Cystic fibrosis mutations R1162X and 2183AA<FONT FACE=Symbol>®</FONT>G in two southern Brasilian states

Abstracts

We screened 79 southern Brazilian patients with cystic fibrosis for the rare cystic fibrosis mutations R1162X and 2183AA<FONT FACE="Symbol">®</FONT>G. Forty-nine patients were born in the State of Paraná (PR) and 30 in the State of Santa Catarina (SC). Two 2183AA<FONT FACE="Symbol">®</FONT>G alleles were found among the SC patients and one among the PR patients. Six R1162X alleles were found among the SC patients and one among the PR patients. Fourteen percent of the alleles found among patients of Italian origin were R1162X, and 7% were 2183AA<FONT FACE="Symbol">®</FONT>G mutations. These mutations, together with <FONT FACE="Symbol">D</FONT>F508, were also studied in a sample of 270 normal non-related subjects of Italian origin who have been born in PR. In this sample we found two <FONT FACE="Symbol">D</FONT>F508 alleles and one 2183AA<FONT FACE="Symbol">®</FONT>G allele. <FONT FACE="Symbol">D</FONT>F508, R1162X and 2183AA<FONT FACE="Symbol">®</FONT>G frequencies were not statistically different from those observed in Italy. Our results demonstrate that it is important to include these mutations in southern Brazilian surveys of cystic fibrosis patients, especially when they are of Italian descent.

Realizou-se a análise de 79 pacientes provenientes do Sul do Brasil para duas mutações raras da fibrose cística (CF), R1162X e 2183AA<FONT FACE="Symbol">®</FONT>G; dentre estes pacientes, 49 eram nascidos no Estado do Paraná (PR) e 30 eram nascidos no Estado de Santa Catarina (SC). Para a mutação 2183AA<FONT FACE="Symbol">®</FONT>G, dois alelos foram detectados entre os pacientes de SC e um alelo nos pacientes de PR. Para a mutação R1162X, seis alelos foram detectados entre os pacientes de SC e um alelo entre os pacientes do PR. Quando estes pacientes foram classificados de acordo com a origem étnica, 14% dos alelos detectados entre os pacientes de origem italiana eram portadores da mutação R1162X e 7% da mutação 2183AA<FONT FACE="Symbol">®</FONT>G. Estas mutações, juntamente com a mutação <FONT FACE="Symbol">D</FONT>F508, também foram analisadas em uma amostra de 270 indivíduos normais de origem italiana não-consangüíneos, os quais eram nascidos no Estado do PR. Nessa amostra foram detectados dois alelos <FONT FACE="Symbol">D</FONT>F508 e um alelo 2183AA<FONT FACE="Symbol">®</FONT>G. As freqüências das mutações <FONT FACE="Symbol">D</FONT>F508, R1162X e 2183AA<FONT FACE="Symbol">®</FONT>G não mostraram desvio estatístico significativo daquelas freqüências observadas no norte da Itália. Nossos resultados demonstram que é importante incluir estas mutações no conjunto de mutações a serem pesquisadas nos pacientes com FC do sul do Brasil, especialmente quando estes pacientes tiverem origem italiana.

Cystic fibrosis mutations R1162X and 2183AA®G in two southern Brazilian States

Lilian Pereira¹, Salmo Raskin¹, Aline A. Freund¹, Patrícia D. Ribas¹, Raquel M.V. Castro¹, Pier F. Pignatti² and Lodércio Culpi¹

¹Departamento de Genética, Universidade Federal do Paraná, 81531-990 Curitiba, PR, Brasil. Send correspondence to L.P. Caixa Postal 19031, Fax: [phone]. E-mail: [e-mail] ²Istituto Di Scienze Biologiche, Università di Verona, Verona, Italy.

ABSTRACT

We screened 79 southern Brazilian patients with cystic fibrosis for the rare cystic fibrosis mutations R1162X and 2183AA®G. Forty-nine patients were born in the State of Paraná (PR) and 30 in the State of Santa Catarina (SC). Two 2183AA®G alleles were found among the SC patients and one among the PR patients. Six R1162X alleles were found among the SC patients and one among the PR patients. Fourteen percent of the alleles found among patients of Italian origin were R1162X, and 7% were 2183AA®G mutations. These mutations, together with DF508, were also studied in a sample of 270 normal non-related subjects of Italian origin who have been born in PR. In this sample we found two DF508 alleles and one 2183AA®G allele. DF508, R1162X and 2183AA®G frequencies were not statistically different from those observed in Italy. Our results demonstrate that it is important to include these mutations in southern Brazilian surveys of cystic fibrosis patients, especially when they are of Italian descent.

