Fanconi anemia (FA) (MIM 227650) is a genetically heterogenous chromosomal instability syndrome associated with multiple congenital abnormalities, progressive pancytopenia, and predisposition to both hematologic malignancies and solid tumors (Morgan et al., 2005). It is a very rare (1–5/million) autosomal recessive or X-linked (FA-B group) disease (Kutler and Auerbach, 2004; Callén et al., 2005). FA is caused by a genetic defect in a cluster of proteins responsible for DNA repair (D’Andrea, 2010).
FA cells are hypersensitive to DNA crosslinking agents, such as diepoxybutane (DEB) and mitomycin C, and this provides a valuable laboratory test for supporting the clinical diagnosis (Auerbach, 2009). About 60–75% of FA patients have variable congenital defects that may affect skeletal morphogenesis as well as any of the major organ systems. Patients often develop pancytopenia during the first few years of life, and complications resulting from bone marrow failure are the major causes of morbidity and mortality (Bagby, 2003).
FA is caused by biallelic mutations in one of at least 15 FA genes that are responsible for the known FA complementation groups [A, B, C, D1 (BRCA2), D2, E, F, G, I, J (BRIP1), L, M, N (PALB2), O (RAD51C), and P (SLX4)] (Hucl and Gallmeier, 2011). FA-A is the most frequent complementation group and represents approximately two-thirds of the patients in the majority of countries, accounting for 60–66% of FA cases. FA-C and FA-G are less frequent, each accounting for 10–15%, respectively, whereas the other groups are rare (Kennedy and D’Andrea, 2005).
The FA-A gene has an open reading frame of 4365 bp encoding a protein of 1455 amino acids. It contains 43 exons and spans 80 kb on chromosome 16q24.3 between microsatellites D16S302 and D16S303 (Ianzano et al., 1997). A previous study including three consanguineous Egyptian families with affected sibs established a panel of families classified as FA-A by complementation analysis, concluding an evidence for linkage of the FA-A to microsatellite markers on chromosome 16q24.3 (Pronk et al., 1995). Strong evidence of allelic association with the disease was detected with the marker D16S303 in the Afrikaner population of South Africa, indicating the presence of a founder effect (Tipping et al., 2001).
A very large and heterogenous spectrum of mutations has been identified in the FANCA gene, with more than 200 different mutations described so far (Tipping et al., 2001; Bouchlaka et al., 2003; Callén et al., 2004; Castella et al., 2011; FA mutation database, http://www.rockefeller.edu/fanconi/mutate). Only two mutations are relatively common (c.1115_1118delTTGG and c.3788_3790delTCT), accounting for 2 and 5% of the FANCA alleles, respectively (Levran et al., 1997).
Levran et al. (2005) in his study on 181 FA patients selected through the International Fanconi Anemia Registry (IFAR) found that about two-thirds of the FA-A gene mutations are distributed in exons 13, 27, 29, 34, and 38. Exon 43 deletion was also reported in several studies (Koc et al., 1999; Tamary et al., 2000).
After reviewing the literature and to the best of our knowledge, FA mutations have not yet been investigated in the Egyptian population, which is characterized by a heterogenous ethnic background and a high rate of consanguinity. The aim of this work was to screen common mutations previously reported in the international literature within exons 27, 34, 38, and 43 of the FANCA gene, with clinical and cytogenetic evaluation.
Patients and methods
Twenty-four Egyptian FA cases were recruited from the outpatient clinic of the Clinical Genetics Department, NRC, Egypt. Informed consent was obtained from their parents following the guidelines of the ethics committees of the NRC. Patients were subjected to thorough clinical evaluation, pedigree analysis, anthropometric assessment, and routine laboratory investigations. All patients had positive chromosomal breakage studies with DEB, confirming the diagnosis of FA as described by Auerbach (2003). Ten healthy unrelated individuals of matching age and sex were included as the control group.
DNA was extracted from peripheral blood leukocytes of patients and controls by means of the salting out technique (Miller et al., 1988).
Amplification of FANCA gene exons
Genomic DNA from both patients and controls was amplified using previously described primers for amplification of the studied regions (Levran et al., 1997).
