Complete genomic structure and mutational spectrum of PHKA2 in patients with x-linked liver glycogenosis type I and II.
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X-linked liver glycogenosis (XLG) is probably the most frequent glycogen-storage disease. XLG can be divided into two subtypes: XLG I, with a deficiency in phosphorylase kinase (PHK) activity in peripheral blood cells and liver; and XLG II, with normal in vitro PHK activity in peripheral blood cells and with variable activity in liver. Both types of XLG are caused by mutations in the same gene, PHKA2, that encodes the regulatory alpha subunit of PHK. To facilitate mutation analysis in PHKA2, we determined its genomic structure. The gene consists of 33 exons, spanning >/=65 kb. By SSCP analysis of the different PHKA2 exons, we identified five new XLG I mutations, one new XLG II mutation, and one mutation present in both a patient with XLG I and a patient with XLG II, bringing the total to 19 XLG I and 12 XLG II mutations. Most XLG I mutations probably lead to truncation or disruption of the PHKA2 protein. In contrast, all XLG II mutations are missense mutations or small in-frame deletions and insertions. These results suggest that the biochemical differences between XLG I and XLG II might be due to the different nature of the disease-causing mutations in PHKA2. XLG I mutations may lead to absence of the alpha subunit, which causes an unstable PHK holoenzyme and deficient enzyme activity, whereas XLG II mutations may lead to in vivo deregulation of PHK, which might be difficult to demonstrate in vitro. (+info)
X-linked late-onset sensorineural deafness caused by a deletion involving OA1 and a novel gene containing WD-40 repeats.
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We have identified a novel gene, transducin (beta)-like 1 (TBL1), in the Xp22.3 genomic region, that shows high homology with members of the WD-40-repeat protein family. The gene contains 18 exons spanning approximately 150 kb of the genomic region adjacent to the ocular albinism gene (OA1) on the telomeric side. However, unlike OA1, TBL1 is transcribed from telomere to centromere. Northern analysis indicates that TBL1 is ubiquitously expressed, with two transcripts of approximately 2.1 kb and 6.0 kb. The open reading frame encodes a 526-amino acid protein, which shows the presence of six beta-transducin repeats (WD-40 motif) in the C-terminal domain. The homology with known beta-subunits of G proteins and other WD-40-repeat containing proteins is restricted to the WD-40 motif. Genomic analysis revealed that the gene is either partly or entirely deleted in patients carrying Xp22.3 terminal deletions. The complexity of the contiguous gene-syndrome phenotype shared by these patients depends on the number of known disease genes involved in the deletions. Interestingly, one patient carrying a microinterstitial deletion involving the 3' portion of both TBL1 and OA1 shows the OA1 phenotype associated with X-linked late-onset sensorineural deafness. We postulate an involvement of TBL1 in the pathogenesis of the ocular albinism with late-onset sensorineural deafness phenotype. (+info)
The timing of XIST replication: dominance of the domain.
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Contiguous replicons are coordinately replicated and may be organized in temporal-spatial domains with early replication domains containing expressed genes and late ones carrying silent genes. XIST is silent on the active, early replicating X chromosome and expressed from the inactive, late replicating homolog. These circumstances potentially deviate from the aforementioned generalization and make studies of replication timing for XIST of special interest. Although earlier investigations of XIST replication in fibroblasts based on analysis of extracted DNA from cells at different stages of the cell cycle suggested that the silent gene does replicate before the expressed allele, studies using FISH technology produced the opposite results. Because the FISH replication studies could not directly distinguish between the active and inactive X chromosomes in the same cell, we undertook a re-investigation of this question utilizing FISH analysis under conditions that allowed us to make that distinction using cells sorted into different cell cycle stages by flow cytometry. The findings reported here indicate that the silent XIST gene on the active X chromosome does replicate before the expressed allele on the inactive X, supporting the view that the time of a gene's replication is determined by the large, multi-replicon domain in which it is located and not necessarily its expression state. (+info)
Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.
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The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes. (+info)
Maternally inherited cardiomyopathy: clinical and molecular characterization of a large kindred harboring the A4300G point mutation in mitochondrial deoxyribonucleic acid.
