Original Paper Acta Haematol 2014;132:15–21 DOI: 10.1159/000355191
Received: April 24, 2013 Accepted after revision: August 5, 2013 Published online: December 13, 2013
Fanconi Anemia Founder Mutation in Macedonian Patients Svetlana Madjunkova a Svetlana A. Kocheva a, b Dijana Plaseska-Karanfilska a
a
Research Center for Genetic Engineering and Biotechnology ‘Georgi D. Efremov’, Macedonian Academy of Sciences and Arts and b Pediatric Clinic, Medical Faculty, University ‘St. Cyril and Methodius’, Skopje, Republic of Macedonia
Abstract Background: Fanconi anemia (FA) is a rare autosomal recessive disorder clinically characterized by developmental abnormalities, progressive bone marrow failure (BMF) and profound cancer predisposition. Approximately 65% of all affected individuals have mutation in the FANCA (Fanconi anemia complementation group A) gene. The mutation spectrum of the FANCA gene is highly heterogeneous. FA-A is usually associated with private FANCA mutations in individual families. Methods: We describe 3 unrelated patients with FA with a similar clinical presentation: BMF, renal anomalies and café-au-lait pigmentation without major skeletal abnormality. The molecular analysis of the FANCA gene using the FA MLPA kit P031A2/P032 FANCA, showed homozygous deletion of exon 3 in all 3 patients. Molecular analysis of the flanking regions of exon 3 precisely defined unique deletion of 2,040 bp and duplication of C (1788_3828dupC). Discussion/Conclusions: These are the first 3 patients homozygous for deletion of FANCA exon 3 described to date. Although not related, the patients originated from the same Gypsy-like ethnic population. We conclude that c.190–256_283 + 1680del2040 dupC mutation in the FANCA gene is a founder mutation in Macedonian FA patients of Gypsy-like ethnic origin. Our finding has very strong implications for these patients in formulating diagnostic and carrier-screening strategy for BMF and FA and to enable comprehensive genetic counseling. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0001–5792/13/1321–0015$38.00/0 E-Mail [email protected] www.karger.com/aha
Introduction
Fanconi anemia (FA) is a rare autosomal recessive disorder (1–5/1,000,000 live births) genetically and phenotypically heterogeneous, defined by cellular hypersensitivity to DNA cross-linking agents [1]. FA cells feature a high frequency of broken and radial chromosomes and are highly sensitive to agents that produce interstrand DNA crosslinks [2, 3]. Covalent bonds linking the two antiparallel strands of DNA are extremely toxic, as they prevent DNA unwinding required for DNA replication and transcription. Clinically, FA is characterized by variable developmental abnormalities that may affect skeletal morphogenesis as well as any of the major organ systems, stem cell loss that causes progressive bone marrow failure (BMF) and male sterility and a profound predisposition to neoplasia mostly in the form of leukemia, solid tumors and squamous cell carcinomas of the gastrointestinal tract and female reproductive system [4–6]. FA is the most frequent inherited cause of BMF and its most important clinical features are hematological aplastic anemia, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [7]. At a molecular level, a fundamental defect in DNA repair underlies the FA phenotype [8]. The FA complementation group consists of 16 FANC genes, namely FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO/RAD51C, FANCP/ Dr. Dijana Plaseska-Karanfilska Macedonian Academy of Science and Arts Research Center for Genetic Engineering and Biotechnology ‘Georgi D. Efremov’ Bul Krste Misirkov 2, 1000 Skopje (Republic of Macedonia) E-Mail dijana @ manu.edu.mk
Downloaded by: East Carolina University 150.216.68.200 - 4/24/2015 11:20:06 AM
Key Words Fanconi anemia · FANCA gene · Founder mutation
Materials Clinical Case Presentation FA patients were identified by the Department of Hematology/ Oncology, Pediatrics Clinic, Medical Faculty, Skopje, Macedonia. Clinical data were obtained regarding age of onset, severity of hematological symptoms, survival, incidence of AML, MDS and somatic abnormalities. The genetic diagnosis was established at the Research Centre for Genetic Engineering and Biotechnology
16
Acta Haematol 2014;132:15–21 DOI: 10.1159/000355191
Table 1. Clinical presentation of FA patients Patient Age/ (initials) sex
J.I. F.N. P.J.
