Hypomorphic Mutations in the Gene Encoding a Key Fanconi Anemia Protein, FANCD2, Sustain a Significant Group of FA-D2 Patients with Severe Phenotype
2007; Elsevier BV; Volume: 80; Issue: 5 Linguagem: Inglês
10.1086/517616
ISSN1537-6605
AutoresReinhard Kalb, Kornelia Neveling, Holger Hoehn, Hildegard Schneider, Yvonne Linka, Sat Dev Batish, C. Hunt, Marianne Berwick, Elsa Callén, Jordi Surrallés, José Antonio Casado, Juan A. Bueren, Ángeles Dasí, Jean Soulier, Éliane Gluckman, C. Michel Zwaan, Rosalina van Spaendonk, Gerard Pals, Johan P. de Winter, Hans Joenje, Markus Grompe, Arleen D. Auerbach, Helmut Hanenberg, Detlev Schindler,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoFANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand–type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%–6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA–non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA. FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand–type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%–6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA–non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA. Fanconi anemia (FA) is a rare genome-instability disorder with the variable presence of congenital malformations, progressive bone-marrow failure (BMF), predisposition to malignancies, and cellular hypersensitivity to DNA-interstrand crosslinking agents.1Joenje H Patel KJ The emerging genetic and molecular basis of Fanconi anaemia.Nat Rev Genet. 2001; 2: 446-457Crossref PubMed Scopus (493) Google Scholar There are at least 13 complementation groups (FA-A [MIM 607139], -B [MIM 300515], -C [MIM 227645], -D1 [MIM 600185], -D2 [MIM 227646], -E [MIM 600901], -F [MIM 603467], -G [MIM 602956], -I [MIM 609053], -J [MIM 609054], -L [MIM 608111], -M [MIM 609644], and -N [MIM 610355]), each of which is associated with biallelic or hemizygous mutations in a distinct gene.2Levitus M Rooimans MA Steltenpool J Cool NF Oostra AB Mathew CG Hoatlin ME Waisfisz Q Arwert F de Winter JP et al.Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes.Blood. 2004; 103: 2498-2503Crossref PubMed Scopus (193) Google Scholar, 3Reid S Schindler D Hanenberg H Barker K Hanks S Kalb R Neveling K Kelly P Seal S Freund M et al.Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer.Nat Genet. 2007; 39: 162-164Crossref PubMed Scopus (465) Google Scholar, 4Xia B Dorsman JC Ameziane N de Vries Y Rooimans MA Sheng Q Pals G Errami A Gluckman E Llera J et al.Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.Nat Genet. 2007; 39: 159-161Crossref PubMed Scopus (359) Google Scholar To date, 12 of the underlying genes have been identified: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, FANCJ/BRIP1, FANCL/PHF9, FANCM/HEF, and FANCN/PALB2.3Reid S Schindler D Hanenberg H Barker K Hanks S Kalb R Neveling K Kelly P Seal S Freund M et al.Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer.Nat Genet. 2007; 39: 162-164Crossref PubMed Scopus (465) Google Scholar, 4Xia B Dorsman JC Ameziane N de Vries Y Rooimans MA Sheng Q Pals G Errami A Gluckman E Llera J et al.Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.Nat Genet. 2007; 39: 159-161Crossref PubMed Scopus (359) Google Scholar, 5Levran O Attwooll C Henry RT Milton KL Neveling K Rio P Batish SD Kalb R Velleuer E Barral S et al.The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia.Nat Genet. 2005; 37: 931-933Crossref PubMed Scopus (295) Google Scholar, 6Meetei AR Medhurst AL Ling C Xue Y Singh TR Bier P Steltenpool J Stone S Dokal I Mathew CG et al.A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M.Nat Genet. 2005; 37: 958-963Crossref PubMed Scopus (348) Google Scholar, 7Levitus M Waisfisz Q Godthelp BC de Vries Y Hussain S Wiegant WW Elghalbzouri-Maghrani E Steltenpool J Rooimans MA Pals G et al.The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.Nat Genet. 2005; 37: 934-935Crossref PubMed Scopus (361) Google Scholar Eight of the FA proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) and other components assemble in a nuclear complex, the FA “core complex,” that is required for the monoubiquitination of FANCD2 at amino acid residue K561.8Meetei AR Sechi S Wallisch M Yang D Young MK Joenje H Hoatlin ME Wang W A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.Mol Cell Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (286) Google Scholar, 9Garcia-Higuera I Taniguchi T Ganesan S Meyn MS Timmers C Hejna J Grompe M D’Andrea AD Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.Mol Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar Monoubiquitination occurs in response to DNA damage and during the S phase of the cell cycle.9Garcia-Higuera I Taniguchi T Ganesan S Meyn MS Timmers C Hejna J Grompe M D’Andrea AD Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.Mol Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar, 10Taniguchi T Garcia-Higuera I Andreassen PR Gregory RC Grompe M D’Andrea AD S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51.Blood. 2002; 100: 2414-2420Crossref PubMed Scopus (385) Google Scholar The monoubiquitinated FANCD2 isoform (FANCD2-L, as opposed to FANCD2-S) is targeted to nuclear foci containing proteins such as BRCA1 [MIM 113705], BRCA2, and RAD51 [MIM 179617] that are involved in DNA-damage signaling and recombinational repair.11Thompson LH Hinz JM Yamada NA Jones NJ How Fanconi anemia proteins promote the four Rs: replication, recombination, repair, and recovery.Environ Mol Mutagen. 2005; 45: 128-142Crossref PubMed Scopus (89) Google Scholar, 12Nakanishi K Yang YG Pierce AJ Taniguchi T Digweed M D’Andrea AD Wang ZQ Jasin M Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.Proc Natl Acad Sci USA. 2005; 102: 1110-1115Crossref PubMed Scopus (305) Google Scholar, 13Niedzwiedz W Mosedale G Johnson M Ong CY Pace P Patel KJ The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair.Mol Cell. 2004; 15: 607-620Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 14Digweed M Rothe S Demuth I Scholz R Schindler D Stumm M Grompe M Jordan A Sperling K Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia.Carcinogenesis. 2002; 23: 1121-1126Crossref PubMed Scopus (70) Google Scholar The precise role of FANCD2 remains unknown, but FANCD2-deficient DT40 cells show defects in homologous recombination-mediated DNA double-strand break (DSB) repair, translesion synthesis, and gene conversion.11Thompson LH Hinz JM Yamada NA Jones NJ How Fanconi anemia proteins promote the four Rs: replication, recombination, repair, and recovery.Environ Mol Mutagen. 2005; 45: 128-142Crossref PubMed Scopus (89) Google Scholar, 15Yamamoto K Hirano S Ishiai M Morishima K Kitao H Namikoshi K Kimura M Matsushita N Arakawa H Buerstedde JM et al.Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination.Mol Cell Biol. 2005; 25: 34-43Crossref PubMed Scopus (115) Google Scholar, 16Mirchandani KD D’Andrea AD The Fanconi anemia/BRCA pathway: a coordinator of cross-link repair.Exp Cell Res. 2006; 312: 2647-2653Crossref PubMed Scopus (64) Google Scholar Therefore, FANCD2 is thought to play a central role in the maintenance of genome stability.11Thompson LH Hinz JM Yamada NA Jones NJ How Fanconi anemia proteins promote the four Rs: replication, recombination, repair, and recovery.Environ Mol Mutagen. 2005; 45: 128-142Crossref PubMed Scopus (89) Google Scholar, 16Mirchandani KD D’Andrea AD The Fanconi anemia/BRCA pathway: a coordinator of cross-link repair.Exp Cell Res. 2006; 312: 2647-2653Crossref PubMed Scopus (64) Google Scholar, 17Levitus M Joenje H de Winter JP The Fanconi anemia pathway of genomic maintenance.Cell Oncol. 2006; 28: 3-29PubMed Google Scholar The human and murine Fancd2 genes show a higher degree of homology than do the corresponding Fanca, Fancc, Fance, Fancf, and Fancg genes.18Blom E van de Vrugt HJ de Winter JP Arwert F Joenje H Evolutionary clues to the molecular function of Fanconi anemia genes.Acta Haematol. 2002; 108: 231-236Crossref PubMed Scopus (14) Google ScholarFancd2-knockout mice suffer from perinatal lethality, microphthalmia, and early epithelial cancers,19Houghtaling S Timmers C Noll M Finegold MJ Jones SN Meyn MS Grompe M Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice.Genes Dev. 2003; 17: 2021-2035Crossref PubMed Scopus (207) Google Scholar but it remains controversial whether the murine FA-D2 phenotype in general is more severe than the corresponding murine knockouts of the other FA genes.19Houghtaling S Timmers C Noll M Finegold MJ Jones SN Meyn MS Grompe M Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice.Genes Dev. 2003; 17: 2021-2035Crossref PubMed Scopus (207) Google Scholar, 20Carreau M Not-so-novel phenotypes in the Fanconi anemia group D2 mouse model.Blood. 2004; 103: 2430Crossref PubMed Scopus (21) Google Scholar Fancd2 is required for survival after DNA damage in Caenorhabditis elegans.21Dequen F St-Laurent JF Gagnon SN Carreau M Desnoyers S The Caenorhabditis elegans FancD2 ortholog is required for survival following DNA damage.Comp Biochem Physiol B Biochem Mol Biol. 2005; 141: 453-460Crossref PubMed Scopus (17) Google Scholar Fancd2-deficient zebrafish embryos display severe developmental defects due to increased apoptosis, which underscores the importance of Fancd2 function during vertebrate ontogenesis.22Liu TX Howlett NG Deng M Langenau DM Hsu K Rhodes J Kanki JP D’Andrea AD Look AT Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis.Dev Cell. 2003; 5: 903-914Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Finally, knock-down of Drosophila Fancd2 causes pupal lethality.23Marek LR Bale AE Drosophila homologs of FANCD2 and FANCL function in DNA repair.DNA Repair (Amst). 2006; 5: 1317-1326Crossref PubMed Scopus (32) Google Scholar In humans, it has been estimated that complementation group FA-D2 accounts for between <1%24Tischkowitz M Dokal I Fanconi anaemia and leukaemia—clinical and molecular aspects.Br J Haematol. 2004; 126: 176-191Crossref PubMed Scopus (114) Google Scholar and 3%25Taniguchi T D’Andrea AD The molecular pathogenesis of Fanconi anemia: recent progress.Blood. 