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.

Mutations in the arginine-rich protein gene (ARP) in pancreatic cancer.

The ARP gene encodes a highly conserved arginine-rich protein from chromosomal band 3p21.1. At the cytogenetic level this region is frequently deleted in a variety of different solid tumors, although not in pancreatic cancer. We have reported the presence of a specific mutation (ATG50-->AGG) or deletion of codon 50 of the ARP gene in different tumor types (Shridhar et al., 1996, 1996a). In the present study, we have observed mutations involving codon 50 in 11 of 37 pancreatic tumors. The frequency of codon 50 mutation is roughly the same in pancreatic tumors as in the other types of tumors previously examined. In addition, we have detected mutations at codon 51 in multiple PCR subclones in two other pancreatic tumors. Mutations in the ARP gene are thus commonly observed in pancreatic cancer, as well as many other cancers.

Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck.

Arginine-rich protein (ARP) is a highly conserved gene that maps to human chromosomal band 3p21.1. This gene contains an imperfect trinucleotide repeat which encodes a string of arginines. We previously detected a specific mutation (ATG50-->AGG) within this region of the gene in 10 of 21 sporadic renal cell carcinomas. Here, we report the detection of the same mutation in 5 of 21 squamous cell carcinomas of the head and neck, 1 of 2 small cell lung cancer cell lines, 6 of 18 non-small cell lung carcinomas, 9 of 22 breast tumors, and 5 of 13 prostate tumors. This mutation was seen in several early stage tumors and may thus be an early event in tumorigenesis. We also detected a mutation at codon 53 of this gene in both primary and metastatic tumors from one patient. Other nucleotide changes were observed in a few PCR subclones, but their frequency was the same in both tumor and control samples, suggesting that many of these changes were PCR or subcloning artifacts rather than mutations in the tumor cells themselves.

A comparison of RFLP maps based on anther culture derived, selfed, and hybrid progenies of Solanum chacoense.

Comparative RFLP linkage maps were constructed using five segregating populations derived from two self-incompatible lines (termed PI 230582 and PI 458314) of diploid tuber-bearing Solanum chacoense Bitt. The analysis was based on 84 RFLP loci identified by 73 different cDNA clones. Distortion of expected Mendelian segregation ratios was observed; less than 10% of the markers showed a skewed segregation in the gametes forming the F1, hybrid population compared with 30% in the selfed population and 46 and 70% in the two populations produced by anther culture. For the anther culture derived populations, most of the skewed loci were scattered throughout the genome, whereas in the populations derived from selfing, they were found primarily in linkage group 1, around the S locus. In this study, we also found that the rate of meiotic recombination could differ between the male and female gametes produced by our parental lines. Thus, male gametes of line PI 458314 showed significantly less recombination as assessed by the total length of the map (206 cM for male gametes vs. 375 cM for female gametes) and the phenomenon was genome-wide. In contrast, the maps from the gametes of PI 230582 had about the same length, but some linkage groups were longer in the female gametes, while others were longer in the male gametes. Key words : Solanum chacoense, RFLP, anther culture, skewed segregation, self-incompatibility, sex differences in recombination.

A gene from human chromosomal band 3p21.1 encodes a highly conserved arginine-rich protein and is mutated in renal cell carcinomas.

We have identified a gene, called ARP for Arginine-rich protein, in human chromosomal band 3p21. It is approximately 600 Kb telomeric to the ACY1 locus (Miller et al., 1989) and encodes a previously unidentified 234 amino acid long, highly basic protein. This gene is highly conserved at the DNA and RNA level. It is found in all species including hamster, rat, mouse, bovine and yeast. We have detected a point mutation (ATG50 to AGG) or deletion of ATG50 in 10 of 21 sporadic renal cell carcinomas. The mutable region is in an imperfect trinucleotide repeat in the coding region which is non-polymorphic among 50 normal individuals examined. The point mutation (ATG50 to AGG) or deletion of codon 50 removes a methionine and increases the stretch of arginines encoded by the AGG repeats in the ARP gene.