BMF Ren Renal Pigmentation PDA VUR Ptosis arquatus ectopy (café-au-lait)
11/F + 17/F + 23/M +
+ + +
+ + +
+ + +
+ – –
+ – –
+ – –
F = Female; M = male; PDA = persistent ductus arteriosus; VUR = vesicouretral reflux.
‘Georgi D. Efremov’, Macedonian Academy of Sciences and Arts. The study was approved by the ethical committee of Academy, and all subjects gave informed consent for their participation in the study in accordance with the Declaration of Helsinki. Table 1 presents, concisely, the clinical findings of the FA patients (2 females, 11 and 17 years of age and 1 male who was 23 years old), with a similar clinical presentation: BMF, ren arquatus and renal ectopy and abnormal skin pigmentation (café-au-lait) without major skeletal abnormality. The pancytopenia first manifested at the age of 8–8.5 years in all 3 patients. All 3 currently follow a 3- to 4-week transfusion regimen. The patients are of symmetrically shortened stature. In the male patient, the physical examination showed hypoplastic thenars and thumbs on both hands as well as syndactyly of the 2nd and 3rd toe on both feet. In one of the female patients, persistent ductus arteriosus vesicoureteral reflux and ptosis were also present. The patients are from the same ethnic population. They are not related and no evidence of consanguinity in their families was reported. Family history was assessed for only one of the patients, F.N. (the 17-year-old). She had had an older sister who died from complications of pancytopenia at the age of 9 years (fig. 1).
Methods Identification and Characterization of the Deletion Genomic DNA was obtained from peripheral blood following standard proteinase K-phenol-chloroform extraction procedures. The molecular analysis of the FANCA gene (NM_000135.2) was done using the Fanconi anemia MLPA kit P031-A2/P032 FANCA (MRC Holland, The Netherlands), following the manufacturer’s protocol. The P031-A2/P032 FANCA kit consists of two probe mixes containing one probe for each FANCA exon as well as reference probes detecting different autosomal chromosomal locations. The products of MLPA reaction were analyzed by capillary electrophoresis on ABI 3130 GeneticAnalyzer (Life Technologies, Calif., USA). The data generated by these probe mixes were then normalized intrasample by dividing the peak area of each probe’s amplification product by the total area of only the reference probes (block normalization) and secondly, inter-sample normalization was achieved by dividing the intranormalized probe ratio in a sample by the average intranormalized probe ratio of all reference probes. Heterozygous deletions of recognition sequences gave a 35–50% reduced relative peak area of the amplification product of that probe, while homozy-
Madjunkova/Kocheva/ Plaseska-Karanfilska
Downloaded by: East Carolina University 150.216.68.200 - 4/24/2015 11:20:06 AM
SLX4 and FANCQ/ERCC4 [9]. FA genes encode proteins implicated in a ubiquitin-signalling pathway, in S-phase checkpoint activation, in translation DNA synthesis and in DNA double-strand break repair by homologous recombination [10]. There is evidence that all 16 gene products operate in a complex molecular network to preserve genomic integrity [9, 11–13]. FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and an associated protein of 100 kDa (FAAP100) form a nuclear, ubiquitin ligase FA core complex. The FA nuclear core complex monoubiquitinates FANCD2/FANCI; this has a central role in DNA replication and repair [14–16]. Biallelic disruption of any one of these genes results in this clinically defined syndrome, the most prevalent ones being FANCA, FANCC, FANCG and FANCD2 [17]. FA-A is the most common group, accounting for approximately 65% of all affected individuals [18]. The FANCA gene, located on chromosome 16q24.3 (
Received: April 24, 2013 Accepted after revision: August 5, 2013 Published online: December 13, 2013
Fanconi Anemia Founder Mutation in Macedonian Patients Svetlana Madjunkova a Svetlana A. Kocheva a, b Dijana Plaseska-Karanfilska a
a
