Investigation of FANCA gene in Fanconi anaemia patients in Iran.
OBJECTIVE
Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC.


METHODS
Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression.


RESULTS
In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations.


CONCLUSIONS
The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more cost-effective than the multiplex ligation-dependent probe amplification (MLPA) procedure.
Ali Akbar Saffar
AA
Moghadam
Medical Biotechnology Institute, National Institute of Genetic Engineering & Biotechnology (NIGEB), Tehran, Iran.
Frouzandeh
F
Mahjoubi
Nahid
N
Reisi
Parvaneh
P
Vosough
D000293:Adolescent; D000328:Adult; D002648:Child; D005199:Fanconi Anemia / Q000453:epidemiology / Q000235:genetics* / Q000473:pathology; D052217:Fanconi Anemia Complementation Group A Protein / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D005838:Genotype; D006801:Humans; D007492:Iran / Q000453:epidemiology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D017384:Sequence Deletion
C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein