Molecular analysis of the most prevalent mutations of the FANCA and FANCC genes in Brazilian patients with Fanconi anaemia

Fanconi anaemia (FA) is a recessive autosomal disease determined by mutations in genes of at least eleven complementation groups, with distinct distributions in different populations. As far as we know, there are no reports regarding the molecular characterisation of the disease in unselected FA patients in Brazil. OBECTIVE: This study aimed to investigate the most prevalent mutations of FANCA and FANCC genes in Brazilian patients with FA. METHODS: Genomic DNA obtained from 22 racially and ethnically diverse unrelated FA patients (mean age ± SD: 14.0 ± 7.8 years; 10 male, 12 female; 14 white, 8 black) was analysed by polymerase chain reaction and restriction site assays for identification of FANCA (D3788-3790) and FANCC (D322G, IVS4+4A ® T, W22X, L496R, R548X, Q13X, R185X, and L554P) gene mutations. RESULTS: Mutations in FANCA and FANCC genes were identified in 6 (27.3%) and 14 (63.6%) out of 22 patients, respectively. The disease could not be attributed to the tested mutations in the two remaining patients enrolled in the study (9.1%). The registry of the two most prevalent gene abnormalities (D3788-3790 and IVS4 + 4 ® T) revealed that they were present in 18.2% and 15.9% of the FA alleles, respectively. Additional FANCC gene mutations were found in the study, with the following prevalence: D322G (11.4%), W22X (9.1%), Q13X (2.3%), L554P (2.3%), and R548X (2.3%) of total FA alleles. CONCLUSION: These results suggest that mutations of FANCA and FANCC genes are the most prevalent mutations among FA patients in Brazil.


Introduction
Fanconi anaemia (FA) is an autosomal recessive disease characterised by a very high frequency of bone marrow failure and many other manifestations, including but not restricted to severe birth defects and marked predisposition to malignancies, especially acute myeloid leukaemia and, to a lesser extent, solid tumours (Young and Alter, 1994;Alter and Young, 1998;Alter, 2003).
FA cells exhibit spontaneous chromosomal instability and hypersensitivity to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C (Auerbach and Wolman, 1976) and the resulting increase in chromosome breakage provides the basis for a diagnostic test (Auerbach, 1993).
The relationship between the complementation group and mutation type and the clinical outcome of the patients with disease is controversial.Patients with FANCA and FANCG mutations appear to constitute a high risk group according to some authors (Faivre et al., 2000).In contrast, The International Fanconi Anaemia Register revealed a significantly earlier onset of bone marrow failure and poorer survival rates for complementation group C compared with groups A and G.Moreover, there was no significant difference in the time for haematologic or nonhaematologic neoplasm development between FA groups of patients (Kutler et al., 2003).
The ethnic origin of the Brazilian population is highly heterogeneous, consisting of indigenous Amerindians and immigrants from Europe, Africa, and Asia (Alves-Silva et al., 2000;Carvalho-Silva et al., 2001).
The ∆3788-3790 and IVS8-2A > G gene mutations were found in a group of Brazilian FA patients seen at a single bone marrow transplantation service, and were described as the most common FANCA and FANCG Brazilian mutations by the International Fanconi Anemia Registry (IFAR) (Levran et al., 1997;Auerbach et al., 2003).As far as we know, there are no reports regarding the molecular analysis of FA in unselected populations of our country.Therefore, this was the aim of the study presented herein.

Eligibility requirements
All unrelated patients with confirmed FA, attended at the Medical Genetic and the Haematology services of UNICAMP and Haemotherapy Centres of several Brazilian Universities, were considered as fully eligible for the present study.The diagnosis of the disease was based on clinical and laboratory data, including haematological analysis, bone marrow aspiration and biopsy, and was confirmed by DEB test.The study protocol was approved by the local Research Ethics Committee.

DEB test
The DEB test was performed at the Cytogenetics Laboratory of the Haematology and Haemotherapy Centre of the State University of Campinas, according to conven-tional methods (Rosendorff and Bernstein, 1988;Auerbach et al., 1989).

Molecular Analysis of FANCA and FANCC genes
Genomic DNA was obtained from peripheral blood of patients enrolled in the study using the salt/chloroform method (Müllenbach et al., 1989).FANCA and FANCC mutations were analysed by polymerase chain reaction (PCR) followed by restriction site assays.
FANCA and FANCC exonic sequences of interest were amplified by PCR using primers described by Levran et al. (1997) and Gibson et al. (1996).PCR was carried out in 25 µL reactions with 250 ng of genomic DNA, 10 ng/µL of each primer, 0.5 mM of each dNTP, and 1.5 units of Taq polymerase in a buffer containing 6.7 mM MgCl 2 .After initial denaturation at 94 °C for 5 min, samples were amplified for 30 denaturation cycles at 94 °C for 1 min, annealing at 51-60 °C, and extension at 72 °C for 1 min, followed by a final 5 min extension at 72 °C.
PCR products were then digested with 5-10 units of the appropriate restriction enzyme for a minimum of 2 h at 37 °C (50C for Bcl I), according to previously described techniques (Gibson et al., 1996;Levran et al., 1997) and analysed on horizontal 7% polyacrylamide gel.
The investigated gene mutations, restriction enzymes, and sizes of the normal and mutant gene fragments obtained from PCR and restriction site assays performed in the study are presented in Table 1.

