FANCA and FANCG are the major Fanconi anemia genes in the Korean population

Fanconi anemia (FA) is a rare disorder characterized by physical abnormalities, bone marrow failure (BMF), increased risk of malignancies, and cellular hypersensitivity to DNA cross‐linking agents. This study evaluated the genetic alterations in three major Fanconi genes (FANCA, FANCC, and FANCG) in 30 FA patients using multiplex ligation‐dependent probe amplification and direct sequencing. Thirteen BMF patients were genetically classified as FA‐A (n = 6, 46%) and FA‐G (n = 7, 54%). Four common founder mutations were identified and included two FANCA mutations (c.2546delC and c.3720_3724delAAACA) and two FANCG mutations (c.307+1G>C and c.1066C>T), which had previously been commonly observed in a Japanese FA population. We also detected four novel deleterious mutations: c.2778+1G>C and c.3627‐1G>A of FANCA, and c.1589_1591delATA and c.1761‐1G>A of FANCG. This study shows that mutations in FANCA and FANCG are common in Korean FA patients and the existence of four common founder mutations in an East Asian FA population. Mutation screening workflow that includes these common mutations may be useful in the creation of an international database, and to better understand the ethnic characteristics of FA.     

Novel mutations of the FANCG gene causing alternative splicing in Japanese Fanconi anemia

Fanconi anemia (FA), an autosomal recessive disorder characterized by a progressive pancytopenia associated with congenital anomalies and high predisposition to malignancies, is a genetically and clinically heterogeneous disease. At least eight complementation groups (FA-A to FA-H) have been identified. Previously, we studied mutations of the FANCA gene, responsible for FA-A, and found pathogenic mutations in 12 of 15 unclassified Japanese FA patients. Here, we further studied an additional 5 FA patients for sequence alterations of the FANCA gene and found pathogenic mutations in 2 of them. We further analyzed mutations of the FANCC and FANCG genes, responsible for FA-C and FA-G, respectively, in the remaining 6 FA patients. Although there was no alterations in the FANCC gene in these 6 patients, two novel mutations of the FANCG gene, causing aberrant RNA splicing, were detected in 2 FA patients. One was a base substitution from G to C of the invariant GT dinucleotides at the splice donor site of intron 3, resulting in the skipping of exon 3, as well as the skipping of exons 3 and 4. The other was a base substitution from C to T in exon 8, creating a nonsense codon (Q356X). This mutation resulted in the exclusion of a sequence of 18 nucleotides containing the mutation from the mRNA, without affecting the splicing potential of either the authentic or the cryptic splice donor site. Collectively, 14 of the 20 unclassified Japanese FA patients belong to the FA-A group, 2 belong to the FA-G group, and none belongs to the FA-C group. Received: December 6, 1999 / Accepted: January 15, 2000     

Spectrum of sequence variations in the FANCA gene: an International Fanconi Anemia Registry (IFAR) study

Fanconi anemia (FA) is an autosomal recessive disorder that is defined by cellular hypersensitivity to DNA cross-linking agents, and is characterized clinically by developmental abnormalities, progressive bone-marrow failure, and predisposition to leukemia and solid tumors. There is extensive genetic heterogeneity, with at least 11 different FA complementation groups. FA-A is the most common group, accounting for approximately 65% of all affected individuals. The mutation spectrum of the FANCA gene, located on chromosome 16q24.3, is highly heterogeneous. Here we summarize all sequence variations (mutations and polymorphisms) in FANCA described in the literature and listed in the Fanconi Anemia Mutation Database as of March 2004, and report 61 novel FANCA mutations identified in FA patients registered in the International Fanconi Anemia Registry (IFAR). Thirty-eight novel SNPs, previously unreported in the literature or in dbSNP, were also identified. We studied the segregation of common FANCA SNPs in FA families to generate haplotypes. We found that FANCA SNP data are highly useful for carrier testing, prenatal diagnosis, and preimplantation genetic diagnosis, particularly when the disease-causing mutations are unknown. Twenty-two large genomic deletions were identified by detection of apparent homozygosity for rare SNPs. In addition, a conserved SNP haplotype block spanning at least 60 kb of the FANCA gene was identified in individuals from various ethnic groups.     

Comprehensive Analysis of Pathogenic Deletion Variants in Fanconi Anemia Genes

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high‐resolution comparative genome hybridization arrays, single‐nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy‐five percent of the FANCA deletions are Alu–Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.     