2006; 107: 4223-4233Crossref PubMed Scopus (297) Google Scholar of all patients with FA. The presence of FANCD2 pseudogenes complicates mutation analysis, which may explain why there has been just one other report of a single human FANCD2 mutation since the original description.26Timmers C Taniguchi T Hejna J Reifsteck C Lucas L Bruun D Thayer M Cox B Olson S D’Andrea AD et al.Positional cloning of a novel Fanconi anemia gene, FANCD2.Mol Cell. 2001; 7: 241-248Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 27Borriello A Locasciulli A Bianco AM Criscuolo M Conti V Grammatico P Cappellacci S Zatterale A Morgese F Cucciolla V et al.A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia.Leukemia. 2007; 21: 72-78Crossref PubMed Scopus (22) Google Scholar As a concerted effort among nine laboratories, we present a comprehensive mutation profile of the FANCD2 gene (Ensembl Genome Browser [accession number ENSG00000144554]). We show that the FA phenotype resulting from FANCD2 deficiency is relatively severe. In contrast to all other FA genes, (1) the mutation spectrum of FANCD2 is dominated by splicing mutations, and (2) residual FANCD2 protein exists in all tested cell lines from FA-D2 patients, which suggests lethality of biallelic null mutations. Anticoagulated peripheral blood and skin-biopsy samples were referred to the participating laboratories for diagnostic testing. Confirmation of the diagnosis of FA, subtyping, and mutation analysis were performed with informed consent according to the Declaration of Helsinki. The study was approved by the institutional review boards of the participating centers. Clinical suspicion of FA was confirmed by the detection of cellular hypersensitivity to DNA-crosslinking agents following published procedures.28Joenje H Fanconi anemia: cytogenetic diagnosis. Free University of Amsterdam, Amsterdam1997Google Scholar, 29Auerbach AD Diagnosis of Fanconi anemia by diepoxybutane analysis.in: Current protocols in human genetics, supplement 37. John Wiley, New York and London2003: 8.7.1-8.7.15Google Scholar, 30Berger R Le Coniat M Gendron MC Fanconi anemia: chromosome breakage and cell cycle studies.Cancer Genet Cytogenet. 1993; 69: 13-16Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 31Seyschab H Friedl R Sun Y Schindler D Hoehn H Hentze S Schroeder-Kurth T Comparative evaluation of diepoxybutane sensitivity and cell cycle blockage in the diagnosis of Fanconi anemia.Blood. 1995; 85: 2233-2237PubMed Google Scholar, 32Heinrich MC Hoatlin ME Zigler AJ Silvey KV Bakke AC Keeble WW Zhi Y Reifsteck CA Grompe M Brown MG et al.DNA cross-linker-induced G2/M arrest in group C Fanconi anemia lymphoblasts reflects normal checkpoint function.Blood. 1998; 91: 275-287PubMed Google Scholar In patients with increasing and/or long-term stable blood counts, the possibility of somatic reversion leading to mosaicism of hematopoietic cells was considered, and cultured fibroblasts were used for mitomycin-C (MMC) sensitivity testing and for complementation studies. A total of 29 fully informative FA-D2 patients (patients 1–29) were included in the present genotype-phenotype study. A fetal case (number 19) and five patients with hematopoietic mosaicism (patients 3, 14, 15, 25, and 26) were excluded from clinical follow-up studies. Four additional FA-D2 patients (patients 30–33) with incomplete clinical data were not part of the phenotype analysis, but results concerning their mutations are indicated