Results
The clinical and laboratory features of 22 FA patients enrolled in the study are presented in Table 2. Their mean age ± SD was 14.0 ± 7.8 years (range: 2-33 years); 10 patients were male and 12 female, 14 were white and 8 black.Sixteen patients presented congenital abnormalities such as microphthalmia (8 cases), microcephaly (4 cases), abnormalities of the thumbs (5 cases), café-au-lait spots (4 cases), hyperpigmentation (4 cases), short stature (2 cases), genital abnormalities (1 case), and renal aplasia (1 case).Short stature was the only physical abnormality identified in FANCA patients.Variable degrees of peripheral cytopenia and positive DEB test were found in all patients enrolled in the study.Cases 1, 2, 16 and 22 are children of consanguineous parents (18%).
FA was attributed to ∆3788-3790 mutation of the FANCA gene in 6 out of 22 (27.3%)patients.The gene abnormality was present in 18.2% of FA alleles.Mutations of the FANCC gene were found in 14 out of 22 (63.6%)patients, with the following prevalence: 15.9% for IVS4+4A → T, 11.4% for ∆G322, 9.1% for W22X, 2.3% for Q13X, 2.3% for L554P, and 2.3% for R548X on FA alleles.The analysed mutations were not found in 2 out of 22 (9.1%) patients enrolled in the study.Figure 1 shows the analysis for IVS4+4A → T mutation.
Two pre-symptomatic patients with FA were identified in our familial studies (Cases 1 and 2).
The median age of the studied group, 14 years old, was higher than those obtained in previous reports, which was 7 years old (McMullin et al., 1991;Auerbach and Allen, 1994;Butturini et al., 1994), and may be attributed to the difficulties in achieving a reference service for FA diagnosis in the country.The congenital abnormalities were  more frequent and severe in FA determined by FANCC gene mutations, particularly in cases with IVS4+4 A → T mutations, in accordance with previous reports (Faivre et al., 2000).Interestingly, the disease was found in 8 (36.4%) patients of African origin in our group, in disagreement with the reports of Rosendorff et al. (1987) and MacDougall et al. (1990), in which the disease was rare in this race.
Mutations in the FANCC and FANCA genes accounted for approximately two-thirds and one-third of our cases.The disease was not determined by the tested mutations in only 9.1% of analysed cases.Therefore, FANCC gene mutations were the most prevalent in our group of patients (14 patients), in disagreement with previous reports that show to a higher prevalence of FANCA mutations in the majority of populations (Tischkowitz and Hodgson, 2003).Levran et al. (1997) and Auerbach et al. (2003) described the ∆3788-3790 and IVS8-2A > G as the most common Brazilian mutations of FANCA and FANCC genes.As far as we know, both studies included Brazilian patients from a single bone marrow transplantation service, and may have included predominantly patients with the most severe disease forms.In our sample there were both patients with slight and severe haematological manifestations.As we did not select patients for study, it is possible that mutations of the FANCC gene represent a particular characteristic of the Brazilian population.
The most prevalent mutation of FANCA gene in our group was ∆3788-3790, in accordance with the report by Levran et al. (1997).The second most common mutation was the IVS4+4 A → T of the FANCC gene (Yamashita et al., 1996;Gillio et al., 1997;Tamary et al., 2003).
In addition, the molecular analysis performed in our study was also important for early diagnosis of the disease, identification of pre-symptomatic and unaffected siblings with FA, as well as genetic counselling for the family.
Another fact that draws our attention is the high number of compound heterozygotes among FA patients.Since FA is considered a rare disease, a high rate of consanguineous parents and a larger number of homozygotes was expected, but not found in study.Our data suggest that FA may not be as rare as thought and taking into account its variable expressivity, many cases may be undiagnosed.
In conclusion, these results present preliminary evidence that FANCA and FANCC gene mutations are the most prevalent mutations among Brazilian FA patients.However, a large study concerning the molecular analysis of the Brazilian FA patients from different areas of the country should be carried out to clarify this issue.

Figure 1 -
Figure 1 -Polymerase chain reaction and restriction site assays for detection of IVS4+4A → T mutation of the FANCC gene in Fanconi anaemia.Ethidium-bromide stained 7% polyacrylamide gel showing fragments of 131 bp and 108 bp corresponding to the mutant and normal alleles, respectively.Lane 1 shows the DNA size marker leader 100 bp.Lane 2 shows the result from an individual with homozygous IVS4+4A → T mutation used as control, and lanes 3 to 5 show the results from heterozygous individuals.

Table 1 -
Fragments observed after digestion with respective restriction enzymes for the distinct analysed mutations in Fanconi anaemia patients.

Table 2 -
Clinical and molecular features of 22 patients with Fanconi anaemia.