Mutations of the Fanconi Anemia Group A Gene (FAA) in Italian Patients

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A-FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of approximately 65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction

Fanconi anemia (FA) is a genetically and phenotypically heterogenous autosomal recessive disease associated with chromosomal instability and hypersensitivity to DNA crosslinkers. Prognosis is poor due to progressive bone marrow failure and increased risk of neoplasia, but revertant mosaicism may improve survival. Mechanisms of reversion include back mutation, intragenic crossover, gene conversion and compensating deletions/insertions. We describe the types of reversions found in five mosaic FA patients who are compound heterozygotes for single base mutations in FANCA or FANCC. Intragenic crossover could be shown as the mechanism of self-correction in the FANCC patient. Restoration to wildtype via back mutation or gene conversion of either the paternal or maternal allele was observed in the FANCA patients. The sequence environments of these mutations/reversions were indicative of high mutability, and selective advantage of bone marrow precursor cells carrying a completely restored FANCA allele might explain the surprisingly uniform pattern of these reversions. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensating missense mutation 15 amino acids downstream of the constitutional mutation, which explains the reversion to MMC resistance of the respective lymphoblastoid cell line. With one exception, our mosaic patients showed improvement of their hematological status during a three- to six-year observation period, indicating a proliferative advantage of the reverted cell lineages. In patients with Fanconi anemia, genetic instability due to defective caretaker genes sharply increases the risk of neoplasia, but at the same time increases the chance for revertant mosaicism leading to improved bone marrow function.     

Identification of a novel large intragenic deletion in a family with Fanconi anemia: First molecular report from India and review of literature

We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature, hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2 Western blot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel homozygous large intragenic deletion (exons 8–27 del) in the FANCA gene in the proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation. We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different countries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is the first molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene. Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper genetic counseling to the family.     

Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA.     

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.     

Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway

Germline intragenic mutations in PTEN are associated with 80% of patients with Cowden syndrome (CS) and 60% of patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS). The underlying genetic causes remain to be determined in a considerable proportion of classic CS and BRRS without a polymerase chain reaction (PCR)-detectable PTEN mutation. We hypothesized that gross gene deletions and mutations in the PTEN promoter might alternatively account for a subset of apparently mutation-negative patients with CS and BRRS. Using real time and multiplex PCR techniques, we identified three germline hemizygous PTEN deletions in 122 apparently mutation-negative patients with classic CS (N=95) or BRRS (N=27). Fine mapping suggested that one deletion encompassed the whole gene and the other two included exon 1 and encompassed exons 1-5 of PTEN, respectively. Two patients with the deletion were diagnosed with BRRS, and one patient with the deletion was diagnosed with BRRS/CS overlap (features of both). Thus 3 (11%) of 27 patients with BRRS or BRRS/CS-overlap had PTEN deletions. Analysis of the PTEN promoter revealed nine cases (7.4%) harboring heterozygous germline mutations. All nine had classic CS, representing almost 10% of all subjects with CS. Eight had breast cancers and/or benign breast tumors but, otherwise, oligo-organ involvement. PTEN protein analysis, from one deletion-positive and five PTEN-promoter-mutation-positive samples, revealed a 50% reduction in protein and multiple bands of immunoreactive protein, respectively. In contrast, control samples showed only the expected band. Further, an elevated level of phosphorylated Akt was detected in the five promoter-mutation-positive samples, compared with controls, indicating an absence of or marked reduction in functional PTEN. These data suggest that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.     

Screening Fanconi anemia lymphoid cell lines of non-A, C, D2, E, F, G subtypes for defects in BRCA2/FANCD1

Biallelic mutations in BRCA2/FANCD1 were recently recognized as a rare cause of Fanconi anemia (FA). Using immunodetection with an antiserum directed against the carboxyterminus of the BRCA2 protein, we screened 38 lymphoid cell lines from FA patients whom we could not previously assign, via retroviral complementation analysis, to any of six known FA complementation groups (FA-A, -C, -D2, -E, -F, or -G). Three of these 38 cell lines lacked the 380-kDa BRCA2 signal on immunoblots. DNA sequencing showed biallelic compound and truncating mutations in two of the immuno-negative cell lines, whereas a monoallelic frameshift mutation and an amino acid substitution were detected in the third cell line. Our data show that less than 10% of unassigned FA cell lines harbor truncating mutations in BRCA2/FANCD1. This finding strongly suggests the existence of (an) additional, as yet unknown FA gene(s).     

