A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two‐thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA‐null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype–phenotype correlations.     

范可尼贫血患者FANCA突变子的构建及其功能

目的研究范可尼贫血（FA）患者FANCA蛋白的表达及其突变子的功能。方法用3例来源于FA-A型患者的外周血淋巴细胞建立的细胞系作为研究对象，分别提取其细胞总蛋白、细胞胞质蛋白和细胞核蛋白，用Westernblot法分析FANCA蛋白的表达及其在细胞胞质和细胞核内的分布，对1例有FANCA截短型蛋白表达的FA患者，构建了质粒突变子并用哺乳动物细胞双杂交方法检测FANCA基因突变子（外显子5缺失）与FANCG蛋白的相互作用。结果用兔抗人抗体检测时，3例FA-A患者均无FANCA蛋白的表达，而用鼠抗人抗体检测时，有1例患者可检测到截短型FANCA蛋白表达，但此截短型FANCA蛋白不能从细胞胞质转运到细胞核，也不能在哺乳动物细胞双杂交系统中与FANCG蛋白相互作用。结论3例FA-A患者中，2例无FANCA蛋白表达，1例可表达截短型FANCA蛋白，但无正常FANCA蛋白功能，进一步证实其FANCA基因突变为致病性病理突变，FANCA基因外显子5参与了与FANCG的相互作用。     

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.     

FAA基因治疗及其基因表达调控序列新型重组载体的构建、鉴定

目的：Y国探索新型逆转录病毒载体Lentivector及其基因表达调控序列在造血细胞中的有效表达和基因治疗中的应用,用分子克隆的方法构建Fanconis Anemis(FA)患者A组基因（FANCA基因）基因治疗新型重组载体（Lentivector)和基因表达调控序列（启动子/增强子）,进一步通过内切酶酶切位点鉴定插入的基因表达调控序列方向和目的基因的片段大小、目的原基因特异引物PCR扩增特异片段鉴定目的基因。方法：首先次质粒Friend基因表达调控序列方向和目的基因的片段大小、目的基因特异引物PCR扩增特异片段鉴定目的基因。方法：首先将质粒Friend基因表达调控序列（Fr-MuLV E/P)强启动子/增强子插入载体Lentivector pRRLsin-18中相应克隆位点,成为pRRLsin-18FrMuLV(test1);用NotI、NcoI双酶 切载体FochA-FANCA,低溶点胶回收FANCA目的基因片段;应用polylinker、adapter方法多步克隆插入新型载体Lentivector pRRLsin-18(test1)中相应特异克隆位点,获得重组载体sin-18FrMuLVE/P-FA(test2),通过酶切鉴定重组载体插入的基因表达调控序列方向和目的基因的片段大小和方向,筛选正向阳性重组质粒,用PCR法进一步证实插入的FANCA基因片段。结果：挑取的阳性克隆经多个酶酶切鉴定,有Friend质粒390bp基因表达调控序列（Fr-MuLVE/P)和4.5kbFANCA目的基因片段酶切位点,用PCR引物扩增出目的基因相应片段,证明为正向重组体。结论：已成功构建表达FANCA基因的重组逆转录病毒载体及其基因表达调控序列,为进一步应用FANCA基因的新型重组载体Lentivector经包装后转染造血干细胞及其表达情况和相应的FAA基因治疗奠定了良好的工作基础。     

Founder Haplotype Analysis of Fanconi Anemia in the Korean Population Finds Common Ancestral Haplotypes for a FANCG Variant

A common ancestral haplotype is strongly suggested in the Korean and Japanese patients with Fanconi anemia (FA), because common mutations have been frequently found: c.2546delC and c.3720_3724delAAACA of FANCA; c.307+1G>C, c.1066C>T, and c.1589_1591delATA of FANCG. Our aim in this study was to investigate the origin of these common mutations of FANCA and FANCG. We genotyped 13 FA patients consisting of five FA‐A patients and eight FA‐G patients from the Korean FA population. Microsatellite markers used for haplotype analysis included four CA repeat markers which are closely linked with FANCA and eight CA repeat markers which are contiguous with FANCG. As a result, Korean FA‐A patients carrying c.2546delC or c.3720_3724delAAACA did not share the same haplotypes. However, three unique haplotypes carrying c.307+1G>C, c.1066C > T, or c.1589_1591delATA, that consisted of eight polymorphic loci covering a flanking region were strongly associated with Korean FA‐G, consistent with founder haplotypes reported previously in the Japanese FA‐G population. Our finding confirmed the common ancestral haplotypes on the origins of the East Asian FA‐G patients, which will improve our understanding of the molecular population genetics of FA‐G. To the best of our knowledge, this is the first report on the association between disease‐linked mutations and common ancestral haplotypes in the Korean FA population.     

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 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.     

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.     

Evaluation of Fanconi anaemia genes FANCA, FANCC and FANCL in cervical cancer susceptibility

Objective

Disrupting the function of any of the 13 Fanconi anaemia (FA) genes causes a DNA repair deficiency disorder, with patients being susceptible to a number of cancer types. Variation in the family of FA genes has been suggested to affect risk of cervical cancer. The current study evaluates the influence of three genes in the FA pathway on cervical cancer risk in Swedish women.

Methods

TagSNPs in FANCA, FANCC and FANCL were selected using the Tagger algorithm in Haploview. A total of 81 tagSNPs were genotyped in 782 cases (CIN3 or ICC) and 775 controls using the Illumina GoldenGate Assay and statistically analyzed for association with cervical cancer.

Results

72 SNPs were successfully genotyped in > 98% of the samples. Nominal associations were detected for FANCA rs11649196 (p = 0.05) and rs4128763 in FANCC (p = 0.02). The associations did not withstand correction for multiple testing.

Conclusions

The current study does not support that genetic variation in FANCA, FANCC or FANCL genes affects susceptibility to cervical cancer in the Swedish population.