ORIGINAL RESEARCH published: 19 February 2021 doi: 10.3389/fgene.2021.610050 FANCA Gene Mutations in North African Fanconi Anemia Patients Abir Ben Haj Ali 1,2, Olfa Messaoud 2*, Sahar Elouej 2,3, Faten Talmoudi 1,2, Wiem Ayed 1,2, Fethi Mellouli 4, Monia Ouederni 4, Sondes Hadiji 5, Annachiara De Sandre-Giovannoli 3, Valérie Delague 3, Nicolas Lévy 3, Massimo Bogliolo 6,7, Jordi Surrallés 6,7, Sonia Abdelhak 2 and Ahlem Amouri 1,2 1 Department of Histology and Cytogenetics, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia, 2 Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia, 3 INSERM, MMG, UMR 1251, Aix Marseille University, Marseille, France, 4 Department of Peadiatric Immuno-Haematology, National Bone Marrow Transplantation, Tunis, Tunisia, 5 Haematology Department, Hedi Chaker Hospital, University of Sfax, Sfax, Tunisia, 6 Research Institute IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain, 7 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain Edited by: Dan Koboldt, Nationwide Children’s Hospital, Populations in North Africa (NA) are characterized by a high rate of consanguinity. United States Consequently, the proportion of founder mutations might be higher than expected and Reviewed by: Olivia J. Veatch, could be a major cause for the high prevalence of recessive genetic disorders like Fanconi University of Kansas Medical Center, anemia (FA). We report clinical, cytogenetic, and molecular characterization of FANCA in United States 29 North African FA patients from Tunisia, Libya, and Algeria. Cytogenetic tests revealed Tommaso Pippucci, Sant’Orsola-Malpighi Polyclinic, Italy high rates of spontaneous chromosome breakages for all patients except two of them. Settara Chandra, FANCA molecular analysis was performed using three different molecular approaches National Institutes of Health (NIH), United States which allowed us to identify causal mutations as homozygous or compound heterozygous *Correspondence: forms. It included a nonsense mutation (c.2749C > T; p.Arg917Ter), one reported missense Olfa Messaoud mutation (c.1304G > A; p.Arg435His), a novel missense variant (c.1258G > A; p.Asp409Glu), olfa.messaoud@pasteur.tn and the FANCA most common reported mutation (c.3788_3790delTCT; p.Phe1263del). Specialty section: Furthermore, three founder mutations were identified in 86.7% of the 22 Tunisian patients: This article was submitted to (1) a deletion of exon 15, in 36.4% patients (8/22); (2), a deletion of exons 4 and 5 in 23% Genetics of Common and Rare (5/22) and (3) an intronic mutation c.2222 + 166G > A, in 27.3% (6/22). Despite the relatively Diseases, a section of the journal small number of patients studied, our results depict the mutational landscape of FA among Frontiers in Genetics NA populations and it should be taken into consideration for appropriate genetic counseling. Received: 24 September 2020 Accepted: 22 January 2021 Keywords: consanguinity, founder mutations, North Africa, molecular diagnosis, Fanconi anemia, FANCA Published: 19 February 2021 Citation: Ben Haj Ali A, Messaoud O, Elouej S, INTRODUCTION Talmoudi F, Ayed W, Mellouli F, Ouederni M, Hadiji S, De Sandre- Fanconi Anemia (FA) is a rare, inherited disorder clinically characterized by various congenital Giovannoli A, Delague V, Lévy N, abnormalities and a predisposition to develop malignancies, especially acute myeloid leukemia, Bogliolo M, Surrallés J, and other cancers (Kutler et al., 2003). The prognosis of the disease is characterized by an evolution Abdelhak S and Amouri A (2021) FANCA Gene Mutations in North toward progressive bone marrow failure (BMF). Without treatment, FA is most often lethal before African Fanconi Anemia Patients. the end of the second decade of life. Cells from FA patients present high levels of spontaneous Front. Genet. 12:610050. chromosomal breakages (Schroeder and Kurth, 1971) that increase dramatically by clastogens such doi: 10.3389/fgene.2021.610050 as Mitomycin C (MMC; Cervenka et al., 1981) and Diepoxybutane (DEB; Auerbach, 2009). Frontiers in Genetics | www.frontiersin.org 1 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients FA is genetically heterogeneous, with at least 22 genes number of patients. Under these circumstances, the conduct, encoding for a group of proteins that cooperate in a unique the analysis and the interpretation of studies on this disease FA/BRCA DNA repair pathway (Nalepa and Clapp, 2018). may sometimes be limited to different degrees by its prevalence. Indeed, the products of these genes are involved in the Also, we have to take into consideration the severe health preservation of genome integrity in response to physiological status of the patients in particular in regard to their extremely stress or to genotoxic agents (Kottemann and Smogorzewska, severe anemia, especially that we give the priority to diagnosis 2013; Ceccaldi et al., 2016). All FA genes are located on (cytogenetic study), thus most of the patients were investigated autosomal chromosomes except FANCB which is X-linked cytogenetically but no sufficient material was available to (Kitao and Takata, 2011). FANCA, FANCC, and FANCG conduct molecular investigation. Furthermore, most of the complementation groups include, respectively, 60, 15, and 10% cases are pediatric cases and we try to avoid any invasive of all FA patients from the USA, whereas mutations in other tests including recurrent blood sampling. Therefore, we were FA genes occur less frequently (<1–4%; Neveling et al., 2009). not able to collect additional samples for DNA investigation. Several reports indicated a higher FA prevalence in some Finally, transplanted and recently transfused patients were ethnic groups like the Spanish Romani (Callén et al., 2005; excluded from the study. As a direct outcome of these, the Castella et al., 2011), the Afrikaner population of South Africa present study was conducted on 22 newly collected families, (Tipping et al., 2001), Ashkenazi Jewish (Whitney et al., 1993), including 29 FA patients who agreed to participate in the and Tunisian populations (Amouri et al., 2014) due to founder molecular genetic testing. effects and isolation. Thus, information on a patient’s ethnic This study was conducted according to the principles of origin may provide evidence for a pathogenic mutation that the Declaration of Helsinki and was approved by the biomedical is likely to be causal (Faivre et al., 2000; Tipping et al., 2001; ethics committee (2017/14/I/LR11IPT05/V0) of Pasteur Institute. Kutler and Auerbach, 2004; Castella et al., 2011). In Tunisia, Informed consent of the legal patients’ representatives were FA-A complementation group is the most prevalent, with a obtained. Peripheral blood samples were collected from the 94% frequency among the FA patients (Bouchlaka et al., 29 FA patients, their parents, and their siblings when available. 2003). A common haplotype shared by FA patients mostly Genomic DNA was extracted using the standard salting-out originating from the South of Tunisia was associated with method (Miller et al., 1988) or the QIAamp DNA mini kit the deletion of exon 15 in FANCA (Bouchlaka et al., 2003; (Qiagen). Amouri et al., 2014). Another founder mutation in the same Clinical examination and familial information details were gene (c.890_893del in exon 10) was identified in Tunisian recorded. Clinical data were obtained based on history, physical Jewish FA patients (Tamary et al., 2000). In neighboring examinations, and reviewing of medical records. countries, Algeria and Libya, no FA molecular studies have Patients were initially screened using PCR to check the been conducted so far. However, several studies on other homozygous deletion of the exon 15 in the FANCA gene genetic conditions have shown that patients originating from (LRG_495) which is a common founder mutation in Southern North Africa (NA) shared many common founder mutations Tunisia (Amouri et al., 2014). (Richard et al., 2008; Messaoud et al., 2010; Bensenouci et al., The 43 coding exons of FANCA gene were amplified using 2016). According to more than 20 years’ experience on the PCR. Intronic oligonucleotides primer pairs were generated investigation of genetic diseases, identification of founder using Primer 31 and in silico PCR (UCSC).2 Direct sequencing mutations that are shared between Tunisia and other NA of PCR products was performed on an ABI prism 3,500 DNA countries even with a single patient were of great impact Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), for launching molecular diagnosis in their respective countries. using the ABI Prism Big Dye Terminator v3.1 Cycle Sequencing This was the case for Xeroderma pigmentosum (Senhaji et al., Ready Reaction Kit (Applied Biosystems). Sequences were then 2013) and for several other diseases (Romdhane et al., 2012; analyzed using Bioedit software and compared to the wild Charoute et al., 2015). sequence using BLAST program available in the NCBI server. The present study aims to investigate the FANCA mutational Multiplex ligation-dependent probe amplification (MLPA) spectrum and to develop a strategy based on molecular was performed to detect large deletions or duplications within approaches applicable to routine clinical use for FA patients the FANCA gene using a SALSA P031-A2/P032 kit (MRC-Holland originating from NA. BV, Amsterdam, The Netherlands) for (F068/15, TFA27, SFA15, F076/15, F031/15, F032/15, and F050/15) patients. PCR products were analyzed using ABI PRISM 3130 Genetic analyzer MATERIALS AND METHODS (Applied Biosystems). For (TFA27, SFA15, F059/15, F058/15, CFA4, CFA3, A total of 87 families with 113 FA patients have been addressed F033/15, and TFA23) patients, available family members to our department in Institut Pasteur de Tunis (IPT) for were genotyped with three microsatellites flanking the FANCA confirmation of FA based on the standard test involving gene (D16S3026‐ D16S3121‐ D16S3407) as previously reported MMC-induced chromosomal breakage analysis. The cytogenetic (Bouchlaka et al., 2003). and molecular status of 30 families with 46 patients have been previously described (Talmoudi et al., 2013; Amouri et al., 2014). http://primer3.ut.ee/ 1 Since FA is a rare disease, it was difficult to recruit a large http://genome.ucsc.edu/cgi-bin/hgPcr 2 Frontiers in Genetics | www.frontiersin.org 2 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients Targeted gene sequencing (TGS) has been conducted for originating from Southern Tunisia, six from Central Tunisia (F072/09, SFA2, GF06/09, and GF78/09) patients. Custom design and two from Northern Tunisia. of the genes’ panel was performed using SureDesign (Agilent Most of patients presented typical clinical features of FA Technologies Inc.); probes were generated to cover the exons including: skin pigmentation abnormalities (86%), microcephaly and 15 bp of the surrounding intronic sequences of a total (52%), triangular face (31%), and skeletal malformations (34%). of 87 candidate genes known to be involved in DNA repair Other clinical abnormalities, such as ear abnormalities and disorders. Library preparation for NGS was performed using hearing problems were presented in two patients (F032/16 and Agilent’s HaloPlexHS (high sensitivity) workflow as a target F076/15). enrichment method. Amplicon libraries were prepared from Chromosomal breakage analysis was successful in all patients patients’ genomic DNA using the HaloPlexHS PCR target except for two patients (GF78/09 and SFA2), for whom blood enrichment system dedicated to Ion Torrent PGM according cell culture showed no growth. For these two patients, results to the manufacturer’s recommendations. Massively parallel of the molecular analysis confirmed the FA diagnosis. For the sequencing was performed on an Ion Torrent PGM (Thermo others, cytogenetic results revealed a high frequency of Fisher Scientific). In our analysis we used the Ion Torrent chromosomal breakages (>3 breaks/cell in MMC treated, up Suite Software v.4 to process our data and we configured the to 0.2 breaks/cell in untreated) in 27 patients when compared settings to reduce the total number of false positive indels to controls (0.1 break/cell in MMC treated, up to 0.03 breaks/ caused by the high frequency of homopolymer sequencing cell in untreated). errors (min_cov_each_strand: 0, min_variant_score: 10, min_ The molecular approach allowed us to identify seven different allele_freq: 0.1, snp_min_coverage: 6 snp and indel; strand_bias: pathogenic variants at homozygous and heterozygous states. 0.98 snp and 0.85 indel). In addition, we visually evaluated These variants include large deletions of one or two exons the read depth and the percentage of mutated reads for each (deletion of exon 15, deletion of exons 4 and 5) respectively, variant using the Integrative Genomic Viewer (IGV). Finally, one nonsense mutation (c.2749C  >  T; p.Arg917Ter) in exon validation by MLPA was made to confirm the identified indels. 28, a small deletion (c.3788_3790del; p.Phe1263del) in exon Data were analyzed using the in-house VarAft software version 38, one likely pathogenic missense variant (c.1304G   >   A; 2.5, which is available online.3 We prioritized rare functional p.Arg435His) in exon 14 that was already associated to FA variants (missense, nonsense, splice site variants, and indels) (Moghrabi et al., 2009), a previously reported intronic mutation and excluded variants with a Minor Allele Frequency c.2222   +   166G   >   A (Bouchlaka et al., 2003) in intron 24, and (MAF)  >  0.01 in dbSNP137 and 138, in the Exome Variant a novel missense variant of uncertain significance (c.1258G  >  A; Server,4 1000 Genomes Project,5 or Exome Aggregation p.Asp409Glu) in exon 14. For variant c.1258G  >  A; p.Asp409Glu, Consortium database (ExAC), Cambridge, MA.6 A number of no functional study, nor cDNA analysis has been performed, online tools were used to predict the functional impact and however, the reported minor allele frequency (MAF) in GnomAD pathogenicity of the variants such as MutationTaster,7 PolyPhen,8 exomes (0.00000796) and GnomAD Genomes (0.0000319) and SIFT,9 I-mutant,10 and CADD.11 in silico evaluations with predictive algorithms (SIFT, POLYPHEN2, Align GVGD) indicate its possible pathogenicity. The last intronic mutation was inherited at a homozygous RESULTS state in