Of a total of 72 cases referred for fragile X testing, 7 (9.7%) were found to be positive for fragile X by either cytogenetics alone or by both cytogenetics and DNA testing, 12 (16.6%) were found to be positive for structural chromosomal abnormality, while 4 (5.5%) were found to exhibit a heteromorphism. Positive chromosomal findings included abnormalities of the sex chromosomes and autosomes, deletions, translocations. Heteromorphism mostly involved an increase in the length of heterochromatic regions of certain chromosomes as well as a pericentric inversion of a chromosome 9, usually considered normal variants. It is concluded that chromosomal abnormalities other than fragile X are found with equal and, in some cases, higher frequency than the frequency of fragile X positivity in patients referred for a question of the Fragile X Syndrome. Our figures consistent with those reported in the literature, underscore the value of routine karyotyping in this population of patients
10.
Fragile X syndrome is inherited as an X-linked dominant inheritance 11,12. Frequencies of fargile X in previous studies have reported as 2.6 to 8.7% among moderate to severely retarded males and 2.9 to 5.4% in mildly retarded females 13. In our study population, frequencies of MR were determined as 3.75% in males and 2.5% in female. Our study consistent with previous studies.
Considering the technical aspects of cytogenetic analysis for FXS, usually a longer exposure to colchicine causes more chromosomal condensation, which turns fragile X easier to be detected by microscopic analysis. We used a shorter exposure (30 to 40 minutes instead of 60 minutes) which is less likely to interfere with the detection of other cytogenetic abnormalities. Although cytogenetic evaluation was sensitive and specific for the diagnosis of FXS, the fact that it is based in a 96-hours cell culture, which can fail, may require re-testing in several occasions. When the culture is successful, the study demands many hours of microscopic analysis, especially for the female specimens. In addition, it is well known that this method may not detect all carrier females, and would miss most male carriers, since only a small proportion of their cells will express the fragile X 14. The best advantage of this method is that it makes it possible to detect other chromosomal abnormalities, including the other fragile X sites with one single test.
Gender of carrier parent, gender of the offspring and the number of CGG repeats are important factors that influence disease expression. The complex pattern of inheritance poses an extraordinary challenge for accurate diagnosis and genetic counselling of affected families. Though a variety of clinical phenotypic characteristics has been described, none are singly or in combination helpful in definitive diagnosis. Though cytogenetic methods and PCR have been used in Turkish studies on Fragile X syndrome, methylation sensitive PCR has not been reported 15,16. In our study with 72 samples three cases with full mutation and another with premutation (carrier state) were detected. The carrier state and healthy normal samples were clearly distinguishable by the size of the amplified PCR product, as also the fragile X positive sample. The basic principle of this method is that it relies on the ability of bisulphite to deaminate C residues in a single strand DNA. A characteristic of the bisulphite treated DNA is that after modification, the sense and antisense strand are no longer complimentary. Thus, the modified strands can be amplified separately by designing primer pairs specific for each of them. The C residues of all CpG dinucleotides flanking the CGG repeats as well as those of the CGG repeats are methylated in affected males and in the inactive X chromosome in females. The same C residues are however, unmethylated in healthy males, normal transmitting males and in the active X chromosome in females 17,18.
The disadvantage of this methylation PCR is that it cannot reliably diagnose affected females with fragile X syndrome due to the fact that the inactive X chromosome is already methylated. Recently modifications have been incorporated into methylation sensitive PCR strategies which reliably differentiate normal from carrier and full mutations, in both females and males 17,18. All our DNA samples in the present study were from male and female children with mental retardation. There is no specific treatment for fragile X syndrome. DNA tests for fragile X syndrome should be done in all mentally retarded children without an obvious cause, along with genetic counselling of the involved families. Methylation sensitive PCR strategy is one of the most comprehensive methods available at present for the accurate diagnosis of fragile X carrier and disease state 18. As a result, in our study, the frequency of normal allels, patients with full mutation and premutation were estimated in Elazig and vicinity. In this population, the frequency of fragile X syndrome that is an important problem for population health was found nearly 9.7% in this study firstly.
We believe that the recording and following of families with FXS will provide prenatal diagnosis and the usage of a possible treatment opportunity in the future. Although cytogenetic analysis in mentally retarded patients help in accurate diagnosis, our data show that all the members with/without clinical findings of FXS in the families with FXS should be screened by the PCR-based method to follow the transmission of the CGG repeats and to give correct genetic counseling to families. Our data suggest that expansion of CGG repeats in the FMR1 gene can be analyzed by Methylation sensitive PCR, an efficient and non-radioactive method that can be used to monitor the expansion of premutation to full mutation, which would eventually lead to reduce the FXS prevalence.