Spectrum of sequence variation in the FANCG gene: an International Fanconi Anemia Registry (IFAR) study

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA cross-linking agents, and predisposition to malignancy. The gene for FA complementation group G (FANCG) was the third FA gene to be cloned, and was found to be identical with human XRCC9, which maps to 9p13. The cDNA is predicted to encode a polypeptide of 622 amino acids, with no sequence similarities to any other known protein or motifs that could point to a molecular function for FANCG/XRCC9. We used single strand conformational polymorphism analysis (SSCP) to screen genomic DNA from a panel of 307 racially and ethnically diverse unrelated FA patients from the International Fanconi Anemia Registry (IFAR) for variants in FANCG. Twenty-seven abnormal SSCP patterns were found; 18 of these variants appear to be pathogenic mutations while nine are likely to be nonpathogenic polymorphisms. Direct sequencing of genomic DNA from seven FA-G probands with one mutant allele not detected in the SSCP study and three additional probands assigned to the FA-G complementation group by retroviral correction with FANCG resulted in the detection of nine additional pathogenic mutations and two common SNPs. Conditions for rapid screening for these mutations by DHPLC for use in a clinical laboratory setting were established. The most common FANCG mutations in the IFAR population were: IVS8-2A>G (seven Portuguese-Brazilian probands), IVS11+1G>C (seven French-Acadian probands), 1794_1803del10 (seven European probands), and IVS3+1G>C (five Korean or Japanese probands). Our data suggest that the Portuguese-Brazilian, French-Acadian, and Korean/Japanese mutations were likely to have been present in a founding member of each of these populations.     

Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia

Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation‐carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.     

Two common founder mutations of the fanconi anemia group G gene FANCG/XRCC9 in the Japanese population

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis with eight complementation groups (FA-A, B, C, D1, D2, E, F and G). To date, seven of the FA genes have been identified. Although extensive analyses in Western countries revealed that the subgroup prevalence and mutational spectrum vary depending on the ethnic background, not much data is available on Asian populations. In the present study, 45 unrelated FA families in Japan were screened for FA gene mutations and 10 families with biallelic pathogenic mutations of FANCG/XRCC9, the gene for FA-G, were identified. A splice mutation IVS3+1G>C was detected in all 9 Japanese families, among whom 4 were homozygous and 5 were heterozygous. Among the heterozygotes, three carried 1066C>T in the second allele. In addition, a family homozygous for 1066C>T with Korean ethnicity was identified. Haplotype analysis by means of 9 microsatellite markers spanning the FANCG locus indicates that IVS3+1G>C and 1066C>T are in complete association with distinct ancestry haplotypes. Our data suggest that IVS3+1G>C arose in the Japanese ancestors at a relatively early time, whereas 1066C>T later on migrated from Korea. The two founder mutations with distinct origins account for most of FANCG mutant alleles in the Japanese population.     

Spectrum of FANCA mutations in Italian Fanconi anemia patients: identification of six novel alleles and phenotypic characterization of the S858R variant

Fanconi anemia (FA) is an autosomal recessive disorder characterized by genomic instability, bone marrow failure, congenital malformations, and cancer predisposition. FA is a genetically heterogeneous disease with at least seven genes so far identified. The role of FA proteins is unknown although they interact in a common functional pathway. Here, we report six novel FANCA sequence changes and review all the mutations identified in Italy. Except for two missense substitutions, all are expected to cause a premature termination of the FANCA protein at various sites throughout the molecule. The premature terminations are due to nonsense and splice site mutations, as well as small insertions and deletions, and large genomic rearrangements. The expected truncated proteins were not detectable on Western blot analyses. The FANCA-S858R variant is instead expressed at lower level than that seen in normal cell lines and is associated with a non-ubiquinated FANCD2 protein, strongly suggesting that the amino acid substitution is a disease-causing mutation. The spectrum of FA mutations is widely in agreement with the heterogeneous ethnic origin of the Italian population.     

Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA.     

Analysis of the Novel Fanconi Anemia Gene SLX4/FANCP in Familial Breast Cancer Cases

SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers—BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2‐negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C‐induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.     

