Association of AKT1 gene variants and protein expression in both schizophrenia and bipolar disorder.

The AKT1 gene has been associated with the genetic aetiology of schizophrenia. Following the overlap model of bipolar disorder and schizophrenia, we aimed to investigate AKT1 genetic variants and protein expression in both diseases. A total of 679 subjects with European ancestry were included: 384 with schizophrenia, 130 with bipolar disorder and 165 controls. Six single nucleotide polymorphisms (SNPs) were investigated for association with the diseases using single- and multi-locus analyses. AKT1 and AKT2 protein levels were measured in post-mortem brain tissues from ante-mortem diagnosed schizophrenia (n = 30) and bipolar disorder subjects (n = 12) and matched controls. The analysis identified a significant global distortion in schizophrenia (P = 0.0026) and a weak association in bipolar disorder (P = 0.046). A sliding window procedure showed a five-SNP haplotype (TCGAG) to be associated with schizophrenia (P = 1.22 x 10(-4)) and bipolar disorder (P = 0.0041) and a four-SNP haplotype (TCGA) with the combined sample (1.73 x 10(-5)). On the basis of selected genotypes, a significant difference in protein expression emerged between subjects (P < 0.02). In conclusion, our findings, by showing the involvement of the AKT1 gene in both schizophrenia and bipolar disorder, support the role of AKT1 in the genetics of both disorders and add support to the view that there is some genetic overlap between them.

A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex.

Cyclic AMP-dependent protein kinase (PKA) activity was involved in a number of brain functions such as cognitive process or aging. The measurement of PKA activity is traditionally based on the use of [(32)P]ATP in phosphorylation of specific protein. Recently non-isotopic PKA assays have been developed, but none has been tested on brain homogenates. This work aimed to adapt a fluorimetric method of PKA activity into a novel assay never applied before in brain homogenate, and to characterize the enzyme activity and ratio in hippocampus and cortex from rats of different ages. Optimal conditions of homogenization and enzyme protection were determined. The method was sensitive and reproducible (intra-assay and interassay variation was 5.0% and 9.0%, respectively). In hippocampal cytosol, PKA activity was 27+/-8 and 80+/-9 nmol/min per mg protein in basal and cAMP-stimulated activity, respectively, and accounted for 80% of total cell PKA activity. The non-PKA activity, assessed by the use of the PKA specific inhibitor (PKI) accounted for 49.0% and 65.0% of endogenous levels in cytosol and membrane, respectively. cAMP-augmenting drugs effects were measured and increase of 53%, 273% and 118% over basal by 10 microM isoproterenol, 100 microM forskolin, 1 microM Sp-AMP, respectively, was observed. With respect to the changes in animal age, PKA activity increased from newborn to the mature rats but decreased in older rats. The PKA ratio was higher in cytosol than in particulate fraction, and was decreased in hippocampal sample from old rats (P<0.05). This last result was interpreted as related to the loss of cognitive capacities in old animals.

[Unverricht-Lündborg disease: clinical and electrophysiologic study of 19 Maghreb families]. / Maladie d'Unverricht-Lündborg: étude clinique et électrophysiologique de 19 familles maghrébines.

We describe clinical, electrophysiological and genetic features in 44 patients with Unverricht-Lündborg disease from 19 families living in North African countries (Tunisia, Algeria and Morocco). The mean age of patients was 25.3 years; mean age was at onset 11.3 years. The disease began more frequently with seizures (91 per cent) or myoclonus (80 p. 100) than ataxia (16 p. 100). Subsequently myoclonus and generalized seizures were present in all patients, cerebellar signs were absent in four cases. EEG findings included normal background activity (90 p. 100), spontaneous fast generalized spikes (93 p. 100) and photosensitivity (70 p. 100). Antiepileptic polytherapy (clonazepam and/or phenobarbital and/or valporic acid) was used in 84 per cent of cases. Antiepileptic drugs were more effective in controlling epileptic seizures (less than one seizure/month in 60 p. 100) than myocloni which persisted daily in 64 p. 100 of cases. Mean duration of the disease was 13.5 years. One patient died of status epilepticus. Consanguinity was noted in 17 families (first degree in 15 families). Linkage to chromosome 21q 22.3 was confirmed in 11 families. We noted an inter and intrafamilial variability of clinical signs and disease course.

