Altered expression of alternatively spliced isoforms of the mRNA NMDAR1 receptor in the visual cortex of strabismic cats.

PURPOSE: Although much has been written about the role of the NMDA receptor's role in experience dependent visual plasticity, the function of the NMDAR1 receptor subunit in the post-plasticity stage of development is still not well understood. However, in the well studied model of strabismic amblyopia where binocularity is reduced, but where most primary visual cortex neurons can be driven by one or other eye, the density of expression of NMDAR1 receptor protein is significantly reduced, compared to normals. This study aims to identify which of eight isoforms of the spliced heterogeneous variants of the NMDAR1 mRNA receptor gene are associated with this decrease in expression as a means of elucidating possible function. METHODS: A series of digoxygenin-labelled oligonucleotide probes based on the human gene sequence have been used for in situ hybridization (ISH) of sections from the striate cortex of four adult cats. The probes were used to uniquely detect the expression of alternatively spliced mRNA variants in 66,487 cells from sections from the area centralis projection of two normal cats and two cats made esotropic as kittens by tenotomy at two weeks of age. RESULTS: As expected, total NMDAR1 mRNA isoform expression was significantly lower in the striate cortex of strabismic compared to normal cats. The proportion of cortical cells expressing the R1-a, R1-b, and R1-1 isoforms in strabismic animals was decreased while the proportion expressing R1-3 was increased, especially in layers V and VI. No significant difference in expression of the R1-2 and R1-4 isoforms was seen comparing strabismic and normal cats. CONCLUSIONS: These results confirm our previous findings and suggest that transcriptional inhibition of specific isoforms of NMDAR1 mRNA may underlie the change in receptor expression. This preferential reduction in the proportion of neurons bearing particular NMDAR1 isoforms, i.e. isoforms R1-a and b, and R1-1 with partial compensation through the expression of the R1-3 isoform, is more likely related to lowered proportion of binocularly activated neurons in the strabismic cat than to changes in eye dominance or the presence of amblyopia in one eye.

The elusive Angelman syndrome critical region.

DNA mapping studies in two families provide further information on the Angelman syndrome critical region, which has recently been defined by the gene UBE3A. The first family has probable familial Angelman syndrome with a maternally imprinted inheritance pattern. A 5 year old girl with this disorder has a 14 year old brother and an 11 year old male cousin who have less typical clinical features. DNA microsatellite analysis has shown that the three share a common segment of the same grandpaternal chromosome 15q11-q13 that overlaps with UBE3A. The child with typical Angelman syndrome has an additional maternal recombination 5' to UBE3A. The second family is a mother and son both of whom have mental retardation but no other features of Angelman syndrome despite an extensive DNA deletion on the telomeric side of UBE3A. Together, the two families identify a region between loci D15S210 and D15S986 which forms part of the Angelman syndrome critical region. A new microsatellite (D15S1234) is described which can be used in place of the LS6-1 marker at locus D15S113.

Establishment of sequence-tagged sites on 15q11-q13 by Alu-vector PCR cloning of YAC-generated fragments.

Angelman syndrome (AS) is caused by the loss of function of undefined gene(s) on human chromosome 15. The majority of subjects have deletions involving maternally-derived chromosome 15q11-q13, and the shortest region of deletion overlap (SRO) has been localized to the region between D15S10 and D15S113. In this study, yeast artificial chromosomes (YACs), 6G-D4, 9H-D2 and 37D-F9, mapping within the AS SRO, were isolated from the ICI YAC library. Alu-vector PCR products were amplified from the YACs and from YACs A229A2 and A33F10 which had been obtained from the St. Louis YAC library. The PCR products were cloned and sequenced, and three new sequence-tagged sites were generated within the AS SRO, facilitating the characterization of gene(s) involved in the Angelman syndrome.

Comparison of high resolution cytogenetics, fluorescence in situ hybridisation, and DNA studies to validate the diagnosis of Prader-Willi and Angelman's syndromes.