Amplification reactions were carried out in a 50 µl mixture containing 150–300 ng genomic DNA, standard PCR buffer, 0.2 mmol/l of each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP), 0.2 µmol/l of each primer, and 0.75 U Taq polymerase. PCR conditions were as follows: initial incubation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 min, annealing at 55°C for 30 min, and primer extension at 72°C for 30 min, followed by final extension at 72°C for 5 min. Amplification was performed in a DNA T1 Thermocycler (Biometra, Goettingen, Germany). The PCR products of amplified exons 27, 34, and 43 (284, 300, and 223 bp, respectively) were run on 2% agarose gel and stained with ethidium bromide for visualization under ultraviolet light.
The amplified exons were subjected to direct sequencing in both directions. Samples were run on 1.5% agarose gel and bands corresponding to the predicted size were cut using a gel extraction kit following the manufacturer’s protocol (QIA quick columns; Qiagen, Foster City, California, USA). Purified samples were subjected to cycle sequencing using a Big Dye Terminator v3.1 Kit (Applied Biosystem, Munich, Germany) and injected into an ABI 3100 Genetic Analyzer (Applied Biosystems, Munich, Germany). The generated sequencing results were blasted on the reference gene on the NCBI gene bank.
A modified mismatch PCR assay was performed for the c.3788_3790delTCT in exon 38 in the reverse orientation to abolish the single MboII site in the normal allele. Restriction analysis using the MboII enzyme was performed following the manufacturer’s instructions (Fermentas, Europe). Digested products were run on 3% agarose gel after staining with ethidium bromide for further visualization under ultraviolet light (Magdalena et al., 2005).
The amplification of a 160 bp fragment in exon 38 of the FANCA gene, followed by digestion with MboII, yielded one fragment of 130 bp and undetected 30 bp in the wild-type sequence. In the presence of 3788–3790del sequence, there will be abolishment of the restriction site.
The study included 24 FA patients (14 female and 10 male) of unrelated consanguineous pedigrees from 22 families. Clinical findings of the FA patients are listed in Table 1. The age of the patients at the time of examination ranged from 4 months to 16 years (mean age 9 years). The age at onset of anemia ranged from 4 months to 12 years (mean age 7 years). Low birth weight was found in 8% of the patients.
All patients had pancytopenia, with average values of 7.7 g/dl for hemoglobin, 2.6×106/µl for RBC, 4×103/µl for WBC, and 45×103/µl for platelet count. Induction of breakage by DEB revealed a large variation in the cellular sensitivities and consequently a wide range of breakages/cell (3.4–12.5, mean 6.3). Hematological and cytogenetic data are shown in Table 2.
Screening for mutations in exons 27, 34, and 43 revealed neither homozygous nor heterozygous mutations in the studied FA patients (Figs 1–3).
Screening for 3788–3790del mutation in exon 38 revealed neither homozygous nor heterozygous mutations in our studied FA patients (Fig. 4).
FA is one of the genetic disorders characterized by increased chromosomal breakage and developmental defects. Hypersensitivity to DEB is a key marker of FA patients, distinguishing them from those with other breakage syndromes (Auerbach et al., 1989; Deakyne and Mazin, 2011).
After reviewing the literature and to the best of our knowledge, FA mutations have not yet been investigated in the Egyptian population.
Although the reported incidence of FA is 1–5/million, the situation is different in closed communities. Carrier incidence was as high as1 : 22 000 in the Afrikaner population of South Africa (Pronk et al., 1995). Higher incidences were reported among Ashkenazi Jews with 1/100 carriers for the mutation FA-C, c.711+4A>T (Kutler and Auerbach, 2004); the highest reported incidence was among Spanish gypsies with 1/64–71carriers for the FANCA mutation (295C>T) (Callén et al., 2005).
Parental consanguinity was found in 100% of our studied cases, higher than that reported in previous studies. Temtamy et al. (2007) reported a high incidence of consanguinity (97%) among previously studied Egyptian FA patients. A Turkish study reported positive consanguinity in 79% of cases (Altay et al., 1997).
Clinical variability in the severity of the disease may be due to interfamilial and intrafamilial variability. Affected sibs were usually found to suffer less anomalies than the proband of the same sibship (Kwee and Kuyt, 1989). However, we could not confirm such a finding among our studied cases.
Studies of other hematological disorders such as β-thalassemia detected many common mutations, especially in the Mediterranean region, shared among Egyptians (El-Kamah et al., 2009).
