ABSTRACT
We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 46-49 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0-4.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.
Subject(s)
Muscular Dystrophy, Duchenne/genetics , Nuclear Proteins , Polymerase Chain Reaction/methods , Sex Determination Processes , Transcription Factors , DNA-Binding Proteins/genetics , Exons/genetics , Female , Gene Deletion , Humans , Male , Sex-Determining Region Y ProteinABSTRACT
The tumour necrosis factor (TNF)2 allele appears to be linked with increased insulin resistance and obesity, conditions often found in overweight patients with polycystic ovary syndrome (PCOS). The significance of TNFalpha polymorphism in relation to the clinical and biochemical parameters associated with PCOS was investigated in 122 well-characterized patients with polycystic ovaries (PCO). Of these, 84 had an abnormal menstrual cycle and were classified as having PCOS, while the remaining 38 had a normal menstrual cycle and were classified as having PCO. There were a further 28 individuals without PCO (non-PCO) and 108 individuals whose PCO status was undetermined (reference population). The promoter region of the TNFalpha gene was amplified by polymerase chain reaction (PCR), and the presence or absence of the polymorphism at -308 was determined by single-strand conformational polymorphism (SSCP) analysis. The less common TNF allele (TNF2) was found as TNF1/2 or TNF2/2 in 11/38 (29%) of PCO subjects, 25/84 (30%) of PCOS subjects, 7/28 (25%) of non-PCO subjects, and 45/108 (42%) of the reference population. There was no significant difference in the incidence of the TNF2 allele between the groups. The relationship of TNF genotype to clinical and biochemical parameters was examined. In both the PCO group and the PCOS group, the presence of the TNF2 allele was significantly associated with lower glucose values obtained from the glucose tolerance testing (P<0.05). The TNF genotype was not significantly associated with any clinical or biochemical parameter measured in the PCO, PCOS or non-PCOS groups. Thus, the TNFalpha -308 polymorphism does not appear to strongly influence genetic susceptibility to polycystic ovaries.
Subject(s)
Polycystic Ovary Syndrome/genetics , Polymorphism, Genetic , Tumor Necrosis Factor-alpha/genetics , Adult , Female , Glucose Tolerance Test , Heterozygote , Humans , Insulin Resistance/genetics , Menstrual Cycle/physiology , Obesity/genetics , Ovulation/genetics , Polycystic Ovary Syndrome/diagnostic imaging , Polymorphism, Single-Stranded Conformational , Promoter Regions, Genetic , UltrasonographyABSTRACT
We report the cDNA sequence of the zinc finger gene, ZNF195, which maps to chromosome 11p15.5. ZNF195 contains an N-terminal KRAB domain and 14 tandemly repeated Krüppel type zinc finger motifs at its C-terminus. Northern analysis shows expression of ZNF195 in adult heart, brain, placenta, skeletal muscle, and pancreas with a predominant transcript size of 4.3 kb. There is little expression in adult lung, liver, and kidney. In fetal lung, liver, kidney, and brain, the predominant transcript is 3.5 kb. Fetal brain also expresses a 4.3-kb transcript. RT-PCR analysis shows that two exons, 4a, which contains an inverted Alu sequence, and 4b, are differentially spliced and absent from the major transcript.
