الفهرس 
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Abstract
Methylmalonic acidemia (MMA) is an autosomal recessive inherited inborn error of metabolism that results from the functional impairment of methylmalonyl-CoA mutase (MCM). The impairment of MCM is due to either defect in the apoenzyme (mut-type MMA) or in biosynthesis of the cofactor, adenosylcobalamin (cbl type).
The present study aims to investigate the mutations of MUT gene in Egyptian patients and molecular analysis of novel mutations to examine their spectrum and explore the possibility of a molecular diagnosis.
This study includes 15 Egyptian patients of unrelated families with methylmalonic acidemia MUT type who were diagnosed and have been followed up previously.
Total RNA was extracted from the peripheral blood of the patient by using GeneJETᵀᴹ Whole Blood RNA Purification Mini Kit) and was converted to cDNA by using the QuantiTect Reverse Transcription Kit. cDNA was amplified by PCR using Go Taq® green master mix, 2X. Five sets of primers (forward and reverse) were designed for amplification of specific sits covering entire length of cDNA of MUT gene. These primers were designed by using web based primer-blast tool, NCBI (National center for biotechnology information). Agarose gel electrophoresis was used for detection of DNA PCR product. Sequencing of the PCR product in both directions was carried out then the results were used for nucleotide blast by the online program on NCBI web site to make alignment with normal MUT gene for detection any mutations. All detected mutations were submitted to online web server, Mutalyzer Name Checker for detection of the effect of the mutation on the amino acids sequence of protein. The amino acids sequence are used then to detect the expected 3-dimension structure of the MCM protein by using the online web server, RaptorX Structure Prediction.
The mutations detected in this study were submitted to Gene Bank with accession numbers MF170176, MF170177, MF170178, MF170179, MF170180, MF170181, MF170182, MF170183, MF170184, MF170185, MF170186 and MF170187.
A total of 12 different mutations, 10 in coding region and 2 in untranslated region UTR-3, were identified. Of these 10 mutations, 5 were missense mutations, 3 were nonsense mutations, 1 was deletion mutation and 1 was synonymous mutation. One novel mutation c.1420 C>T (p.Arg474*) in exon 7 was identified in this study and other mutations have been reported previously.
Patient no.1 has a homozygous synonymous mutation c.636G>A (p.Lys212=) in exon 3 which also was identified in other 8 patients and considered as the most common mutation in Egypt. He has two homozygous missense mutations c.1495G>A (p.Ala499Thr), c.2011A>G (p.Ile671Val) which have been reported previously. In addition, he has a homozygous nonsense mutation c.2179C>T (p.Arg727*) in exon 13.
Patient no.2 did not show any mutation in his entire length of MUT cDNA. This result suggested that the mutation may be intronic affecting on splicing or the stability of mRNA, or in the transcription factors.
Patient no.3 has a novel homozygous nonsense mutation c.1420 C>T (p.Arg474*) in exon 7 which is reported firstly in this study. He also has a homozygous missense mutation c.2011A>G (p.Ile671Val) in exon 12.
Patient no.4 has a homozygous missense mutation c.322C>T (p.Arg108Cys) in exon 2.
Patient no.5 has a homozygous nonsense mutation c.91C>T (p.Arg31*) in exon 2. He also has a homozygous synonymous mutation c.636G>A, two homozygous missense mutations c.1495G>A and c.2011A>G, and a homozygous silent mutation c.*51C>G in UTR-3.
Patient no.6 has a homozygous missense mutation c.2150G>T (p.Gly717Val) in exon 13.
Patient no.7 has a homozygous missense mutation c.643 G>A (p.Gly215Ser) in exon 3.
Patient no.8 and 9 are homozygous for synonymous mutation c.636G>A (p.Lys212=) and missense mutation c.2011A>G (p.Ile671Val).
Patient no.10 has a heterozygous synonymous mutation c.636G>A (p.Lys212=) and a heterozygous missense mutation c.1495G>A (p.Ala499Thr) which also has been identified in patient no.12.
Patient no.11, 13 and 15 are homozygous for synonymous mutation c.636G>A (p.Lys212=).
Patient no.14 has a homozygous deletion mutation c.613_615delGAA (p.Glu205del) in exon 3. He also has a homozygous synonymous mutation c.636G>A and a homozygous silent mutation c.*192delA in UTR-3.
Identification of these mutations in MMA Egyptian patients will facilitate differential confirmatory diagnosis, which is important for appropriate treatments. It will also aid carrier detection, genetic counseling, and subsequent prenatal diagnosis among Egyptian families who have history of disease.