Neurogenetics Spectrum of Sq related spinal muscular atrophies (lower motor neuron syndromes) in Sudanese and Saudi populations; a multicentre clinical and Molecular Study.

Abstract

Abstract Background Spinal muscular atrophy is one of the most common autosomal recessive neurogcnetics disorders in East Africa and Middle East countries, also considered as a leading genetic killer under the age of two years, with high carriers‘ frequency exceeds 1:50 in Europe. The most common presentation is proximal muscle weakness as a consequence of anterior hom cells degeneration. A mutation in the telomeric copy of the survival motor neuron gene (SMNI) considered as main underlying causative factor. Most carriers of SMA have one chromosome 5 with a normal SMNI gene and one with a deleted SMNIcopy. Although no large-scale population-based study has been established in the region; a previous estimation has indicated that incidence of SMA is much higher in Arab population compared with other ethnic groups, partly because of high prevalence of tribal and consanguineous marriages. Objectives General objective The project has been designed to introduce the molecular medicine advanced technology to improve the accuracy in the diagnosis of lower motor neuron disorders presented in a form of SMA as translational reseach for our cun'ent knoweledge of the pathogenic chromosomal region (5ql3). The specific objectives were: '2' To Introduce the SMA Molecular diagnostics and the carrier detection methods in Sudan. '1' To review the role of SMNI and other candidate genes (NAIPs genes in susceptibility to SMA in Middle East countries and Arab ethnic groups). To establish a method could help detection of other possible mutations in SMA nondeleted patients (~5% of SMA Patients). '3' To explore the possibility of increasing the sensitivity of the diagnostic molecular technology for 5qSMA cases from 95 to~99%. '2' To correlate the genotype (SMNI and SMN2 copy number) with the phenotype and carrier status in diagnosed patients within special type of SMA families. ¢ 0.0 Material and methods Materials bower Motor neurone/5qSMA diagnosis retrospective study included 259 of DNA samples each sample extracted from clinically symptomatic patient during the year 2005 and 2006. Material for SMA carrier analysis study included Total number of 2000 healthy volunteers. Material for SMNI-Exon7 nondeleted patients’ analysis study Included 80 patients showed SMA clinical phenotype. Material for Sq SMA families without SMNI common deletion Included Ten (10) families (36 individuals) for SMA patients didn't show SMNI-exon7 deletion. Methods DNA extraction fi'om peripheral blood using QIAGEN (QIAamp DNA Blood Midi Kit) has been used for all samples included in this project; an altemative rapid extraction method l.raCode paper has been used for heterozygousity carriers testing. Amplification ofSMNI exon7 and 8 PCR performed in l2.5 ul total volumes containing l00 ng of DNA; the PCR products then digested with Dral and Ddel enzyme to visualize and identify the SMNI copy. Multiplex-PCR has been used as quantitative-PCR to determine the presence of single SMNlcopy (gene dosage scan) and stndardized as SMA carriers’ detection method as we used it also to detennine SMNI/2 copies and NAIF deletions in SMA family study. SMNI Long-Range PCR (LR-PCR) Selective amplification of SMNI gene by LR-PCR method was initiated to detect the single nucleotide mutation possibilities on SqSMA eases without SMNI common deletions. Fluorescenct~Based PCR SSCA (Single Strand Conformational Analysis) has been established as pre- sequencing methods to determine SMNI-exons with possible mutation. Sequencing has been performed using a dye terminator cycle sequencing kit (Perkin Elmer, Wanington, UK) and an automatic Sequencer (Applied Biosystem 373) for specific SMNI exons purified from the LR-PCR product. Outcomes and Results patients presented with lower motor neuron pheotype showed SMNl—Exon7 deletion therefore had a oonfinnation of Sq SMA diagnosis were (8l.08%). SMNI gene was absent or interrupted in 97 out of 137 (70.8%) of SMA types l patients, although SMA type II patients showed deletion in (93.1%) i.e. 8| out of 87; while (9l.4%) i.e. 32 out of 35 type-lll patients showed the same deletion mutation. The initial controls of the healthy carriers screening study included [87 normal individuals showed carrier frequency of 5%, i.e. one carrier in each 20 persons compared to one in 50-80 in other parts of the world. Therefore we expanded the healthy volunteers number to include i869 subjects. Only 43 i.e. (2.3%) individuals showed single copy of SMNIthis make the carrier frequency one in 34. 5 Ali Alsanousi-October/2010 ____ _ SMNI-Exon7 nondeleted SMA came study: included 80 patients classified into three classes. 5% of them showed SMN]-Exon8 deletions. Class] patients (SMNI-Exon7 nondeleted independent patients) results showed 21.05% of those patients showed deletion mutation of NAIP gene Exons.Class2 (SMN1 -Exon7 Nondeleted patient investigated in correlation with their family SMN genetic profile) showed deletion mutation in NAIP in 6.25%. Class 3 (included patients wit11 Exon7- SMN2 deletion) showed NAIP gene mutation in 50% of patients included from this group. Futher molecular analysis of the SMA families investigated for SMN!/SMN2 copies number using gene dosage scan analysis showed (100%) patients in this group were SMNI- Exon7 nondeleted. Gene dosage scan classified class 2 into further 3 classes; i.e. patients showed single copy of SMN! and single copy of SMN2 (IT/1C) represented 10% of this group. Patients showed 2 copies of SMN] and no copy of SMN2 (2T/0C) represented 30% of this group. While the majority of this group (60%) showed 2 copies of SMN] and one copy of SMN2 (2T/1C). We have established in this study a newly invented method using the Long range PCR to determine SMN! by amplification of 13 Kb of the SMN! genes i.e. (ex0n2a to exon8).The selective amplification of SMN! successfully performed using LR-PCR as substituting method for SMNI gene cloning from cDNA. Using 10% of class 2 patients (i.e. patients form showed single copy of SMNI in gene dosage scan).SMNl has been visualized then purified for specific Exon SSCA/SSCP which is optimized using I5 samples with known gene dosage scan profile. Two samples (13.3%) showed abnormal migration curve in SSCP therefore selected for optimimtion of the sequence analysis study. Both abnomially migrating SMN-Exon6 samples showed a normal or nonmuatated sequence. ln conclusions The project presented both the SMA diagnostic method together with a method for carriers testing with the highest sensitivity and specificity; then could be considered a comer stone to stop the spread of this fatal diabling disease. Homozygous deletion of Sll/flVI-exons7 was found in 93.1 to 91% while SMNI-exon8 deletion detected in 87% of patients included therefore could present a confimiatory diagnosis for SMA with high confidence. Healthy volunteers’ carriers study revealed relatively higher carrier frequency in comparison with other ethnics (2.34% i.e. one in 34). SMA families study gave an evidence of a possible underlying genetics mechanism related to S1vflV gene conversion and Sq 13 region rearrangements indicated by the gene dose scan analysis and NAIP gene deletions in SMA nondeleted patients. Ali Alsnnnusi-October/2010 6 Ali Alsanousi-October/2010