Eighty seven referrals with Prader-Willi syndrome and 49 with Angelman's syndrome were studied. High resolution cytogenetics was performed on all probands. Molecular studies, performed on the proband and both parents in each case, utilised multiple probes from within and distal to the 15(q11-13) region in order to establish the presence of DNA deletion or uniparental disomy. In addition, FISH, with probes at D15S11 and GABR beta 3 from the Prader-Willi syndrome/Angelman's syndrome region, was performed on a subset of 25 of these patients. In the referral group with Prader-Willi syndrome, 62 patients had a normal karyotype and 25 were deleted on high resolution cytogenetics. Twenty nine were found to be deleted with DNA techniques. In the Angelman's syndrome group, 37 had a normal karyotype and 12 were deleted on high resolution cytogenetics while 26 were deleted on molecular studies. The diagnosis was reassessed in 35 referrals with Prader-Willi syndrome and 11 with Angelman's syndrome following a non-deleted, non-disomic result. Of individuals who were neither deleted nor disomic on DNA studies, a false positive rate of 11.4% (4/35) for Prader-Willi syndrome and 16.7% (2/12) for Angelman's syndrome was found for a cytogenetically detected deletion. The false negative rate for deletion detected on high resolution cytogenetics was 19.5% (12/62) for Prader-Willi syndrome and 35% (13/37) for Angelman's syndrome. Thus high resolution cytogenetics was shown to be unreliable for deletion detection and should not be used alone to diagnose either syndrome. There were no discrepancies with FISH in 25 cases when FISH was compared with the DNA results, indicating that FISH can be used reliably for deletion detection in both syndromes.

A variety of genetic mechanisms are associated with the Prader-Willi syndrome.

An extensive set of chromosome 15 DNA polymorphisms and densitometric analysis with four markers mapping to the Prader-Willi chromosome region (PWCR) of chromosome 15 have been used to characterize a cohort of 30 subjects with classical Prader-Willi syndrome (PWS). Molecular analysis enabled the classification of the PWS subjects into four groups: (A) 18 subjects (60%) had deletions of paternal 15q11-13 involving a common set of DNA markers. Two subjects had differently sized deletions, one larger and one smaller than the other cases. (B) Eight (27%) had maternal uniparental disomy for chromosome 15. (C) One (3%) had a marker chromosome carrying an extra copy of the PWCR. The marker chromosome was demonstrated to be of paternal origin and the two intact chromosomes were maternally derived. This case represents an apparent exception to the generally held view that PWS is associated with an absence of paternally inherited gene(s) located in the PWCR. (D) The remaining three cases (10%) had none of the above abnormalities. This last subgroup of patients has not previously been well characterized but could represent limited deletions not detectable with the markers used or abnormalities in the imprinting process. These cases represent potentially valuable resources to elucidate more precisely the fundamental disorders responsible for PWS.

Familial unbalanced translocation t(8;15)(p23.3;q11) with uniparental disomy in Angelman syndrome.

A 29-year-old male with Angelman syndrome and an unbalanced reciprocal translocation, 45,XY,-8, -15, +der(8),t(8;15)(p23.3;q11)pat, was evaluated with DNA studies. These showed the underlying mechanism to be paternal uniparental disomy. This is the second case reported of Angelman syndrome that has resulted from a familial unbalanced reciprocal translocation.

The Australian type of nondeletional G gamma-HPFH has a C-->G substitution at nucleotide -114 of the G gamma gene.

Nondeletional hereditary persistence of fetal haemoglobin (HPFH) results in the continued production of 2-25% haemoglobin F (Hb F) in the adult who is heterozygous for this mutation. This increase is associated with single-base mutations in the promoter region of either the G gamma- or A gamma-globin genes. Affected positions include -202, -175, -161, -158 and -114 of the G gamma gene, and -202, -198, -196, -195, -175, and -117 of the A gamma gene. There is now evidence that these mutations produce their effect by changing the binding of certain regulatory proteins. We describe a novel C-->G transversion at position -114 of the G gamma gene which is associated with the phenotype of G gamma-HPFH.

Physical mapping studies at D15S10: implications for candidate gene identification in the Angelman syndrome/Prader-Willi syndrome chromosome region of 15q11-q13.