INTRODUCTION

Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disease among Caucasians. It occurs in approximately one in 2500 live-births in populations of European ancestry, and the frequency of heterozygotes is estimated at one in 25 Caucasians (Romens et al., 1989; Riordan et al., 1989; Kerem et al., 1989).

The incidence of CF in the Brazilian population is unknown. The incidence of CF is expected to have regional differences due to the various patterns of racial mixture, immigration and selection pressure (Raskin et al., 1993, 1997a,b).

The CF gene, that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, is located on the long arm of chromosome 7, at position 7q31. It has a length of 250 kb and contains 27 exons. This is involved in active ion transport through the apical membrane of epithelial cells (Riordan et al., 1989; Tsui and Buchwald, 1992).

The DF508 3-bp deletion is the most frequent CF mutation. However, more than 1000 different mutations of the CFTR gene have been reported (Mercier et al., 1994; Zielenski and Tsui, 1995; CFGAC, 1999). The frequency of DF508 among Brazilian CF patients is about 47% (Raskin et al., 1993). This frequency varies among states, being 53% in Minas Gerais, 52% in São Paulo, 49% in Rio Grande do Sul, 44% in Paraná and 27% in Santa Catarina. Presence of DF508 in Brazil is much higher than in the United States or England (CFGAC, 1999). As expected, the frequencies found in Portugal, Spain and Italy are similar to those found in Brazil, due to the colonization of Brazil by southern Europeans (Raskin et al., 1993). CF haplotype studies also suggest that a high number of CF mutations are present in Brazilian CF patients (Raskin et al., 1997a,b).

Other authors have reported frequencies of the DF508 in Brazil: 50.8% in Rio Grande do Sul (Marostica et al., 1998), 35% in Rio de Janeiro (de Miranda et al., 1993) and 33% in São Paulo (Martins et al., 1993), but these studies generally involved only a small number of patients.

In northeastern Italy the three most frequent CF mutations are DF508 (48%), R1162X (10%) and 2183AA®G (9%) (Bonizzato et al., 1995; Rendine et al., 1997). The R1162X and 2183AA®G mutations are rare in other regions, with respective world frequencies of 0.8 and 0.3%.

R1162X is a nonsense mutation characterized by the substitution of cytosine for thymine (C®T) at position 3616 of the DNA in exon 19. As a consequence, an amino acid arginine is substituted by a stop codon at position 1162 (CFGAC, 1993).

2183AA®G is a frameshift mutation characterized by the substitution of adenine for guanine (A®G) at DNA position 2183 and the deletion of an adenine at position 2184 in exon 13 (R domain) (Bozon et al., 1994; CFGAC, 1994).

The Brazilian Caucasian population is not ethnically homogeneous. While in the northern states the Portuguese contribution prevails, different waves of European immigration have caused a higher ethnic diversity in the southern regions (Salzano and Freire-Maia, 1967).

Italian immigration to Brazil consisted mainly of inhabitants of the Veneto region, which accounted for 48% of the Italian immigrants. In certain southern Brazilian regions, this percentage has been as high as 90%, which is the case for rural centers in the States of Paraná, PR and Santa Catarina, SC (Hutter, 1987). Considering the relatively low frequency of the DF508 mutation in SC and PR (Raskin et al., 1993), we decided to screen Brazilians for R1162X and 2183AA®G.

MATERIAL AND METHODS

Characterization of the sample

The CF patient sample consisted of 79 families, 49 from PR and 30 from SC. Blood samples were collected from the patient, the father and mother. Average patient age was 7.05 ± 6.59 years and ranged from two months to 32 years at the time of blood collection. We also examined 270 normal individuals of Italian descent by both maternal and paternal lines. Average subject age was 52.02 ± 17.44 years and ranged from 16 to 89 years at the time of blood collection.

The data was analyzed by a chi-square test, with Yates correction when necessary.

Methodology and molecular analysis

DNA extraction was performed by the phenol-chloroform method (Sambrook et al., 1989) modified by Lahiri and Nurnberger Jr. (1991). The extracted DNA was amplified by polymerase chain reaction (PCR), digestion with restriction enzymes, when necessary, and polyacrylamide gel electrophoresis. Methodology for these mutations were essentially as described in the references: the DF508 mutation(Raskin et al., 1992); the R1162X mutation (Gasparini et al., 1992); the 2183AA®G mutation (Bozon et al., 1994).

