Expanded roles of the Fanconi anemia pathway in preserving genomic stability

Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle.     

Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

The maintenance of genome stability is critical for survival, and its failure is often associated with tumorigenesis. The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links (ICLs), and a germline defect in the pathway results in FA, a cancer predisposition syndrome driven by genome instability. Central to this pathway is the monoubiquitination of FANCD2, which coordinates multiple DNA repair activities required for the resolution of ICLs. Recent studies have demonstrated how the FA pathway coordinates three critical DNA repair processes, including nucleolytic incision, translesion DNA synthesis (TLS), and homologous recombination (HR). Here, we review recent advances in our understanding of the downstream ICL repair steps initiated by ubiquitin-mediated FA pathway activation.     

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj^−/− mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2^−/− mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway.     

Analysis of 65 Turkish patients with congenital aplastic anemia (Fanconi anemia and non-Fanconi anemia): Hacettepe experience

During the last 14 years, 65 unrelated patients were diagnosed as having constitutional aplastic anemia (CAA). In 52 of 65 patients the diepoxybutane (DEB) test was positive. Comparison of several hematological and clinical parameters in Fanconi anemia (FA) (DEB⁺) and non-Fanconi anemia (non-FA) (DEB^-) patients disclosed no statistically significant differences. The study indicated that in Turkey there were no peculiarities in associated congenital abnormalities in FA and non-FA. The rate of consanguinity was 78% in FA and 46% in non-FA, suggesting that also among the non-FA group recessively inherited disorders are hidden. The mean age at diagnosis in FA was 7.7 ± 4.4 (1.8–12) and in non-FA 7.8 ± 3.8 (2–15) years. Nine out of 52 FA and five out of 13 non-FA patients died during the follow-up period. Five of the 52 FA patients developed malignancies, three of them had acute myeloblasts leukemia (AML), one a squamous cell carcinoma of the gingiva, and another a hepatocellular carcinoma. Peliosis hepatica occurred in three of the FA and one of the non-FA patients. A total of seven patients stayed in remission without any medication. The remaining 58 patients were given 2–5 mg/kg of oxymetholone and 5 mg prednisolone treatment. Because of sustained remission, oxymetholone therapy was terminated in four of the 45 FA and two of the 13 non-FA patients. Detailed examination of the pedigrees of all of patients indicated the presence of multiple congenital anomalies. In seven of 52 FA and one of 13 non-FA patients there was increased risk for AML and/or other cancers among family members.     

Cytogenetic differentiation of Fanconi anemia, “idiopathic” aplastic anemia,and Fanconi anemia heterozygotes

We have analyzed chromosome breaks in 8 patients with Fanconi anemia (FA), 42 with “idiopathic” aplastic anema (AA), 15 first-degree relatives of FA patients, and 13 controls. Their lymphocytes were treated in culture with three concentrations of mitomycin-C (MMC). A 60-fold increase in breaks was observed in FA patients as compared to AA patients, regardless of severity of clinical signs. The MMC-stress test was standardized to clearly differentiate FA from other pancytopenias in doubtful cases. Also, the effect of storage of MMC in solution was investigated. The data on SCEs of 12 subjects tested, 10 mo apart, showed an inverse relationship between length of storage of MMC and chromosome damage. The 10-month-old solution induced only one half as many SCEs as it induced at 4 months. Further, the usefulness and power of diepoxybutane (DEB) in detection of FA heterozygotes was investigated in 12 first-degree relatives of patients with Fanconi anemia and 12 healthy controls. The mean number of chromosome breaks per mitosis by DEB stress in obligate heterozygotes was 0.08 in comparison to 0.06 in controls. Four of twelve control subjects showed proportions of breaks almost identical to or higher than those of FA heterozygotes, ie, 0.12, 0.10, 0.10, and 0.11 breaks per mitosis. The responses of healthy controls to DEB could be separated into two groups: one with mean chromosome breaks of 0.11 per mitosis, and a second with mean breaks of 0.04 per mitosis. Thus, it appears that heterozygote detection by DEB stress of cultured lymphocytes is not unequivocal.     

Prenatal diagnosis of Fanconi anemia

Prenatal diagnosis was performed on a fetus at risk for Fanconi anemia. A high spontaneous (0.30 breaks/cell) and diepoxybutane-induced (0.69 breaks/cell) chromosome breakage rate indicated an affected fetus and the pregnancy was terminated. The anatomic findings in the aborted fetus together with cytogenetic findings in cultured fetal skin fibroblasts confirmed the prenatal diagnosis.     

