Validation of Fanconi anemia complementation Group A assignment using molecular analysis

Abstract

Purpose: Fanconi anemia is a genetically heterogeneous chromosomal breakage disorder exhibiting a high degree of clinical variability. Clinical diagnoses are confirmed by testing patient cells for increased sensitivity to crosslinking agents. Fanconi anemia complementation group assignment, essential for efficient molecular diagnosis of the disease, had not been validated for clinical application before this study. The purpose of this study was (1) confirmation of the accuracy of Fanconi anemia complementation group assignment to Group A (FANCA) and (2) development of a rapid mutation detection strategy that ensures the efficient capture of all FANCA mutations. Methods: Using fibroblasts from 29 patients, diagnosis of Fanconi anemia and assignment to complementation Group A was made through breakage analysis studies. FANCA coding and flanking sequences were analyzed using denaturing high pressure liquid chromatography, sequencing, and multiplex ligation-dependent probe amplification. Patients in which two mutations were not identified were analyzed by cDNA sequencing. Patients with no mutations were sequenced for mutations in FANCC, G, E, and F. Results: Of the 56 putative mutant alleles studied, 89% had an identifiable FANCA pathogenic mutation. Eight unique novel mutations were identified. Conclusion: Complementation assignment to Group A was validated in a clinical laboratory setting using our FANCA rapid molecular testing strategy. Genet anconi anemia (FA; MIM no. 227650), the most common inherited bone marrow disorder, has an overall prevalence of 1-5 per million and an estimated carrier frequency of 1 in 200 to 1 in 300 in most populations. 1,2 Demonstrating either an autosomal or X-linked recessive mode of inheritance, FA is characterized by childhood progressive bone marrow failure and predisposition to acute myelogenous leukemia; older patients are at increased risk for squamous cell carcinomas of the head, neck, and genitourinary tract. [3][4][5][6][7] Congenital abnormalities are present in approximately 70% of FA patients and may include radial ray defects; café au lait spots or hypopigmentation; short stature; microphthalmia; malformations of kidneys, gastrointestinal tract, and heart; mental retardation; and hearing defects. 8 Because of the high degree of phenotypic variability exhibited by FA patients, diagnosis may be difficult on the basis of clinical manifestations alone. Because FA patient-derived lymphocytes and fibroblasts exhibit hypersensitivity to DNA crosslinking agents such as diepoxybutane (DEB) 9 and mitomycin C (MMC), 10 resulting in a high rate of chromosomal breakage and radial formation, analysis on the basis of this hypersensitivity has been routinely used to confirm clinical diagnosis.Molecular diagnosis of FA has been challenging because of the genetic heterogeneity associated with the disease; FA is multigenic, with 13 complementation groups and associated genes having been characterized (A, B, C, D1, D2, E, F, G, I, J, L, M, N). [11][12][13][14][15][16][17][18][19][20][21][...

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

“…For the majority of patients, FA is suspected only after the onset of pancytopenia. Genetic studies in FA-A patients of various ethnic groups have revealed a variety of mutations, including large deletions, nucleotide substitutions, and small deletions/insertions across the entire gene without a mutation-clustered region (15,16). A precise diagnosis can be made with careful history, physical examination, and a positive chromosomal breakage test in the majority of cases.…”

Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study

“…All living affected individuals were specifically screened for all of the major IBMFS; genotyping was performed when possible (Ameziane et al , 2008; Moghrabi et al , 2009). Affected individuals who had not received a transplant had bone marrow aspirations, biopsies and cytogenetic studies.…”

Fanconi's anemia, type A presenting as VACTERL association with atresia right external auditory canal

“…Other congenital abnormalities include congenital hip dislocation, scoliosis with vertebral anomalies, cafe´au lait spots (63-79%), low birth weight, microphthalmia, microcephal, renal anomalies (30%) (especially renal agenesis and renal ectopia), microcephaly, hypogenitalia (51%) with hypoplastic gonads, small uterus in females and hypospadias in males, gastrointestinal anomalies (including esophageal atresia, duodenal atresia and imperforate anus), congenital heart disease (especially patent ductus arteriosus), ventricular septal defect, pulmonic valvar stenosis and coarctation. 6,7 The concurrence of VACTERL features has been described in FANC A, C, D1, F and G. 4 VACTERL-H syndrome, which is an X-linked condition with hydrocephalus, usually aqueductal stenosis, has been associated with FANC B.…”

“…4,5 Radial ray anomalies occur in 63-79% of patients, especially anomalies of the thumb (49-66%). Other congenital abnormalities include congenital hip dislocation, scoliosis with vertebral anomalies, cafe´au lait spots (63-79%), low birth weight, microphthalmia, microcephal, renal anomalies (30%) (especially renal agenesis and renal ectopia), microcephaly, hypogenitalia (51%) with hypoplastic gonads, small uterus in females and hypospadias in males, gastrointestinal anomalies (including esophageal atresia, duodenal atresia and imperforate anus), congenital heart disease (especially patent ductus arteriosus), ventricular septal defect, pulmonic valvar stenosis and coarctation.…”

“…3 This is a relatively common condition with a carrier rate of the gene mutation of 1 in 200-300 in the general population. 4 It is an autosomal recessive, rarely Xlinked condition that is associated with progressive bone marrow failure, congenital anomalies, growth retardation, hyperpigmentation of the skin, cafe´au lait spots and a predisposition to malignancies, especially acute myelocytic leukemia and squamous cell carcinoma. The red blood cells in FA There are 13 gene loci described in FA, listed below with their gene and gene frequency:…”