in the text, tables, and figures. Three end points were evaluated to determine hematologic severity: time to hematological onset, defined as “cell count of at least one lineage constantly below normal range”; period from BMF to hematological stem-cell transplantation (HSCT); and time to HSCT. Kaplan-Meier estimates were computed for the length of overall survival. Birth was taken as the date of FA onset for all these calculations. Comparisons were made with patients in the International Fanconi Anemia Registry (IFAR), as reported elsewhere,33Kutler DI Auerbach AD Fanconi anemia in Ashkenazi Jews.Fam Cancer. 2004; 3: 241-248Crossref PubMed Scopus (33) Google Scholar by means of log-rank test statistics. Multivariate and competing-risk analyses were not possible because of the limited number of informative patients. Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines (LCLs) were established using cyclosporin A, as described elsewhere.34Neitzel H A routine method for the establishment of permanent growing lymphoblastoid cell lines.Hum Genet. 1986; 73: 320-326Crossref PubMed Scopus (532) Google Scholar All blood-derived cell cultures were maintained in RPMI 1640 medium with GlutaMAX (Gibco) supplemented with 15% fetal bovine serum (FBS) (Sigma). Fibroblast strains were established using standard cell-culture procedures and were propagated in Earle’s minimal essential medium with GlutaMAX (Gibco) and 15% FBS. All cultures were kept in high-humidity incubators in an atmosphere of 5% (v/v) CO2 and, in the case of fibroblasts, 5% (v/v) O2 by replacing ambient air with nitrogen.35Schindler D Hoehn H Fanconi anemia mutation causes cellular susceptibility to ambient oxygen.Am J Hum Genet. 1988; 43: 429-435PubMed Google Scholar MMC treatment was for 48 h at 12 ng/ml (fibroblasts) or 15 ng/ml (LCLs) to cause cell-cycle arrest, or for 12 h at 100 ng/ml to induce monoubiquitination of the protein, FANCD2-L. In some cases, RNA stabilization was achieved by the addition of cycloheximide (CHX) to cell cultures at a final concentration of 250 μg/ml 4.5 h before RNA isolation. For construction of the D2-IRES (internal ribosomal entry site)–neo retroviral expression vector S11FD2IN, the D2-IRES-puro plasmid pMMP-FANCD226Timmers C Taniguchi T Hejna J Reifsteck C Lucas L Bruun D Thayer M Cox B Olson S D’Andrea AD et al.Positional cloning of a novel Fanconi anemia gene, FANCD2.Mol Cell. 2001; 7: 241-248Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar was cut using SalI. The ends were blunted, and the fragment containing the FANCD2 ORF was cut out with EcoRI and was ligated into S11IN, which was cut with BamHI, was blunted, and was cut again with EcoRI (fig. 1). S11 vectors are based on the spleen focus–forming virus and are derived from the GR plasmid.36Hildinger M Abel KL Ostertag W Baum C Design of 5′ untranslated sequences in retroviral vectors developed for medical use.J Virol. 