Psychiatric and cognitive symptoms in Huntington's disease are modified by polymorphisms in catecholamine regulating enzyme genes.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder characterized by motor, psychiatric, and cognitive manifestations. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene but the exact pathogenesis remains unknown. Dopamine imbalance has previously been shown in HD, and furthermore dopamine is thought to be implicated in cognition, behavioral and motor disturbances. A substantiated inverse correlation between motor onset and the elongated CAG repeat in the HTT has been established. This relation does not account for the full variability of the motor onset, and efforts have been put into finding genetic modifiers of motor onset, however, mostly with unsuccessful outcome. In this study, we took an alternative approach focusing on symptom complexes and searched for modifiers of cognitive impairment and psychiatric symptoms in a well-described cohort of Danish HD gene-expansion carriers. We show that cognitive impairment and psychiatric symptoms in HD are modified by polymorphisms in the monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT) genes and by the 4p16.3 B haplotype. These results support the theory of dopamine imbalance in HD, and point toward more personalized treatment modalities of HD in the future.

4p16.3 haplotype modifying age at onset of Huntington disease.

Huntington disease (HD) is caused by an expanded CAG repeat sequence in the HD gene. Although the age at onset is correlated to the CAG repeat length, this correlation only explains approximately half of the variation in onset age. Less variation between siblings indicates that the variation is, in part, explained by genetic modifiers. We analyzed polymorphic loci within or close to the HD gene on the HD chromosome in Danish HD patients. We found one specific haplotype segregating with later age at onset, compared with patients with similar CAG repeat length and another haplotype. The nine Danish families in the study carrying this haplotype most likely have a common founder. Several of the polymorphic loci displayed alleles that may be specific to the late-onset haplotype, implicating that from this study we cannot determine which of the loci tested (or other polymorphic loci in this chromosomal area) do in fact contain genetic modifiers of age at onset.

A novel presenilin 2 mutation (V393M) in early-onset dementia with profound language impairment.

BACKGROUND: Mutations in the Presenilin 2 gene (PSEN2) are rare causes of Alzheimer's disease (AD). Pathogenic mutations in the genes associated with autosomal dominant inherited AD have been shown to alter processing of the amyloid precursor protein (APP) resulting in a relative increase of the amount of Abeta42 peptide. METHODS AND RESULTS: We present a patient with neuropathologically confirmed early-onset AD characterized by profound language impairment. The patient was heterozygous for a novel missense mutation in exon 11 of the PSEN2 gene leading to a predicted amino acid substitution from valine to methionine in position 393, a conserved residue. However, in vitro expression of PSEN2 V393M cDNA did not result in detectable increase of the secreted Abeta42/40 peptide ratio. The mutation was not found in 384 control individuals tested. CONCLUSIONS: The possible pathogenic nature of the mutation is not clarified. We discuss the limitations of functional PSEN2 studies and the challenges associated with genetic counselling of family members at risk.

Five novel mutations in fourteen patients with Fabry Disease.

Fabry disease is an X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A. The mutations responsible for Fabry disease are diverse and include large rearrangements as well as single base substitutions, and they are dispersed throughout the seven exons of the gene. In this study, we found five novel mutations in four different exons. We have detected the mutations by the PCR-SSCP method and then analysed them by direct sequencing. Three of the novel mutations were deletions: 1205delA, 1238del26 and 5236del18. We also found one novel nonsense mutation: W162X. The final novel mutation was an insertion combined with a deletion: 10995ins24del4.

Analysis of FMR1 (CGG)n alleles and FRAXA microsatellite haplotypes in the population of Greenland: implications for the population of the New World from Asia.

The fragile X syndrome is caused by the expansion of a polymorphic (CGG)n tract in the promoter region of the FMR1 gene. Apparently the incidence of fragile X syndrome is rare in the population of Greenland. In order to examine population-related factors involved in stability of the (CGG)n sequence, DNA samples obtained randomly from the Greenlandic population were analysed for size and AGG interspersion pattern of the FMR1 (CGG)n region and associated DXS548-FRAXAC1 haplotypes. In addition a large Greenland family with unstable transmission in the premutation range was analysed. The (CGG)n allele sizes in the Greenland population showed a narrow distribution similar to that reported for Asian populations. DNA sequencing of alleles with 36 CGG repeats revealed an AGG(CGG)6 insertion previously reported exclusively in Asian populations and a high frequency of alleles with a (CGG)10AGG(CGG)9AGG(CGG)9 or (CGG)9AGG(CGG)9AGG(CGG)6AGG(CGG)9 sequence pattern was found. Thus the data confirm the Asian origin of the Greenlandic (Eskimo) population and indicates that some (CGG)n alleles have remained stable for 15-30,000 years, since the population of the New World arrived from Asia via the Bering Strait.