Risk of cancer in heterozygous relatives of patients with Fanconi anemia

PurposeFanconi anemia (FA) is a cancer-prone inherited bone marrow failure syndrome caused by biallelic pathogenic variants in one of >22 genes in the FA/BRCA DNA repair pathway. A major concern is whether the risk of cancer is increased in individuals with a single pathogenic FA gene variant.MethodsWe evaluated the risk of cancer in the relatives of patients with FA in the National Cancer Institute Inherited Bone Marrow Failure Syndrome cohort. We genotyped all available relatives and determined the rates, types of cancer and the age of patients at cancer diagnosis. We calculated the observed-to-expected (O/E) cancer ratios using data from the Surveillance, Epidemiology, and End Results Program adjusted for age, sex, and birth cohort.ResultsThe risk of cancer was not increased among all FA relatives and FA heterozygotes (O/E ratios of 0.78 and 0.79, respectively). In particular, the risk of cancer was not increased among FANCA or FANCC heterozygotes (O/E ratios of 0.92 and 0.71, respectively). Relatives did not have typical FA cancers, and age at cancer diagnosis was not younger than expected.ConclusionUnderstanding the risk of cancer in individuals with single pathogenic FA variants is critical for counseling and management. We did not find increased risk of cancer in these individuals. These findings do not extend to the known cancer predisposition autosomal dominant FA genes, namely BRCA1, BRCA2, PALB2, BRIP1, and RAD51C.     

An atypical case of Fanconi anemia in elderly sibs

We describe a 56-year-old woman suspected of Fanconi anemia on the basis of the following clinical findings: microcephaly, short stature, congenital deafness, and the clinical findings in her deceased brother. Hematologic or other signs of malignancy were absent. The diagnosis was confirmed by demonstrating hypersensitivity of her lymphocytes to mitomycin C (MMC). Cell fusion experiments indicated that the patient belongs to complementation group A. The patient's brother died at the age of 50 of heart and renal failure, and anemia. He had clinical findings similar to those of his sister, and a horseshoe kidney. From 31 years on he had thrombocytopenia and leucopenia. Both patients had insulin-dependent diabetes mellitus. A chromosomal breakage test carried out elsewhere before his death failed to demonstrate MMC hypersensitivity of his lymphocytes, which led to the investigation of his sister. To our knowledge these two cases are the oldest Fanconi anemia patients reported thus far. Am. J. Med. Genet. 68:362–366, 1997. © 1997 Wiley-Liss, Inc.     

Diagnosis of Fanconi anemia in patients without congenital malformations: An international Fanconi anemia registry study

Data were analyzed from 419 Fanconi anemia (FA) patients enrolled in the American Registry of the International Fanconi Anemia Registry (IFAR) to determine whether Fanconi anemia (FA) patients without major congenital malformations (CM) have distinguishing characteristics that can lead to an earlier diagnosis. These included 377 patients reported by physicians to the IFAR and 42 patients examined by us. The number of FA patients in each group without CM was 128 and 16, respectively; one third of all patients lacked CM. We found that height, weight, and head circumference were ≥5th centile in 26.6%, 18.0%, and 8.6% of FA patients without CM referred to the IFAR, and in 43.8%, 25.0%, and 43.8% of FA patients without CM examined by us. Minor anomalies were reported in 9.4% of FA patients without CM referred to the IFAR and 100% of FA patients without CM examined by us. Most FA patients without CM have alterations in growth parameters, skin pigmentation abnormalities, or microphthalmia. Increased awareness of the complete spectrum of FA by clinicians will enable an earlier diagnosis to be made. Am. J. Med. Genet. 68:58–61, 1997 © 1997 Wiley-Liss, Inc.     

淋巴瘤细胞系中范可尼贫血/BRCA途径缺陷的探讨

目的 寻找维持基因稳定性的范可尼贫血(Fanconi anemia,FA)/BRCA途径发生缺陷与T和B细胞淋巴瘤潜在发病机制的关系.方法 筛选19种来源于不同亚型的淋巴瘤细胞系,Western印迹分析相关FA蛋白的表达,细胞生长抑制实验检测丝裂霉素C(mitomycin C,MMC)敏感性,流式细胞仪检测MMC诱导的细胞周期阻滞,染色体断裂试验和DNA测序检测基因的突变.结果 在两种淋巴瘤细胞系HT和Sudhl4中,存在FANCN蛋白表达缺失,这种缺失与MMC诱导的G2期细胞阻滞、细胞生长抑制增强和较高的染色体断裂发生率结果一致.DNA测序发现HT细胞系中,FANCN基因外显子5a片段中存在点突变C.1769 C>T,P.A590V;Sudhl4细胞系中未发现FANCN的任何突变.结论 EANCN基因C.1769 C>T突变可能是导致FANCN蛋白表达缺失或者是使该蛋白不稳定及功能丧失的原因.     

Fanconi anaemia: genetics,molecular biology,and cancer – implications for clinical management in children and adults

Fanconi anaemia (FA) is an inherited disease with congenital and developmental abnormalities, cross‐linker hypersensitivity and extreme cancer predisposition. With better understanding of the genetic and molecular basis of the disease, and improved clinical management, FA has been transformed from a life‐limiting paediatric disease to an uncommon chronic condition that needs lifelong multidisciplinary management, and a paradigm condition for the understanding of the gene‐environment interaction in the aetiology of congenital anomalies, haematopoiesis and cancer development. Here we review genetic, molecular and clinical aspects of FA, and discuss current controversies and future prospects.     

