STANDARD OPERATING PROCEDURE FOR MOLECULAR GENETICS TEST SPINAL MUSCULAR ATROPHY (SMA) AT HUMAN GENOME CENTER, USM

HEALTH CAMPUS, KUBANG KERIAN.

Prepared by : Ernie Suhaiza Radzi

Checked by : Approved by:

Dr. Teguh Haryo Sasongko Prof. Madya Dr. Zilfalil Bin Alwi

Protocol 3
Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder, which has been mapped to chromosome 5q11.2-q13.3. Childhood-onset proximal SMA is characterized by degeneration of anterior horn cells of the spinal cord leading to symmetrical limb and trunkparalysis. SMA is the common autosomal recessive disease of early life after cystic fibrosis, with an incidence of between 1 in 6,000 to 1 in 10,000 live births. Depending on the clinical severity, SMA is classified into type I, II and III. Type I, Werdnig-Hoffman disease is lethal in infants and manifests at birth or within 6 months of age. These patients are floppy and never able to sit unaided. Type II patients sit, but never walk unaided and survival depends on how much the respiratory muscles are affected. Type III SMA, also known as Kugelberg-Welander disease, is a relatively mild disorder with onset in adolescence or later. The patients survive until adults.

Analysis: SMN and NAIP genes deletion PCR and restriction enzymes based analysis for the SMN gene (exon 7 and 8) and the NAIP gene (exon 5).

1) SMN exon 7 and exon 8 deletion analysis The presence or absence of the SMN1 gene in SMA patients is analyzed by PCR-restriction enzyme digestion method as described by van der Steege et al. (1995). The primer sets used for the amplification are as follows: Exon 7 8 Primer R111 X7-Dra 541C960 541C1120 Primer sequences AGA CTA TCA ACT TAA TTT CTG ATC A CCT TCC TTC TTT TTG ATT TTG TTT GTA ATA ACC AAA TGC AAT GTG AA CTA CAA CAC CCT TCT CAC AG Size 188bp 188bp

The PCR Mixture is as follows: Reagents 1) ddH2O 2) 10X PCR Buffer 3) 25mM MgCl2 4) 10mM dNTP 5) Primer (F) 6) Primer (R) 7) 5U/ul Taq DNA Polymerase 8) DNA template TOTAL The PCR condition for both SMN exon 7 and exon 8 includes: Step Initial denaturation Denaturation Annealing Extension Final extension Temperature 94C 94C 56C 72C 72C Duration 7 min 1 min 1 min 1 min 7 min Cycle(s) 1 28 1 Volumes 11.3 ul 5.0 ul 4.0 ul 0.5 ul 1.0 ul 1.0 ul 0.2 ul 2.0 ul 25 ul

The products will be electrophoresed in a 2.5% agarose gel and stained with Sybr GreenI. PCR-amplified fragments of exon 7 (188bp) and 8 (188bp) are then subjected to DraI and DdeI digestions, respectively. Digestion with Dra I (exon 7) and Dde I (exon 8): Reagents 1) ddH2O 2) 10X Buffer 3) PCR product 4) Dra 1 5) dde 1 TOTAL Exon 7 6 ul 2 ul 10 ul 2 ul 20 ul Exon 8 6 ul 2 ul 10 ul 2 ul 20 ul

The digested products will be electrophoresed in a 3% agarose gel and stained with Sybr Green1. The enzymes cleaved SMN2 fragment, but not SMN1, allowing distinction of those two genes. 10ul fr RE +1 ul sybr +1 ul loading dye Exon 7 1) Volt 100 v 2) Minutes 50 Chen et al., 2007 and van der Steege et al., 1995 Exon 8 100 v 50

Transportation procedures

1) Appoinment/information Any cases that related to SMA, please contact Dr.Teguh Haryo Sasongko (09-7676794 / 012 9874175) or Assoc. Prof. Dr.Zilfalil Alwi (09-7676531/ 019-9875767) or 2) Consent Form and Clinical Summary Form. Please fill-up the Consent Form and Clinical Summary Form to be send together with the blood samples. These forms will be included in the self-addressed envelope. 3) Transportation media Transportation media that will be used is the container/tube that contains anti-coagulant such as EDTA. 4) Amount samples needed and how to send the samples. Two EDTA containers (provided) with 1.5ml in each tube. To make sure the samples are in a safe and good condition, please wrap the containers with the plastic (provided). Label NON-HAZARDOUS BLOOD SAMPLES FOR RESEARCH PURPOSE ONLY The blood samples should be sent to us as soon as possible at room temperature (on working days) at this address: Dr.Teguh Haryo Sasongko Pusat Genom Manusia, Pusat Pengajian Sains Perubatan, Kampus Kesihatan USM, 16150 Kubang Kerian, Kelantan. Tel: 09-7676794 /6796)