Molecular and behavioral analysis of the R6/1 Huntington's disease transgenic mouse.

Transgenic mice expressing exon 1 of the human Huntington's disease (HD) gene carrying a 115 CAG repeat (line R6/1) are characterized by a neurologic phenotype involving molecular, behavioral and motor disturbances. We have characterized the R6/1 to establish a set of biomarkers, which could be semi-quantitatively compared. We have measured motor fore- and hindlimb coordination, fore- and hindpaw footprinting, general activity and anxiety, feetclasping, developmental instability. Molecular investigations involved measurements of cannabinoid receptor 1 mRNA, met-enkephalin peptide, dopamine and cyclic AMP-regulated phosphoroprotein 32 kDa and neuronal inclusions. Molecular and behavioral testing was performed on female hemizygotic R6/1 transgenic mice and female wildtype littermates between 6 and 36 weeks of age. We show that the cannabinoid receptor 1 receptor is severely and rapidly downregulated in the R6/1 mouse between the 8(th) to the 10(th) week of age. At 14 weeks of age the first transgenic mice showed a behavioral phenotype measured by feetclasping. However, there was great variation between the individual animals. At 11 weeks of age the mice demonstrated progressively increasing developmental instability as measured by fluctuating asymmetry. Weight differences were evident by 22 weeks of age. Mice tested at 23 and 24 weeks of age showed significant impairments in open field and plus-maze analysis respectively. We observed no significant abnormalities in stride length of the R6/1 mouse model. As the analyzed parameters are easily detected and measured, the R6/1 mouse appears to be a good model for evaluating new drugs or types of therapy for HD.

Haplotype and AGG-interspersion analysis of FMR1 (CGG)(n) alleles in the Danish population: implications for multiple mutational pathways towards fragile X alleles.

The AGG interspersion pattern and flanking microsatellite markers and their association with instability of the FMR1 (CGG)(n) repeat, involved in the fragile X syndrome, were analyzed in DNA from filter-paper blood spots randomly collected from the Danish newborn population. Comparison of DXS548-FRAXAC1 haplotype frequencies in the normal population and among fragile X patients suggested strong linkage disequilibrium between normal alleles and haplotype 7-3 and between fragile X alleles and haplotype 2-1 and 6-4. Comparison of the AGG interspersion pattern in 143 alleles, ranging in size from 34-62 CGG, and their associated haplotypes indicates the existence of at least three mutational pathways from normal alleles toward fragile X alleles in the Danish population. Two subgroups of normal alleles, with internal sequences of (CGG)(10)AGG(CGG)(19) and (CGG)(9)AGG(CGG)(12) AGG(CGG)(9), possibly predisposed for expansion, were identified in the data set. When alleles larger than 34 CGG were investigated, comparing the length of 3' uninterrupted CGG triplets (uCGG), we found that alleles associated with haplotype 2-1 and 6-4 contain significantly longer stretches of uCGG than alleles associated with haplotype 7-3. Thus, the data support that (CGG)(n) instability is correlated to the length of uCGG.

Machado-Joseph disease in three Scandinavian families.

Machado-Joseph disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder characterized by varying age of onset and pronounced phenotypic heterogeneity. The clinical core features include gait ataxia, external ophthalmoplegia, nystagmus, and bulging eyes. Recently, Kawagushi et al. (1994) cloned the MJD1 gene on chromosome 14 and MJD turned out to be the fifth neurodegenerative disease caused by an unstable CAG repeat expansion. We have studied two large Danish families and one Norwegian family with MJD. Three features not previously associated with MJD are reported: dementia, generalized muscle and joint pain, and in one case neuropathological examination revealed atrophy of the inferior olives. We found a significant inverse correlation between age of onset and the length of the CAG repeat expansion, and anticipation is described through four succeeding generations. Instability of the CAG repeat expansion was most pronounced at paternal transmission.

Huntington's disease does not appear to increase the risk of diabetes mellitus.