RESULTS AND DISCUSSION

Sample of normal subjects of Italian descent

Among the 540 alleles analyzed we found two DF508 alleles, one 2183AA®G allele and no R1162X allele. These alleles were all found in heterozygous individuals.

In northeastern Italy, the frequency of the DF508 mutation is 48% (Bonizzato et al., 1995). Since our sample consisted of northern Italian descendants, we expected to find about five mutant alleles among the 270 normal individuals. The observed difference was not statistically significant (P > 0.2).

The frequency of the 2183AA®G mutation in northeastern Italy is 9% of the CF alleles. Thus, we should find approximately one 2183AA®G allele in every 500 alleles in normal subjects of Italian descent. We found one heterozygote (0.2%), a frequency not significantly different from that observed in northern Italy (P > 0.6).

Sample of patients from Paraná and Santa Catarina

Seven R1162X alleles among the 158 alleles analyzed were found. This frequency varied significantly when the patients were classified by state of birth. Among patients born in PR, only one R1162X allele was found among the 98 analyzed, while among those born in SC, six alleles were found among the 60 examined. In the same sample we found three 2183AA®G. One 2183AA®G allele was found in PR and two in SC, in one homozygote (Table I).

The heterogeneity observed in the patient sample in comparison to the homogeneity observed in the control sample may be a consequence of the selection of the control group. They were of Italian descent, free from mixture, at least as far as we know. However, the patient sample was intensely mixed, since they were not selected by ethnic origin, but for having CF and for being born in PR or SC. All patients who had the R1162X and the 2183AA®G mutations were also of Italian descent (Table I). When patients were classified into three familial categories (Italian/Italian (I x I); Italian/other (I x O); other/other (O x O)), we found the following frequencies for R1162X (I x I = 14% (N = 26); I x O = 2% (N = 32) and O x O = 0% (N = 40) and for 2183AA®G (I x I = 7%; I x O and O x O = 0%).

There was no significant difference when the northeastern Italian frequency of the R1162X mutation was compared to the frequency found among all patients (I x I) from both states (14.29%) (P > 0.2) and among (I x O) PR patients (3%) (P > 0.1). However, the frequency observed among (I x I) SC patients (37%) was significantly higher (P < 0.001) than that found in northeastern Italy.

The frequency of the 2183AA®G mutation (7.15%), among all the patients (I x I) from both states, was not significantly different from that observed in northeastern Italy (9.3%), (P > 0.4). If the comparison is made for each state separately, the differences are not statistically significant (P > 0.2 for PR and P > 0.6 for SC) .

Genotype x phenotype in the patient sample

In relation to R1162X carriers, our results agree with previous studies (Rolfini and Cabrini, 1993). Their study proposed that the truncated CFTR protein could be partially functional in pulmonary tissues. Therefore, only mild or moderate pulmonary disease would occur. Probably this happens because when this mutation is present, the protein still contains the regulatory domain, the first nucleotide binding site and both transmembrane domains (Table I).

The phenotypes of the 2183AA®G carriers did not agree with Bozon et al. (1994) and Bonizzato et al. (1995), who suggested that this mutation could lead to severe symptoms. However, it did agree with Castaldo et al. (1996), who believe that this mutation causes only moderately severe disease (Table I).

The heterogeneity observed in the patients was discussed by Wolf (1997), who explained that genetic heterogeneity may occur when different mutations converge to give a similar phenotype, and phenotypic heterogeneity occurs when mutations are within the same gene, and diverge to give different phenotypes. Although many examples of this heterogeneity have been cited, the mechanisms involved are still poorly understood.

Anyane-Yeboa and Heggarty (1998) discussed the discordant expression of CF mutations in siblings. They suggested that there are factors other than mutations of the CFTR gene which account for the discrepant CF phenotype in siblings.

One possible explanation is interaction of modifier genes. Rozmahel et al. (1996) have identified modifier genes in CFTR-deficient mice. Zielenski et al. (1998) continued the study of Rozmahel. They mapped the CF-modifier (CFM) loci in the human genome and tested these CFM in patients. Their data showed a strong association between the markers in the 19q13 region and meconium ileus status in CF sibpairs, but the association was not so strong for pulmonary function.

