenatal diagnosis and genetic counselling of cystic fibrosis (CF). As this approach based on denaturing gradient gel electrophoresis is adaptable to different populations, it greatly increases the sensibility and specificity of CF prenatal diagnosis.

Relation between HFE mutations and mild iron-overload expression.

The identification of the HFE gene involved in hemochromatosis allows genetic tests based on mutation analysis to be performed. However, discrepancies in the correlation between HFE genotypes and iron-loading status have arisen. We investigated 708 patients with various signs or symptoms suggesting a putative iron overload that, nevertheless, did not reach the current criteria for hemochromatosis diagnosis. Most of the patients (91.4%) included in our study displayed one of three classical iron marker values above the threshold defined for iron overloading. HFE mutation analysis allowed us to identify 45.7% of carrier chromosomes in the studied group of patients that showed higher frequencies of HFE mutations compared with controls. In addition, the frequencies of compound C282Y/H63D heterozygous, H63D/H63D homozygous, and C282Y heterozygous genotypes were higher than those in HH probands and controls; they accounted for 16, 5.6, and 22.5% of the patients, respectively. All genotypic groups had a significantly higher value of serum ferritin concentration compared to the normal value; only the C282Y homozygotes and compound heterozygotes with H63D had a transferrin saturation significantly higher than the normal value. On the whole the H63D homozygous and compound heterozygous patients constitute an intermediate phenotypic group between HH and controls. Some of them may reach the critical overloading defined for HH diagnosis along with a potential risk of developing complications, whereas others only show a partial phenotypic expression.

Nramp2 analysis in hemochromatosis probands.

The mechanism that leads to iron overload in hereditary hemochromatosis is not yet fully understood and genes other than HFE may be involved. Nramp2 is an intestinal iron transporter, upregulated by dietary iron deficiency, which also colocalizes with transferrin in recycling endosomes. The purpose of the present study was to analyze the coding region of the Nramp2 gene in 14 hemochromatosis probands which did not carry any HFE mutations on both chromosomes. We confirmed the existence of a polymorphism (1254 T --> C), which presumably is not associated with hereditary hemochromatosis, but we did not find any mutation. On the other hand, we identified 17 splice variants of the Nramp2 mRNA. Eight corresponded to activation of cryptic splicing sequences between exons 3 and 4. They were observed in a majority of hemochromatosis probands and control subjects. This indicates the existence of an important splicing instability in this region. At this stage, the biological significance of these variants is unclear. Our study did not find evidence for the involvement of the Nramp2 gene in hereditary hemochromatosis. The remaining question is whether hemochromatosis probands in our study have iron overload because of environmental factors or due to mutation in gene(s) other than HFE and Nramp2.

Mutation analysis in the HFE gene in patients with hereditary haemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, Canada).

A mutation analysis of the HFE gene followed, when applicable, by sequencing was performed on 47 patients with hereditary haemochromatosis (HH) living in Saguenay-Lac-Saint-Jean. The C282Y and H63D mutations were present on 50% and 20.3% of the HH chromosomes respectively. These frequencies were very different from those found in other populations and could be, at least partially, the result of a founder effect. No new mutation was identified among the remaining 28.1% of the HH chromosomes. Five of the eight probands with no mutation in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis.

Clinical and molecular aspects of juvenile hemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, canada).

We report the clinical, biochemical, and genetic characteristics of 13 hemochromatosis patients from Saguenay-Lac-Saint-Jean in whom the first symptoms appeared before age 30. Although the mean age at onset of the first symptoms was 21. 5 years, their mean age at diagnosis was 23.8 years; the diagnosis was particularly delayed among women. Seventy-seven percent of the patients had hypogonadotrophic hypogonadism and 69% heart failure and/or cardiac arrhythmias. Genetic analysis of the HFE gene revealed heterozygosity for the C282Y mutation in 2 patients and for the S65C mutation in 2 others and homozygosity for the H63D mutation in 1 patient. The remaining 8 patients had no identified mutation in the HFE gene, although sequencing of all seven codons and intron-exon junctions was performed (5 patients). All 13 patients fulfill the clinical criteria of juvenile hemochromatosis and represent the largest cluster thus far reported.

Identical intragenic microsatellite haplotype found in cystic fibrosis chromosomes bearing mutation G551D in Irish, English, Scottish, Breton and Czech patients.

Mutation G551D of exon 11 of the cystic fibrosis transmembrane conductance regulator gene is one of the most common mutations in patients of European origin. In order to test the hypothesis that the mutation is identical by descent in these patients, we have studied haplotypes for the three intragenic microsatellite markers IVS8CA, IVS17bTA and IVS17bCA from 92 patients bearing this mutation, who had been referred to laboratories in Ireland, Scotland, England, France (Brittany) and the Czech Republic. In all cases we found that only haplotype 16-7-17 is associated with mutation G551D. Our results support the hypothesis of identity by descent of all cystic fibrosis chromosomes bearing mutation G551D in these patient populations, and suggest that given the combined mutation rate of the microsatellite markers, there is a low probability (p < 0.05) that the haplotype where mutation G551D first occurred remained unaltered for more than 170 generations.

Association of 1078 del T cystic fibrosis mutation with severe disease.

Apart from the high frequency of the delta F508 mutation (81.81%) in Breton cystic fibrosis chromosomes, one mutation, 1078 del T, is also observed frequently (4.96%) in this group, in comparison with the rest of the French where it occurs with a frequency of 0.57%. These two mutations account for more than 86.5% of the total CF mutations identified on Breton chromosomes. We have conducted an unblinded retrospective analysis of 25 patients with the 1078 del T mutation and compared their phenotypes with those of a group of 70 delta F508 homozygous patients. Both groups of patients had the same ethnic origin and were regularly attending the same CF centre in Brittany, which makes this sample highly homogeneous despite the small size. The 1078 del T mutation appeared to be associated with severe presentation of the disease with, however, a trend to reduced mortality and less Pseudomonas aeruginosa colonisation.