Mucin variable number tandem repeat polymorphisms and severity of cystic fibrosis lung disease: significant association with MUC5AC

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2×10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation.     

Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients,carriers, and controls

Summary Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the CF gene due to lack of specificity.     

The study of X-rays and TCDD effects on satellite associations may suggest a simple model for application in environmental mutagenesis

Summary Satellite associations were used as parameters to test nucleolar organizer activity. Assuming that toxic and/or mutagenic agents may affect the ribosomal genes, satellite associations in human lymphocytes were analysed following exposure to X-rays and compared with the satellite association pattern of subjects exposed to TCDD. A significant decrease in the satellite association frequency in D group chromosomes was found both in irradiated lymphocytes and in subjects exposed to Dioxin. The findings seem to be in accordance with the hypothesis based on random damage of functional nucleolar organizing regions.     

Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity of CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normala cells, which may be useful for the diagnosis of carriers of this defective gene.     

Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion

To explain the very high frequency of cystic fibrosis (CF) mutations in most populations of European descent, it has been proposed that CF heterozygotes have a survival advantage when infected with Vibrio cholerae or Escherichia coli, the toxins of which induce diarrhea by stimulation of active intestinal chloride secretion. Two assumptions underlie this hypothesis: (1) chloride conductance by the CF transmembrane conductance regulator (CFTR) is the rate-limiting step for active intestinal chloride secretion at all levels of expression, from approximately zero in patients with CF to normal levels in people who are not carriers of a mutation; and (2) heterozygotes have smaller amounts of functional intestinal CFTR than do people who are not carriers, and heterozygotes therefore secrete less chloride when exposed to secretagogues. The authors used an intestinal perfusion technique to measure in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF. Patients with CF had essentially no active chloride secretion in the basal state, and secretion was not stimulated by a prostaglandin analogue. However, CF heterozygotes secreted chloride at the same rate as did people without a CF mutation. If heterozygotes are assumed to have less-than-normal intestinal CFTR function, these results mean that CFTR expression is not rate limiting for active chloride secretion in heterozygotes. The results do not support the theory that the very high frequency of CF mutations is due to a survival advantage that is conferred on heterozygotes who contract diarrheal illnesses mediated by intestinal hypersecretion of chloride.     

Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis

Summary The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22) is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.     

Satellite association patterns in postpartum women and in newborn infants

Different features of the satellite associations between the acrocentric chromosomes of 100 postpartum mothers, between 14 and 43 years of age, and 100 neonates (51 males and 49 females) from Puriscal, Costa Rica, were compared (400 cells were examined, 2 from each individual). Several types of satellite associations showed higher frequencies in the mothers than in the neonates. The associations most frequently found in the two groups were those of two chromosomes, followed by those of three chromosomes. The D/G ratio was 1.43 in the children and 1.40 in the mothers. The number of associations per cell in the mothers was almost twice that of the children. The mean number of acrocentric chromosomes per association was 2.2 in the children and 2.35 in the mothers. No significant difference was found between observed and expected values of D and G chromosomes in the two groups. Thus, the probable factors responsible for the increase of satellite associations in the mothers similarly influenced the two types of acrocentric chromosomes.     

Cultured epithelial cells from patients with cystic fibrosis have an increased expression of the 14 kDa Ca2(+)-binding protein CFA

The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The steady state level of CFA was in general higher in epithelial cells from CF patients compared to control cells and was found to increase during cell aging. The latter difference could be attributed to an increased rate of CFA synthesis rather than to an impairment of CFA degradation or secretion, as shown by pulse chase experiments.     

Genetic comparisons of patients with cystic fibrosis with or without meconium ileus. Clinical Centers of the French CF Registry.

Cystic fibrosis (CF) is an autosomal disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Neonatal meconium ileus (MI) occurs in 10-20 percent of newborns with CF. The purpose of this study was to determine the allelic frequencies of the CF mutation in French patients with and without MI and the incidence of MI in 7 homozygotes or compound heterozygotes for mutation of the CFTR gene. Our study confirms the positive association between delta F508, the most frequent CF mutation, G542X mutation and MI and a negative association with G551D.     