The Angelman syndrome (AS) and Prader-Willi syndrome (PWS) loci have been mapped to chromosome 15q11-q13. Chromosomal deletions of differing parental origin in the two syndromes have been interpreted as being due to genetic imprinting. Molecular analysis of patients with varying deletions has localized the AS locus to the interval between D15S113 and GABRB3 and the PWS locus between D15S13 and D15S113. In the present study, DNA cloning and physical mapping techniques have been used to characterize the AS/PWS chromosome region in the vicinity of D15S10, a locus that is telomeric to D15S113 and centromeric to GABRB3. A CpG island near TD3-21 at D15S10 has been cloned, allowing the identification of a widely expressed 4.5-kb transcript and providing a novel DNA marker, OP3, at this locus. OP3 and TD3-21 have been used to construct a long-range physical map extending over approximately 2800 kb. Clusters of rare-cutting restriction sites on this map locate four other CpG islands. Since these CpG islands lie within the minimum deletion intervals for AS and PWS, they mark the possible locations of candidate genes for the two syndromes.

Novel patterns of inheritance of genetic disease are illustrated by the Angelman syndrome.

OBJECTIVE: To characterise the molecular abnormalities present in a cohort of patients with the Angelman syndrome. METHODS: DNA samples from 10 patients with the Angelman syndrome were investigated with molecular probes. Family studies were performed by means of DNA polymorphism analysis and densitometric estimation of allele copy number to determine the underlying mutation and its parental origin. RESULTS: Nine probands were shown to have molecular (DNA) deletions involving chromosome 15q11-q13. Polymorphism analyses demonstrated that all deletions were maternal in origin. Five of the nine had normal karyotypes, with deletions only detected after DNA study. One patient had inherited both chromosomes 15 from her father. This represented an example of paternal uniparental disomy of chromosome 15. CONCLUSIONS: Development of the Angelman syndrome can result from either deletion of the maternally-derived copy of chromosome 15q11-q13 or the presence of two paternally derived copies of chromosome 15, that is, uniparental disomy. DNA testing allows the identification of deletions that are not seen on cytogenetic analysis and can provide additional information regarding the parental origin of the deletion. Uniparental disomy is most readily established by DNA studies.

Fluorescence in-situ hybridisation and molecular studies used in the characterisation of a Robertsonian translocation (13q15q) in Prader-Willi syndrome.

A patient with classical Prader-Willi syndrome was found to have a Robertsonian translocation 45,XY,t(13q15q)mat. On CBG banding, the translocation chromosome had a large centromere with one primary constriction. Using fluorescence in situ hybridisation, positive signals were obtained with chromosome 13 and chromosome 15 centromere probes, proving that the translocation was dicentric. NOR banding was negative in this chromosome, suggesting that the breakpoints were at 13p11 and 15p11. DNA studies showed that, while there was no deletion involving 15(q11-13), maternal uniparental disomy for chromosome 15 was present. We compare our findings with the five other cases of familial Robertsonian translocation PWS that have been reported.

[Preventive effect of re du qing on hepatocytes and mitochondria damaged by lipid peroxidation in experimental rabbits with endotoxin-induced disseminated intravascular coagulation].

In this study, the general Shwartzman reaction of rabbits induced by Escherichia Coli endotoxin was made as DIC models. The experiments showed that the levels of lipid peroxide (LPO) in hepatic tissue and mitochondria in the model group were increased significantly compared with the control group (P less than 0.01), while superoxide dismutase (SOD) activity in hepatic tissue and glutathione peroxidase (GSH-Px) activity in hepatic tissue and mitochondria were decreased significantly (P less than 0.01). The levels of LPO in hepatic tissue and mitochondria in Re Du Qing (RDQ) group and vitamin E (VE) group were decreased significantly (P less than 0.01 and P less than 0.05 respectively) compared with the model group. The levels of LPO in the RDQ group did not differ from the control group (P greater than 0.05), but the levels of LPO in the VE group were still higher than those in the control group significantly (P less than 0.05). The SOD activity in hepatic tissue and GSH-Px activity in hepatic tissue and mitochondria in both RDQ group and VE group were also significantly higher than those in the model group (P less than 0.01). These data suggest that the levels of oxygen free radicals were increased in hepatocytes and mitochondria. This is related to the decreased activities of SOD and GSH-Px in the course of pathogenesis of endotoxin-induced DIC. This study indicates that lipid peroxidation might be one of the important mechanisms resulting in hepatocellular and mitochondria from oxidative damage.