In summary, we found no significant differences between samples of normal subjects of Italian origin from PR and SC and the northeastern Italian population in terms of the CF mutations DF508, R1162X and 2183AA®G. In the patient sample, the overall frequency of R1162X and 2183AA®G mutations did not differ significantly from those found in northeastern Italy in subjects of Italian ancestry, except for the R1162X mutation, whose frequency was higher in SC than in northeastern Italy. The R1162X and 2183AA®G mutations are associated with mild or moderate lung disease and pancreatic insufficiency. Our data suggest that the R1162X and 2183AA®G mutations should be included as part of the mutation pool to be analyzed in CF patients of northeastern Italian descent. This suggestion applies also for other countries with a substantial Italian immigration.

ACKNOWLEDGMENTS

We thank Dr. Eleidi A. Chautard-Freire-Maia for critically reading the manuscript. The research was supported by fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) as well as by CNPq and Fundação da Universidade Federal do Paraná grants.

RESUMO

Realizou-se a análise de 79 pacientes provenientes do Sul do Brasil para duas mutações raras da fibrose cística (CF), R1162X e 2183AA®G; dentre estes pacientes, 49 eram nascidos no Estado do Paraná (PR) e 30 eram nascidos no Estado de Santa Catarina (SC). Para a mutação 2183AA®G, dois alelos foram detectados entre os pacientes de SC e um alelo nos pacientes de PR. Para a mutação R1162X, seis alelos foram detectados entre os pacientes de SC e um alelo entre os pacientes do PR. Quando estes pacientes foram classificados de acordo com a origem étnica, 14% dos alelos detectados entre os pacientes de origem italiana eram portadores da mutação R1162X e 7% da mutação 2183AA®G. Estas mutações, juntamente com a mutação DF508, também foram analisadas em uma amostra de 270 indivíduos normais de origem italiana não-consangüíneos, os quais eram nascidos no Estado do PR. Nessa amostra foram detectados dois alelos DF508 e um alelo 2183AA®G. As freqüências das mutações DF508, R1162X e 2183AA®G não mostraram desvio estatístico significativo daquelas freqüências observadas no norte da Itália. Nossos resultados demonstram que é importante incluir estas mutações no conjunto de mutações a serem pesquisadas nos pacientes com FC do sul do Brasil, especialmente quando estes pacientes tiverem origem italiana.

REFERENCES

Anyane-Yeboa, K. and Heggarty, E. (1988). Discordant expression of cystic fibrosis (CF) mutations in siblings. Am. J. Hum. Genet. 63 (Suppl. 4): A349.

Bonizzato, A., Bisceglia, L., Marigo, C., Nicolis, E., Bombieri, C., Castellani, C., Borgo, G., Zelante, L., Mastella, G., Cabrini, G., Gasparini, P. and Pignatti, P.F. (1995). Analysis of the complete coding region of the CFTR gene in a cohort of cystic fibrosis patients from northeastern Italy: identification of 90% of the mutations. Hum. Genet. 95: 397-402.

Bozon, D., Zielenski, J., Rininsland, F. and Tsui, L.C. (1994). Identification of four mutations in the cystic fibrosis transmembrane conductance regulator gene: I148T, L1077P, Y1092X, 2183AA®G. Hum. Mut. 3: 330-332.

Castaldo, G., Rippa, E., Raia, V., Salvatore, D., Massa, C., Deritis, G. and Salvatore, F. (1996). Clinical features of cystic fibrosis patients with rare genotypes. J. Med. Genet. 33: 73-76.

CFGAC (1993). Cystic Fibrosis Genetic Analysis Consortium. Newsletter, 11/04/93.

CFGAC (1994). Cystic Fibrosis Genetic Analysis Consortium. Newsletter, 30/03/94.

CFGAC (1999). http://www.genet.sickkids.on.ca

de Miranda, A.B., Llerena Junior, J., Dallalana, L.T., Moura-Neto, R.S., Suffys, P.N. and Degrave, W.M. (1993). Use of PCR for the determination of the frequency of the delta F508 mutation in Brazilian cystic fibrosis patients. Mem. Inst. Oswaldo Cruz88: 309-312.

Gasparini, P., Borgo, G., Mastella, G., Bonizzato, A., Dognini, M. and Pignatti, P.F. (1992). Nine cystic fibrosis patients homozygous for the CFTR nonsense mutation R1162X have mild or moderate lung disease. J. Med. Genet. 29: 558-562.

