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 FMR1 GENE ANALYSIS

Betaine-Based-PCR Protocol. The primers used to amplify the CGG repeat in the 5’UTR of the FMR1 gene, the polymerase chain reaction (PCR) mixture and cycling conditions have been described elsewhere [11]. This amplification permits detection of normal and of some premutated alleles. For the correct sizing, PCR products were subjected to electrophoresis on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in the presence of ROX500 size standard (Applied Biosystems). Methylation Sensitive MLPA (MSMLPA). The MS-MLPA (SALSA MS-MLPA ME029 probemix) was performed following the manufacturer’s instructions (www.mlpa.com). DNA samples diluted in TE buffer (10 mM Tris- HCl, pH 7.5; 1 mM EDTA) were subjected to denaturation for 10 min. in denaturing buffer provided by the manufacturer. Hybridization with the FMR1-specific probes and 20 control probes was performed overnight at 60°C. Each sample was divided for the ligation reaction, which is the copy number test (CNT) for deletions/duplications detection, and for the ligation-digestion reaction, the methylation test (MT) for methylation assessment at CpG islands. In both tests, the hybridized probes were ligated by a specific ligase mix, provided by the manufacturer. In the methylation, test the methylation-sensitive restriction enzyme HhaI (Pharmacia Biotech, Uppsala, Sweden) was added. The final step consisted of PCR amplification of the obtained fragments. The PCR buffer, PCR primers, the enzyme dilution buffer and the polymerase were provided in the kit. The PCR products obtained were analyzed on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems) in the presence of ROX500 size standard (Applied Biosystems). All patient samples were analyzed simultaneously with at least two normal male samples and a mutant male carrier of a full mutation. Copy number changes were determined by comparison to the normal controls. The MT results were easy to interpret as the five FMR1 exon 1 methylation-sensitive fragments (see Figure 1) were absent in the normal controls after HhaI digestion, but were present, as a result of hypermethylation, in a patient carrying a full mutation.

Number 27 VOL. 27 (2), 2024	Number 27 VOL. 27 (1), 2024
Number 26 Number 26 VOL. 26(2), 2023 All in one	Number 26 VOL. 26(2), 2023
Number 26 VOL. 26, 2023 Supplement	Number 26 VOL. 26(1), 2023
Number 25 VOL. 25(2), 2022	Number 25 VOL. 25 (1), 2022
Number 24 VOL. 24(2), 2021	Number 24 VOL. 24(1), 2021
Number 23 VOL. 23(2), 2020	Number 22 VOL. 22(2), 2019
Number 22 VOL. 22(1), 2019	Number 22 VOL. 22, 2019 Supplement
Number 21 VOL. 21(2), 2018	Number 21 VOL. 21 (1), 2018
Number 21 VOL. 21, 2018 Supplement	Number 20 VOL. 20 (2), 2017
Number 20 VOL. 20 (1), 2017	Number 19 VOL. 19 (2), 2016
Number 19 VOL. 19 (1), 2016	Number 18 VOL. 18 (2), 2015
Number 18 VOL. 18 (1), 2015	Number 17 VOL. 17 (2), 2014
Number 17 VOL. 17 (1), 2014	Number 16 VOL. 16 (2), 2013
Number 16 VOL. 16 (1), 2013	Number 15 VOL. 15 (2), 2012
Number 15 VOL. 15, 2012 Supplement	Number 15 Vol. 15 (1), 2012
Number 14 14 - Vol. 14 (2), 2011	Number 14 The 9th Balkan Congress of Medical Genetics
Number 14 14 - Vol. 14 (1), 2011	Number 13 Vol. 13 (2), 2010
Number 13 Vol.13 (1), 2010	Number 12 Vol.12 (2), 2009
Number 12 Vol.12 (1), 2009	Number 11 Vol.11 (2),2008
Number 11 Vol.11 (1),2008	Number 10 Vol.10 (2), 2007
Number 10 10 (1),2007	Number 9 1&2, 2006
Number 9 3&4, 2006	Number 8 1&2, 2005
Number 8 3&4, 2004	Number 7 1&2, 2004
Number 6 3&4, 2003	Number 6 1&2, 2003
Number 5 3&4, 2002	Number 5 1&2, 2002
Number 4 Vol.3 (4), 2000	Number 4 Vol.2 (4), 1999
Number 4 Vol.1 (4), 1998	Number 4 3&4, 2001
Number 4 1&2, 2001	Number 3 Vol.3 (3), 2000
Number 3 Vol.2 (3), 1999	Number 3 Vol.1 (3), 1998
Number 2 Vol.3(2), 2000	Number 2 Vol.1 (2), 1998
Number 2 Vol.2 (2), 1999	Number 1 Vol.3 (1), 2000
Number 1 Vol.2 (1), 1999	Number 1 Vol.1 (1), 1998

About the journal ::: Editorial ::: Subscription ::: Information for authors ::: Contact