all FA patients originating from Kairouan (Central Tunisia). Furthermore, all these patients shared the same In this study, we report on the clinical, cytogenetic, and haplotype (206-81-200; Figure 1). molecular investigation of 22 families including 29 FA patients. All patients from Sidi Bouzid and Gafsa were homozygous Our cohort is composed of 8 girls and 21 boys (sex ratio = 2.625) for the founder mutation, deletion of exon 15, except for one aged from 1 to 28 years. Inbreeding examination showed that patient originating from Gafsa (GF14/12) and another Libyan 25 patients (86.20%) were born from consanguineous unions. patient (F075/03) who were both homozygous for the novel Eight families are multiplex (having more than one affected missense variant c.1258G  >  A; p.Asp409Glu. child). Geographical distribution showed that five patients are Haplotype findings showed that the patient (SFA15) shares from Libya, one from Algeria, one Algerian/Tunisian patient the same homozygous haplotype described to be associated (father: from Algeria; mother: from Tunisia) and 22 are Tunisians. with the founder deletion of exon 15 in Tunisian patients The latest group (Tunisian patients) is composed of 14 patients (Amouri et al., 2014). On the other hand, the same haplotype was identified at a heterozygous state in the Algerian patient (TFA27) carrying the deletion in one allele. This patient is http://varaft.eu/index.php 3 probably compound heterozygous. These results suggest a http://evs.gs.washington.edu/EVS/ 4 common ancestor between Tunisian, Libyan, and Algerian FA http://www.1000genomes.org/ 5 patients bearing the exon 15 deletion but do not exclude the http://exac.broadinstitute.org 6 hypothesis of another ancestor, especially for Algerians http://www.mutationtaster.org/ 7 (Figure 2). http://genetics.bwh.harvard.edu/pph2/ 8 http://sift.bii.a-star.edu.sg/ 9 Deletion of exons 4 and 5 has been identified in five patients 10 http://folding.biofold.org/i-mutant/i-mutant2.0.html originating from Sfax. Four of them belong to two related 11 https://cadd.gs.washington.edu families (family 10 and family 11) and were homozygous for Frontiers in Genetics | www.frontiersin.org 3 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients FIGURE 1 | Pedigrees of FA nuclear families originating from Tunisian center (Kairouan) showing genotyping results of three microsatellites markers flanking FANCA gene. The presence of a common haplotype in all patients indicates a founder effect. FIGURE 2 | Haplotype analysis for a Libyan SFA15 and an Algerian TFA27 patients. The Libyan patient SFA15 carries homozygously the founder haplotype (226- 95-218) already described among Tunisian patients as associated with exon 15 deletion. The Algerian patient TFA27 carries both the founder haplotype associated with exon 15 deletion in the heterozygous state and another haplotype (230-99-216). this large deletion. The other patient (GF06/09) was heterozygous c.2222  +  166G > A are founder mutations in Sidi Bouzid/Gafsa, and the second mutation is still unknown. Sfax, and Kairouan, respectively (Figure 3). Our findings showed that deletion of exon 15, the large A summary of genealogical, clinical, cytogenetic, and molecular deletion of exons 4 and 5, and the intronic mutation characteristics of the 29 studied patients is presented in Table 1. Frontiers in Genetics | www.frontiersin.org 4 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients B A FIGURE 3 | (A) Geographic distribution of mutations in the FANCA gene among Algerian and Libyan patients; (B) Geographic distribution of mutations in the FANCA gene including three founder mutations among Tunisian patients: c.2222  +  166G  >  A in Kairouan (Central Tunisia), deletion of exon 15 in Sidi Bouzid (Southern Tunisia) and Gafsa (Southern Tunisia), and deletion of exons 4 and 5 in Sfax (Southern Tunisia). DISCUSSION Clinical heterogeneity within the same family or between patients having the same mutation was also observed in our This study has been carried out with 29 FA patients. Their study. This could be explained by environmental, genetic, and/ clinical features vary from one patient to another, but the or epigenetic factors (Koc et al., 1999). It should be noted common characteristics were: short stature, microcephaly, that there is little genotype–phenotype correlation, apart from skin pigmentation, and skeletal abnormalities. Clinically, the greater severity of group D2 and the association of groups we found that facial dysmorphia was a common feature in D1/BRCA2 and N with the occurrence of multiple and very the majority of our patients. This could be related to the early cancers (Mialou, 2011). predominance of the FANCA gene. Indeed, a correlation In addition, a few previous publications