Hemochromatosis gene variants in three different ethnic populations: effects of admixture for screening programs

Genetic testing for hemochromatosis may have important implications for diagnosis and screening of the disease. However, the relative importance of mutations in the gene for hereditary hemochromatosis, HFE, may vary among populations, when the mutant allele frequencies and their penetrance in a particular genetic and environmental background are taken into account. We present data on the allele and genotype frequencies and population structure of two HFE genetic variants in three different ethnic groups from a highly mixed urban population (S?o Paulo, Brazil). Allele frequencies for both the C282Y and H63D HFE mutations showed significant differences among the studied populations (for the C282Y mutation, Euro-Brazilian 3.7%, admixed 0.7%, Afro-Brazilian 0.5%; and for the H63D mutation, Euro-Brazilian 20.3%, admixed 13.0%, Afro-Brazilian 6.4). The data substantiate a European origin for these mutations. Furthermore, they provide a basis for a more rational strategic planning of population screening programs for the disease.     

Analysis of the CCR5 gene coding region diversity in five South American populations reveals two new non-synonymous alleles in Amerindians and high CCR5*D32 frequency in Euro-Brazilians

The CC chemokine receptor 5 (CCR5) molecule is an important co-receptor for HIV. The effect of the CCR5*D32 allele in susceptibility to HIV infection and AIDS disease is well known. Other alleles than CCR5*D32 have not been analysed before, neither in Amerindians nor in the majority of the populations all over the world. We investigated the distribution of the CCR5 coding region alleles in South Brazil and noticed a high CCR5*D32 frequency in the Euro-Brazilian population of the Paraná State (9.3%), which is the highest thus far reported for Latin America. The D32 frequency is even higher among the Euro-Brazilian Mennonites (14.2%). This allele is uncommon in Afro-Brazilians (2.0%), rare in the Guarani Amerindians (0.4%) and absent in the Kaingang Amerindians and the Oriental-Brazilians. R223Q is common in the Oriental-Brazilians (7.7%) and R60S in the Afro-Brazilians (5.0%). A29S and L55Q present an impaired response to β-chemokines and occurred in Afro- and Euro-Brazilians with cumulative frequencies of 4.4% and 2.7%, respectively. Two new non-synonymous alleles were found in Amerindians: C323F (g.3729G > T) in Guarani (1.4%) and Y68C (g.2964A > G) in Kaingang (10.3%). The functional characteristics of these alleles should be defined and considered in epidemiological investigations about HIV-1 infection and AIDS incidence in Amerindian populations.     

Spectrum of CFTR mutations in Mexican cystic fibrosis patients: identification of five novel mutations (W1098C, 846delT, P750L, 4160insGGGG and 297–1G→A)

We have analyzed 97 CF unrelated Mexican families for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Our initial screening for 12 selected CFTR mutations led to mutation detection in 56.66% of the tested chromosomes. In patients with at least one unknown mutation after preliminary screening, an extensive analysis of the CFTR gene by single stranded conformation polymorphism (SSCP) or by multiplex heteroduplex (mHET) analysis was performed. A total of 34 different mutations representing 74.58% of the CF chromosomes were identified, including five novel CFTR mutations: W1098C, P750L, 846delT, 4160insGGGG and 297-1G-->A. The level of detection of the CF mutations in Mexico is still lower than that observed in other populations with a relatively low frequency of the deltaF508 mutation, mainly from southern Europe. The CFTR gene analysis described here clearly demonstrated the high heterogeneity of our CF population, which could be explained by the complex ethnic composition of the Mexican population, in particular by the strong impact of the genetic pool from southern European countries.     

Interleukin–10 promoter haplotypes are differently distributed in the Brazilian versus the Dutch population

The frequency of five different single nucleotide polymorphisms of the promoter interleukin-10 (IL-10) gene (-3575, -2849, 2763, -1082, -819) was compared between two healthy populations, one originating from the Netherlands and one from Rio de Janeiro, Brazil. A total of 321 Caucasian Dutch individuals and 293 Brazilians, grouped as Afro-Brazilians and Euro-Brazilians, were genotyped using PCR-RFLP. The frequencies of the genotypes in the Brazilian population were different (P<0.05) from the frequencies in the Dutch population in all but one (-2763) genotype. The comparison of genotype frequencies between Afro- and Euro-Brazilians did not demonstrate any differences. The haplotype combination of the most-distant three polymorphisms showed strong linkage disequilibrium. All eight possible combinations were observed in Brazilians, but only seven in Dutch Caucasians. The haplotype frequencies were also significantly different between Brazilians when compared with Dutch and also between Euro-Brazilians and Dutch. No differences were observed in haplotype frequencies between Afro-Brazilians and Euro-Brazilians. The -3575T/-2849G/-2763C is more frequent, while the AAA haplotype was much less represented in the Brazilian than in the Dutch population. The haplotype TAC, which was described in African-Americans, was observed only in Brazilians, almost exclusively among those of European origin. The results corroborate the data indicating that the Brazilian population exhibits a genetic admixture of Africans, Europeans, and Amerindians, and the data may serve as a background for clinical and immunological studies involving the IL-10 locus.     