Research Center for Genetic Engineering and Biotechnology ‘Georgi D. Efremov’, Macedonian Academy of Sciences and Arts and b Pediatric Clinic, Medical Faculty, University ‘St. Cyril and Methodius’, Skopje, Republic of Macedonia
Abstract Background: Fanconi anemia (FA) is a rare autosomal recessive disorder clinically characterized by developmental abnormalities, progressive bone marrow failure (BMF) and profound cancer predisposition. Approximately 65% of all affected individuals have mutation in the FANCA (Fanconi anemia complementation group A) gene. The mutation spectrum of the FANCA gene is highly heterogeneous. FA-A is usually associated with private FANCA mutations in individual families. Methods: We describe 3 unrelated patients with FA with a similar clinical presentation: BMF, renal anomalies and café-au-lait pigmentation without major skeletal abnormality. The molecular analysis of the FANCA gene using the FA MLPA kit P031A2/P032 FANCA, showed homozygous deletion of exon 3 in all 3 patients. Molecular analysis of the flanking regions of exon 3 precisely defined unique deletion of 2,040 bp and duplication of C (1788_3828dupC). Discussion/Conclusions: These are the first 3 patients homozygous for deletion of FANCA exon 3 described to date. Although not related, the patients originated from the same Gypsy-like ethnic population. We conclude that c.190–256_283 + 1680del2040 dupC mutation in the FANCA gene is a founder mutation in Macedonian FA patients of Gypsy-like ethnic origin. Our finding has very strong implications for these patients in formulating diagnostic and carrier-screening strategy for BMF and FA and to enable comprehensive genetic counseling. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0001–5792/13/1321–0015$38.00/0 E-Mail [email protected] www.karger.com/aha
Introduction
Fanconi anemia (FA) is a rare autosomal recessive disorder (1–5/1,000,000 live births) genetically and phenotypically heterogeneous, defined by cellular hypersensitivity to DNA cross-linking agents [1]. FA cells feature a high frequency of broken and radial chromosomes and are highly sensitive to agents that produce interstrand DNA crosslinks [2, 3]. Covalent bonds linking the two antiparallel strands of DNA are extremely toxic, as they prevent DNA unwinding required for DNA replication and transcription. Clinically, FA is characterized by variable developmental abnormalities that may affect skeletal morphogenesis as well as any of the major organ systems, stem cell loss that causes progressive bone marrow failure (BMF) and male sterility and a profound predisposition to neoplasia mostly in the form of leukemia, solid tumors and squamous cell carcinomas of the gastrointestinal tract and female reproductive system [4–6]. FA is the most frequent inherited cause of BMF and its most important clinical features are hematological aplastic anemia, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [7]. At a molecular level, a fundamental defect in DNA repair underlies the FA phenotype [8]. The FA complementation group consists of 16 FANC genes, namely FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO/RAD51C, FANCP/ Dr. Dijana Plaseska-Karanfilska Macedonian Academy of Science and Arts Research Center for Genetic Engineering and Biotechnology ‘Georgi D. Efremov’ Bul Krste Misirkov 2, 1000 Skopje (Republic of Macedonia) E-Mail dijana @ manu.edu.mk
Downloaded by: East Carolina University 150.216.68.200 - 4/24/2015 11:20:06 AM
Key Words Fanconi anemia · FANCA gene · Founder mutation
Materials Clinical Case Presentation FA patients were identified by the Department of Hematology/ Oncology, Pediatrics Clinic, Medical Faculty, Skopje, Macedonia. Clinical data were obtained regarding age of onset, severity of hematological symptoms, survival, incidence of AML, MDS and somatic abnormalities. The genetic diagnosis was established at the Research Centre for Genetic Engineering and Biotechnology
16
Acta Haematol 2014;132:15–21 DOI: 10.1159/000355191
Table 1. Clinical presentation of FA patients Patient Age/ (initials) sex
J.I. F.N. P.J.
