Balkan Journal of Medical Genetics

ASSOCIATION BETWEEN LOSS OF Dp140 AND COGNITIVE IMPAIRMENT IN DUCHENNE AND BECKER DYSTROPHIES
Chamova T1,*, Guergueltcheva V1, Raycheva M1, Todorov T2,3, Genova J2, Bichev S4, Bojinova V5, Mitev V3, Tournev I1,6, Todorova A2,3
*Corresponding Author: Teodora Chamova, M.D., Ph.D., Clinic of Neurology, University Hospital “Alexandrovska”, 1 Georgi Sofiiski str., Sofia 1431, Bulgaria; Tel.: +359-889802252; Fax: +359-2-9526787; E-mail: teodoratch@abv.bg
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MATERIALS AND METHODS

Fifty three patients were included in this study. The participating families were informed about the aim of the study and written informed consent was obtained from all patients and/or parents. They were diagnosed on the base of clinical symptoms, family history, EMG data, creatine kinase levels. The mean age of the patients was 15.87 ± 14.13 years, varying between 6 and 62. Their motor impairment was evaluated by the North star ambulatory assessment scale. All participants underwent a formal cognitive assessment conducted by a neuropsychologist at the hospital site. None of them had a family history of neuropsychiatric disorders. The battery consisted of tests, evaluating general intelligence, verbal memory and executive functions. General intelligence was assessed by the Wechsler Intelligence Scale for children (HAWIK-R Bulgarian version, based on WISCR) measuring full scale (FSIQ), verbal (VIQ), and performance IQ (PIQ). The verbal scale of WISC-R estimates verbal intelligence and it is based on completion of information, similarities, arithmetic, and vocabulary subtests. The performance scale of WISCR assesses non verbal intelligence and is computed from picture-completion, picture-arrangement, blockdesign, and object-assembly subtests. Verbal memory was assessed by the Rey auditory verbal learning test sented five times, always in the same order, with an assessment of recall after each presentation as well as a second list (B) that was assessed only once. A recognition memory condition, consisting of 30 words was also included. The Tower of London (TOL) test was applied for the assessment of executive functioning, specifically to detect deficits in planning. The test consisted of two boards with pegs and several beads with different colors. The examiner used the beads and the boards to present the examinee with problemsolving tasks. Performances were considered either pathological or normal according to cut-off scores obtained by comparison with the existing norms. All the participants were genetically confirmed either by MLPA (multiplex ligation-dependent probe amplification) or direct sequencing. The DNA samples were obtained from peripheral blood, using a DNA extraction kit (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany), following the manufacturer’s instructions. The MLPA analysis [SALSA MLPA P034/ P035 kit (www.mlpa.com)] was used as a first step to screen our patients for deletions/duplications along the DMD gene. The analysis was performed according to the manufacturer’s instructions [www.mlpa. com]. The obtained polymerase chain reaction (PCR) products were analyzed on an ABI PRISM™ 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) in the presence of ROX500 size standard (Applied Biosystems). Each patient sample was analyzed simultaneously with at least two normal male samples. The MLPA data interpretation, in order to assess copy number changes (deletions) in comparison to the normal controls, was performed by the Excel program by Coffalyser MLPA data analysis software [www.mlpa.con]. The entire coding region of the DMD gene, including exon/intron boundaries, was sequenced in patients with no detectable mutation on MLPA. The PCR products were purified by a PCR Product Pre-Sequencing Kit (Affymetrix Inc., Santa Clara, CA, USA), containing 4 U exonuclease I (10 U/μL) and 0.8 U shrimp alkaline phosphatase (2 U/μL). The sequencing reaction was performed by ABI PRISM™ BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI PRISM™ 310 genetic analyzer (Applied Biosystems). The sequencing profile was obtained by Sequencing Analysis v.3.4.1 software (Applied Biosystems). Twenty patients with mutations, terminating in exon 44 or starting at exon 45, were tested by PCR amplification of microsatellites STR44, SK12, SK21 and P20 DXS269, in order to evaluate the integrity of the Dp140 promoter region. The following single nucleotide polymorphism (SNPs) were chosen along intron 44 of the DMD gene: 5’-STR44 (CA)n Gen- Bank M81257, IVS44SK12 (GT)n, IVS44SK21 (GT) n, P20 DXS269 (TC)n(TG)n GenBank M86524-3’ [www.dmd.nl]. The PCR amplification was performed in 25 μL total volume, containing 0.4 μM primers, 0.2 mM dNTPs, 1 × supplied PCR reaction buffer (Genet Bio, Chungnam, Korea) and 0.5 U Prime Taq (Genet Bio). The annealing temperature was 58°C. The obtained PCR products were analyzed on an ABI PRISM™ 310 genetic analyzer (Applied Biosystems) in the presence of ROX500 size standard (Applied Biosystems) for 6-FAM-labeled primers or on ALFexpress (Pharmacia Biotech, Stockholm, Sweden) for Cy5-labeled primers. Statistical analyses were conducted using statistical package IBM SPSS Statistics 19.0. A p value below 0.05 was considered significant. The following statistical methods were used: descriptive statistics; student T-test for testing hypotheses for differences between two independent and two related samples; non parametric Mann-Whitney test for testing hypotheses for differences between two independent samples.

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