Mutational spectrum of autosomal recessive limb-girdle muscular dystrophies in a cohort of 112 Iranian patients and reporting of a possible founder effect.

BACKGROUND: Limb-girdle muscular dystrophies are a group of genetically heterogeneous diseases that are inherited in both autosomal dominant (LGMDD) and autosomal recessive forms (LGMDR), the latter is more common especially in populations with high consanguineous marriages like Iran. In the present study, we aimed to investigate the genetic basis of patients who are suspicious of being affected by LGMDR. DNA samples of 60 families suspected of LGMD were extracted from their whole blood. Four short tandem repeat (STR) markers for each candidate genes related to LGMD R1 (calpain3 related)- R6 (δ-sarcoglycan-related) were selected, and all these 24 STRs were applied in two sets of multiplex PCR. After autozygosity mapping, Sanger sequencing and variant analysis were done. Predicting identified variants' effect was performed using in-silico tools, and they were interpreted according to the American College of Medical Genomics and Genetics (ACMG) guideline. MLPA was used for those patients who had large deletions. Fresh muscle specimens were taken from subjects and were evaluated using the conventional panel of histochemical stains. RESULTS: forty out of sixty families showed homozygote haplotypes in CAPN3, DYSF, SGCA, and SGCB genes. The exons and intron-exon boundaries of the relevant genes were sequenced and totally 38 mutations including CAPN3 (n = 15), DYSF (n = 9), SGCB (n = 11), and SGCA (n = 3) were identified. Five out of them were novel. The most prevalent form of LGMDs in our study was calpainopathy followed by sarcoglycanopathy in which beta-sarcoglycanopathy was the most common form amongst them. Exon 2 deletion in the SGCB gene was the most frequent mutation in this study. We also reported evidence of a possible founder effect in families with mutations in DYSF and SGCB genes. We also detected a large consanguineous family suffered from calpainopathy who showed allelic heterogeneity. CONCLUSIONS: This study can expand our knowledge about the genetic spectrum of LGMD in Iran, and also suggest the probable founder effects in some Iranian subpopulations which confirming it with more sample size can facilitate our genetic diagnosis and genetic counseling.

Autozygosity mapping of methylmalonic acidemia associated genes by short tandem repeat markers facilitates the identification of five novel mutations in an Iranian patient cohort.

Isolated Methylmalonic acidemia/aciduria (MMA) is a group of inborn errors of metabolism disease which is caused by defect in methylmalonyl-CoA mutase (MCM) enzyme. The enzyme has a key function in the catabolism of branched chain amino acids (BCAA, isoleucine, and valine), methionine, and threonine. MCM is encoded by a single gene named "MUT". Other subtypes of MMA are caused by mutations in cblA (encoded by MMAA) and cblB (encoded by MMAB), which is involved in the synthesis of methylmalonyl-coenzyme A cofactor. Different types of mutations have been identified as the cause of MMA. However, the mutation spectrum of MMA in Iran has not been studied so far. Here, we aimed to investigate the MMA causative mutations in the Iranian population. Using STR (Short Tandem Repeat) markers, we performed autozygosity mapping to identify the potential pathogenic variants in 11 patients with clinical diagnosis of MMA. Nineteen STR markers which are linked to the MUT, MMAA and MMAB genes (the genes with known causative mutations in MMA) were selected for PCR-amplification using two recently designed multiplex PCR panels. Next, the families that were diagnosed with homozygous haplotypes for the candidate genes were directly sequenced. Five novel mutations (c.805delG, c.693delC, c.223A > T, c.668A > G and c.976A > G in MUT) were identified beside other 4 recurrent mutations (c.361insT in MUT, c.571C > T and c.197-1 G > T in MMAB and c.1075C > T in MMAA). In silico analyses were also performed to predict the pathogenicity of the identified variants. The mutation c.571C > T in MMAB was the most common mutation in our study.

A rare form of limb girdle muscular dystrophy (type 2E) seen in an Iranian family detected by autozygosity mapping.

Sarcoglycanopathies (SGPs) constitute a subgroup of autosomal recessive limb girdle muscular dystrophies (LGMDs) which are caused by mutations in sarcoglycan (SGs) genes. SG proteins form a core complex consisting of α, ß, γ and δ sarcoglycans which are encoded by SGCA, SGCB, SGCG and SGCD genes, respectively. Genetic defect, in any of these SG proteins, results in instability of the whole complex. This effect can be helpful in interpreting muscle biopsy results. Autozygosity mapping is a gene mapping approach which can be applied in large consanguineous families for tracking the defective gene in most autosomal recessive disorders. In the present study, we used autozygosity mapping, to find the gene responsible for muscular dystrophy. Proband was a 10-year-old boy referred to our center for ruling out DMD (Duchenne muscular dystrophy). According to the pedigree and clinical reports, we assessed him for SGPs. Haplotyping, using the four short tandem repeat (STR) markers for each of the SG genes, showed that the phenotype may segregate with SGCB gene; and observing two crossing overs which occurred within the gene suggested that the mutation might be in the first two exons of SGCB gene. Mutation analysis showed a 26 bp duplication (10 bp before the initiation codon till 13 bp after the ATG start codon). This will cause a frameshift in protein synthesis.

NPM1 Mutation Detection in Acute Myeloid Leukemia: A Method Comparison Study.

BACKGROUND: Mutations in the nucleophosmin (NPM1) gene have been used as molecular biomarkers for prognostication of patients with adult acute myeloid leukemia (AML). METHODS: We designed a rapid and sensitive method using the allele-specific-refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect the most common mutations of NPM1 gene, which are mostly four base pair insertions and compared its efficacy with direct sequencing and capillary electrophoresis which served as the gold standards. RESULTS: The incidence of mutation was 22% (33% of patients with normal karyotypes had mutation compared with 16% of patients with abnormal karyotypes) based on the results obtained with capillary electrophoresis analysis and direct sequencing. All of the specimens determined to be mutation-positive by the gold standard tests were also positive by the ARMS-PCR method. Significantly, the ARMS-PCR test also helped determine the mutation status of an extra set of patients who had low call rates on capillary electrophoresis and appeared normal on direct sequencing. DISCUSSION: The low mutation rate in some patients hindered its detection in the gold standard assays because of the interference of the mutation signal by high background noise. The low sensitivity of the gold standard assays for detecting low copy number mutations rates thus increase their risk of producing false negative results that adversely affects prognostication and therapy. Our results suggest that the mutation detection rate of the ARMS-PCR assay is better than existing tests. This is most probably because of the fact that in an ARMS-PCR-based method, the mutated variant is specifically amplified, based on a mutation-specific primer. CONCLUSIONS: We conclude that the high sensitivity of the ARMS-based method together with its rapidity and low expense should make it a suitable choice for clinical laboratories.