have examined the study between the phenotype of FA patients and the different correlations between the types of specific FANCA mutations complementation groups revealed a significantly higher and phenotype. In fact, a Spanish study showed that no frequency of facial dysmorphia in patients from groups A, association was found between the type of FANCA mutations G, and C (Faivre et al., 2000). Similarly, congenital and hematological diseases or somatic malformations (Castella malformations (skeletal and skin) were observed in most et al., 2011). The discrepancies observed between these studies patients. This is not surprising since a previous study conducted may reflect specific characteristics of the population, making on patients with two founder mutations (deletion of exon it difficult to rely on the Fanconi group or the type of mutation 15 and the intronic mutation) showed that malformations to define the risk of disease complications. were present in 96% of cases and consisted mainly of skeletal Our results at the clinical and cytogenetic level showed the malformations. Skin pigmentation abnormalities were present absence of any correlation between the severity of the disease in 92% of cases (Bouchlaka et al., 2003). In addition, an and the level of cellular sensitivity to MMC which fits with epidemio-clinical study carried out on Tunisian FA patients the observations reported in the literature (Castella et al., 2011; showed that skin, head, and skeletal abnormalities were Zen et al., 2011; Talmoudi et al., 2013). Therefore, the cytogenetic common among these patients (90, 71, and 51%), respectively approach can be considered as an important diagnostic tool (Hadiji Mseddi et al., 2012). for aplastic anemia, especially for asymptomatic cases for whom Frontiers in Genetics | www.frontiersin.org 5 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients TABLE 1 | Clinical, cytogenetic, and genetic features in North African Fanconi anemia patients. Family Patient Sex/Age Parents Origin Clinical manifestations Percentage of Type of mutations Zygosity Detection number code (years) relatedness chromosomal methods instability (CI %) -Hyperpigmentation (c.2749C > T; Algeria/Tunisia ‐Microcephaly Family 1 F068/15 F/10 No 100% p.Arg917Ter) CH MLPA/DS (Kef) ‐Thrombocytopenia Deletion of exon 15 ‐Anomalies of the thumb ‐Microcephaly Family 2 TFA27 M/1 2nd degree Algeria 70% Deletion of exon 15 Hz MLPA/DS -Hyperpigmentation ‐Thrombocytopenia (c.2749C > T; F031/16 F/5 100% Hz MLPA/DS ‐Café au lait spots p.Arg917Ter) Family 3 No Libya -Ear abnormalities (c.2749C > T; F032/16 M/6 -An extra thumb 100% Hz MLPA/DS p.Arg917Ter) -Café au lait spots -Microcephaly (c.1258G > A; Family 4 F075/03 M/8 1st degree Libya 90% Hm DS -Hyperpigmentation p.(Asp409Glu)) -Thrombocytopenia -Ecchymotic tasks No growth of (c.1304G > A; Family 5 GF78/09 M/2 1st degree Libya ‐A triangular facies Hm TGS blood cell culture p.Arg435His) -Red hair -Hypoplasia of the right thumb ‐Microcephaly Family 6 SFA15 M/11 2nd degree Libya ‐Thrombocytopenia 100% Deletion of exon 15 Hm MLPA/DS ‐Anomalies of the thumb ‐A triangular facies GF09/09 F/28 98% North of Tunisia -Hyperpigmentation (c.3788_3790delTCT; Family 7 1st degree ‐A triangular facies Hm DS GF27/09 F/17 (Mateur) 100% p.Phe1263del). ‐Café au lait spots South of Tunisia ‐Microcephaly (c.1258G > A; Family 8 GF14/12 M/5 No 100% Hm DS (Gafsa) ‐Café au lait spots p.(Asp409Glu)) South of Tunisia -Facial dysmorphism Deletion of exons 4 Family 9 GF06/09 M/19 1st degree 90% Hz TGS (Sfax) -Café au lait spots and 5 ‐Facial dysmorphism F072/09 F/8 -Red hair 100% ‐Hyperpigmentation -Red hair South of Tunisia -Facial dysmorphism Deletions of exons 4 Family 10 1st degree Hm TGS (Sfax) ‐Anomalies of the thumb No growth of and 5 SFA2 F/7 -Café au lait spots blood cell culture -Hyperpigmentation -Short stature 1st degree South of Tunisia -Café au lait spots Deletions of exons 4 Hm MLPA/DS (Sfax) -Hyperpigmentation and 5 -Microcephaly F076/15 M/19 83.6% -Triangular facies Family 11 ‐Microphthalmia ‐Microtia ear F050/15 F/6 -Toe abnormalities 100% -Triangular facies -Red hair Family 12 F073/15 M/10 1st degree South of Tunisia -Triangular facies 100% Deletion of exon 15 Hm DS (Gafsa) -Café au lait spots South of Tunisia -Microcephaly Family 13 F022/17 M/10 1st degree 100% Deletion of exon 15 Hm DS (Sidi Bouzid) ‐Hyperpigmentation South of Tunisia -Café au lait spots Family 14 F008/17 M/12 1st degree 100% Deletion of exon 15 Hm DS (Gafsa) -Pre-axial polydactyly (Continued) Frontiers in Genetics | www.frontiersin.org 6 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients TABLE 1 | Continued Family Patient Sex/Age Parents Origin Clinical manifestations Percentage of Type of mutations Zygosity Detection number