Balkan Journal of Medical Genetics

CLINICAL AND MOLECULAR DATA ON MENTAL RETARDATION IN BULGARIA
Todorov T1#, Todorova A1#, Avdjieva D2, Dimova P3, Angelova L4, Tincheva R2 and Mitev V1
*Corresponding Author: Tihomir Todorov, Department of Medical Chemistry and Biochemistry, Sofia Medical University, 2 “Zdrave” str., Sofia 1431, Bulgaria; Tel./Fax: +359 29530715; tisho.todorov@abv.bg
page: 11

PROTOCOLS FOR SNRPN GENE ANALYSIS

Methylation-Specific PCR. DNA was modified by bisulfite treatment using MethylCode Bisulfite Conversion Kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. The standard protocol was used to amplify maternal methylated and paternal unmethylated alleles [15]. The amplicons were separated in 3% agarose gel and visualized after ethidium bromide staining. The lack of paternal unmethylated fragment (164 bp) leads to PWS, the lack of maternal methylated fragment (131 bp) leads to AS. Methylation-Sensitive MLPA (MS-MLPA). The MS-MLPA (SALSA MS-MLPA ME028-A1 Prader-Willi/ Angelman probemix) was performed following the manufacturer’s instructions [14]. This test is performed as a CNT and a MT in presence of methylation-sensitive restriction enzyme HhaI (Pharmacia Biotech). The protocol is as described above for FMR1 gene. The HhaI cleaves only unmethylated genomic DNA. If the CpG islands are methylated, the HhaI does not digest them; the fragment is amplified by PCR and detected by capillary electrophoresis. If the CpG sites are unmethylated, there is no PCR product in the electrophoretic profile. The CNT permits detection of deletions in PWS/AS cases, while the MT permits clarification of UPD in PWS, as two maternal methylated alleles are present.

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