Hutter, L.M. (1987). A Imigração Italiana no Brasil (Séculos XIX e XX). In: A Presença Italiana no Brasil (Boni, L.A., ed.). Escola Superior de Teologia/Fondazione Giovanni Agnelli, Porto Alegre, pp. 74-101.

Kerem, B.S., Romens, J.M., Buchanan, J.A., Markiewikz, D., Cox, T.K., Chakravarti, A., Buchwald, M. and Tsui, L.C. (1989). Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080.

Lahiri, D.K. and Nurnberger Jr., J.I. (1991). A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res. 19: 5444.

Marostica, P.J., Raskin, S. and Abreu-e-Silva, F.A. (1998). Analysis of the delta F508 mutation in Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients. Braz. J. Med. Biol. Res. 31: 529-532.

Martins, C.S., Ribeiro, F. and Costa, F.F. (1993). Frequency of the cystic fibrosis delta F508 mutation in a population from São Paulo State, Brazil. Braz. J. Med. Biol. Res. 26: 1037-1040.

Mercier, B., Raguénes, O., Estivil, X., Morral, N., Kaplan, G.C., Mclure, M., Grebe, T.A., Kessler, D., Pignatti, P.F., Marigo, C., Bombieri, C., Audrézete, M.P., Verlingue, C. and Férec, C. (1994). Complete detection of mutations in cystic fibrosis patients of native American origin. Hum. Genet. 94: 629-632.

Raskin, S., Philips III, J.A., Kaplan, G., McClure, M. and Vnencak-Jones, C. (1992). Cystic fibrosis genotyping by direct PCR analysis of Guthrie blood spots. PCR Methods Appl. 2: 154-156.

Raskin, S., Philips III, J.A., Krishnamani, M.R.S., Vnencak-Jones, C., Parker, R.A., Rozov, T., Cardieri, J.M., Marostica, P., Abreu, F., Giugliani, R., Reis, F., Rosario, N.A., Ludwig, N. and Pilotto, R.F. (1993). DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards. Am. J. Med. Genet. 46: 665-669.

Raskin, S., Philips III, J.A., Krishnamani, M.R.S., Vnencak-Jones, C., Parker, R.A., Dawson, E., Rozov, T., Cardieri, J.M., Marostica, P., Abreu, F., Giugliani, R., Reis, F., Rosario, N.A., Ludwig, N. and Culpi, L. (1997a). Regional distribution of cystic fibrosis-linked DNA haplotypes in Brazil: Multicenter study. Hum. Biol. 69: 75-78.

Raskin, S., Philips III, J.A., Krishnamani, M.R.S., Vnencak-Jones, C., Parker, R.A., Rozov, T., Cardieri, J.M., Marostica, P., Abreu, F., Giugliani, R., Reis, F., Rosario, N.A., Ludwig, N. and Culpi, L. (1997b). Cystic fibrosis in the Brazilian population: DF508 mutation and KM-19/XV-2C haplotype distribution. Hum. Biol. 69: 499-508.

Rendine, S., Calafell, F., Cappello, N., Gagliardini, R., Caramia, G., Rigillo, N., Silvetti, M., Zanda, M., Miano, A., Battistini, F., Marianelli, L., Taccetti, G., Diana, M.C., Romano, L., Romano, C., Giunta, A., Padoan, R., Pianaroli, A., Raia, V., De Rtis, G., et al. (1997). Genetic history of cystic fibrosis mutations in Italy. I. Regional distribution. Ann. Hum. Genet. 61: 411-424.

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Rolfini, R. and Cabrini, G. (1993). Nonsense mutation R1162X of the cystic fibrosis transmembrane regulator gene does not reduce messenger RNA expression in nasal epithelial tissue. J. Clin. Invest. 92: 2683-2687.

Romens, J.M., Iannuzzi, M.A.C., Kerem, B.S., Drum, M.L., Melmer, G., Deam, M., Rozmahel, R., Cole, J.L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J.R., Tsui, L.C. and Collins, F.S. (1989). Identification of cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065.

Rozmahel, R., Wilschanski, M., Matin, A., Plyte, S., Oliver, M., Auerbach, W., Moore, A., Forstner, J., Durie, P., Nadeau, J., Bear, C. and Tsui, L.C. (1996). Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat. Genet. 12: 280-287.

