Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis

One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For a disease such as cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation based PGD protocol is impracticable. An elegant way to overcome this problem is to set up an indirect diagnosis using polymorphic markers allowing the identification of the pathogenic haplotype instead of the mutation. We present here a new PGD protocol for CF. Our strategy is based on a multiplex fluorescent PCR co-amplifying the DeltaF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA). Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DeltaF508 mutation. The use of intragenic markers reduces the risk of misdiagnosis due to meiotic recombination. In 97% of the single lymphoblasts (151/155) tested a PCR signal was obtained. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop out (ADO) was observed. Three cases were performed. Case one was at risk of transmitting mutations DeltaF508 and R1162X, case 2 DeltaF508 and R1066C and case 3 DeltaF508 and 1341+1A. Considering these three cases and the re-analysis of the affected embryos, we have analysed 62 blastomeres from which we had PCR signal for 58 (94%) and a complete haplotype for 49 (84%). With the degree of polymorphism of the markers used in this work (48 and 39%) and the fact that we co-amplified the F508 locus our test should be suitable for nearly 80% of the couples requesting PGD for CF. This fluorescent multiplex PCR indirect diagnosis provides also a safer test since it allows the confirmation of the diagnosis, the detection of contamination and could give an indication on the ploidy of the embryos tested.     

Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers

We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, ΔF508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A → G, R1162X, and 3849 + 10kbC → T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF. © 1996 Wiley-Liss, Inc.     

Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.     

Informativity of intragenic microsatellites for carrier detection and prenatal diagnosis of cystic fibrosis in the Italian population

Molecular diagnosis of cystic fibrosis (CF) in the Italian population, based on the detection of the deltaF508 mutation (51.2% of CF chromosomes), provides full informativity for prenatal diagnosis (PDN) in about 28% of families at risk. Identification of the predominant non-deltaF508 mutations allows the characterization of about 70% of CF chromosomes, making approximately 48% of couples fully informative. In families where at least one chromosome remains uncharacterized, allele segregation is still determined using RFLPs closely linked to the CF gene. The recent identification of three polymorphic clusters of dinucleotide repeats (IVS8/ GT, IVS17b/TA and IVS17b/CA) led us to evaluate whether their analysis might improve feasibility studies for prenatal diagnosis or hetero-zygote identification. One hundred nuclear families with a CF child, reflecting the general Italian deltaF508 mutation distribution, were geno-typed for the three microsatellites. In this study microsatellite analysis using IVS8/GT and IVS17b/TA allowed the identification of both parental CF chromosomes in 94% of couples; inclusion in the study of the less polymorphic repeat locus, IVS17b/CA, slightly improved this percentage (97%). Hence, a strategy involving primarily the detection of the delta F508 mutation and secondarily microsatellite analysis makes possible PDN of CF in virtually all Italian CF families.     

Multiplex sequence variation detection throughout the CFTR gene appropriate for preimplantation genetic diagnosis in populations with heterogeneity of cystic fibrosis mutations

Cystic fibrosis (CF) is one of the most important genetic diseases requiring prevention programmes. Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis, and is especially appropriate for couples with an unsuccessful reproductive history. For clinical application, protocols must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes. We have developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR and denaturing gradient gel electrophoresis analysis, along with analysis of a fluorescently labelled intragenic microsatellite (IVS8CA). The experiments were carried out on 390 single lymphocytes from three CF patients, one heterozygote and one non-CF individual. PCR efficiency of the exons ranged from 90 to 100%, and ADO from 0 to 3.8%. IVS8CA was co-amplified with a PCR efficiency of 92.4 and 10.8% ADO. The present method overcomes the need for separate assays for each CFTR gene mutation. Additionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons) along with analysis of a microsatellite, which is useful (when informative) for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells.     

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.     

Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination

Two healthy sisters with a familial history of mental retardation were referred to our centre for preimplantation genetic diagnosis (PGD). Their two brothers showed severe mental retardation. The molecular basis for their disorder could not be identified, but one of the sisters and the mother presented a highly skewed pattern of X-inactivation reinforcing the likelihood of an X-linked mode of inheritance. Both sisters requested PGD to avoid the abortion of potentially affected male fetuses. PGD for sex by fluorescent in-situ hybridization was carried out for the first sister and resulted in the birth of a female child. The second sister and her partner, whose niece had cystic fibrosis (CF), were tested for CF mutations, and were both found to be deltaF508 heterozygous. We developed an efficient single cell PCR protocol for the simultaneous amplification of the CF (deltadeltaF508) locus as well as the X-linked amelogenin gene and its highly homologous pseudogene on the Y chromosome. Two PGD cycles were carried out to screen against male and deltaF508 homozygous deleted embryos. In each case several embryos could be selected for transfer and the second cycle resulted in a twin pregnancy followed by the birth of two healthy female infants.     

Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal / carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for ΔF508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, ΔF508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G>T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes. © 1996 Wiley-Liss, Inc.     

Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR

We present a method for rapid and accurate identification of the normal and DeltaF508 alleles of the cystic fibrosis (CF) gene in single human cells that utilizes LATE (linear after the exponential)-PCR, a newly invented form of asymmetric PCR. Detection of the single-stranded amplicon is carried out in real time, using allele-specific molecular beacons. The LATE-PCR method permits controlled abrupt transition from exponential to linear amplification and thereby enhances the fluorescent signals and reduces variability between replicate samples relative to those obtained using typical real-time PCR. Of 239 single lymphoblasts generating amplification signals, 227 (95%) exhibited signals that met objective quantitative criteria required for diagnosis. Among these samples, 222 were genotyped correctly, for an assay accuracy of 98%. The small number of diagnostic errors was due to allele drop-out among heterozygous lymphoblasts, 4/119 (3.4%), and contamination among homozygous DeltaF508 lymphoblasts, 1/57 (1.8%). LATE-PCR offers a new strategy for preimplantation genetic diagnosis and other fields in which accurate quantitative detection of single copy genes is important.     

Spectrum of Mutations in the CFTR Gene in Cystic Fibrosis Patients of Spanish Ancestry

We analyzed 1,954 Spanish cystic fibrosis (CF) alleles in order to define the molecular spectrum of mutations in the CFTR gene in Spanish CF patients. Commercial panels showed a limited detection power, leading to the identification of only 76% of alleles. Two scanning techniques, denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism/hetroduplex (SSCP/HD), were carried out to detect CFTR sequence changes. In addition, intragenic markers IVS8CA, IVS8-6(T)n and IVS17bTA were also analyzed. Twelve mutations showed frequencies above 1%, p.F508del being the most frequent mutation (51%). We found that eighteen mutations need to be studied to achieve a detection level of 80%. Fifty-one mutations (42%) were observed once. In total, 121 disease-causing mutations were identified, accounting for 96% (1,877 out of 1,954) of CF alleles. Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. This updated information on the spectrum of CF mutations in Spain will be useful for improving genetic testing, as well as to facilitate counselling in people of Spanish ancestry. In addition, this study contributes to defining the molecular spectrum of CF in Europe, and corroborates the high molecular mutation heterogeneity of Mediterranean populations.     

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.     

CFTR gene: molecular analysis in patients from South Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator (CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DeltaF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventy-seven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a non-radioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The DeltaF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.     

Single-sperm analysis for haplotype construction of de-novo paternal mutations: application to PGD for neurofibromatosis type 1

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the neurofibromin gene. Approximately, 50% of cases are caused by de-novo mutations. Even when the NF1 mutation is known, accuracy of PGD is highly enhanced by simultaneous analysis of linked markers. In a childless couple referred to PGD, the male carried a de-novo mutation, precluding the possibility of typing relatives to establish the mutation-associated haplotype. We developed a single-sperm haplotype analysis strategy to establish the haplotype linked to the NF1 mutation. METHODS: Spermatozoa from freshly ejaculated semen were used as a substrate for multiplex PCR on single sperm. RESULTS: In addition to the NF1 mutation, six informative polymorphic markers flanking the NF1 gene (D17S1294, D17S1849, D17S841, D17S975, NF1TG2 and NF1AC5) were linked to individual alleles in single sperm from the affected male. CONCLUSIONS: Single-sperm analysis established the haplotypes of both mutant and wild-type NF1 alleles and enabled the implementation of a PGD protocol using polymorphic marker analysis. This method is generally applicable to PGD for any disease in which the haplotype of paternal mutations cannot be determined by typing relatives.     

