Journal of Heredity Advance Access originally published online on July 12, 2006
Journal of Heredity 2006 97(4):313-317; doi:10.1093/jhered/esl016
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Polymorphic Markers Suggest a Gene Flow of CFTR Gene from Sub-Saharan/Arabian and Mediterranean to Brazilian Population
From the Laboratório de Genética Humana, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365 Manguinhos, 21040-900 Rio de Janeiro, Brasil (GMK Cabello and PH Cabello); Centro de Genética Médica, Instituto Fernandes Figueira, FIOCRUZ, Av. Rui Barbosa 716, 22250-020 Rio de Janeiro, Brasil (Llerena); and Departamento de Medicina Tropical, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, 21040-900 Rio de Janeiro, Brasil (Fernandes)
Address correspondence to G. M. K. Cabello at the address above, or e-mail: gkalil{at}ioc.fiocruz.br.
The analysis of 2 diallelic loci (M470V and T854T) and a microsatellite IVS8(T)n of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has shown different haplotype distribution in Brazilian cystic fibrosis (CF) chromosomes carrying different CF mutations. The 
F508 mutation was in absolute linkage disequilibrium with 1-1 haplotype (M470V-T854T). Most of F508 chromosomes (84%) were found to carry the IVS8-9T. The most frequent haplotypes IVS8-7T and 2-1 (M470V-T854T) were found associated with Non-F508 mutations. Although there is a remarkable linkage disequilibrium between these markers with CFTR locus, the mutations R334W (7T-1-2 and 7T-2-1) and the 3120 + 1G 
A (7T-1-2 and 9T-1-2) are associated with two different haplotypes probably introduced in the Brazilian population by migration. These findings suggest that recombination events from the original haplotype and gene flow among different ethnic groups (sub-Saharan and Mediterranean) might have resulted in CF mutations associated with different haplotypes by independent introductions.
Since the foundation of the Cystic Fibrosis Genetic Analysis Consortium in 1989, there has been an extraordinary advance in molecular genetics of cystic fibrosis (CF). More than 1400 mutations and sequence variations have been described (CFMDB 2006); being the most frequent, a deletion of 3 bp at codon 508 (
F508) (Kerem and others 1989) accounted for two-thirds of the worldwide CF chromosomes. Other mutations are rare, but their frequencies are fairly variable among different ethnic and geographically located populations (CFGAC 1994; CFMDB 2006). The current knowledge of the nature of mutations identified among Europeans has enabled several groups to analyze their association with short tandem repeat polymorphisms and single-nucleotide polymorphisms and the correspondent defined haplotypes. Both types of markers provide useful information regarding the origin and evolution of the different cystic fibrosis transmembrane conductance regulator (CFTR) mutations (Morral and others 1993, 1994; Claustres and others 1996). Diallelic markers are relatively stable and can be used to define the haplotypic frameworks on which CFTR mutation occurred. Microsatellite markers that mutate faster and display a high number of alleles are powerful tools to measure genetic variability at the CFTR locus and to estimate the age of the CF mutations (Morral and others 1993).
In populations with high ethnic heterogeneity, the analysis of association between polymorphic markers and CFTR locus may provide information about disease-causing mutations and may allow indirect carrier detection for diagnosis in CF.