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(Received April 27, 1998)

References

• Anyane-Yeboa, K. and Heggarty, E. (1988). Discordant expression of cystic fibrosis (CF) mutations in siblings. Am. J. Hum. Genet 63 (Suppl. 4): A349.
• Bonizzato, A., Bisceglia, L., Marigo, C., Nicolis, E., Bombieri, C., Castellani, C., Borgo, G., Zelante, L., Mastella, G., Cabrini, G., Gasparini, P. and Pignatti, P.F. (1995). Analysis of the complete coding region of the CFTR gene in a cohort of cystic fibrosis patients from northeastern Italy: identification of 90% of the mutations. Hum. Genet 95: 397-402.
• Castaldo, G., Rippa, E., Raia, V., Salvatore, D., Massa, C., Deritis, G. and Salvatore, F. (1996). Clinical features of cystic fibrosis patients with rare genotypes. J. Med. Genet 33: 73-76.
• CFGAC (1993). Cystic Fibrosis Genetic Analysis Consortium Newsletter, 11/04/93.
• CFGAC (1994). Cystic Fibrosis Genetic Analysis Consortium Newsletter, 30/03/94.
• CFGAC (1999). http://www.genet.sickkids.on.ca
• de Miranda, A.B., Llerena Junior, J., Dallalana, L.T., Moura-Neto, R.S., Suffys, P.N. and Degrave, W.M. (1993). Use of PCR for the determination of the frequency of the delta F508 mutation in Brazilian cystic fibrosis patients. Mem. Inst. Oswaldo Cruz88: 309-312.
• Gasparini, P., Borgo, G., Mastella, G., Bonizzato, A., Dognini, M. and Pignatti, P.F. (1992). Nine cystic fibrosis patients homozygous for the CFTR nonsense mutation R1162X have mild or moderate lung disease. J. Med. Genet 29: 558-562.
• Hutter, L.M. (1987). A Imigraçăo Italiana no Brasil (Séculos XIX e XX). In: A Presença Italiana no Brasil (Boni, L.A., ed.). Escola Superior de Teologia/Fondazione Giovanni Agnelli, Porto Alegre, pp. 74-101.
• Kerem, B.S., Romens, J.M., Buchanan, J.A., Markiewikz, D., Cox, T.K., Chakravarti, A., Buchwald, M. and Tsui, L.C. (1989). Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080.
• Lahiri, D.K. and Nurnberger Jr., J.I. (1991). A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 19: 5444.
• Marostica, P.J., Raskin, S. and Abreu-e-Silva, F.A. (1998). Analysis of the delta F508 mutation in Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients. Braz. J. Med. Biol. Res 31: 529-532.
• Martins, C.S., Ribeiro, F. and Costa, F.F. (1993). Frequency of the cystic fibrosis delta F508 mutation in a population from Săo Paulo State, Brazil. Braz. J. Med. Biol. Res 26: 1037-1040.
• Mercier, B., Raguénes, O., Estivil, X., Morral, N., Kaplan, G.C., Mclure, M., Grebe, T.A., Kessler, D., Pignatti, P.F., Marigo, C., Bombieri, C., Audrézete, M.P., Verlingue, C. and Férec, C. (1994). Complete detection of mutations in cystic fibrosis patients of native American origin. Hum. Genet 94: 629-632.
• Raskin, S., Philips III, J.A., Kaplan, G., McClure, M. and Vnencak-Jones, C. (1992). Cystic fibrosis genotyping by direct PCR analysis of Guthrie blood spots. PCR Methods Appl. 2: 154-156.
• Raskin, S., Philips III, J.A., Krishnamani, M.R.S., Vnencak-Jones, C., Parker, R.A., Rozov, T., Cardieri, J.M., Marostica, P., Abreu, F., Giugliani, R., Reis, F., Rosario, N.A., Ludwig, N. and Pilotto, R.F. (1993). DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards. Am. J. Med. Genet 46: 665-669.
• Raskin, S., Philips III, J.A., Krishnamani, M.R.S., Vnencak-Jones, C., Parker, R.A., Dawson, E., Rozov, T., Cardieri, J.M., Marostica, P., Abreu, F., Giugliani, R., Reis, F., Rosario, N.A., Ludwig, N. and Culpi, L. (1997a). Regional distribution of cystic fibrosis-linked DNA haplotypes in Brazil: Multicenter study. Hum. Biol 69: 75-78.
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Publication Dates

• Publication in this collection
  01 Feb 2000
• Date of issue
  Sept 1999

History

• Received
  27 Apr 1998