1999; 73: 4083-4089Crossref PubMed Google Scholar Sequencing of the retroviral plasmid S11FD2IN revealed three reported polymorphisms in the FANCD2 ORF—c.1122A→G, c.1509C→T, and c.2141C→T26Timmers C Taniguchi T Hejna J Reifsteck C Lucas L Bruun D Thayer M Cox B Olson S D’Andrea AD et al.Positional cloning of a novel Fanconi anemia gene, FANCD2.Mol Cell. 2001; 7: 241-248Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar—and another silent base substitution, c.3978C→T. Stable retroviral packaging cells were generated by infection of PG13 cells and selection in G418 (Sigma), as described elsewhere.37Hanenberg H Xiao XL Dilloo D Hashino K Kato I Williams DA Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.Nat Med. 1996; 2: 876-882Crossref PubMed Scopus (479) Google Scholar In addition, the cDNAs encoding enhanced green fluorescent protein (GFP) and FANCA cDNAs were separately cloned into the vector S11IN for transduction of the cells under study, with GFP serving to monitor complete selection and FANCA serving as negative complementation control. Retroviral transduction of cultured cells followed published protocols.38Hanenberg H Hashino K Konishi H Hock RA Kato I Williams DA Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells.Hum Gene Ther. 1997; 8: 2193-2206Crossref PubMed Scopus (172) Google Scholar, 39Hanenberg H Batish SD Pollok KE Vieten L Verlander PC Leurs C Cooper RJ Gottsche K Haneline L Clapp DW et al.Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool.Exp Hematol. 2002; 30: 410-420Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Selection of transduced cells was in G418 (Sigma) at a final concentration of 0.8–1.2 mg/ml for ∼10 d. Transduced cells were analyzed for their sensitivity to MMC, with use of flow cytometry, to assess survival rates and cell-cycle arrest.39Hanenberg H Batish SD Pollok KE Vieten L Verlander PC Leurs C Cooper RJ Gottsche K Haneline L Clapp DW et al.Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool.Exp Hematol. 2002; 30: 410-420Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 40Chandra S Levran O Jurickova I Maas C Kapur R Schindler D Henry R Milton K Batish SD Cancelas JA et al.A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients.Mol Ther. 2005; 12: 976-984Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar FANCD2 immunoblotting was performed as described elsewhere,9Garcia-Higuera I Taniguchi T Ganesan S Meyn MS Timmers C Hejna J Grompe M D’Andrea AD Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.Mol Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar with minor modifications. Detection was by the chemiluminescence technique with use of standard enhanced chemoluminescence reagent (Amersham) or SuperSignalWestFemto (Pierce). Primers used for cDNA amplification are shown in table 1, and those additionally used for cDNA sequencing are shown in table 2 (GenBank accession numbers NM_033084 and AF340183).Table 1FANCD2 cDNA Amplification PrimersForwardReversePCR FragmentDesignationBinding PositionSequence (5′→3′)DesignationBinding PositionSequence (5′→3′)PCR Product Size (bp)1FA-D2, Fr.1 F−47 to −27GCGACGGCTTCTCGGAAGTAAFA-D2, Fr.1 R998 to 976CTGTAACCGTGATGGCAAAACAC9982FA-D2, Fr.2 F763 to 787GACCCAAACTTCCTATTGAAGGTTCFA-D2, Fr.2 R1996 to 1975CTACGAAGGCATCCTGGAAATC1,2343FA-D2, Fr.3 F1757 to 1777CGGCAGACAGAAGTGAATCACFA-D2, Fr.3 R2979 to 2958GTTCTTGAGAAAGGGGACTCTC1,2234FA-D2, Fr.4 F2804 to 2829TTCTACATTGTGGACTTGTGACGAAGFA-D2, Fr.4 R3942 to 3922GTCTAGGAGCGGCATACATTG1,1395FA-D2, Fr.5(L) F3761 to 3781CAGCAGACTCGCAGCAGATTCFA-D2, Fr.5(L) R4700 to 4679GACTCTGTGCTTTGGCTTTCAC940 Open table in a new tab Table 2FANCD2 cDNA Sequencing PrimersDesignationBinding Position Sequence (5′→3′)sFA-D2, 244 F244 to 263ACCCTGAGGAGACACCCTTCsFA-D2, 545 F545 to 566GGCTTGACAGAGTTGTGGATGGsFA-D2, 1011 F1011 to 1033CAGCGGTCAGAGCTGTATTATTCsFA-D2, 1308 