Allelic exclusion in rat kappa immunoglobulin chains: extent of Jk rearrangement in normal B lymphocytes

The frequency of normal rat peripheral B lymphocytes stained for surface immunoglobulin kappa allotypes a and b in (a X b) F1 heterozygotes was assessed by two-colour immunofluorescence on a fluorescence-activated cell sorter. The upper limit to the frequency of double- stainers was 8% among all kappa-positive cells, though it was not resolved how far cytophilic antibody accounted for these. Cells expressing each allotype singly were isolated and the extent of rearrangement of the genes encoding the joining-kappa segment on the expressed and non-expressed chromosome were independently assessed. The expressed allele was found to be virtually completely rearranged while the non-expressed allele showed approximately 45-60% rearrangement. The implications of this substantial non-productive rearrangement for models of allelic exclusion are discussed.     

Intercellular NOR-Ag-variability in man. II. Search for determining factors,clonal analysis

Summary Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts.In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.     

Frequency of satellite association of human chromosomes is correlated with amount of Ag-staining of the nucleolus organizer region.

Methaphase chromosomes from karyotypically normal adult humans (three males, six females) and one male with a 13p - chromosome were stained by quinacrine and then by the Ag-AS silver staining method to reveal nucleolus organizer regions (NORs). Each person had a characteristic number of Ag-stained chromosomes per cell, always fewer than 10. Determination of the mean Ag-size of each chromosome showed that each of the 10 individuals had a unique distribution of Ag-stain. Within each individual, there was some variation from cell to cell in the number of acrocentric chromosomes that were Ag-stained; this was not random, and the same chromosomes (those that had at most a small amount of Ag-stain) tended to be unstained in every cell. Satellite associations were scored on the same cells. Chromosomes that had no Ag-stain were involved in satellite association less than 20% as often as those that had some Ag-stain. Chromosomes that had a small amount of Ag-stain were involved in association about 50% as often as those that had a large amount of stain. Regression analysis of the 50 (of a total of 100) acrocentric chromosomes which could be individually identified by quinacrine markers showed that the frequency with which a chromosome was involved in satellite association was strongly correlated with the amount of Ag-stained material in the NOR.     

Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57Bl/6J mice

The osteopetrotic, microphthalmic (mi/mi) mouse lacks functional osteoclasts and has also been reported to be deficient in mast cells and natural-killer (NK) cells. The later deficiencies could be secondary to the osteopetrotic marrow, or a direct result of the mi allele. Therefore, heterozygotes were examined for these cell types, since these mice do not exhibit osteopetrosis. Adult +/mi animals have approximately 50%, and mi/mi animals examined by histologic techniques or tissue histamine levels have 0-10%, of the peritoneal, dermal, and intestinal mast cells compared with that of +/+ animals. Leukocyte histamine, indicative of the number of basophils, demonstrates the same pattern. Histamine content per mast cell in +/+ and +/mi animals is identical. The number of large granular lymphocytes (LGL) in splenic leukocyte preparations from +/mi animals is 50% that of +/+ animals, and these cells are undetectable in preparations from mi/mi mice. NK activity against YAC-1 cells paralleled the number of LGL present. The resorptive response of neonatal calvaria to parathyroid hormone was delayed in the case of cultured +/mi bone compared with that of +/+ bone, but the final rate of calcium release was identical. These data indicate that 1) the presence of one mi allele can affect the development of four distinct cell types, and 2) osteopetrosis alone does not account for the lack of mast cells, basophils, and NK cells in mi/mi mice.     

Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks, as a possible means for identifying individuals who are mildly hypersensitive to ionizing radiation, such as some ATM heterozygotes. We compared levels of gamma-H2AX foci after irradiation in cells from six apparently normal individuals as well as from individuals from two separate AT families including the proband, mother, father and three unaffected siblings in each family. After a 1-Gy single acute (high-dose-rate) gamma-ray dose delivered to noncycling contact-inhibited monolayers of cells, clear differences were seen between samples from normal individuals (ATM(+/+)) and probands (ATM(-/-)) at nearly all sampling times after irradiation, but no clear distinctions were seen for cells from normal compared to obligate heterozygotes (ATM(+/-)). In contrast, after 24 h of continuous irradiation at a dose rate of 10 cGy/h, appreciable differences in numbers of foci per cell were observed for cells from individuals for all the known ATM genotypes compared with controls. Four unaffected siblings had mean numbers of foci per cell similar to that for the obligate heterozygotes, whereas the other two had mean values similar to that for normal controls. We determined independently that those siblings with mean numbers of foci per cell in the range of ATM heterozygotes carried the mutant allele, while both siblings with a normal number of foci per cell after irradiation had normal alleles. A more limited set of experiments using lymphoblastoid cell strains in the low-dose-rate assay also revealed distinct differences for normal compared to ATM heterozygotes from the same families and opens the possibility of using peripheral blood lymphocytes as a more suitable material for an assay to detect mild hypersensitivities to radiation among individuals.     

Tumour necrosis factor-alpha and heat-shock protein 70-2 gene polymorphisms in a family with rheumatoid arthritis

OBJECTIVES: The aim of this work was to investigate the prevalence of TNF-alpha-308 polymorphism among the 29 members of a family with RA and the association between the MHC-linked biallelic HSP70-2 gene and the TNF-alpha polymorphism. Five of the members with RA were diagnosed by using the revised 1987 ACR criteria, and 1 member suffered from SLE. METHODS: The variations in the TNF-alpha and the HSP70-2 genotypes were analyzed by PCR-RFLP, using NcoI and PstI restriction enzymes. RESULTS: Two of the 29 members were homozygotes for allele A, 18 were heterozygotes (TNF A/G) and 9 of them were homozygotes for allele G. Nineteen of the 29 were heterozygotes for HSP70-2 (A/G), 10 of them were homozygotes for the G allele, and none were homozygotes for allele A. Four of the 5 the RA patients carried the A allele for TNF-alpha all 5 were heterozygotes for HSP70-2 genotypes. CONCLUSION: The carriage of the A allele for TNF-alpha of -308 SNP in 4 of the 5 RA patients, and the high prevalence (68.0%) of TNF A allele carriers in this family confirms the important role of this candidate gene in the pathomechanism of RA, and might be of prognostic value for future clinical observations. Further, to test for association a much larger set of genetically independent patients and controls is needed.     

Susceptibility to typhoid fever is associated with a polymorphism in the cystic fibrosis transmembrane conductance regulator (CFTR)

The cystic fibrosis transmembrane conductance regulator (CFTR) is the affected protein in cystic fibrosis (CF). The high rate of CF carriers has led to speculation that there must be, similar to the sickle cell haemoglobin advantage in malaria, a selective advantage for heterozygotes. Such a selective advantage may be conferred through reduced attachment of Salmonella typhi to intestinal mucosa, thus providing resistance to typhoid fever. We tested this hypothesis by genotyping patients and controls in a typhoid endemic area in Indonesia for two highly polymorphic markers in CFTR and the most common CF mutation. We found an association between genotypes in CFTR and susceptibility to typhoid fever (OR=2.6). These analyses suggest that the role CFTR plays in vitro in S. typhi infection is also important for infection in the human population.Electronic Supplementary Material Supplementary material is available for this article at     

Cytogenetic damage in plastic industry workers exposed to polyvinyl chloride

Cytogenetic investigations on peripheral blood lymphocytes of 40 workers exposed to polyvinyl chloride in the plastic industry were undertaken. These were compared with an equal number of occupationally unexposed and matched controls in relation to age, sex and smoking habits. The mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchanges (SCE) and satellite associations (SA) were analysed. All the parameters showed a significant increase (p <0.01) in the exposed sample compared with the controls, viz MI, 3.64-6.30, CA 1.02-3.77, SCE 3.40-7.83 and SA 5.57-12.05. The occurrence of the DG type of satellite association B was highest and that of 3D type lowest. The frequencies of all the parameters increased with the duration of exposure, but MI declined after 15 years of exposure.