Fragile X syndrome is a result of an unstable expansion of (CGG)n trinucleotide sequences in the FMR-1 (Fragile X Mental Retardation 1) gene site at Xq27. In a normal person, n ranges from 6 to 40 repeats with an average of 30 repeats, whereas in a mutated FMR1 gene the sequence is repeated several times over (stuttering gene). Full mutation occurs when n equals 200 repeats or more. Where n equals 50 to 200 repeats, it is a premutation. Fragile X occurs when the FMR-1 gene is unable to make normal amounts of usable Fragile X Mental Retardation Protein, or FMRP. The amount of FMRP in the body is one factor that determines the severity of the Fragile X syndrome. A person with nearly normal levels of FMRP usually has mild or no symptoms, while a person with very little or no normal FMRP has more severe symptoms. The mechanism for the role of the FMRP gene is still being researched upon. However, it has been observed that large numbers of repeats (more than 200) inactivates the gene through a process of methylation and when the gene is inactivated, the cell may make little or none of the needed FMRP. Inheritance is X-linked with reduced penetrance and the frequency of occurrence goes up through generations. The phenotypic manifestations of fragile-X syndrome vary and are largely dependent on the size of the mutation or premutation. The identification of the fragile site on G banded metaphases is a time consuming and delicate process requiring experience and skill, however, molecular diagnosis using DNA analysis and Southern blotting, even though expensive, is more specific in determining the presence or absence of the gene. This study was aimed to establish a rapid polymerase chain reaction (PCR) based - touch down PCR, as a screening method for fragile X syndrome. A total of six cases were analysed. Of these, one was a known case of Fragile X (T1) diagnosed by conventional cytogenetics, two were from the latter’s family members namely, his mother (T2) and father (T3), and the other two (T4 and T5) were randomly selected from patients presenting with dysmorphic features and delayed development respectively. One normal control (TC) was included. Cytogenetic analyses for detection of the fragile site was carried out in all cases. Two culture systems were used, namely the synchronised lymphocyte culture and the folate - thymidine deficient culture. Stained metaphases from the fragile X cultures were screened for the presence of the fragile site on the X chromosome. G-banded karyotyping was done using an image analyser to exclude presence of chromosomal abnormalities. DNA was extracted from these samples and amplified by touch-down PCR. Cytogenetic analysis revealed a folate-sensitive fragile site in the affected male, but none in the other five samples. G-banded karyotyping exhibited no additional chromosomal abnormalities. All extracted DNA samples were successfully amplified. Five of the samples showed presence of the product at the expected band at 552bp, excluding the presence of an expansion of CGG segment of the FMR-1 gene. The absence of a band in an affected individual, suggested a fully mutated allele of FRAXA (Folate Sensitive Fragile Site at Xq28). We succeeded in establishing a slightly modified touch-down PCR analysis. Our study indicates that PCR testing offers a rapid and specific method for screening of normal allele and full mutation of the fragile X gene. We suggest this technique to be applied as a complementary tool for cytogenetic analysis to detect the FRAXA gene.
Cytogenetic investigations were carried out on 117 women with primary amenorrhea who had been referred to our Genetics Laboratory by clinicians throughout Malaysia, after exclusion of other causes of the disorder. Thirty-six cases (31%) showed numerical or structural abnormalities of the sex chromosomes. These can be broadly classified into 4 main types, namely, presence of a Y chromosome (14%), X-chromosome aneuploidies (8%), structural anomalies of the X-chromosome (7%) and lastly, presence of a marker chromosome (2%). Mosaics constituted 17% of the abnormalities observed, always in association with a 45,X cell line. There was no observable correlation between the phenotype of the patients and their respective abnormal karyotypes. The aetiological role of sex chromosomal abnormalities in these amenorrheic women is discussed.
A cytogenetic survey 01 124 children in lour special schools for the mentally handicapped was carried out to determine the contribution of chromosomal abnormalities to the aetiology of mental retardation in these children. All the children were karyotyped employing the G·banding technique 01 43 (34.7%) with an abnormal chromosome complement, 40 had Down's Syndrome, and 3 had other chromosomal abnormalities, namely a translocation 1;17, a mosaic male/trisomy 18 and a Klinefelter's syndrome. Polymorphic variants involving chromosomes 1, 9, and 14 were also observed. Two other children showed variants of the Y chromosome (one a small Y and the other a metacentric Y). The possible contribution by these abnormal variants to mental retardation is discussed. Details of the abnormal cytogenetic findings are reported.
An Indian family with all 3 sons having the fragile X syndrome is reported. The frequency of fragile X cells observed ranged from 4·16%. The phenotypically normal mother, although an obligate carrier, did not express any fragile X chromosomes in her Iymphocyte cultures. The range of mental retardation in affected
hemizygous males and heterozygous females as well as the significance of the fragile X chromosome in prenatal diagnosis are discussed.
The haematological findings and case history of 3 patients with the association of acute myeloid leukemia and translocation involving the long arm of chromosome no. 11 are presented. The recipient chromosome for the translocated material from chromosome 11 differs in all the three cases being namely chromosomes 1, 10 and 17.
An RT-PCR assay detected the t(4;11) translocation in two infants with acute lymphoblastic leukemia (ALL). Case P76 was a 10-month-old, female infant, who presented with a WBC of 137.4 x 10(9)/l and a pre-pre-B ALL immunophenotype. Case P120 was a 6-month-old female infant, with a WBC > 615 x 10(9)/l and a pre-pre-B ALL immunophenotype. RT-PCR of cDNA from both these cases generated a 656 bp and a 542 bp respectively, which sequencing confirmed as t(4;11) fusion transcripts. The primers and conditions selected for this assay are compatible with a one-step multiplex PCR for the main translocations in childhood ALL.