Low frequency of the deltaAF508 mutation of the CFTR gene in a highly admixed population in Bahia, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease in the European (Caucasian) population, with an incidence of 1:2000 to 1:8000. The deltaF508 mutation (66%) is predominant among more than 1300 different mutations of the CFTR gene. The population of the state of Bahia, in northeastern Brazil, is highly admixed (mainly African and Portuguese descendants), and so far, no study has been carried out to assess the molecular basis of CF in this population. We determined the deltaF508 mutation frequency in 503 individuals from the general population of Salvador, the capital of the state of Bahia, and in 144 CF patients from several cities in Bahia. In the general population samples we found 4 individuals heterozygous for the deltaF508 mutation (allele frequency of 0.4%). This frequency was lower than that found in the state of Rio de Janeiro, in southeastern Brazil, and similar to that reported for the state of Paraná, in the far south. In the CF patients we found 9 heterozygous individuals and 8 homozygous individuals (allele frequency of 8.68%) for the deltaF508 mutation. This frequency is considerably lower than the average frequency of CF in the world population and in the Brazilian CF population of European ancestry (47%). These data could be explained by the intense admixture among the population in Bahia, and they suggest a heterogeneous molecular basis for CF in this area of Brazil.     

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.     

Spectrum of CFTR mutations on Réunion Island: impact on neonatal screening

The large heterogeneity in the cystic fibrosis (CF) gene is the main difficulty for genotype characterization. Numerous studies have reported considerable variations in frequencies of CF transmembrane conductance regulator (CFTR) mutations in different populations, such as African, Asian, or European populations. To completely characterize the spectrum of mutations in the CFTR gene in the Réunion Island population, we screened 228 CF chromosomes using denaturing high-pressure liquid chromatography and denaturing gradient gel electrophoresis following by direct sequencing. We identified 27 mutations, accounting for 93% of CF chromosomes. They included three novel mutations (M1T, 3121-3C-->G, and L1324P), which are described in this paper. The detection of such a high proportion of Réunion Island CFTR mutations is important for improving neonatal screening of CF on Réunion Island.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

Cystic fibrosis in Afro-Brazilians: <Emphasis Type="Italic">XK</Emphasis> haplotypes analysis supports the European origin of <Emphasis Type="Italic">p.F508del</Emphasis> mutation

Cystic fibrosis (CF) is a common autosomal recessive disorder, being the p.F508del the most frequent mutation. Also, a nearby restriction fragment length polymorphism (RFLP) named XK (KM19 and XV2C) is non-randomly associated with specific CF alleles. Our aim was to analyze the occurrence of the p.F508del mutation and XK haplotypes in Afro-Brazilians CF patients and controls, since these data is available for the other two main ethnic groups found in Brazil (Euro-Brazilians and Brazilian Amerindians), contributing for the whole comprehension of these haplotypes in the Brazilian population. A total of 103 patients and 54 controls were studied. PCR and PCR-RFLP methodologies were used to identify the presence of the p.F508del and the XK haplotype in the subjects. The combined data show that 84.2% of p.F508del mutation is associated with haplotype B and only 15.8% with haplotype A; no other haplotypes were found to be associated with this mutation. Our data suggest that the occurrence of p.F508del mutation and haplotype B in Afro-Brazilian patients occurs probably due to admixture with Euro-descendants. Therefore this mutation and haplotype could be used as a admixture marker.     

XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.     

Analysis of cystic fibrosis gene mutations and associated haplotypes in the Croatian population

The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: DeltaF508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 + 1G --> T; G85E; Y569C; E585X; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (DeltaF508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.     

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.     

Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians.

To determine the nature and frequency of non-delta F508 cystic fibrosis (CF) mutations among diverse populations, we have sequenced exons 9-12 and 19-23 of the CF transmembrane conductance regulator (CFTR) gene from 128 CF chromosomes (39 U.S. Caucasian, 27 African-American, 42 Northern Irish, and 20 Israeli chromosomes). These regions were chosen because they encode the two putative ATP-binding folds of CFTR, domains which appear to have functional significance. In addition, CFTR exons 1 and 2 were analyzed in the American patients. Mutations were found on 49 of the 128 CF chromosomes. Nineteen different mutations were observed; six were novel, while the remaining 13 had been reported previously by our group or by other investigators. Six of nine different mutations found in African-American patients were unique to that population. However, the vast majority of the mutations found in U.S. Caucasians (eight of nine), Northern Irish (four of five), and Israelis (three of three) also occurred in other Caucasian groups. The preponderance of previously reported mutations in these three groups suggested that a subset of the non-delta F508 mutations occur in common among Caucasians. A survey of mutation frequencies in other Caucasian groups confirmed this observation. Unfortunately, this subset accounts for less than half of non-delta F508 CF mutations in most groups. These data suggest that screening for delta F508 and this select group of mutations will efficiently and economically maximize the number of CF mutations identified in Caucasian groups. However, it will be difficult to detect more than 90% of mutant CFTR alleles except in ethnically and geographically discrete populations where CF is the result of founder effect.     