F1308 to 1327GTCGCTGGCTCAGAGTTTGCsFA-D2, 1574 F1574 to 1596CCCCTCAGCAAATACGAAAACTCsFA-D2, 2142 F2142 to 2162GGTGACCTCACAGGAATCAGGsFA-D2, 2381 F2381 to 2404GAGAGATTGTAAATGCCTTCTGCCsFA-D2, 2679 F2679 to 2699TGACCCTACGCCATCTCATAGsFA-D2, 3268 F3268 to 3288GCCCTCCATGTCCTTAGTAGCsFA-D2, 3573 F3573 to 3594GCACACAGAGAGCATTCTGAAGsFA-D2, 4049 F4049 to 4069ACACGAGACTCACCCAACATGsFA-D2, 4303 F4303 to 4323GAGTCTGGCACTGATGGTTGCsFA-D2, 367 R367 to 347CATCCTGCAGACGCTCACAAGsFA-D2, 621 R621 to 600CAGGTTCTCTGGAGCAATACTGsFA-D2, 951 R951 to 929CTGTAACCGTGATGGCAAAACACsFA-D2, 1158 R1183 to 1158TCTGAGTATTGGTGCTATAGATGATGsFA-D2, 1414 R1414 to 1396CCTGCTGGCAGTACGTGTCsFA-D2, 1704 R1704 to 1684GAATACGGTGCTAGAGAGCTGsFA-D2, 2253 R2253 to 2232CTCCTCCAAGTTTCCGTTATGCsFA-D2, 2526 R2526 to 2505GTTTCCAAGAGGAGGGACATAGsFA-D2, 3346 R3346 to 3328GGACGCTCTGGCTGAGTAGsFA-D2, 3674 R3674 to 3653GTAGGGAATGTGGAGGAAGATGsFA-D2, 4159 R4159 to 4139CCAGCCAGAAAGCCTCTCTACsFA-D2, 4409 R4409 to 4387GGGAATGGAAATGGGCATAGAAG Open table in a new tab A total of seven large amplicons (superamplicons) were generated with primers that are unique to certain regions of the functional FANCD2 gene. These primers and the sizes of the superamplicons I–VII are shown in table 3. The superamplicons served as templates in place of genomic DNA. They were used to amplify the genomic sequence before sequencing; an exception to this was superamplicon V, which was used for direct sequencing. Genomic primers for the amplification of all FANCD2 exons and adjacent intron regions and their sizes are displayed in table 4. Additional genomic mutation-specific primers are shown in table 5.Table 3FANCD2 Superamplicon PrimersForwardReverseSuperampliconContaining Exon(s)DesignationSequence (5′→3′)DesignationSequence (5′→3′)PCR Product Size (bp)I1 and 2hFANCD2_exon1_FTATGCCCGGCTAGCACAGAAhFANCD2_super_1_2_RGGCCCACAGTTTCCGTTTCT4,346II3hFANCD2_super_3_3_FGTGTCACGTGTCTGTAATCTChFANCD2_super_3_3_RCTGGGACTACAGACACGTTTT2,323III7, 8, and 9hFANCD2_super_7_14_FTGGGTTTGGTAGGGTAATGTChFANCD2_exon9_RTACTCATGAAGGGGGGTATCA4,595IV10, 11, 12, 13, and 14hFANCD2_exon10_FGCCCAGCTCTGTTCAAACCAhFANCD2_super_7_14_RTTAAGACCCAGCGAGGTATTC5,635V13, 14, 15, 16, and 17FA-D2, sup13-I17 FCATGGCAGGAACTCCGATCTTGFA-D2, sup13-I17 FCTCCCTTAAAAGCTCAAAGCTCAAGTTC8,858VI19 and 20hFANCD2_super_19_22_FACGTAATCACCCCTGTAATCChFANCD2_exon20_RTGACAGAGCGAGACTCTCTAA2,749VII21, 22, and 23FA-D2, 21_23, FGCTTCTAGTCACTGTCAGTTCACCAGFA-D2, 21_23, RACGTTGGCCAGAAAGTAATCTCAG2,518VIII23, 24, 25, and 26hFANCD2_super_23_29_FGGCCTTGTGCTAAGTGCTTTThFANCD2_exon26_RTCAGGGATATTGGCCTGAGAT3,252IX27, 28, and 29hFANCD2_exon27_FGCATTCAGCCATGCTTGGTAAhFANCD2_super_23_29_RCACTGCAAACTGCTCACTCAA3,371X30hFANCD2_super_30_32_FCCAAAGTACTGGGAGTTTGAGhFANCD2_exon30_RTACCCAGTGACCCAAACACAA2,186XVI31 and 32hFANCD2_exon31_FCCATTGCGAACCCTTAGTTTChFANCD2_super_30_32_RACCCTGGTGGACATACCTTTT299XII33 and 34hFANCD2_super_33_36_FGAGCAATTTAGCCTGTGGTTTThFANCD2_exon34_RTATAGCAAGAGGGCCTATCCA3,457XIII35 and 36hFANCD2_exon35_FTTAGACCGGGAACGTCTTAGThFANCD2_super_33_36_RTCTGGGCAACAGAACAAGCAA2,040XIV43ahFANCD2_super_43_44_FAGGGTCCTGAGACTATATACChFANCD2_exon43a_RAGCATGATCTCGGCTCACCA2,040XV44hFANCD2_exon44_FCACCCAGAGCAGTAACCTAAAhFANCD2_super_43_44_RACCATCTGGCCGACATGGTA464 Open table in a new tab Table 4FANCD2 Exon PrimersForwardReverseExonDesignationSequence (5′→3′)DesignationSequence (5′→3′)PCR Product Size (bp)1hFANCD2_exon1_FTATGCCCGGCTAGCACAGAAhFANCD2_exon1_RTCCCATCTCAGGGCAGATGA3242hFANCD2_exon2_FCCCCTCTGATTTTGGATAGAGhFANCD2_exon2_RTCTCTCACATGCCTCACACAT2583hFANCD2_exon3_FGACACATCAGTTTTCCTCTCAThFANCD2_exon3_RAAGATGGATGGCCCTCTGATT3544hFANCD2_exon4_FTGGTTTCATCAGGCAAGAAACThFANCD2_exon4_RAATCATTCTAGCCCACTCAACT2534/5FA-D2, exon 4 II FGAGAAGGAAAACTATGGTAGGAAACFA-D2, exon 5 II RGTGTAAGCTCTGTTTTCCTCAGAG5095hFANCD2_exon5_FGCTTGTGCCAGCATAACTCTAhFANCD2_exon5_RAGCCCCATGAAGTTGGCAAAA2986hFANCD2_exon6_FGAGCCATCTGCTCATTTCTGThFANCD2_exon6_RGCTGTGCTAAAGCTGCTACAA3417hFANCD2_exon7_FAATCTCGGCTCACTGCAATCThFANCD2_exon7_RCAGAGAAACCAATAGTTTTCAG2808hFANCD2_exon8_FTAGTGCAGTGCCGAATGCATAhFANCD2_exon8_RAGCTAATGGATGGATGGAAAAG3339hFANCD2_exon9_FTTCACACGTAGGTAGTCTTTCThFANCD2_exon9_RTACTCATGAAGGGGGGTATCA32310hFANCD2_exon10_FGCCCAGCTCTGTTCAAACCAhFANCD2_exon10_RCATTACTCCCAAGGCAATGAC229FA-D2, exon10, FGTCTGCCCAGCTCTGTTCAAACFA-D2, exon10, RATTACTCCCAAGGCAATGACTGACTG23211hFANCD2_exon11_FGTGGGAAGATGGAGTAAGAGAhFANCD2_exon11_RAGCTCCATTCTCTCCTCTGAA341FA-D2, exon11, FCAGTTCAGTACAAAGTTGAGGTAGTGFA-D2, exon11, RCCGGATTAGTCAGTATTCTCAGTTAG26712hFANCD2_exon12_FTGCCTACCCACTATGAATGAGhFANCD2_exon12_RTCTGACAGTGGGATGTCAGAA21113hFANCD2_exon13_FCAGGAACTCCGATCTTGTAAGhFANCD2_exon13_RATGTGTCCATCTGGCAACCAT321FA-D2, exon 13 F P1+2CCGATCTTGTAAGTTCTTTTCTGGTACGFA-D2, exon 13 R P1+2TGGCAACCATCAGCTATCATTTCCAC30214hFANCD2_exon14_FCGTGTTTCGCTGATGTGTCAThFANCD2_exon14_RTGGAGGGGGGAGAAAGAAAG18615hFANCD2_exon15a_FGTGTTTGACCTGGTGATGCTThFANCD2_exon15a_RGGAAGGCCAGTTTGTCAAAGT325hFANCD2_exon15b_FGTGGAACAAATGAGCATTATCChFANCD2_exon15b_RCTTATTTCTTAGCACCCTGTCAA204FA-D2, exon 15 F uniqGGAACAAATGAGCATTATCCATTCTGTGFA-D2, exon 15 R/ P1CTCAATGGGTTTGAACAATGGACTG36316hFANCD2_exon16_FAGGGAGGAGAAGTCTGACATThFANCD2_exon16_RTTCCCCTTCAGTGAGTTCCAA332FA-D2, exon 16 F P1GTCTGACATTCCAAAAGGATAAGCAACFA-D2, exon 16 RCTTGAGACCCAGGTCAGAGTTC34417hFANCD2_exon17_FGATGGGTTTGGGTTGATTGTGhFANCD2_exon17_RGATTAGCCTGTAGGTTAGGTAT422FA-D2, exon 17 F P1+2CTGGCATATTCCTAAATCTCCTGAAGFA-D2, exon 17 RGCCTGTAGGTTAGGTATAAAGAAGTG47218hFANCD2_exon18_FGGCTATCTATGTGTGTCTCTTThFANCD2_exon18_RCCAGTCTAGGAGACAGAGCT28219hFANCD2_exon19_FCGATATCCATACCTTCTTTTGChFANCD2_exon19_RACGATTAGAAGGGAACATGGAA32820hFANCD2_exon20_FCACACCAACATGGCACATGTAhFANCD2_exon20_RTGACAGAGCGAGACTCTCTAA23921hFANCD2_exon21_FAAAGGGGCGAGTGGAGTTTGhFANCD2_exon21_RGAGACAGGGTAGGGCAGAAA33922hFANCD2_exon22_FATGCACTCTCTCTTTTCTACTThFANCD2_exon22_RGTAACTTCACCAGTGCAACCAA27923hFANCD2_exon23_FTTCCCTGTAGCCTTGCGTATThFANCD2_exon23_RACAAGGAATCTGCCCCATTCT35624hFANCD2_exon24_FCTCCCTATGTACGTGGAGTAAhFANCD2_exon24_RCCCCACATACACCATGTATTG25825hFANCD2_exon25_FAGGGGAAAGTAAATAGCAAGGAhFANCD2_exon25_RGTGGGACATAACAGCTAGAGA35026hFANCD2_exon26_FGACATCTCTCAGCTCTGGATAhFANCD2_exon26_RTCAGGGATATTGGCCTGAGAT32427hFANCD2_exon27_FGCATTCAGCCATGCTTGGTAAhFANCD2_exon27_RCCAATTACTGATGCCATGATAC32428hFANCD2_exon28_FTCTACCTCTAGGCAGTTTCCAhFANCD2_exon28_RGATTACTCCAACGCCTAAGAG354FA-D2, exon 28 FTCTACCTCTAGGCAGTTTCCAFA-D2, exon 28 RGATTACTCCAACGCCTAAGAG35429hFANCD2_exon29_FCTTGGGCTAGAGGAAGTTGTThFANCD2_exon29_RTCTCCTCAGTGTCACAGTGTT38430hFANCD2_exon30_FGAGTTCAAGGCTGGAATAGCThFANCD2_exon30_RTACCCAGTGACCCAAACACAA348FA-D2, exon 30 FCATGAAATGACTAGGACATTCCTGFA-D2, exon 30 FGCAAGATGAATATTGTCTGGCAATACG31931hFANCD2_exon31_FCCATTGCGAACCCTTAGTTTChFANCD2_exon31_RACCGTGATTCTCAGCAGCTAA34132hFANCD2_exon32_FCCACCTGGAGAACATTCACAAhFANCD2_exon32_RAGTGCCTTGGTGACTGTCAAA33633hFANCD2_exon33_F
Referência(s)