FANCA Gene in Pakistani Fanconi Anemia Patients: Screening of Mutations in Exon 28 and Exon 29

Objective: This kind of study was conducted first time in Pakistan. Its objective was to ascertain the associated clinical features and analyze the FANCA exon 28 and exon 29 mutations in Pakistani Fanconi anemia (FA) patients. Methods: A total of 38 patients with Fanconi anemia were recruited presenting in the Armed forces institute of pathology (AFIP) Rawalpindi Pakistan. They were enrolled in this study on the basis of comprehensive clinical evaluation and positive Diepoxybutane (DEB)/Mitomycin C Chromosomal breakage test. Genomic DNA was extracted from peripheral blood of patients and age and gender-matched controls. Mutation analysis of FANCA gene was done by conventional Polymerase chain reaction (PCR) and DNA sequencing. Various online tools and software were used for analysis of the obtained data and identification of the sequence alterations in FANCA gene in exon 28 and exon 29 of FA patients. Results and Discussion: The current study on screening of FANCA mutational analysis in exon 28 and exon 29 revealed four novel mutations. These include three missense variants (p.F876L, p.L883H, and p.K921I) in exon 28 and a novel homozygous frameshift variant (p.S947FfsX950) in exon 29. In addition two new intronic variants were also found in this set of patients. Conclusion: The sequence variants identified in this study in 10 (26.31%) FA patients in two out of forty-three FANCA gene exons (i.e., exon 28 and exon 29) strongly emphasize the importance of large-scale molecular studies on FANCA gene in Pakistani population.

Spontaneous chromatid break as clonal evolution in myelodysplastic syndrome patients / Quebra espontânea de cromátides como evolução clonal em pacientes com síndrome mielodisplásica

ABSTRACT Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by peripheral cytopenias due to ineffective erythropoiesis and an increased risk for evolving into acute myeloid leukemia (AML). Chromosomal abnormalities represent the most important marker of risk stratification for AML transformation. Chromatid break (chtb) is a discontinuity of a single chromatid. We report the case of a patient with MDS whose cytogenetic analysis showed spontaneous chromatid breakage (chrb): 46,XY,add(13)(q34),chtb(15)(q24) [3]/47,XY,chtb(2)(q22),del(5)(q35),del(7)(q32),+8,del(11q)(q23),del(q22)[cp17]. He was considered a high-risk patient due to the complex karyotype and the presence of chtb. We suggest that this chromosomal abnormality may be considered as a marker of genomic instability in MDS.

RESUMO A síndrome mielodisplásica (SMD) é uma desordem clonal das células-tronco hematopoiéticas caracterizada por citopenias periféricas devido à hematopoiese ineficaz e pelo aumento do risco de evolução para a leucemia mieloide aguda (LMA). As alterações cromossômicas representam o marcador mais importante da estratificação de risco para a transformação de LMA. Quebra das cromátides (chtb) é uma descontinuidade de uma única cromátide. Relatamos o caso de um paciente com SMD, cuja análise citogenética mostrou chtb espontâneo: 46,XY,add(13)(q34),chtb(15)(q24)[3]/47,XY,chtb(2)(q22),del(5) (q35),del(7)(q32),+8,del(11q)(q23),del(q22)[cp17]. O paciente foi considerado de alto risco devido ao cariótipo complexo e à presença de chtb. Sugerimos que essa anormalidade cromossômica possa ser considerada como marcador de instabilidade.

Análisis de roturas cromosómicas en un paciente con sospecha clínica de anemia de Fanconi / Chromosomal breakage analysis in a patient with presumptive diagnosis of Fanconi anemia