code (years) relatedness chromosomal methods instability (CI %) -Microcephaly F492– M/11 -Triangular facies 100% 1/2016 South of Tunisia ‐ Hyperpigmentation Family 15 1st degree -Microcephaly Deletion of exon 15 Hm DS F492– (Gafsa) M/9 -Triangular facies 100% 2/2016 -Café au lait spots -Triangular facies South of Tunisia Family 16 F025/17 F/9 1st degree -Café au lait spots 90% Deletion of exon 15 Hm DS (Sidi Bouzid) -Anomalies of the thumb -Microcephaly F059/16 M/7 -Thrombocytopenia 81% South of Tunisia -Anomalies of the thumb Family 17 1st degree Deletion of exon 15 Hm DS (Sidi Bouzid) -Café au lait spots F060/16 M/5 90% -Microcephaly -Microcephaly F058/15 M/9 -Café au lait spots 85% Center of Tunisia -Anomalies of the thumb Family 18 1st degree -Microcephaly c.2222 + 166G > A Hm DS (Kairouan) F059/15 M/5 -Café au lait spots 92% ‐Anomalies of the thumb Center of Tunisia -Microcephaly Family 19 CFA4 M/6 1st degree 100% c.2222 + 166G > A Hm DS (Kairouan) -Café au lait spots Center of Tunisia -Triangular facies Family 20 CFA3 M/4 1st degree 100% c.2222 + 166G > A Hm DS (Kairouan) -Café au lait spots Center of Tunisia Family 21 F033/15 M/3 1st degree -Café au lait spots 100% c.2222 + 166G > A Hm DS (Kairouan) Center of Tunisia -Microcephaly Family 22 TFA23 M/8 1st degree 100% c.2222 + 166G > A Hm DS (Kairouan) -Café au lait spots CH, compound heterozygous; Hm, homozygous; Hz, heterozygous; M, male; F, female; DS, direct sequencing; MLPA, multiplex ligation-dependent probe amplification; TGS: targeted gene sequencing. References for described mutations: • Solanki et al., 2016: c.2749C > T; p.Arg917Ter • Amouri et al., 2014: Deletion of exon 15 • Moghrabi et al., 2009: c.1304G > A; p.Arg435His • Magdalena et al., 2005: c.3788_3790delTCT • Ameziane et al., 2008: Deletion of exons 4 and 5 • Bouchlaka et al., 2003: c.2222 + 166G > A absence of clinical signs and any familial history would made Libya considering their common genetic and the clinical diagnosis very challenging, hence showing the historical background. importance of chromosomal breakage assessment for the As previously shown, the deletion of exon 15 in the FANCA establishment of an accurate and rapid diagnosis. Indeed, testing gene is a founder mutation present at a frequency of 54% for induced chromosomal breakage is commonly used for a among FA-A Tunisian patients (Amouri et al., 2014). This precise diagnosis in the frame of a proper management of FA deletion was found to be the most frequent mutation in our patients (bone marrow transplant) and their families (genetic cohort (11/29 patients: 37.93%). All the Tunisian patients bearing counseling and prenatal diagnosis). Though molecular methods this deletion were from Sidi Bouzid and Gafsa. This is not are developed to screen FA patients, cytogenetic investigations surprising as we have previously reported that the geographic with MMC and DEB induction is still “the gold standard” for distribution of 85% of families bearing this deletion are from diagnosing FA. Southern Tunisia thus suggesting a founder effect (Amouri As showed with previous reports, FANCA is the most frequent et al., 2014). Furthermore, FA cases of Maghrebian origin living gene (94%) associated with FA in Tunisia (Bouchlaka et al., in France (Amouri et al., 2014) and Spain (unpublished data) 2003). This result suggests that FANCA could be also the most shared this deletion. Our findings, together with what causal gene of FA in other NA countries like Algeria and we published in 2014, represent strong evidence that this Frontiers in Genetics | www.frontiersin.org 7 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients deletion is the most common mutation among NA population within FANCA (Levran et al., 1998; Morgan et al., 1999; Moghrabi and this observation has to be taken into consideration mainly et al., 2009). Besides the genetic structure of the FANCA gene, for migrated families. Similar findings have been observed in the high frequency of deletions noted among NA population NA countries for several diseases. For instance, congenital is likely resulting from the presence of founder mutations. myasthenic syndrome patients living in France and originating Screening by PCR and Sanger sequencing of FANCA 43 exons from Tunisia, Algeria, Morocco, and Libya share the same allowed the identification of 5 different nucleotide changes in haplotype associated with the point founder mutation 14 FA cases (47%): one reported missense mutation (c.1304G>A; (c.1293insG) in CHRNE which has been also found in patients p.Arg435His), a novel missense variant (c.1258G > A; p.Asp409Glu), from the Maghreb (Richard et al., 2008). a nonsense mutation (c.2749C > T; p.Arg917Ter), a small deletion Another large deletion including exon 4 and 5 has