A PCR amplification method reveals instability of the dodecamer repeat in progressive myoclonus epilepsy (EPM1) and no correlation between the size of the repeat and age at onset.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is a rare, autosomal recessive disorder characterized by onset at age 6-16 years, generalized seizures, incapacitating myoclonus, and variable progression to cerebellar ataxia. The gene that causes EPM1, cystatin B, encodes a cysteine proteinase inhibitor. Only a minority of EPM1 patients carry a point mutation within the transcription unit. The majority of EPM1 alleles contain large expansions of a dodecamer repeat, CCC CGC CCC GCG, located upstream of the 5' transcription start site of the cystatin B gene; normal alleles contain two or three copies of this repeat. All EPM1 alleles with an expansion were resistant to standard PCR amplification. To precisely determine the size of the repeat in affected individuals, we developed a detection protocol involving PCR amplification and subsequent hybridization with an oligonucleotide containing the repeat. The largest detected expansion was approximately 75 copies; the smallest was approximately 30 copies. We identified affected siblings with repeat expansions, of different sizes, on the same haplotype, which confirms the repeat's instability during transmissions. Expansions were observed directly; contractions were deduced by comparison of allele sizes within a family. In a sample of 28 patients, we found no correlation between age at onset of EPM1 and the size of the expanded dodecamer. This suggests that once the dodecamer repeat expands beyond a critical threshold, cystatin B expression is reduced in certain cells, with pathological consequences.

Allelic heterogeneity of Mediterranean myoclonus and the cystatin B gene.

Mediterranean myoclonus is a progressive myoclonus epilepsy with autosomal recessive inheritance. Another form has been described in Finland, the so-called Baltic myoclonus. Mediterranean myoclonus and Baltic myoclonus are also known as Unverricht-Lundborg disease. Linkage analyses have shown that the genes for both these forms of myoclonus are closely linked to 21q22.3 DNA markers, suggesting that they are caused by mutations at the same locus (EPM1). Recently, two heterozygous mutations were found in the cystatin B gene in patients with Unverricht-Lundborg disease. We report recombinational and linkage disequilibrium mapping of EPM1, and cystatin B gene sequencing, in 14 consanguineous pedigrees with Mediterranean myoclonus. Linkage to 21q22.3 DNA markers was observed in all these families. Haplotype analysis suggests that a common mutation segregates within these pedigrees, and that this mutation is different from the common one responsible for the Finnish form of Unverricht-Lundborg disease. No mutation was found in the exons or splice junctions of the cystatin B gene in the 14 pedigrees.

Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder with onset between 6 and 13 years followed by variable progression to mental deterioration and cerebellar ataxia. It is a rare disorder but more common in Finland (1 in 20,000) and the western Mediterranean. Two point mutations in the cysteine proteinase inhibitor gene cystatin B (CSTB), proved that this gene is responsible for EPM1 (ref. 3). An extensive search in the CSTB gene revealed mutations accounting only for 14% of the 58 unrelated EPM1 alleles studied. Here we report that the majority of EPM1 alleles contain expansions of a dodecamer (12-mer) repeat located about 70 nucleotides upstream of the transcription start site nearest to the 5' end of the CSTB gene. Normal alleles contain 2 or 3 copies of this repeat whereas mutant alleles contain more than 60 such repeats and have reduced levels of CSTB messenger RNA in blood but not in cell lines. 'Premutation' CSTB alleles with 12-17 repeats show marked instability when transmitted to offspring.

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1).

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.

Absence of linkage between chromosome 11p15 markers and manic-depressive illness in a Belgian pedigree.

OBJECTIVE: The original finding of genetic linkage in an Old Order Amish pedigree has been contradicted by the results of several subsequent studies. Using the same genetic parameter values, diagnostic criteria, and 11p15 genetic markers as those used to study the initial Amish population, the authors performed a linkage study of a four-generation informative pedigree in Belgium. METHOD: Recombinant DNA technology was used to analyze three markers for the chromosome 11p15 location: the genes for tyrosine hydroxylase (TH) and insulin (INS) and the c-Harvey-ras oncogene (HRAS). Diagnoses of the relatives of a proband with bipolar affective disorder were determined with the Schedule for Affective Disorders and Schizophrenia--Lifetime Version and based on the Research Diagnostic Criteria. Relatives were considered affected if they had bipolar disorder, unipolar disorder, or cyclothymia; a diagnostic hierarchy was developed to include unipolar disorder and cyclothymia in the linkage analysis. RESULTS: Pairwise analyses of the disease locus and each of the three polymorphisms excluded the possibility of close linkage between manic-depressive illness and the three chromosome 11p15 markers. Multipoint linkage analysis combining the information from all three genes also excluded linkage. CONCLUSIONS: The conflict between the original results from the Amish study and the many negative reports on chromosome 11 linkage of manic-depression has been interpreted as indicating genetic heterogeneity, but heterogeneity has not been documented for the 11p15 locus. Conversely, the linkage approach has major drawbacks, so other genetic strategies should also be considered.