VACTERL with hydrocephalus in twins due to Fanconi anemia (FA): Mutation in the FAC gene

We present a dizygotic twin pair each with ventriculomegaly, a radial ray defect and multiple malformations in keeping with the VACTERL association. Molecular studies demonstrated that both are homozygous for IVS4 + 4 A → T, a mutation in the Fanconi anemia complementation group C gene. This is the first molecular proof that VACTERL with hydrocephalus may be the result of severe Fanconi anemia. Am. J. Med. Genet. 68:86–90, 1997 © 1997 Wiley-Liss, Inc.     

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.     

The Ashkenazi Jewish Fanconi anemia mutation: Incidence among patients and carrier frequency in the at-risk population

Fanconi anemia (FA) is an autosomal recessive disease for which at least four complementation groups exist. Recently the gene that corrects the defect in Fanconi anemia complementation group C cells (FACC) has been cloned. We have previously identified a common mutation in the FACC gene, which accounts for a majority of FA cases in Ashkenazi Jewish individuals. We here describe the use of allele-specific oligonucleotide (ASO) hybridization to determine the frequency of this mutation among additional Jewish FA patients and to determine the carrier frequency in the Jewish population. The common IVS4 + 4A → T allele was found on 19/23 (83%) Jewish FA chromosomes, indicating that it is indeed responsible for most cases of FA among Ashkenazi Jews. The carrier frequency was 2/314 for Jewish individuals and the mutant allele was not detected in 130 non-Jewish controls. © 1994 Wiley-Liss, Inc.     

Genetic subtyping of Fanconi anemia by comprehensive mutation screening

Fanconi anemia (FA) is a recessively inherited syndrome with predisposition to bone marrow failure and malignancies. Hypersensitivity to cross-linking agents is a cellular feature used to confirm the diagnosis. The mode of inheritance is autosomal recessive (12 subtypes) as well as X-linked (one subtype). Most genetic subtypes have initially been defined as "complementation groups" by cell fusion studies. Here we report a comprehensive genetic subtyping approach for FA that is primarily based on mutation screening, supplemented by protein expression analysis and by functional assays to test for pathogenicity of unclassified variants. Of 80 FA cases analyzed, 73 (91%) were successfully subtyped. In total, 92 distinct mutations were detected, of which 56 were novel (40 in FANCA, eight in FANCC, two in FANCD1, three in FANCE, one in FANCF, and three in FANCG). All known complementation groups were represented, except D2, J, L, and M. Three patients could not be classified because proliferating cell cultures from the probands were lacking. In cell lines from the remaining four patients, immunoblotting was used to determine their capacity to monoubiquitinate FANCD2. In one case FANCD2 monoubiquitination was normal, indicating a defect downstream. In the remaining three cases monoubiquitination was not detectable, indicating a defect upstream. In the latter four patients, pathogenic mutations in a known FA gene may have been missed, or these patients might represent novel genetic subtypes. We conclude that direct mutation screening allows a molecular diagnosis of FA in the vast majority of patients, even in cases where growing cells from affected individuals are unavailable. Proliferating cell lines are required in a minority (<15%) of the patients, to allow testing for FANCD2 ubiquitination status and sequencing of FANCD2 using cDNA, to avoid interference from pseudogenes.     

A male newborn with VACTERL association and Fanconi anemia with a FANCB deletion detected by array comparative genomic hybridization (aCGH)

We report on a male newborn with multiple congenital abnormalities consistent with the diagnosis of VACTERL association (vertebral, anal, cardiac, tracheo-esophageal fistula, renal, and limb anomalies), who had Fanconi anemia (complementation group B) recognized by the detection of a deletion in chromosome Xp22.2 using an oligonucleotide array. The diagnosis of Fanconi anemia was confirmed by increased chromosomal breakage abnormalities observed in cultured cells that were treated with cross-linking agents. This is the first report in the literature of Fanconi anemia complementation group B detected by oligonucleotide array testing postnatally.     

A homozygous nonsense mutation early in exon 5 of BRCA2 is associated with very severe Fanconi anemia

Fanconi anemia (FA) due to biallelic mutations in the BRCA2 gene is very rare and associated with an extremely high risk of early-onset of aggressive childhood malignancies, predominantly brain tumors, leukemia, and nephroblastoma. Here, we present a consanguineous family with three affected children of the D1 subtype of FA and describe the clinical consequences of the earliest known biallelic nonsense/stop-gain germ-line mutation in BRCA2, exon 5 c.469A>T, that leads to a premature stop of translation, p.Lys157*. The three patients were born with severe intrauterine growth restrictions and different degrees of congenital malformations. Altogether, they developed eight distinct malignancies and died within their first three years of life. Treatment with a reduced chemotherapy regimen was only performed in patient 2 for his first tumor, a nephroblastoma, which the patient tolerated well for eight months, until he developed myelodysplastic syndrome (MDS) and then acute myeloid leukemia (AML). Finally, the third patient experienced a hepatoblastoma, an unclassified brain tumor and MDS in parallel and died in her second year of life. Our report re-emphasizes the aggressiveness and fatality of the FA-D1 children with biallelic BRCA2 nonsense mutations, that are both located before exon 11, which contains binding domains for the RAD51 recombinase.