been (c.3788-3790delTCT), and an intronic mutation (c.2222 + 166G>A). found in patients originating from Sfax (17%; Figure 4). This Some identified variants were described in other populations deletion was firstly described in two patients from Northwestern (Magdalena et al., 2005; Moghrabi et al., 2009; Solanki et al., Europe in heterozygous states (Ameziane et al., 2008). A 2016). However, none of them were described before in NA molecular study applied for Spanish patients revealed the patients, except for the intronic mutation c.2222 + 166G > A presence of this large deletion (Castella et al., 2011). This (Bouchlaka et al., 2003). This mutation inherited in all FA mutation could probably be introduced in Tunisia when patients originating from Kairouan (central Tunisia) represents Andalusians began crossing the strait of Gibraltar to seek refuge the second most frequent mutation in our cohort (20.68%). in NA in 1492 but a haplotype analysis should be performed This pathogenic variant always segregates with the same haplotype, to confirm or discard this hypothesis. already described (Bouchlaka et al., 2003). Our finding suggests Our results showed large intragenic deletions as the most that this intronic mutation has a common ancestor in Kairouan. frequent mutations found in homozygous and heterozygous The identification of a founder mutation specific to a given states in 55.17% of the patients. This frequency is higher than geographic origin facilitates the diagnosis and reduces the that observed in other populations [31.25% in India (Solanki diagnosis cost. In fact, various laboratories have adapted their et al., 2016); 25% in Brazilian patients (Pilonetto et al., 2017)]. strategies to optimize the chances of detecting the causal A meta-analysis has shown that between 15 and 40% of mutation while keeping costs to a minimum and decreasing pathogenic mutations in FANCA are caused by large deletions the time required to get a reliable result. (Ameziane et al., 2012; Gille et al., 2012). This is explained The identification of a founder mutation specific to a given by the presence of a large number of Alu repeat sequences geographic origin facilitates the diagnosis and reduces the FIGURE 4 | CNV plots for patients (GF06/09 and F072/09) showing exon 4 and 5 deletion. The plots indicate a heterozygous deletion for patient GF06/09 while it is homozygous for patient F072/09. The two plots also show a decrease of the exon 1 signal but the quality control does not retain it as a deletion. Frontiers in Genetics | www.frontiersin.org 8 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients diagnosis cost. Indeed, in Tunisia, among 174 genetic diseases was the case for the recurrent mutation p.V548AfsX25 affecting with an identified molecular defect, 73 (41.9%) are due to XPC and leading to Xeroderma pigmentosum. This mutation was founder mutations (Romdhane and Abdelhak, 2011; Elloumi firstly described in Algerian and Moroccan patients in a Zghal and Chaabouni Bouhamed, 2018; Romdhane et al., 2019). homozygous state (Khan et al., 2006), then another study This high frequency of founder mutations could be explained demonstrated its high prevalence in Tunisian patients and confirmed by the high rates of inbreeding and endogamy that characterize a founder effect in this population (Ben Rekaya et al., 2009). NA populations: 34.04% in Algeria, 37.6% in Libya and 29.9% The molecular approach applied in this study allowed us to in Tunisia (Abudejaja et al., 1987; Zaoui and Biémont, 2002; identify causative variants for almost all patients (25 Ben Halim et al., 2013). The most frequent form of intermarriage patients: 86.20%). However, the second mutated allele failed to is between first cousins, particularly paternal first cousins and be identified for the rest of patients (four patients: 13.79%). includes double first-cousin marriage (Ben Halim et al., 2013). Taking together, we can conclude that the combination between Considering the first cousins’ marriages, the risk of recessive MLPA Sanger sequencing and, possibly, TGS methods for FANCA hereditary diseases is multiplied by an average of 8 times, mutations screening seems to be the appropriate molecular which doubles the total frequency of congenital and genetic approach for a precise diagnosis of FA patients in NA (Figure 6). diseases (Romdhane et al., 2014; Ben Halim et al., 2016). In For patients (TFA27, GF06/09, F031/16 and F032/16) whose our study, 86% of our families are consanguineous (Figure 5), second mutated allele was not identified, it could be explained which may explain the elevated number of deleterious mutations by (i) the presence of a second undetected pathogenic variant found at a homozygous state in the affected patients (82.75%). in intronic or