DIRECT MOLECULAR ANALYSIS OF THE FRAGILE-X SYNDROME IN A SAMPLE OF EGYPTIAN AND GERMAN PATIENTS USING NONRADIOACTIVE PCR AND SOUTHERN BLOT FOLLOWED BY CHEMILUMINESCENT DETECTION

Molecular genetic analysis of individuals from 6 Egyptian; and 33 Germ an families with fragile X syndrome and 240 further patients with ment al retardation was performed applying a completely non-radioactive sys tem. The aim of our study was the development of a non-radioactive det ection method and its implementation in molecular diagnosis of the fra gile X syndrome. Furthermore, we wanted to assess differences in the m utation sizes between Egyptian and German patients and between Egyptia n and German carriers of a premutation. Using non-radioactive polymera se chain reaction (PCR), agarose gel electrophoresis and blotting of t he PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)(5) and chemiluminescent detection, we ide ntified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of t he CGG repeat between the Egyptian and German patients and carriers, r espectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whe reas Southern blot analysis with the digoxigenin labelled probe StB 12 .3 revealed presence of a full mutation. Our newly established nonradi oactive genomic blotting method is based on the conventional radioacti ve Southern blot analysis. Labelling of the probe StB 12.3 with digoxi genin via PCR allowed the detection of normal, premutated and fully mu tated alleles. For exact sizing of small premutated or large normal al leles, we separated digoxigenin labelled PCR products through denaturi ng polyacrylamide gelelectrophoresis (PAGE) and transfered them to a n ylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody . The number of trinucleotide repeat units can be determined by scorin g the detected bands against a digoxigenated M13 sequencing ladder. Ou r newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detectio n of full fragile X mutations and premutations.