RFLP analyses and segregation of molecular markers in plants produced by in vitro anther culture, selfing, and reciprocal crosses of two lines of self-incompatible Solanum chacoense.

RFLP analyses were used to characterize several plant populations of Solanum chacoense Bitt. developed to investigate the generation of new S alleles at the self-incompatibility locus. The plant material consisted of two diploid parental lines, their anther culture derived (AC) progenies, their selfed progenies, and their reciprocal F1 hybrids. The RFLP analyses on the AC plants (121 individuals in total) permitted unambiguous identification of their origin. In particular, a distinction between plants originated from reduced (n) or unreduced (2n) microspores could be made. All the AC plants produced by gametic embryogenesis showed distinct RFLP patterns, whereas a number of clones (i.e., plants with identical RFLP patterns) were found among those regenerated via callus. The analyses conducted on the selfed progenies (69 plants) and the F1 hybrids (66 plants) showed only one case of accidental outcross. Segregation studies of the RFLP markers revealed significant deviations from expected Mendelian ratios in both AC-derived populations, as well as in the selfed progenies. Such deviations, however, were rare in the reciprocal F1 hybrids. These results are discussed in relation to the possible presence of genetic sieves operating during AC, illegitimate selfing, or during normal fertilization.

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar.

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.

Potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3 and rhinovirus 14.

Hands often become contaminated with respiratory viruses, either directly or through contact with contaminated surfaces. Spread of such viruses could then occur by touching the nasal mucosa or the conjunctivae. In this quantitative study, we compared the survival of mucin-suspended human parainfluenza virus 3 (HPIV-3) and rhinovirus 14 (RV-14) and the transfer of the viruses to and from the fingers of adult volunteers. When each finger pad was contaminated with 10 microliters of either HPIV-3 (1.3 x 10(5) to 5.5 x 10(5) PFU) or RV-14 (2.1 x 10(4) to 1.1 x 10(5) PFU), less than 1.0% of HPIV-3 and 37.8% of RV-14 remained viable after 1 h; after 3 h, nearly 16% of RV-14 could still be detected, whereas HPIV-3 became undetectable. Tests on the potential spread of viruses from contaminated hands or surfaces were conducted 20 min after contamination of the donor surface by pressing together donor and recipient surfaces for 5 s. Transfer of HPIV-3 from finger to finger or finger to metal disk could not be detected, but 1.5% of infectious HPIV-3 was transferred from disk to finger. Irrespective of the type of donor or recipient surface, 0.7 to 0.9% of RV-14 was transferred. The relatively rapid loss of HPIV-3 infectivity on hands suggests that their role in the direct spread of parainfluenza viruses is limited. However, the findings of this study further reinforce the view that hands can be vehicles for rhinovirus colds. These results also suggest a role for nonporous environmental surfaces in the contamination of hands with respiratory viruses.

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt.

In this study, a novel approach was used to characterize the genetic architecture of plants produced by in vitro anther culture of two lines of self-incompatible Solanum chacoense Bitt. (2n=2x=24). We used cytological observations to determine the ploidy level of the regenerated plants and scanned genomic DNA of the anther donor plants to identify heterozygous sequences. Restriction fragment length polymorphism (RFLP) analyses permitted the visualization of DNA variations. Several heterozygous DNA markers were found within single anther donor plants. Completely homozygous lines could be easily identified. Somatically derived plants could be separated from diploid plants produced from 2n (unreduced) microspores. Our results demonstrate first division restitution (FDR) as the mechanism operating during the production of 2n microspores in one of our S. chacoense line. Potential applications of RFLP analyses for genetic mapping, identification of lethal alleles and quantitative trait loci (QTL) with haploid or homozygous diploid plants and determination of gene-centromere distance with diploid plants derived from 2n microspores will be discussed.