Genetic testing of Mucopolysaccharidoses disease using multiplex PCR- based panels of STR markers: in silico analysis of novel mutations.

The Mucopolysaccharidoses (MPS) are group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade glycosaminoglycans (GAGs) in the lysosomes. GAGs are sulfated polysaccharides involving repeating disaccharides, uronic acid and hexosamines including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). Hyaluronan is excluded in terms of being non-sulfated in the GAG family. Different types of mutations have been identified as the causative agent in all types of MPS. Herein, we planned to investigate the pathogenic mutations in different types of MPS including type I (IDUA gene), IIIA (SGSH) and IIIB (NAGLU) in the eight Iranian patients. Autozygosity mapping was performed to identify the potential pathogenic variants in these 8 patients indirectly with the clinical diagnosis of MPSs. so three panels of STR (Short Tandem Repeat) markres flanking IDUA, SGSH and NAGLU genes were selected for multiplex PCR amplification. Then in each family candidate gene was sequenced to identify the pathogenic mutation. Our study showed two novel mutations c.469 T > C and c.903C > G in the IDUA gene, four recurrent mutations: c.1A > C in IDUA, c.220C > T, c.1298G > A in SGSH gene and c.457G > A in the NAGLU gene. The c.1A > C in IDUA was the most common mutation in our study. In silico analysis were performed as well to predict the pathogenicity of the novel variants.

Development and validation of a novel panel of 16 STR markers for simultaneous diagnosis of ß-thalassemia, aneuploidy screening, maternal cell contamination detection and fetal sample authenticity in PND and PGD/PGS cases.

Prenatal diagnosis (PND) may be complicated with sample mix-up; maternal cell contamination, non-paternity and allele drop out at different stages of diagnosis. Aneuploidy screening if combined with PND for a given single gene disorder, can help to detect any common aneuploidy as well as aiding sample authenticity and other probable complications which may arise during such procedures. This study was carried out to evaluate the effectiveness of a novel panel of STR markers combined as a multiplex PCR kit (HapScreen™ kit) for the detection of ß-thalassemia, aneuploidy screening, ruling in/out maternal cell contamination (MCC), and sample authenticity. The kit uses 7 STR markers linked to ß-globin gene (HBB) as well as using 9 markers for quantitative analysis of chromosomes 21, 18, 13, X and Y. Selection of the markers was to do linkage analysis with ß-globin gene, segregation analysis and to perform a preliminary aneuploidy screening of fetal samples respectively. These markers (linked to the ß-globin gene) were tested on more than 2185 samples and showed high heterozygosity values (68.4-91.4%). From 2185 fetal cases we found 3 cases of non-paternity, 5 cases of MCC, one case of sample mix-up and one case of trisomy 21 which otherwise may have end up to misdiagnosis. This kit was also successfully used on 231 blastomeres for 29 cases of pre-implantation genetic diagnosis (PGD) and screening (PGS). The markers used for simultaneous analysis of haplotype segregation and aneuploidy screening proved to be very valuable to confirm results obtained from direct mutation detection methods (i.e. ARMS, MLPA and sequencing) and aneuploidy screening.

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.