Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis.     

Cluster of cystic fibrosis cases in a limited area of Brittany (France)

Cystic fibrosis in the northern sector of the French "département" of Finistère is 1:1787 live births. Within this sector a concentration of the disease was found in a small area. The minimal frequency in this area, from 1946 to 1972, was calculated as 1 per 377 live births, the gene frequency being 0.0515. Genealogic analysis, going back to the beginning of the 18th century, showed a relationship between 8 of the 10 families to which the patients belonged. The origin of the deleterious genes may be explained by at least five primary ancestral couples living in the 18th century. Random drift is the most probable explanation for the concentration of cystic fibrosis in this region.     

The ΔF508 mutation in mild adult forms of cystic fibrosis (CF)

Twenty CF chromosomes from ten patients with mild adult form of cystic fibrosis were tested for delta F508. This mutation was found to be significantly less frequent than in the severe form of the disease.     

Cystic fibrosis mutation frequencies in an Irish population

The incidence of cystic fibrosis (CF) at birth in Ireland is 1/1461. Neonate CF genetic testing is not routinely performed in Ireland. Currently, screening is only carried out where there is clinical evidence or a family history to suggest disease. Here we report the frequencies of common CF mutations occurring in an Irish population composed of samples collected from western, mid-western and southern regions of Ireland. Rarer CF mutations were also identified in a selected number of CF patients. In addition, a number of polymorphisms were identified, some of which are reported to be functionally and phenotypically important.     

Cystic fibrosis mutation frequencies in upstate New York

Upstate New York patients (100) with cystic fibrosis (i.e., 200 CF chromosomes), 72 from the CF center in Syracuse and 28 from a Buffalo CF center, were analyzed for their CF-causing mutations using restriction enzyme digest, single-strand conformation analysis (SSCA), and Heteroduplex (HA) analysis. Polymerase chain reaction (PCR) amplified products from all 27 CFTR exons using primers that included flanking intron junction sequence were investigated. More than 120 known cystic fibrosis transmembrane conductance regulator (CFTR) disease-causing mutations were screened. Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). A detection rate of 96% of the combined Syracuse and Buffalo population CF chromosomes was obtained. Hum Mutat 10:436–442, 1997. © 1997 Wiley-Liss, Inc.     

Allele frequencies of Mp6D-9 and GATT markers in 32 Turkish cystic fibrosis families

The allele frequency of GATT and Mp6D-9 markers was investigated in 32 cystic fibrosis (CF) families. The GATT₆ allele was found to be significantly associated with the ΔF508 mutation. The Mp6D-9 allele 2/GATT₆ haplotype was the major haplotype of the mutant alleles. Further analysis of CF alleles for population-specific mutations is underway so that a more direct approach can be taken, especially for families seeking prenatal diagnosis.     

Phenotypic heterogeneity in cystic fibrosis

We have confirmed heterogenity in CF using a different combination of primary clinical variables than those used in previous studies. Subgroupings of individuals with similar levels of sweat chloride were independent of the clustering based on level of pancreatic enzyme supplementation and degree of pulmonary involvement. Data from families with multiple CF children are consistent with the hypothesis that the genetic etiology of CF involves two or more genes that modify the expression of the primary gene defect.     

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cysticfibrosis (CF) not only depends on the genotype, but apart from a combination ofenvironmental and stochastic factors predominantly also on modifier gene effects. It hasbeen proposed that genes interacting with CF transmembrane conductance regulator (CFTR)and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed theimpact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CFdisease outcome. SNPs that potentially alter gene function were genotyped in 95well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysiswas used to assess the relationship between sequence variants and the repeatedmeasurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes.Twenty-five SNPs were used for statistical analysis, where we found strong associationsfor one SNP in PPP2R4 with the lung clearance index (P≤0.01), thespecific effective airway resistance (P≤0.005) and the forced expiratoryvolume in 1 s (P≤0.005). In addition, we identified one SNP inSNAP23 to be significantly associated with three lung function parameters aswell as one SNP in PPP2R1A and three in KRT19 to show a significantinfluence on one lung function parameter each. Our findings indicate that directinteracters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residualfunction of p.Phe508del-CFTR while variants in KRT19 may modulate the amount ofp.Phe508del-CFTR at the apical membrane and consequently modify CF disease.     

Heterozygote advantage in cystic fibrosis: mosquito tests

Tests to demonstrate a preference by mosquitoes for stinging controls as opposed to obligate heterozygotes for Cystic Fibrosis proved negative. If a heterozygote advantage caused a lower malarial incidence in carriers in South West Africa, it must have worked through the malarial parasite being adversely affected by a serum factor. This remains to be tested.     

Isoelectric focusing of serum in genetic counselling of families with cystic fibrosis

The high incidence of carriers of cystic fibrosis in the general population allows application of a less than perfect test to genetic counselling of relatives of children with the disorder and their spouses. In the absence of a definitive carrier detection test, we employ isoelectric focusing of serum in this way and include the a priori chance of carrier status in calculating the risk. The test will eventually be replaced but for the present is preferable in our hands to counselling based simply on the known gene frequency.     

The molecular basis of cystic fibrosis in South Africa

The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.     

Novel CFTR mutations in black cystic fibrosis patients

Cystic fibrosis (CF) is considered as a rare disease in black Africans. In fact, this disease is likely to be underestimated since clinical features consistent with CF diagnosis are often ascribed to environmental factors such as malnutrition. Very little is known about CFTR mutations in affected patients from Central Africa. We report here four novel mutations, i.e., IVS2 + 28 (intron 2), 459T > A (exon 4), EX17a_EX18del (exons 17-18), and IVS22 + IG > A (intron 22), in such patients. An update of CFTR mutations reported in black patients from various ethnies is included. These data might be helpful for genetic counselling regarding CF in black patients.     

