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

RESULTS AND DISCUSSION

The clinical data, genetic tests performed and detected mutations are presented in Table 1 for FXS patients, Table 2 for RTT patients and Table 3 for PWS/AS patients. FXS. Parents frequently stated that the disease onset has been provoked by severe illnesses, such as whooping cough, pneumonia, recurrent otitis. Sometimes, parents have noticed that their child’s development regressed after an infectious disease. Karyotype analysis showed normal results in all but patient 18 in whom the result was compatible with FXS: 46,fra(X)(q27.3)Y[18]/46,XY[22]. The PCR protocol failed to detect a normal fragment in three patients (9, 18 and 31 in Table 1),who were shown to have a hypermethylated CGG pattern by MLPA analysis (Figure 1). Their mothers showed single normal alleles and premutated expanded alleles of ~70, ~90 and ~180 CGG repeats, respectively (Figure 2) (the results were confirmed by Southern blotting, data not shown). In patient 31, a mosaic pattern normal/premutation/full mutation was detected as a smear located from the zone of normal fragments through the premutated zone to full expansion detected as hypermethylation. Patient 3 showed a normal allele along the CGG repeated region, but this allele was three repeats longer than the normal allele of his mother, who also carried a premutation of about 90 repeats [10]. The hypermethylated MLPA profile of this patient confirmed a mosaic genotype of full mutation/ normal allele [10]. These results were confirmed by classical Southern blotting (data not shown). The MLPA CNT did not show any deletion/ duplication on the FMR1 gene. In addition, the MLPA probemix contains 14 specific probes for the FMR2 gene (OMIM #300806), mutations which are associated with fragile X MR E (FRAXE) (OMIM #309584). Both FMR1 and FMR2 genes were simultaneously analyzed by CNT and MT and no pathological changes were detected on the FMR2 gene. The remaining 28 FXS boys were subjected to genetic tests on the ARX and MECP2 genes. The reason is that mutations in the ARX gene have been associated with MR and epilepsy [6,7], but the results from a large series of screened patients have been disappointing [16]. On the other hand, mutations in the MECP2 gene can cause moderate MR and obesity [17]. We detected no mutations in the ARX gene or in the MECP2 gene. RTT. Twenty-two of the mentally retarded girls in this group were born after a complicated pregnancy and delivery by Caesarean section. Various vaccinations or severe infections were reported by the parents as a triggering factor for disease onset. Karyotype analysis gave normal results in all but patient 6 who carried a deletion of the short arm of the X chromosome 46,XX,del(X)(p1.22). Sequencing of the MECP2 gene revealed seven mutations in this group (19.4%). Five different types of substitutions were detected on exon 4 of this gene: c.473C>T, p.Thr158Met; c.763C>T, p.Arg255Stop; c.808C>T, p.Arg270Stop (found in two girls); c.880C>T, p.Arg294 Stop, and c.916C>T, p.Arg306Cys. A deletion of 44 bp was also found by sequencing of exon 4 of this gene: c.1157_1200 del44, p.Leu386fs. These mutations have been reported in the MECP2 gene mutation database [18]. The other 29 patients were subjected to the MLPA analysis for large deletions of the MECP2, CDKL5 and ARX genes. Two large deletions, encompassing exon 3 and a part of exon 4 (c.27-?_*?del) of the MECP gene were detected in patients 23 and 35. This deletion is available at the MECP2 mutation database [18]. The MLPA profile is presented in Figure 3. The deleted fragments were assessed by the relative peak ratios, calculating the peak area in comparison to the mean peak area of both neighboring control peaks, and compared to the control sample. The relative ratios obtained for the probes in exon 3 and a part of exon 4 were <0.6, which we interpreted as a deletion. The calculated relative peak ratios are provided in the legend to Figure 3. We screened for mutations in the CDKL5 gene in 27 patients without success. The PWS/AS as a differential diagnosis in some of the patients (5, 14, 24, 25, 34 and 36) were also excluded. PWS/AS. Karyotype analysis showed normal results except in patient 17 in whom fluorescent in situ hybridization analysis showed results compatible with deletion: 46,XX,ishdel(15)(q11.2q11.2) (D15S10x1),15p11.2 (D15Z1x2),15q22(PMLx2). The methylation-specific PCR revealed that seven patients lacked the paternal unmethylated fragment (PWS) and that two patients lacked the maternal methylated fragment (AS) (Figure 4). To determine more precisely the mutation type (deletion or UPD), we analyzed the nine genetically confirmed cases by MS-MLPA and found six deletions and three to have UPD (Figures 5A and 5B). The calculated relative peak ratios in the deletion cases were less than 0.6 and are presented in the legend to Figure 5A. Eight patients in this group remain with a still unclear molecular defect. The suspected diagnosis of FXS in the male patients was excluded by analysis of the FMR1 gene. The ARX gene was sequenced in the three PWS boys and no mutations were detected. Patient 1 (Table 3) was additionally sequenced for mutations in the MECP2 gene, but no pathological changes were detected. Altogether, we were able to clarify the molecular basis in 22 of the 85 MR patients (26.0%), ~10.6% of whom were PWS/AS, the same percentage (~10.6%) were RTT and ~4.8 were FXS. The percentage of the genetically proved diagnosis in the group of FXS is relatively small (12.5%), which might be due to the clinical criteria for patient selection, including a number of autistic cases in this group. The test for FXS is always the first step in molecular diagnostics of boys with MR. It is worth mentioning, that genetically proved diagnosis among our RTT patients (25.0%) is relatively high and all these cases are due to MECP2 mutations. Despite of the type of mutation, all these cases are very similar from a clinical point of view and well recognized in Bulgaria.

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