La anemia de Fanconi (AF) es un síndrome de inestabilidad cromosómica caracterizado por diversos rasgos dismórficos, pancitopenia progresiva y predisposición a neoplasias hematológicas. El ensayo de sensibilidad a la mitomicina C (MMC) proporciona un marcador celular único para el diagnóstico de la enfermedad. Con el objetivo de introducir este ensayo de roturas cromosómicas, se aplicó la técnica en dos muestras procedentes de un paciente con sospecha clínica de AF y un sujeto control. Las muestras de sangre periférica fueron cultivadas según los protocolos establecidos para los estudios citogenéticos. Se prepararon cuatro frascos de cultivo por cada muestra. A uno de ellos se le añadió solo cloruro de sodio (cultivo control) y a los restantes se les añadieron concentraciones crecientes de MMC (50, 150 y 300 nM). Fueron analizadas cincuenta metafases por cada frasco. La exposición de los linfocitos del paciente a todas las concentraciones de MMC provocó diferencias significativas en el número de células con roturas cromosómicas respecto a la misma exposición en el control (p <0.005). Se comprobó el éxito del ensayo teniendo en cuenta que a 300 nM en el control sano solo aparece el 32 por ciento de células con roturas. Es interesante resaltar que en la muestra del paciente a la concentración más elevada, se apreció la presencia de 2 líneas celulares, una con pocas o ninguna rotura (38 por ciento) similar a las que aparecen en las células no-AF; y otra con múltiples roturas (62 por ciento) típicas de las células AF. Esto indicó la presencia de mosaicismo somático en los linfocitos T del paciente. De acuerdo con los resultados obtenidos se confirmó la sospecha clínica de que se trata de un paciente AF, por la hipersensibilidad a la acción de la MMC que presenta mosaicismo somático en linfocitos T...

Fanconi anemia (FA) is a chromosomal instability syndrome characterized by various dysmorphic features, progressive pancytopenia and predisposition to hematological malignancies. The assay sensitivity to mitomycin C (MMC) provides a unique cell marker for the diagnosis of the disease. In order to introduce this chromosomal breakage test the technique was applied in two samples from a patient with clinical suspicion of AF and a control subject. Peripheral blood samples were cultured by protocols established for cytogenetic studies. Four flasks were prepared for each sample culture. Only sodium chloride was added to one of the flasks (control) and to the remaining flasks increasing concentrations of MMC (50, 150 and 300 nM) were added. Fifty metaphases were analyzed for each bottle. Exposure of lymphocytes from the patient at all concentrations of MMC caused significant differences in the number of cells with chromosome breaks with respect to the same exposure in the control (p <0.005). Assay success was proved considering that in 300 nM in healthy control only 32 percent shows cell breakage. It is interesting to remark that in the patient sample with highest concentration, the presence of two cell lines were observed, one with little or no breakage (38 percent) similar to those found in no-F cells and other with multiple breaks (62 percent), typical of AF cells. These results indicated the presence of somatic mosaicism in patient´s T lymphocytes. The results obtained confirmed the clinical suspicion that this is an AF patient, due to the hypersensitivity to the action of MMC and the presence of somatic mosaicism in T lymphocytes...

Evaluation of concentration and storage effects of mitomycin C in the diagnosis of Fanconi anemia among idiopatic aplastic anemia patients.

BACKGROUND: Fanconi anemia (FA) is a rare autosomal recessive genetic disorder that shows an increased sensitivity to the intercalating agents such as mytomycin C (MMC), measured as chromosomal aberrations. This study was conducted to differentiate between FA and “idiopathic” aplastic anemia on the basis of induced chromosomal breakage study with MMC. MATERIALS AND METHODS: MMC stress tests in different final concentrations of 20 and 50 ng/ml of MMC were conducted on peripheral blood lymphocytes from 32 patients with aplastic anemia and 13 healthy controls. Fifty nanograms per milliliter of MMC from old, fresh and frozen stocks was used to check the sensitivity of diagnosis on FA-diagnosed patients. Statistical analysis was used for the assessment of aberrations, including chromatid and chromosome breaks and exchanges. RESULTS: Eight patients (25%) with a very high percentage of chromosomal breakage were diagnosed as FA on the basis of the chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation, while another two lacked such anomalies or had minor physical problems. Freshly made MMC has shown more sensitivity to detect FA patients compared with frozen or 1-week-old MMC stock. CONCLUSIONS: The study indicates that freshly made MMC stress test provides an unequivocal means of differentiation between FA and “idiopathic” aplastic anemia. Further, the study, the first of its kind from Iran, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of FA to implement appropriate therapy.

Cytoprotective effect of honey against chromosomal breakage in fanconi anemia patients in vitro.