Antibodies to gastroenteritis viruses in cystic fibrosis patients

Infectivity of certain enteric viruses including rotavirus is profoundly affected by proteolytic enzymes. To test whether cystic fibrosis patients, possessing chronically decreased levels of pancreatic enzymes, show altered susceptibility to gastroenteritis viruses, we examined sera from patients and controls for antibodies to two major pathogens. In cystic fibrosis patients, normal rotavirus antibody levels were found and Norwalk virus antibody prevalence was unchanged.     

Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening

Although there have been numerous reports from around the world of mutations in the gene of chromosome 7 known as CFTR (cystic fibrosis transmembrane conductance regulator), little attention has been given to integrating these mutant alleles into a global understanding of the population molecular genetics associated with cystic fibrosis (CF). We determined the distribution of CFTR mutations in as many regions throughout the world as possible in an effort designed to: 1) increase our understanding of ancestry-genotype relationships, 2) compare mutational arrays with disease incidence, and 3) gain insight for decisions regarding screening program enhancement through CFTR multi-mutational analyses. Information on all mutations that have been published since the identification and cloning of the CFTR gene's most common allele, DeltaF508 (or F508del), was reviewed and integrated into a centralized database. The data were then sorted and regional CFTR arrays were determined using mutations that appeared in a given region with a frequency of 0.5% or greater. Final analyses were based on 72,431 CF chromosomes, using data compiled from over 100 original papers, and over 80 regions from around the world, including all nations where CF has been studied using analytical molecular genetics. Initial results confirmed wide mutational heterogeneity throughout the world; however, characterization of the most common mutations across most populations was possible. We also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations. Data for these analyses were filtered for reliability and methodological strength before being incorporated into the final analysis. Statistical assessment of these variables revealed that there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs. From comprehensive assessment of these data, we offer recommendations that multiple CFTR alleles should eventually be included to increase the sensitivity of newborn screening programs employing two-tier testing with trypsinogen and DNA analysis.     

Cystic fibrosis mutations in French Canadians: Three CFTR mutations are relatively frequent in a Quebec population with an elevated incidence of cystic fibrosis

The French-Canadian population in the Saguenay-Lac St. Jean region of northeastern Quebec has an elevated frequency of cystic fibrosis (CF). The average incidence of cystic fibrosis was 1 in 891 births and the prevalence of CF carriers was estimated to be 1 in 15. We tested for 10 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 133 French-Canadian CF families from Quebec. Ninety-one families were from the Saguenay-Lac St. Jean region and 42 families were referred from other regions of Quebec. We detected the CFTR mutation in 93 and 92% of the CF chromosomes in the Saguenay-Lac St. Jean and the major-urban Quebec families, respectively. The two groups of French-Canadian families were significantly different for the proportions of CFTR mutations. The three most common mutations in the Saguenay-Lac St. Jean families were ΔF508 (58%), 621 + 1G → T (23%), and A455E (8%); and in the major-urban Quebec families were ΔF508 (71%), 711 + 1G → T (9%), and 621 + 1G → T (5%). These results provide evidence for the role of founder effect in the elevated incidence of cystic fibrosis in the Saguenay-Lac St. Jean population.     

Genotyping of cystic fibrosis families with linked DNA probes

Forty-six British families, containing at least one child affected with cystic fibrosis, were typed for restriction fragment length polymorphisms (RFLPs) by the probes pmet H, pmet D, pJ3.11 and 7c22. Thirty-five (76%) were fully informative for prenatal diagnosis and carrier detection, while in the remainder prenatal exclusion of an affected fetus could be carried out in half the pregnancies. The frequencies of individual alleles did not differ between cystic fibrosis and normal chromosomes. However, the previously noted excess of one haplotype on chromosomes carrying the cystic fibrosis gene was confirmed.     

Molecular basis of cystic fibrosis in the Republic of Macedonia

Petreska L, Koceva S, Plaseska D, Chernick M, Gordova-Muratovska A, Fustic S, Nestorov R, Efremov GD. Molecular basis of cystic fibrosis in the Republic of Macedonia. Clin Genet 1998: 54: 203–209. 0 Munksgaard, 1998
Eighty-three cystic fibrosis (CF) patients and their families, belonging to various ethnic groups living in the Republic of Macedonia were studied for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and for the associated extragenic marker loci XV-2c and KM 19. The DNA methodology used included characterization of CFTR mutations in 19 exons (and flanking sequences) of the gene and analysis of distribution of the XV-2c/KM19 haplotypes among normal (N) and CF chromosomes by polymerase chain reaction (PCR) amplification followed by dot blot hybridization, restriction digestion, single-strand conformational polymorphism, constant denaturing gel electrophoresis, denaturing gradient gel elec-trophoresis, and sequencing. We identified 58.4% (97/166) of the CF chromosomes. Nine different CFTR gene mutations, including three novel ones. were found. Eight known and one new CFTR intragene polymorphisrns were also characterized. The haplotype analysis of the XV-2c/TaqI and KM19/PstI polymorphic loci have shown that haplotype C is the most frequently found haplotype among the non-AF508 CF chromosomes from Macedonia (36.5%). The results demonstrate the broad heterogeneity of CF origin in this part of the Balkan Peninsula.     

Bimodal month of birth distribution in cystic fibrosis

The month of birth of 923 patients in England and Wales dying with cystic fibrosis as an underlying cause was found to be distributed independently of sex and year of birth during 1954–1976. When adjusted for the monthly variations in births in the national population, birth frequencies were bimodally distributed. This confirmed results obtained previously in the Netherlands and Australia. Peak births occurred in late February and August, regardless of whether a single six-monthly cycle or two annual cycles were assumed. It was not possible to choose the better of these models, each yielding similar parametric estimates.