We have analyzed 3 polymorphic loci, consisting of 2 diallelic variants (M470V and T854T) (Kerem and others 1990) and the microsatellite IVS8(T)n (Chu and others 1991) within about 35 kbp of the CFTR gene. CF mutations are extremely variable in populations from different Brazilian regions (Raskin and others 1993, 2003; Cabello and others 1999, 2001; Bernardino and others 2000; Streit and others 2003; Araújo and others 2005; Cabello GMK, Cabello PH, Otsuki, and others 2005). The genetic profile of the Rio de Janeiro population in southeastern Brazil is very peculiar, expressing the ethnic admixture of the country. A previous screening of the whole coding region and flanking intronic sequences from the 23 exons of the CFTR gene in 190 chromosomes allowed us to identify 11 different mutations: F508 (28.4%), G85E (4.7%), 3120 + 1G A (3.7%), R334W (2.6%), G542X (2.1%), P205S (1.0%), G551D (0.5%), R1162X (0.5%), Y1092X (0.5%), S549R (0.5%), and S4X (0.5%) (Cabello GMK, Cabello PH, Otsuki, and others 2005). The F508 mutation accounts for 28.42% of the CF alleles and is significantly lower than those found in other Brazilian states (Raskin and others 1993, 2003; Cabello and others 1999, 2001; Bernardino and others 2000; Streit and others 2003).
This study aimed to analyze genetic CFTR polymorphisms to estimate allele and haplotypes frequencies and to measure linkage disequilibrium between markers with the CFTR gene, to trace the origin and evolution of different CF mutations.
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Materials and Methods |
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CF Patients
Genomic DNA was isolated from peripheral blood leukocytes according to Miller and others (1988) from 84 unrelated CF patients and, whenever possible, from their parents. An informed consent was signed during the medical interview for all patients and relatives. The National Brazilian Committee for Research on Human Subjects, a department of the Ministry of Health, has approved the project. The number of chromosomes analyzed for each polymorphism is shown in the respective tables. Screening for mutations have been described elsewhere (Cabello and others 1999, 2001; Cabello GMK, Cabello PH, Otsuki, and others 2005).
Analyzes of Diallelic Markers
Two intragenic polymorphisms, the M470V/HphI (1540 A/G) in exon 10 and T854T/AvaII (2694 T/G) in exon 14a (Kerem and others 1990; Dörk and others 1992), were analyzed in 82 CF chromosomes by polymerase chain reaction (PCR) amplification and digestion with the appropriated restriction enzyme as described (Kerem and others 1990; Dörk and others 1992). All restriction digests of PCR products were separated using 10% polyacrylamide gel electrophoresis, and alleles defined by cleavage are shown in Table 1.
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Analyzes of the Intron 8 Polythymidine Tract Length Variants (Microsatellite IVS8(T)n)
The genomic region flanking the polythymidine tract at the acceptor/branch site of exon 9, IVS8(T)n, was amplified by the allele-specific PCR using previously described methodology (Friedman and others 1997). Amplification products were submitted to 3% agarose gel electrophoresis and visualized under UV light. A total of 168 CF chromosomes were typed for the IVS8(T)n polymorphism. Alleles defined by allele-specific PCR assay are shown in Table 1.
Statistical Analysis
The program PHASE (http://www.stat.washington.edu/stephens/software.html) was used to reconstruct most probable haplotype pairs for each individual (Stephens and others 2001).
Haplotype frequencies and linkage disequilibrium were estimated by maximized likelihood method implemented by EH program (http://linkage.rockefeller.edu/ott/eh.htm). Based on sample data, the EH program estimates allele frequencies for each marker, haplotype frequencies with allelic association (H1) and without association (H0), and also provides log likelihood, 
2, which is the difference in 2 ln(likelihood), and number of degrees of freedom under hypotheses H0 and H1 (Terwillinger and Ott 1994). Linkage disequilibrium coefficients for each pair of loci were estimated using the GDA program (http://hydrodictyon.eeb.uconn.edu/people/plewis/software.php). This analysis calculates the gametic linkage disequilibrium for 2 loci each with 2 alleles when Hardy–Weinberg equilibrium is assumed (maximum likelihood analysis) in order to distinguish between the 2 types of double heterozygous. Significance was assessed with a 2 statistic.
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Results |
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Three polymorphic loci consisting of 2 diallelic variants (M470V and T854T) and the microsatellite IVS8(T)n within the CFTR gene were analyzed. Because multiple mutations were described in CFTR gene, for analysis purposes, we considered the CFTR locus as diallelic—
F508 and "Non-F508." The latter corresponds to all other mutated alleles, making possible the association analysis between F508 and Non-F508 with alleles at marker loci.
