Balkan Journal of Medical Genetics

INTELLECTUAL ABILITY IN THE DUCHENNE MUSCULAR DYSTROPHY AND DYSTROPHIN GENE MUTATION LOCATION
Milic Rasic V1,2,*, Vojinovic D1, Pesovic J3, Mijalkovic G1, Lukic V1, Mladenovic J1, Kosac A1, Novakovic I4, Maksimovic N4, Romac S3, Todorovic S1, Savic Pavicevic D3
*Corresponding Author: Vedrana Milic Rasic, M.D., Ph.D., Clinic for Neurology and Psychiatry for Children and Youth, Dr. Subotica 6A, 11000 Belgrade, Serbia. Tel: +381-11-265-8355. Fax: +381-11-264-5064. E-mail: vedrana.milic.npk@gmail.com
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MATERIALS AND METHODS

Patient Data. Forty-one patients with DMD were recruited retrospectively at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia, during the period between 1992 and 2013. The diagnosis of DMD was based on the clinical onset of the disease before 5 years of age, initial or clear neurological signs of decline of motor function at the age of 6 years, decline of motor function or positive family history of DMD for boys younger than 6 years, elevated serum creatine kinase levels and confirmed mutation in the DMD gene. All recruited patients were unrelated except of one sibling pair. All patients and/or their parents gave informed consent concerning the use of the data for research. This study was approved by the Ethics Committee of the Clinic for Neurology and Psychiatry for Children and Youth, Belgrade, Serbia. Methods: Genetic Analysis. Deletions and duplications were detected via multiplex ligationdependent probe amplification (MLPA) using two probe mixes, P034 and P035 (MRC Holland, Amsterdam, The Netherlands) [17] at the Center for Human Molecular Genetics at the Faculty of Biology in Belgrade, Serbia. A few samples were analyzed via multiplex polymerase chain reaction (PCR) [18-20] at the Institute of Human Genetics at the Faculty of Medicine in Belgrade, Serbia, were included in the analyses as the mutation location allowed us to unequivocally assign the altered dystrophin isoforms. Mutations were described using Human Genome Variation Society (HGVS) nomenclature [21]. The positions of the mutations were determined in relation to reference sequence NM_004006 (GenBank) at cDNA level and in relation to reference sequence UniProtKB:11532 (Uni ProtKB/Swiss-Prot) at protein level. The mutation effect on the reading frame was determined using software DMD gene reading frame checker (available at http://www. dmd.nl/). Since the effect of mutation location on FSIQ was expected to be different, all mutations were divided into two structural groups according to previously applied classifications [22,23]. The mutations localized upstream from exon 30 (1-30) and the mutations localized upstream from exon 45 (1-45) were defined as proximal mutations, while the mutations downstream from exon 30 (31-79) or exon 45 (46-79) were considered as distal mutations. Considering the complex organization of DMD gene and dystrophin isoforms produced from the inner promoters, mutations were assigned to altered expression of dystrophin isoforms (Dp427, Dp260, Dp140, Dp116, Dp71 and Dp40), and were divided into various groups. We took into consideration that the Dp140 transcript has a long 5’ untranslated region (5’UTR), consisting of exons 45-50, so that the effect of mutations in this region on Dp140 expression could not be confidently predicted. Therefore, we also analyzed clustering, in which mutations within this region (Dp140utr) were assumed to be coding exon mutations affecting only expression of Dp427 and Dp260 but not Dp140, while mutations in the promoter and protein coding region of Dp140 (Dp140pc) were assigned to affect expression of Dp140 [14]. As the promoter that regulates Dp140 expression lies within intron 44 of the DMD gene, all patients who had a deletion breakpoint within intron 44 were tested by PCR for the presence of the Dp140 promoter using the following primers: IN44F (5’-GCC CTA AGT GCT TCC AGA AA-3’) and IN44R (5’-CTC ACA GCT CCT GCA TCA GA-3’). This original approach allowed us to accurately group patients with the affected Dp140 expression. To assess the cumulative effect of dystrophin isoforms on FSIQ, the patients were divided into three groups with respect to the preservation or loss of Dp140 and Dp71/Dp40. Cognitive Assessment. All DMD patients were psychologically tested. Taking into account the patients’ age, different psychological instruments were used for cognitive status assessment. In order to assess intelligence level in children younger than 16 years and 11 months, the Wechsler Intelligence Scale for Children (WISC) was used [24]. The test generates FSIQ, which represents overall cognitive ability. Patients with FSIQ ≤70 were considered mentally disabled, while patients with FSIQ ranging between 70 and 85 (70

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