Analysis of linkage disequilibrium between different cystic fibrosis mutations and three intragenic microsatellites in the Italian population

Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVSl7bTA and IVSl7bCA, located in intron 17b and a CA repcat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and different CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all AF508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1IG→, which might have been originated more recently than δF508. © 1995 Wiley- Liss, Inc.     

Fcgamma receptor IIA genotype and susceptibility to P. aeruginosa infection in patients with cystic fibrosis

It has been suggested that genes other than CFTR could modulate the severity of lung disease in cystic fibrosis (CF). Neutrophil Fcgamma receptor II (FcgammaRII) is involved in host defense against microorganisms and in inflammatory response. We evaluated the association between genetic variability of this gene and both airway infection with Pseudomonas aeruginosa and severity of lung disease in patients with CF. We studied 167 Italian unrelated patients with CF and 50 control subjects. The distribution of FcgammaRIIA genotypes in CF patients was compared with that in control subjects and the different genotypes were related with the presence or absence of P. aeruginosa infection and markers of disease severity in CF patients. The distribution of FcgammaRIIA genotypes was not significantly different between CF patients and controls. We observed that in CF patients with the same CFTR genotype (DeltaF508/DeltaF508), those carrying the R allele of FcgammaRIIA had an increased risk of acquiring chronic P. aeruginosa infection (P=0.042, R.R.: 4.38; 95% CI: 1.17/22.4). Moreover, the frequency of R/R genotype in patients with chronic P. aeruginosa infection seems to be higher than that of control subjects and patients without chronic infection. The observation that CF patients carrying the R allele of FcgammaRIIA are at higher risk of acquiring chronic P. aeruginosa infection suggests that the FcgammaRII loci genetic variation is contributing to this infection susceptibility.     

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.     

Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.     

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.     

Cystic fibrosis in a southern Brazilian population: characteristics of 90% of the alleles

Cystic fibrosis (CF) is a genetic disease that frequently leads to death in infancy among Europeans and their descendants. The goals of the present study were to analyze the molecular aspects of CFTR gene characterizing mutations, their frequencies, and the haplotypes formed by four CFTR gene intragenic markers, IVS8-6(T)n, IVS8CA, IVS17bTA and IVS17bCA, in a southern Brazilian population of Caucasian origin. DNA samples from 56 non-related CF patients were analyzed using scanning techniques (single strand conformation polymorphism and denaturing gradient gel electrophoresis), restriction fragment length polymorphism and direct DNA sequencing to identify the mutations. Our results revealed a total of 25 different CF mutations representing nearly 90% of CF alleles, two being novel mutations. Microsatellite haplotypes were defined for CF and normal alleles. The mutational spectrum and the associated haplotypes described for the first time in this study should prove relevant for genetic counselling and CF population screening in Brazil. Moreover, our results suggest the presence of a major Mediterranean component in the contemporary Brazilian CF patient pool.     

Analysis of CA/GT microsatellite polymorphism in IVS8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a study of Italian CF families

Forty-six CF Italian patients and their parents were screened for a highly polymorphic microsatellite consisting of a variable number of CA/GT repeats in intron 8 of the CFTR gene. A strong degree of association was found between alleles 2 and 6 and the CF mutation delta F508. Moreover, considering the haplotypes at the closely linked locus D7S23 and the microsatellite's alleles, a strong linkage disequilibrium was again found for delta F508 and also for non-delta F508 CF chromosomes and the eight commonest haplotypes (B2, B6, C7, A6, A7, B7, D2 and D7). These data, compared with those described in the Spanish population, further support the common origin of the delta F508 mutation in Southern European populations.