Tables 2 and 3 show the allelic frequencies of each diallelic polymorphism for 82 CF chromosomes. Frequencies of F508 and Non-F508 alleles for these CF chromosomes are also shown. Haplotypes frequencies and measure of the strength of association between alleles defined by the CFTR locus and M470V-T854T markers were estimated by likelihood maximization method implemented in the EH program. Considering the association between the CFTR locus and these 2 markers, 4 haplotypes of the 8 possible haplotypes were found (Tables 2 and 3). The haplotype F508-1-1 was observed in 30.5% of CF chromosomes, which corresponds to 100% of the F508 chromosomes. All other haplotypes Non-F508-1-1, Non-F508-1-2, and Non-F508-2-1 were found in 69.5% of the CF chromosomes, with frequencies of 7.3%, 20.7%, and 41.5%, respectively. Strong linkage disequilibrium between markers and CFTR locus could be observed (2 ln(L) = 62.58, P < 0.00001).
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Allelic frequencies of the microsatellite IVS8(T)n for 168 CF chromosomes are shown in Tables 4 and 5. Frequencies of
F508 and Non-F508 alleles for this sample are shown. Analysis of association between mutations of CFTR locus and alleles at IVS8(T)n locus by the maximized likelihood method showed that 5 haplotypes of the 6 possible haplotypes were present. The most common 9T allele associated with F508 allele was observed in 24.4% of CF chromosomes, which corresponds to 83.6% of the F508 chromosomes. Nevertheless, it is noteworthy that the allele 7T associated with F508 allele also occurred in 16.4% of F508 chromosomes (Tables 4 and 5). The proportion of allele 7T was slightly higher than allele 9T among Non-F508 chromosomes (48.7% and 42.1%, respectively). Allele 5T was found in very low frequency on Non-F508 chromosomes (9.2%). Haplotypes frequencies and measure of the strength of association among alleles (2 ln(L) = 16.92, P < 0.0001), estimated by the EH program, are shown in Tables 4 and 5. Estimates of linkage disequilibrium coefficients between the CFTR locus and IVS8(T)n were performed using the GDA program. The results showed strong linkage disequilibrium between 9T with F508 (2 = 26.34, P < 0.00001) and 7T with Non-F508 (2 = 22.47, P < 0.0001), as shown in Table 6.
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Table 7 is a collection of the most probable 3-marker haplotypes obtained from 7 of the 11 identified CFTR mutations on CF patients from Rio de Janeiro (Cabello GMK, Cabello PH, Otsuki, and others 2005). These mutations were found distributed in 5 different IVS8(T)n-M470V-T854T haplotypes. The
F508, R334W, and 3120 + 1G A mutations were found linked with 2 different haplotypes as shown in Table 7. The most common 9T-1-1 haplotype among Caucasians was also the most frequent haplotype linked to F508 and G542X mutations in our patients.
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Discussion |
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In the present study, we have extended our previous marker haplotype analysis of CFTR locus in Brazilian CF patients (Cabello GMK, Cabello PH, Lopez-Camelo, and others 2005) by including 3 additional intragenic polymorphisms. The analysis of 2 diallelic loci, M470V and T854T, within about 35 kb of the CFTR gene (Rommens and others 1989) could reveal the presence of 1-1 haplotype in absolute linkage disequilibrium with the
F508 allele. This haplotype was found in only 10.5% of Non-F508 chromosomes. Remaining 1-2 and 2-1 haplotypes were found in 29.8% and 59.7%, respectively, of Non-F508 chromosomes. This finding is in agreement with European studies (Dörk and others 1992; Claustres and others 1996), which the F508 mutation arose in a same original haplotype (1-1) from the Caucasian population introduced in Brazil by European immigrants. The splicing site length polymorphism in intron 8, IVS8(T)n, herein analyzed, displayed strong association with CFTR locus.