Factors Affecting the Outcome of Related Allogeneic Hematopoietic Cell Transplantation in Patients with Fanconi Anemia

Hematopoietic cell transplantation (HCT) can cure bone marrow failure in patients with Fanconi Anemia (FA), and it is generally accepted that these patients should receive low-intensity conditioning because of the underlying DNA repair defect in their cells. Outcomes for recipients of matched related HCT have generally been favorable, but only a few studies have scrutinized the factors that may affect the eventual outcome of these patients. This retrospective analysis of 94 pediatric patients with FA who underwent related HCT at King Faisal Specialist Hospital & Research Center was carried out to attempt to identify factors that may affect outcome. Results showed overall survival (OS) probabilities of 92.5%, 89%, and 86% at 1, 5, and 10 years, respectively. In univariate analysis, use of higher dose cyclophosphamide (CY) (60 mg/kg) conditioning was associated with a better 10-year OS than lower dose CY (20 mg/kg) conditioning (91% versus 82%, respectively; P = .035), and use of radiation-containing regimens was associated with a significantly lower 10-year OS than nonradiation regimens (76% versus 91%, respectively; P = .005). Of the 4 regimens used in this study, the fludarabine-based regimen was associated with the highest survival (95.2%; P = .034). The use of the higher dose CY (60 mg/kg) was associated with a significantly increased incidence of hemorrhagic cystitis (HC) (20% versus 5.6% respectively; P = .049). Three patients (3%) developed squamous cell carcinoma (2 oropharyngeal and 1 genitourinary), at 9.4, 5.4, and 13.3 years after HCT; 2 of them had radiation-containing conditioning. In conclusion, our data suggest that although using a higher dose CY (60 mg/kg) conditioning regimen may be associated with better survival, it is also associated with a significantly increased risk of HC. The addition of fludarabine to the low-dose CY (20 mg/kg) is associated with the best survival. On the other hand, radiation-containing regimens are associated with significantly lower survival.     

Evaluation of Rare Variants in the New Fanconi Anemia Gene ERCC4 (FANCQ) as Familial Breast/Ovarian Cancer Susceptibility Alleles

Recently, it has been reported that biallelic mutations in the ERCC4 (FANCQ) gene cause Fanconi anemia (FA) subtype FA‐Q. To investigate the possible role of ERCC4 in breast and ovarian cancer susceptibility, as occurs with other FA genes, we screened the 11 coding exons and exon–intron boundaries of ERCC4 in 1573 index cases from high‐risk Spanish familial breast and ovarian cancer pedigrees that had been tested negative for BRCA1 and BRCA2 mutations and 854 controls. The frequency of ERCC4 mutation carriers does not differ between cases and controls, suggesting that ERCC4 is not a cancer susceptibility gene. Interestingly, the prevalence of ERCC4 mutation carriers (one in 288) is similar to that reported for FANCA, whereas there are approximately 100‐fold more FA‐A than FA‐Q patients, indicating that most biallelic combinations of ERCC4 mutations are embryo lethal. Finally, we identified additional bone‐fide FA ERCC4 mutations specifically disrupting interstrand cross‐link repair.     

Haplotype Structure of FSHB, the Beta-Subunit Gene for Fertility-Associated Follicle-Stimulating Hormone: Possible Influence of Balancing Selection

Follicle-stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor-binding beta-subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76-96.6% of each population's sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organism's fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.     

Evidence of a Common Founder for SCA12 in the Indian Population

Spinocerebellar ataxia type 12 (SCA12) is an autosomal dominant cerebellar ataxia associated with the expansion of an unstable CAG repeat in the 5′ region of the PPP2R2B gene on chromosome 5q31–5q32. We found that it accounts for ～16% (20/124) of all the autosomal dominant ataxia cases diagnosed in AIIMS, a major tertiary referral centre in North India. The length of the expanded allele in this population ranges from 51–69 CAG triplets. Interestingly, all the affected families belong to an endogamous population, which originated in the state of Haryana, India. We identified four novel SNPs and a dinucleotide marker spanning ～137 kb downstream of CAG repeat in the PPP2R2B gene. Analysis of 20 Indian SCA12 families and ethnically matched normal unrelated individuals revealed one haplotype to be significantly associated with the affected alleles (P= 0.000), clearly indicating the presence of a common founder for SCA12 in the Indian population. This haplotype was not shared by the American pedigree with SCA12. Therefore, the SCA12 expansion appears to have originated at least twice.     