regulatory regions; (ii) another FA gene could Similar values between the consanguinity rate and the percentage be mutated in TFA27, and the presence of FANCA exon 15 of homozygous mutations clearly show the impact of inbreeding deletion could be due to its higher prevalence in the NA population. on the genomic structure by increasing the number of regions The assignment of NA patients to their genetic FA subtypes of homozygosity and hence homozygous pathogenic variants. and the identification of their respective mutations provide Some of the identified FANCA variants (Deletion of exon valuable data for a better knowledge of the genetic basis of 15; p.Arg917Ter; p.Arg409Glu) are shared among Tunisian, FA in NA and for a more appropriate management of patients Libyan, and Algerian patients. These findings enhance the at both preventive (plan for genetic counseling and prenatal previous observations that NA patients share a common ethnic diagnosis) and therapeutic (selection of suitable donors for background and consequently the same mutational spectrum hematopoietic stem cell transplantation) levels. (Romdhane et al., 2012). NA is characterized by its mixed population history due to migratory/invasive flows which have resulted in high levels of DATA AVAILABILITY STATEMENT genetic and phenotypic variability. Our results contain private mutations, ethnic specific mutations, and recurrent founder Processed data related to Sanger sequencing, haplotype analysis mutations, thus reflecting a great genetic heterogeneity of FANCA and MLPA are available in the article. Raw data related to TGS mutations within the NA population. Indeed, prioritizing mutation are available from the corresponding author upon reasonable screening by relying on deleterious gene variants already identified request. Indeed, in Tunisia, genetic data are considered as personal in neighboring populations can be particularly efficient. This private data, for these reasons we have submitted the minimal FIGURE 5 | Pedigrees of two investigated FA families showing different degrees of inbreeding loops. Frontiers in Genetics | www.frontiersin.org 9 February 2021 | Volume 12 | Article 610050 Ben Haj Ali et al. Fanconi Anemia in North African Patients FIGURE 6 | A proposed strategy for molecular diagnosis of FA in North African patients mainly from Tunisia, Algeria, and Libya. dataset as supporting files but we are not allowed to submit results. AB and FT extracted DNA and carried out the the full raw data. The full raw data may be made available experimental work. OM, SE, AS-G, VD, and NL analyzed upon request by other investigators and after approval of our IRB. molecular data. AB drafted the manuscript. AB, SA, AA, OM, MB, and JS designed the project and edited the manuscript. All authors contributed to the article and approved the ETHICS STATEMENT submitted version. This work was supported by the Ethical Committee of the Institut Pasteur de Tunis (IPT) N° 2017/14/I/LR11IPT05/V0. FUNDING Written informed consent for publication of both clinical data and photographs was obtained from our patients’ families. This work was supported by the Tunisian Ministry of Public Informed consent of the legal representatives of the patient Health, the Tunisian Ministry of Higher Education and Scientific was obtained. Research (LR16IPT05). AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS AA, FM, MO, and SH established the clinical diagnosis. AB We would like to thank patients and their families for their and FT collected the samples. 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A. 98, 5734–5739. doi: 10.1073/ pnas.091402398 Copyright © 2021 Ben Haj Ali, Messaoud, Elouej, Talmoudi, Ayed, Mellouli, Ouederni, Whitney, M. A., Saito, H., Jakobs, P. M., Gibson, R. A., Moses, R. E., and Hadiji, De Sandre-Giovannoli, Delague, Lévy, Bogliolo, Surrallés, Abdelhak and Grompe, M. (1993). A common mutation in the FACC gene causes Amouri. This is an open-access article distributed under the terms of the Creative Fanconi anaemia in Ashkenazi Jews. Nat. Genet. 4, 202–205. doi: 10.1038/ Commons Attribution License (CC BY). The use, distribution or reproduction in ng0693-202 other forums is permitted, provided the original author(s) and the copyright owner(s) Zaoui, S., and Biémont, C. (2002). Fréquence et structure des mariages consanguins are credited and that the original publication in this journal is cited, in accordance dans la région de Tlemcen (Ouest algérien) [Frequency of consanguineous with accepted academic practice. No use, distribution or reproduction is permitted unions in the Tlemcen area (West Algeria)]. Sante 12, 289–295. which does not comply with these terms. Frontiers in Genetics | www.frontiersin.org 12 February 2021 | Volume 12 | Article 610050