BMF Ren Renal Pigmentation PDA VUR Ptosis arquatus ectopy (café-au-lait)
11/F + 17/F + 23/M +
+ + +
+ + +
+ + +
+ – –
+ – –
+ – –
F = Female; M = male; PDA = persistent ductus arteriosus; VUR = vesicouretral reflux.
‘Georgi D. Efremov’, Macedonian Academy of Sciences and Arts. The study was approved by the ethical committee of Academy, and all subjects gave informed consent for their participation in the study in accordance with the Declaration of Helsinki. Table 1 presents, concisely, the clinical findings of the FA patients (2 females, 11 and 17 years of age and 1 male who was 23 years old), with a similar clinical presentation: BMF, ren arquatus and renal ectopy and abnormal skin pigmentation (café-au-lait) without major skeletal abnormality. The pancytopenia first manifested at the age of 8–8.5 years in all 3 patients. All 3 currently follow a 3- to 4-week transfusion regimen. The patients are of symmetrically shortened stature. In the male patient, the physical examination showed hypoplastic thenars and thumbs on both hands as well as syndactyly of the 2nd and 3rd toe on both feet. In one of the female patients, persistent ductus arteriosus vesicoureteral reflux and ptosis were also present. The patients are from the same ethnic population. They are not related and no evidence of consanguinity in their families was reported. Family history was assessed for only one of the patients, F.N. (the 17-year-old). She had had an older sister who died from complications of pancytopenia at the age of 9 years (fig. 1).
Methods Identification and Characterization of the Deletion Genomic DNA was obtained from peripheral blood following standard proteinase K-phenol-chloroform extraction procedures. The molecular analysis of the FANCA gene (NM_000135.2) was done using the Fanconi anemia MLPA kit P031-A2/P032 FANCA (MRC Holland, The Netherlands), following the manufacturer’s protocol. The P031-A2/P032 FANCA kit consists of two probe mixes containing one probe for each FANCA exon as well as reference probes detecting different autosomal chromosomal locations. The products of MLPA reaction were analyzed by capillary electrophoresis on ABI 3130 GeneticAnalyzer (Life Technologies, Calif., USA). The data generated by these probe mixes were then normalized intrasample by dividing the peak area of each probe’s amplification product by the total area of only the reference probes (block normalization) and secondly, inter-sample normalization was achieved by dividing the intranormalized probe ratio in a sample by the average intranormalized probe ratio of all reference probes. Heterozygous deletions of recognition sequences gave a 35–50% reduced relative peak area of the amplification product of that probe, while homozy-
Madjunkova/Kocheva/ Plaseska-Karanfilska
Downloaded by: East Carolina University 150.216.68.200 - 4/24/2015 11:20:06 AM
SLX4 and FANCQ/ERCC4 [9]. FA genes encode proteins implicated in a ubiquitin-signalling pathway, in S-phase checkpoint activation, in translation DNA synthesis and in DNA double-strand break repair by homologous recombination [10]. There is evidence that all 16 gene products operate in a complex molecular network to preserve genomic integrity [9, 11–13]. FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and an associated protein of 100 kDa (FAAP100) form a nuclear, ubiquitin ligase FA core complex. The FA nuclear core complex monoubiquitinates FANCD2/FANCI; this has a central role in DNA replication and repair [14–16]. Biallelic disruption of any one of these genes results in this clinically defined syndrome, the most prevalent ones being FANCA, FANCC, FANCG and FANCD2 [17]. FA-A is the most common group, accounting for approximately 65% of all affected individuals [18]. The FANCA gene, located on chromosome 16q24.3 (
