Clinical and family studies in genetic hemochromatosis: microsatellite and HFE studies in five atypical families.

A candidate gene (HFE) has been described for hereditary hemochromatosis on chromosome 6. The study of well-defined atypical hemochromatosis families using genetic markers may increase our understanding of the sensitivity and the specificity of genotyping in hemochromatosis. One hundred and thirteen Canadian families with genetic hemochromatosis were surveyed to find atypical families as possible examples of people with genetic recombinations. All families underwent clinical investigations including iron studies and HLA typing. Each individual was typed at three polymorphic microsatellite loci (D6S105, D6S1260, and D6S299) on chromosome 6. Sixteen subjects were studied for the two missense mutations described for the candidate gene for hemochromatosis (C282Y, H63D). There were eight HLA-identical siblings found in four different families (five men, three women; age range 30-72) with normal transferrin saturation and ferritin levels. There were two patients identified who were homozygous for the C282Y mutation without biochemical evidence of iron overload, and two patients with no evidence of the mutation with significant iron overload. Our conclusions are as follows: 1) finding HLA-identical siblings without iron overload does not confirm a genetic recombination, 2) difficulties in phenotypic definition of disease and the description of new iron overload syndromes that may differ from classical genetic HC cause complicated genetic studies, and 3) finding iron-loaded patients without a C282Y mutation and patients that are homozygous for the C282Y mutation without evidence of iron overload may limit the use of genotyping in population screening for hemochromatosis.

Structural analysis of the HLA-A/HLA-F subregion: precise localization of two new multigene families closely associated with the HLA class I sequences.

Positional cloning strategies for the hemochromatosis gene have previously concentrated on a target area restricted to a maximum genomic expanse of 400 kb around the HLA-A and HLA-F loci. Recently, the candidate region has been extended to 2-3 Mb on the distal side of the MHC. In this study, 10 coding sequences [hemochromatosis candidate genes (HCG) I to X] were isolated by cDNA selection using YACs covering the HLA-A/HLA-F subregion. Two of these (HCG II and HCG IV) belong to multigene families, as well as other sequences already described in this region, i.e., P5, pMC 6.7, and HLA class 1. Fingerprinting of the four YACs overlapping the region was performed and allowed partial localization of the different multigene family sequences on each YAC without defining their exact positions. Fingerprinting on cosmids isolated from the ICRF chromosome 6-specific cosmid library allowed more precise localization of the redundant sequences in all of the multigene families and revealed their apparent organization in clusters. Further examination of these intertwined sequences demonstrated that this structural organization resulted from a succession of complex phenomena, including duplications and contractions. This study presents a precise description of the structural organization of the HLA-A/HLA-F region and a determination of the sequences involved in the megabase size polymorphism observed among the A3, A24, and A31 haplotypes.

A new non-HLA multigene family associated with the PERB11 family within the MHC class I region.

In an effort to initiate steps designed to characterize the idiopathic hemochromatosis disease gene, the HLA-A/HLA-F region where this gene is in disequilibrium linkage with some polymorphic markers has been overlapped by a yeast artificial chromosome (YAC) contig. In order to achieve the physical mapping of these YACs and of the corresponding genomic region, we subcloned one of the YACs involved. A computer-assisted analysis of the sequence of one subclone led to the isolation of a potential exon that proved to belong to a new expressed messenger named HCGIX. After Southern blot analysis, the corresponding cDNA clone was found to belong to a new multigene family whose members are dispersed throughout the HLA class I region and are closely associated with members of another recently described multigene family designated PERB11. The data reported here suggest that these two multigene families form a cluster that have been dispersed together throughout the telomeric part of the major histocompatibility complex and have been involved in the genesis of this human class I region.

Physical map of the HLA-A/HLA-F subregion and identification of two new coding sequences.

As part of an effort to characterize the hemochromatosis gene, we selected three non-chimeric yeast artificial chromosomes (YACs) overlapping with the YAC B30 previously described and forming an 800 kilobase contig covering the HLA-A/HLA-F region. The precise physical map of these YACs and of the corresponding genomic region were established. Nine concentrated sites of CpG cutter elements, potentially HTF islands, were mapped. In addition, several probes have been generated as tools for mapping and examining transcripts produced in the region. This allowed for the characterization and localization of two new coding sequences, provisionally named HCG (for hemochromatosis candidate gene) and numbered VIII and IX.

Obstructive azoospermia with agenesis of vas deferens or with bronchiectasia (Young's syndrome): a genetic approach.

Two groups of infertile men with obstructive azoospermia were screened for cystic fibrosis (CF) gene mutations (delta F508, exons 3, 4, 7, 10, 11, 14a, 17b, 19, 20, 21). The first group was composed of 26 patients with congenital agenesis of vas deferens (CAVD). The second group was composed of 12 patients with obstructive azoospermia associated with chronic suppurating respiratory disease (Young's syndrome). Of the group with CAVD, 77% of patients showed at least one mutation in the CF transmembrane conductance regulator (CFTR) gene. The delta F508 mutation occurred most frequently (54%), and the second most frequent mutation to occur was R117H (27%). Six patients were double heterozygotes. In Young's syndrome, no CF mutations were detected. CAVD can be considered as an incomplete clinical form of CF. However, the differences observed in CF mutations between CF and CAVD suggest that they are different disorders resulting from mutations in the same gene. Young's syndrome is a very different clinical entity.

Serum alpha-L-fucosidase: a new marker for the diagnosis of primary hepatic carcinoma?

alpha-L-fucosidase, a lysosomal enzyme which catabolizes fucoproteins, was studied in sera from 30 controls, 32 patients with primary hepatic carcinomas, 24 patients with secondary metastatic liver carcinomas and 36 patients with cirrhosis. Serum alpha-L-fucosidase was increased in primary hepatic carcinomas (145.5 +/- 12 nkat per liter) with a high statistical significance versus controls (51.4 +/- 4.5 - p less than 10(-7], secondary metastatic liver carcinomas (58.9 +/- 6.4 - p less than 10(-5] and cirrhotics (71.3 +/- 6 - p less than 10(-5). A level exceeding 110 was a useful marker for the diagnosis of primary hepatic carcinoma with 75% sensitivity and 90% specificity.