On the basis of the relatively high frequency of FANCA in some neighboring Mediterranean countries such as Italy, Spain, Tunisia, and Palestine, as well as among Ashkenazi Jewish patients (Savino et al., 1997; Tamary et al., 2000, 2004; Bouchlaka et al., 2003; Callén et al., 2004), and on the basis of the previous Egyptian study by Pronk et al. (1995) that included a limited number of Egyptian patients, it was assumed that our studied cases might likely belong to complementation A group.
A very large and heterogenous spectrum of mutations has been identified in the FANCA gene, with more than 200 different mutations described so far, including all types of possible point mutations such as frameshift mutation, small insertions or deletions, splicing defect, and nucleotide substitutions (Gregory et al., 2001; Tipping et al., 2001; Callén et al., 2004; FA mutation database, http://www.rockefeller.edu/fanconi/mutate/).
Levran et al. (2005) in his study on 181 FA patients selected from the IFAR found that about two-thirds of the FA-A gene mutations are distributed in exons 13, 27, 29, 34, and 38.
The studied exons were selected on the basis of ethno-geographical backgrounds. Exon 27 was investigated, as the 2574CG (S858R) mutation was detected in 30% of unrelated FA Israelinon-Ashkenazi Jews (Tamary et al., 2000).
Similarly, a single-nucleotide deletion in exon 34 was reported in six different patients in a study including 46 unrelated Afrikaners of South Africa (Tipping et al., 2001). Moreover, the IFAR reported four different missense mutations in exon 34 among 97 patients of different ethnic origins (Levran et al., 1997). Also, Gille et al. (2012) reported another three different mutations in the same exon in 17 instances among 54 FA patients from different ethnicities.
Considering the long historical admixture between Egyptian and Turkish populations, and the relative prevalence of exon 43 deletions among the latter, its sequencing was included in our study. Exon 43 deletion was detected in patients from Turkey and Iran from among 40 patients studied by the European Concerted Action on FA Research (EUFAR) (Wijker et al., 1999), and in another study on Turkish patients by Koc et al. (1999). Exon 43 mutations were also detected in two patients among unrelated 13 studied FA Israeli Jews (Tamary et al., 2000).
Taking into consideration the migratory flows, the possibility of common ancestors, and/or founder effects resulting in sharing the same mutation between African and European patients as in previous reports of other diseases such as β-thalassemia (El-Kamah et al., 2009), we screened the coding sequences of FANCA exons 27, 34, and 43 among our FA patients. No mutations were reported within the studied exons, similar to other studies conducted on FA-A patients from Italy, Tunisia, Japan, and Spain (Bouchlaka et al., 2003; Yagasaki et al., 2004; Castella et al., 2011).
A mismatch PCR assay for the c.3788–3790del mutation of the FANCA gene was also performed as it constituted 20.7% of the mutated alleles in a study on 67 Spanish (Mediterranean) FA-A patients (Castella et al., 2011). The aforementioned mutation was found to be present in up to 51% in a study by Levran et al. (2005) and in 32% of Brazilian FA patients’ alleles as reported by Magdalena et al. (2005). The IFAR reported the 3788–3790del mutation in 10% of 350 non-FANCC patients (Levran et al., 1997).
In contrast to previous studies, c.3788–3790del mutation in exon 38 of the FANCA gene could not be detected in any of our studied patients.
Although the c.3788_3790delTCT and c.1115_1118delTTGG mutations are relatively common, representing 5 and 2% of reported FANCA alleles, respectively, within several unrelated patients descending from different populations (Levran et al., 1997), there is no reported evidence for common mutations in any particular population except in closed communities such as Gypsies and Ashkenazi Jews (Tamary et al., 2000). Heterogeneity of the FANCA gene mutations could be attributed to variations in geographic and ethnic origin. Heterogeneity was even found within populations, with 12 different mutations reported in Italian (Savino et al., 1997) and eight in German patients (Wijker et al., 1999).
Further studies are needed to explain the clinical discordance and define the spectrum of Egyptian FA mutations, thus probably expanding the understanding of the molecular pathogenesis in FA patients.
In conclusion, screening for c.3788_3790delTCT mutation in exon 38 in the FANCA gene as well as for any reported or new mutations in exons 27, 34, and 43 yielded no detectable mutations among Egyptian patients, and further studies are highly recommended.
Conflicts of interest
There are no conflicts of interest.
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