Application of oligonucleotide array CGH in the detection of a large intragenic deletion in POLG associated with Alpers Syndrome

Mutations in the polymerase γ (POLG) gene are among the most common causes of mitochondrial disease and more than 160 POLG mutations have been reported. However, a large proportion of patients suspected of having POLG mutations only have one (heterozygous) definitive pathogenic mutation identified. Using oligonucleotide array CGH, we identified a compound heterozygous large intragenic deletion encompassing exons 15–21 of this gene in a child with Alpers syndrome due to mtDNA depletion. This is the first large POLG deletion reported and the findings show the clinical utility of using array CGH in cases where a single heterozygous mutation has been identified in POLG.     

Mutations in Fanconi anemia genes and the risk of esophageal cancer

The incidence of esophageal squamous cell carcinoma (ESCC) is very high in northeastern Iran. Previously, we reported a strong familial component of ESCC among Turkmens, who constitute approximately one-half of the population of this region. We hypothesized that the genes which cause Fanconi anemia might be candidate genes for ESCC. We sequenced the entire coding regions of 12 Fanconi anemia genes in the germline DNA of 190 Turkmen cases of ESCC. We identified three heterozygous insertion/deletion mutations: one in FANCD2 (p.Val1233del), one in FANCE (p.Val311SerfsX2), and one in FANCL (p.Thr367AsnfsX13). All three patients had a strong family history of ESCC. In addition, four patients (out of 746 tested) were homozygous for the FANCA p.Ser858Arg mutation, compared to none of 1,373 matched controls (OR?=?16.7, 95% CI?=?6.2-44.2, P?=?0.01). The p. Lys3326X mutation in BRCA2 (also known as Fanconi anemia gene FANCD1) was present in 27 of 746 ESCC cases and in 16 of 1,373 controls (OR?=?3.38, 95% CI?=?1.97-6.91, P?=?0.0002). In summary, both heterozygous and homozygous mutations in several Fanconi anemia-predisposing genes are associated with an increased risk of ESCC in Iran.     

Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients

Mutations in the methyl-CpG-binding protein-2 (MECP2) gene on Xq28 have been found to be a cause of Rett syndrome (RS). In a previous mutation screening, we found MECP2 mutations in 81% of Swedish classical Rett women. In this study, we have analyzed 22 patients for MECP2 deletions using multiplex-ligation-dependent probe amplification (MLPA). Clinically, 11 of the patients who were classical Rett women, 3 were forme fruste, 1 was congenital RS, and 7 were Rett variants. As inclusion criteria, we used DNA from patients in whom previous sequencing results showed no mutations in coding portions of the MECP2 gene. MLPA is a method based on multiplex PCR. In one PCR, as many as 40 probes are amplified with the same primers. The specificity of the amplification products is determined by the site-specific hybridization of each probe construct, prior to amplification. Each PCR product has a unique length, which makes it possible to identify it by size separation. In 3 of 11 (27%) classical Rett women, we detected large deletions in MECP2 using MLPA. All these patients had deletions covering two exons; in 2 cases the deletion involved exons 3 and 4 and, in one case, exons 1 and 2 were missing. In the forme fruste, congenital and Rett-variant patients, we found no large deletions. We have found that MLPA is useful when it comes to finding large deletions compromising whole exons in MECP2. Used as a complementary method to DNA sequencing, it revealed new MECP2 mutations in classical RS patients.     

Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies. An erratum to this article can be found at     

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ～60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ～30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.     

Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions

Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutations. PAP is a method for nucleic acid amplification in which 3' blocked oligonucleotides (P*) are activated by pyrophosphorolysis when annealed to the target template and subsequently extended. A key advantage to this technology is that PAP reactions produce little or no primer-dimer or false priming. As a result of this enhanced specificity, MD-PAP is easy to optimize. Herein, we utilize MD-PAP to determine gene dosage of each exon of the human factor IX gene by comparison with one endogenous internal control from the ATM gene. Estimated dosage is proportional to the actual template copy number over a minimum dynamic range from 1 to 16 copies. A blinded analysis detected 100% of 43 heterozygous deletions of exons in the human factor IX gene.     

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.     

Molecular spectra of HPRT deletion mutations in circulating T-lymphocytes in Fanconi anemia patients

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.     

Isolation of a cDNA representing the Fanconi anemia complementation group E gene

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.     

Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene

Introduction: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)). Methods: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants. Results and discussion: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.