Molecular Characterization of Dystrophin Gene in Iraqi Patients with Muscular Dystrophy.

Abstract

Background: Dystrophinopathies are the commonest forms of muscular dystrophy and comprise clinically recognized forms, Duchenne Muscular Dystrophy (DMD), and Becker Muscular Dystrophy (BMD). Mutations in the dystrophin gene which consist of large gene deletions (65%), duplications (5%) and point mutations (30%) are responsible for reducing the amount of functional dystrophin protein in skeletal muscle fibers.  This study concentrate mainly at the spectrum of deletions in the 'distal hot spot' region of the DMD/BMD gene in Iraqi DMD/BMD patients using multiplex PCR technique

Objectives: The aim of this study was to investigate the rate, and distribution of deletions in 10 exons of Dystrophin gene in a group of Iraqi dystrophinopathy patients using the multiplex polymerase chain reaction (MPCR).

Patients materials and methods: This is a case prospective study which include 27 clinically diagnosed DMD/BMD patients and six suspected carriers in Medical city /Baghdad . A written consent was obtained from each family for going the research as well as ethical committee approval. Forty six apparently healthy individual were included as a control group. Blood samples were collected in 5-6 ml EDTA tubes by venepuncture. The DNA was extracted by using the Wizard Genomic purification kit (Promega/USA), and the quantity was estimated by UV-spectrophotometer (Cecil CE7800) . Ten exons of the dystrophin gene were examined ( 19 ,45 ,46 ,47 ,48 ,49 ,50 ,51 ,52,53)   using synthesized primers with complementary sequences and set in five different multiplex PCR groups. The products of PCR amplifications were subjected to electrophoresis and visualized by The UVCI140 & 200 series (advanced CCD gel imaging system from Major Science. It consists of a CCD camera, UV transilluminator UV- light system). All done in college of medicine /Baghdad university.

Results: The rate (relative frequency) of subjects with any positive exonal deletion (among the10 selected and tested exons was significantly higher (85.2%) among patients compared to that among the suspected carriers (33.3%).The distribution of exonal deletions among patients compared to suspected carriers were statistically significant. The frequency of deletions detected in male patients (~82%) was higher than frequencies mentioned in the other studies of comparison. The control group show no deletion in all tested exons.

Conclusions: Multiplex PCR technology was utilized to demonstrate the frequency of 10 exons deletions in a limited group of Iraqi DMD/BMD patients. The overall distribution of deletion mutations in the distal ‘hot spot’ region was higher than that of DMD/BMD cases investigated elsewhere. The study also serves as a good starting point for further investigations into the genetic aspects of the Iraqi DMD/BMD population.