In our sample, F508 chromosomes were found associated with both 9T allele and 7T allele differing from the absolute disequilibrium between F508 and 9T allele found in European chromosomes (Dörk and others 1994). A recombination event from the original haplotype in which the F508 allele arose is the most probable explanation for this finding. Interestingly, the 7T allele was also observed in F508 chromosomes in Arabic population (Desgeorges and others 1997), and an alternative explanation for the presence of this allele in our sample could be the putative introduction of 7T via this population in Brazil.
When all 3 polymorphic markers were considered, the most common F508 mutation was found to be associated with 2 haplotypes, 9T-1-1 and 7T-1-1. The 9T-1-1 haplotype was also found to be associated with G542X mutation. This finding is in agreement with our previous study with reference to 4 additional CFTR markers, in which these mutations were found associated with B-6-1 haplotype composed by multilocus system XV2C-KM19-GATT-TUB9 (Cabello GMK, Cabello PH, Lopez-Camelo, and others 2005). These data are in accordance with European studies, where the F508 mutation originally arose in a chromosome bearing the 6-repeat allele and a B haplotype (Chehab and others 1991; Dörk and others 1992; Claustres and others 1996). It is worth to note that in a previous study uncommon multilocus haplotypes A-6-1 and D-6-1 (XV2C-KM19-GATT-TUB9) were also found associated with F508 mutation (Cabello GMK, Cabello PH, Lopez-Camelo, and others 2005). In addition, in the present analysis, an uncommon haplotype 7T-1-1, composed by multilocus system IVS8(T)n-M470V-T854T, was observed. These findings are in accordance with the hypothesis of recombination events from the original haplotype in which the F508 mutation could have occurred. Probably, these uncommon haplotypes were introduced in Brazilian population by ethnic admixture because uncommon haplotypes for F508 chromosomes were also observed previously in Mediterranean countries (Dörk and others 1992). It is noteworthy that the Brazilian population is mainly the result of a 3-way ethnic admixture between Europeans, Africans, and Amerindians. The most frequent 7T (IVS8(T)n) and 2-1 (M470V-T854T) haplotypes were found associated with a great number of Non-F508 mutations. Moreover, the R334W and 3120 + 1G A mutations were associated with 2 different haplotypes (7T-1-2 or 7T-2-1 and 9T-1-2 or 7T-1-2, respectively). These same mutations were found linked to different haplotypes XV2C-KM19 background in previous study (Cabello GMK, Cabello PH, Lopez-Camelo, and others 2005). Although recombination events cannot be excluded, the occurrence of R334W mutation associated with 2 different haplotypes in our population could suggest an origin in different genetic background introduced in Brazilian population by migrations. Recombination and genetic admixture likely explain our finding on different haplotypes associated with the 3120 + 1G A mutation that most likely derived from a sub-Saharan/Arabian common ancestor (Dörk and others 1998).
The present study provides information about an association among 3 CFTR polymorphic markers with CFTR locus. These findings agree with our previous observation, using other 4 CFTR polymorphisms (Cabello GMK, Cabello PH, Lopez-Camelo, and others 2005), suggesting that a gene flow between different ethnic groups, mainly sub-Saharan/Arabian and Mediterranean in Brazilian population, could result in CF mutations associated with different haplotypes probably by independent introductions.
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Acknowledgments |
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We would like to thank all the CF families for their collaboration in these investigations and medical staff of the Medical Genetics Center, Fernandes Figueira Institute. We are grateful to Tamara Gomes Kalil for proofreading. Financial support for this research was supplied by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Financiadora de Estudos e Projetos, and Programa de Apoio à Pesquisa Estratégica em Saúde/Fundação Oswaldo Cruz.
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Footnotes |
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Corresponding Editor: Roger Reeves
Received October 11, 2005
Accepted April 28, 2006
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References |
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