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OBJECTIVES: The purpose of this study was to describe the clinical and molecular features of a large family with maternally inherited cardiomyopathy (MICM). BACKGROUND: Recently, several mitochondrial deoxyribonucleic acid (mtDNA) point mutations have been associated with MICM. However, the distinctive clinical and morphologic features of MICM are not fully appreciated. This is partially due to the small size of the reported pedigrees, often lacking detailed clinical and laboratory information. METHODS: Clinical and genetic analysis of the family was carried out. RESULTS: Echocardiography showed mostly symmetrical hypertrophic cardiomyopathy in 10 family members. The illness had an unfavorable course. Progressive heart failure occurred in three subjects, who eventually died; one individual underwent heart transplantation. Electrocardiographic or echocardiographic signs of cardiac hypertrophy in the absence of significant clinical complaints were observed in five subjects. Neurologic examination was normal. The mutation was detected in blood from all available subjects. Abundance of mutated molecules ranged between 13% and 100% of total mtDNA genomes. The severity of the disease could not be foreseen by the proportion of mutation in blood. CONCLUSIONS: This report contributes a better description of the clinical aspects of MICM and provides important clues to distinguish it from hypertrophic cardiomyopathy. We suggest that mtDNA mutations, particularly in the transfer ribonucleic acid for isoleucin, should be systematically searched in patients with MICM. The identification of an underlying maternally inherited mitochondrial DNA defect in familial cases of cardiomyopathy may considerably influence the management and genetic counseling of affected patients. (+info)
Implications of sperm chromosome abnormalities in recurrent miscarriage.
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PURPOSE: Our purpose was to assess the existence of sperm chromosome abnormalities in recurrent pregnancy loss in an assisted reproduction program. METHODS: In this prospective study, 12 sperm samples from couples undergoing in vitro fertilization with two or more first-trimester spontaneous abortions were analyzed. Diploidy and disomy in decondensed sperm nuclei were assessed for chromosomes 13, 18, 21, X, and Y using two- and three-color fluorescence in situ hybridization. RESULTS: Sex chromosome disomy in sperm samples from recurrent abortion couples was significantly increased compared to that from internal controls (0.84% vs 0.37%). In a subpopulation of seven couples who underwent oocyte donation, mean frequencies for sex chromosome disomy (1%) were even higher and diploidy (0.43%) was also significantly increased. CONCLUSIONS: These results suggest an implication of sperm chromosome abnormalities in some cases of recurrent pregnancy loss. (+info)
Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor.
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The vast majority of the known biological effects of the renin-angiotensin system are mediated by the type-1 (AT1) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.0001). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 +/- 0.5 to 208 +/- 1 mmHg (P < 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 +/- 0.01 to 0.05 +/- 0.002 mM/day (P < 0.0001)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3', 5'-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3', 5'-monophosphate by approximately 4-fold (P < 0.0001) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT2 receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II. (+info)
Analysis of red/green color discrimination in subjects with a single X-linked photopigment gene.
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Many subjects despite having only a single X-linked pigment gene (single-L/M-gene subjects) are able to make chromatic discriminations by Rayleigh matching, especially when large fields are used. We used a combination of psychophysics (Rayleigh match), electroretinograms (ERG), and molecular genetic techniques to rule out several possible explanations of this phenomenon. Use of rods for chromatic discrimination was unlikely since strong adapting fields were employed and the large-field match results were not consistent with rod participation. A putative mid- to long-wavelength photopigment that escapes detection by current molecular genetic analysis was ruled out by finding only a single L/M photopigment in flicker ERGs from 16 single-L/M-gene subjects. Large-field match results were not consistent with participation of S cones. Amino acid sequence polymorphisms in the S-pigment gene that might have shifted the S cone spectrum towards longer wavelengths were not found on sequencing. The mechanism of chromatic discrimination in the presence of a single photopigment therefore remains unknown. Further possible explanations such as variations in cone pigment density and retinal inhomogeneities are discussed. (+info)