CFTR molecular analysis reveals infrequent allele frequencies in nine cystic fibrosis patients from São Paulo State, Brazil

Although cystic fibrosis (CF) is the most common autosomal recessive disorder in whites, it is thought to be relatively rare or, alternatively, underdiagnosed in Latin America. In Brazil, different groups have shown a DF508 mutation frequency ranging from 30.7% to 50.8%. Such variation may be explained by the ethnic differences observed in this country, which is genetically very heterogeneous. We describe the molecular analysis for 32 mutations of the CFTR gene in nine unrelated patients with cystic fibrosis from S?o Paulo State, Brazil. The main observation of this study was the absence of 30 out of the 32 mutations in 12 alleles among these patients. Except for mutations DF508 and N1303K, no other mutation could be detected in any of the studied patients. In one of two alleles, a DF508 mutation was detected in four patients (22% of the total sample) and an N1303K mutation was detected in two patients (11% of the total sample). One patient was a compound heterozygote for DF508/N1303K. Although the sample studied here was small, it may be possible that our patients have infrequent alleles once these can occur at higher frequencies in selected populations and also show relevant regional differences. Additional investigations in a larger sample are currently in progress in our laboratory to confirm our results, and further studies are still needed to determine the frequencies of CF gene mutations in different regions and ethnic groups in the Brazilian population.     

A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype

We have analysed 1173 cystic fibrosis (CF) chromosomes from Switzerland for eight mutations in the CF transmembrane conductance regulator (CFTR) gene. This permitted the identification of 88.5% of all mutations present. A novel insertion mutation in exon 20 of the CFTR gene, 3905insT, was discovered. This mutation accounted for 4.8% of CFTR gene mutations in Switzerland and has since been identified in other populations of probable Swiss descent. It is associated with a highly variable clinical phenotype but always with pancreatic insufficiency. Haplotype analysis with three intragenic microsatellites in the CFTR gene showed that the mutation is associated with a haplotype rarely identified on other CFTR alleles and, therefore, that the frequency of the mutation in Switzerland is explained by a founder effect of a relatively recent mutation event. Received: 17 February 1997 / Accepted: 26 March 1977     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.     

Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis

We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.     

XV-2c and KM.19 haplotype analysis in Chilean patients with cystic fibrosis and unknown CFTR gene mutations

Cystic fibrosis (CF) is caused by mutations in the CFTR gene. More than 1600 mutations have been described, with frequencies that differ worldwide according to the ethnic origin of patients. A small group of mutations are recurrent on several populations. It has been shown that they each tend occur on specific chromosome 7 haplotypes, supporting the notion of a single origin for them. Less than 50% of mutations in Chilean patients have been identified to date. To indirectly assess the possible presence of a predominant founder mutation in the remaining unknown alleles, we evaluated 2 polymorphic markers, XV-2c and KM.19, tightly linked to the CFTR locus. The study was done in Chilean CF patients with unknown or deltaF508 (DeltaF508) CFTR mutations and their haplotypes were compared to affected family-based controls. DeltaF508 showed marked linkage disequilibrium with XV-2c/KM.19 haplotype B, with 90% of alleles on that haplotype. There was no difference in haplotype distribution between unknown mutations and normal controls. These results support a European origin for DeltaF508 alleles in Chilean patients, and make unlikely the presence of a predominant founder mutation in the so-far unknown alleles.     

Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele

Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.     

Cystic fibrosis: low frequency of DF508 mutation in 2 population samples from Rio de Janeiro, Brazil

Blood samples from 44 unrelated cystic fibrosis (CF) patients from Rio de Janeiro, Brazil, were analyzed for the 8 European CF mutations. Six homozygous and 15 heterozygous carriers of the DF508 mutation were found, corresponding to 47.7% of CF patients (allele frequency 0.3068). The G542X and G551D mutations were also observed with allele frequencies of 0.0227 and 0.0114, respectively. An analysis of the DF508 mutation in 291 randomly chosen, healthy individuals was performed, and only 3 heterozygous carriers were identified. These results show that the frequency of the DF508 allele in Rio de Janeiro is much lower than the world average; this may be due to the extremely heterogeneous ethnic admixture of the study population. By combining the results of these 2 different samples (CF patients and random population) and admixture data from Rio de Janeiro, we can estimate the CF incidence in this population to be 1:3542 individuals. However, taking into account the Rio de Janeiro ethnic admixture, we can find an estimate of 1:6902 individuals.