A founder variant in the South Asian population leads to a high prevalence of FANCL Fanconi anemia cases in India

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease‐causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.     

Genetic Variations and Haplotype Structures of the ABCB1 Gene in a Japanese Population: An Expanded Haplotype Block Covering the Distal Promoter Region, and Associated Ethnic Differences

As functional ABCB1 haplotypes were recently reported in the promoter region of the gene, we resequenced the ABCB1 distal promoter region, along with other regions (the enhancer and proximal promoter regions, and all 28 exons), in a total of 533 Japanese subjects. Linkage disequilibrium (LD) analysis based on 92 genetic variations revealed 4 LD blocks with the same make up as previously described (Blocks ?1, 1, 2 and 3), except that Block 1 was expanded to include the distal promoter region, and that a new linkage between polymorphisms ?1789G>A in the distal promoter region and IVS5 + 123A>G in intron 5 was identified. We re‐assigned Block 1 haplotypes, and added novel haplotypes to the other 3 blocks. The reported promoter haplotypes were further classified into several types according to tagging variations within Block 1 coding or intronic regions. Our current data reconfirm the haplotype profiles of the other three blocks, add more detailed information on functionally‐important haplotypes in Block 1 and 2 in the Japanese population, and identified differences in haplotype profiles between ethnic groups. Our updated analysis of ABCB1 haplotype blocks will assist pharmacogenetic and disease‐association studies carried out using Asian subjects.     

A Multicenter Retrospective Analysis of the Clinical Features of Pernicious Anemia in a Korean Population

To determine the approximate incidence and clinical features of pernicious anemia in a Korean population, we retrospectively analyzed clinical data for patients with pernicious anemia who were diagnosed between 1995 and 2010 at five hospitals in Chungnam province. Ninety-seven patients were enrolled, who accounted for 24% of patients with vitamin B₁₂ deficiency anemia. The approximate annual incidence of pernicious anemia was 0.3 per 100,000. The median age was 66 (range, 32-98) yr, and the male/female ratio was 1.25. Anemia-associated discomfort was the most common symptom (79.4%), followed by gastrointestinal and neurological symptoms (78.4% and 38.1%, respectively). Pancytopenia was found in 36 patients (37.1%), and autoimmune disorders were found in 15 patients (15.5%). Antibody to intrinsic factor was detected in 62 (77.5%) of 80 patients examined, and antibody to parietal cells was detected in 35 (43.2%) of 81 patients examined. Of the 34 patients who underwent tests for Helicobacter pylori, 7 (12.5%) were positive. The anemia-associated and gastrointestinal symptoms resolved completely in all patients after intramuscular injection of cobalamin, whereas neurological symptoms remained in some. In conclusion, pernicious anemia is less frequent in Koreans than in Western populations; however, the clinical features of this disorder in Koreans do not differ from those of Western cases.     

Association of Methylenetetrahydrofolate Reductase (MTHFR 677C>T and 1298A>C) Polymorphisms and Haplotypes with Silent Brain Infarction and Homocysteine Levels in a Korean Population

Purpose

Methylenetetrahydrofolate reductase (MTHFR) is the main regulatory enzyme for homocysteine metabolism. In the present study, we evaluated whether the MTHFR 677C>T and 1298A>C gene polymorphisms are associated with SBI and plasma homocysteine concentration in a Korean population.

Materials and Methods

We enrolled 264 patients with SBI and 234 healthy controls in South Korea. Fasting plasma total homocysteine (tHcy) concentrations were measured, and genotype analysis of the MTHFR gene was carried out.

Results

The plasma tHcy levels were significantly higher in patients with SBI than in healthy controls. Despite a significant association between the MTHFR 677TT genotype and hyperhomocysteinemia, the MTHFR 677C>T genotypes did not appear to influence susceptibility to SBI. However, odds ratios of the 1298AC and 1298AC + CC genotypes for the 1298AA genotype were significantly different between SBI patients and normal controls. The frequencies of 677C-1298A and 677C-1298C haplotypes were significantly higher in the SBI group than in the control group.

Conclusion

This study demonstrates that the MTHFR 1298A>C polymorphism is a risk factor for SBI in a Korean population. The genotypes of 677C>T and 1298A>C polymorphisms interact additively, and increase the risk of SBI in Korean subjects.