BACKGROUND: Natural honey is widely used all over the world as a complementary and alternative medicine in various disorders including Fanconi anemia (FA). FA is a rare genetic chromosomal instability syndrome caused by impairment of DNA repair and reactive oxygen species (ROS) imbalance. This disease is also related to bone marrow failure and cancer. The aim of this study was to evaluate the cytoprotective effect of honey on mitomycin C (MMC-) induced chromosomal damage in peripheral lymphocytes from FA patients. MATERIALS AND METHODS: Treatment of these complications with alkylation agents MMC may enhance chromosomal breakage. We have evaluated the effect of honey on MMC- induced chromosomal breakage in FA blood cells using chromosomal breakage assay. The basal chromosomal breakage count was higher among FA patients than healthy subjects. RESULTS: The addition of MMC alone gave a significantly higher of chromosomal breakage in FA patients than control group (P < 0.0001). Pre- treatment with honey significantly inhibited breakage induced by MMC in FA patients by its antioxidant effect. CONCLUSION: Honey can prevent MMC- induced chromosomal breakage by its antioxidant effect.

Continuous exposure of three successive generations of mice to electromagnetic fields: implication on double minute frequency.

Background: Epidemiological studies indicate increased risk of leukemia, lymphoma, and brain tumor among electrical workers exposed to electromagnetic field (EMF). Other investigator reported that continuous exposure of four successive generations of mice to EMF in doses of 1 kV to 5 kV caused tumor formation in offspring. The objective of this study was to evaluate the effect of continuous exposure of three successive generations of mice (Mus musculus L) to EMF of 3 kV, 4 kV, and 5 kV and its implication of chromosomal breakage, as detected by double minute formation. Methods: Four couples of mice of Swiss Webster strain, 3-4 months of age, and 7-40 gram of body weight were exposed to EMF at the doses of 3 kV, 4 kV, and 5 kV, and one couple served as control. Double minute formation was examined in all offspring, except one couple of each group to be exposed with the same doses of EMF to get the F2 generation, and so forth until F3 generation. Twenty metaphases of chromosomes were examined and frequencies of double minute were calculated in the three generations of all group. Results: Frequencies of double minute in F1, F2, and F3 of mice exposed to EMF of 3 kV were respectively 0.78 ± 0.08; 0.83 ± 0.09; and 0.80 ± 0.05. In the 4 kV group were 0.083 ± 0.11; 0.73 ± 0.03; and 0.96 ± 0.15, and in the 5 kV group were 0.96 ± 0.25; 0.75 ± 0.05; and 0.99 ± 0.33, whereas no double minute chromosomes were noted in control group. Frequencies of the double minute in mice exposed to EMF were significantly higher than control group. Conclusions: Continuous exposure of mice during three successive generations to EMF at doses of 3 kV, 4 kV, and 5 kV causes increased chromosomal breakage as detected as double minute chromosome formation.

A Clinical Study of Fanconi's Anemia / 대한소아혈액종양학회지

BACKGROUND: Fanconi's anemia(FA) is an autosomal recessive disease characterized by aplastic anemia and congenital malformations. As up to 30% of patients have no physical stigmata, the modern diagnosis of FA rests on chromosomal breakage of patient's cells induced by chemical clastogens such as diepoxybutane(DEB) or mitomycin-C(MMC). METHODS: We reviewed the clinical manifestations, laboratory findings, diagnostic methods, treatment and outcome of 6 patients diagnosed to have a FA at the Chonnam University Hospital for the last 6 years. RESULTS: Six cases(16.2 %) were found to have FA among 37 aplastic children who were diagnosed during the same period. The mean age at diagnosis was 6.3 years which was the usual onset of hematologic findings. All patients had features of aplastic anemia, and had one or more anomalies, such as low birth weight, hyperpigmentation, cafeau-lait spots, mental retardation, developmental delay, peculiar face(broad nasal bases, epicanthal folds, micrognathia), polydactyly, microcephaly, short stature, and dislocation of hip. We found increased breaks in cultured cells with DEB and MMC in 5 cases tested. The median duration of follow-up was 30 months. Oxymetholone and prednisolone treatment was partially beneficial in three cases. Immunosuppressive treatment with ALG/ATG was not successful in two cases tried. Four cases are living now, without transfusion in three. Two patients were died of disseminated fungal infection and transplant-related problems, respectively. CONCLUSIONS: Fanconi's anemia should be sought carefully in any patients with aplastic anemia because the prognosis, treatment modality, and the approach to bone marrow transplantation are quite different when the hematologic disorder is inherited rather than acquired.