Huntington's disease (HD) is an autosomal, dominantly inherited, neurodegenerative disorder characterised by neurological, cognitive and psychiatric symptoms. HD has been associated with diabetes mellitus, which is, to some extent, supported by studies in transgenic HD mice. In transgenic mice, the severity of the diabetic phenotype appears to correlate with the length of a polyglutamine expansion in the protein huntingtin. In the present study, we investigated the association between diabetes mellitus and HD by performing an oral glucose-tolerance test (OGTT) to evaluate the glucose-tolerance status and OGTT-related insulin release in 14 HD patients. Furthermore, we expressed N-terminal huntingtin fragments with different polyglutamine lengths in an insulinoma-cell line (INS-1E) to investigate how mutant huntingtin influences glucose-stimulated insulin release in vitro. We found no difference between a group of early- and middle-stage HD patients and a large group of control individuals in any of the assessed variables. However, the glucose-stimulated induction of insulin release was significantly reduced in the insulinoma-cell line expressing highly expanded huntingtin compared to cells expressing huntingtin with modestly elongated polyglutamine stretches. These data indicate that insulin release from beta-cells expressing mutant huntingtin appears to be polyglutamine length-dependent, and that polyglutamine lengths within the range normally found in adult onset HD do not influence insulin release. This challenges the assumption of an increased risk of diabetes among HD patients, although our results do not exclude a changed glucose tolerance in end-stage HD patients or in patients with juvenile onset HD. It also raises the question of which extent transgenic mice models reflect the pathology of human HD in this regard.

Impaired glucose tolerance in the R6/1 transgenic mouse model of Huntington's disease.

Previous reports have highlighted a possible link between Huntington's disease (HD) and diabetes mellitus (DM), but the association has not been characterised in detail. A transgenic mouse model for HD, the R6/2 mouse, also develops diabetes. In the present study, we examined the R6/1 mouse, which carries a shorter CAG repeat than the R6/2 mouse, and found that, although not diabetic, the mice showed several signs of impaired glucose tolerance. First, following i.p. glucose injection, the blood glucose concentration was approximately 30% higher in young R6/1 mice (10 weeks) compared to wild-type mice (P = 0.004). In older mice (38 weeks), glucose tolerance was further impaired in both R6/1 and wild-type animals. Second, during glucose challenge, the R6/1 mice reached higher plasma insulin levels than wild-type mice, but the peripheral insulin sensitivity was normal as measured by injection of human or mouse insulin or when evaluated by the quantitative insulin sensitivity check index (QUICKI). Third, the beta cell volume was 17% and 39% smaller at 10 and 38 weeks of age, respectively, compared to age-matched wild-type littermates and the reduction was not caused by apoptosis at either age. Finally, we demonstrated the presence of the HD gene product, huntingtin (htt), in both alpha- and beta-cells in R6/1 islets of Langerhans. Since pancreatic beta cells and neurons share several common traits, clarification of the mechanism associating neurodegenerative diseases with diabetes might improve our understanding of the pathogenic events leading to both groups of diseases.

Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs.

Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435-fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.

46,XY/45,X mosaicism in an amniotic fluid cell culture: suppression of abnormal cell line after subcultivation.

An abnormal cell population, 45,X, appeared in 3 of 4 cell lines established from an amniotic fluid specimen obtained from a normal mid-trimester pregnancy. Two of the cell lines were subjected to repeated chromosome analyses until VII passage. The abnormal cells were suppressed after repeated trypsinisations; simultaneously, fibroblast-like cells outgrew the cultures, which were previously predominated by epithelial-like cells. Polyploidy was found in 0 to 12% of the cells, the highest level existing in the early passages. The question of whether chromosomally abnormal cells present in primary cultures and the early subcultures reflect the karyotype of the fetus is discussed.

Behaviour of cell cultures from human amniotic fluid.

The growth pattern of cell cultures originating from 11 amniotic fluid specimens have been observed. From each specimen 2 to 12 primary cultures were set up. In most cases growth started simultaneously in the primary cultures originating from one sample. The primary cultures lasted from 7 to 30 days. A variation was found both between cultures from different pregnancies as well as among cultures obtained from single amniotic fluids. The growth period from setting up the cultures until harvest of the cell lines for biochemical analysis ranged from 20 to 54 days. No connexion was noticed between the time spent in primary culture and the behaviour of the cell line before harvest. The effects of two types of serum (fetal calf serum and pooled human serum) on the behaviour of the cultures were compared. The cells grown in human serum were harvested a few days before those grown in fetal calf serum. The influence of different batches of medium was also examined; no significant effect of the growth pattern was found. The appearance of epithelial-like and fibroblast-like cells in cultures from 6 specimens was observed concurrently. At the time of harvest the cell lines originating from the same amniotic specimen contained the cell types in different proportions.

Lysosomal alpha-galactosidase in endothelial cell cultures established from a Fabry hemizygous and normal umbilical veins.

An endothelial cell line has been established from the umbilical vein obtained after abortion of a male fetus suffering from Fabry disease. This X-linked inborn error of glycosphingolipid catabolism results from deficiency of the lysosomal hydrolase alpha-galactosidase A. The clinical manifestations of the disease are mainly caused by glycosphingolipid depositions in the endothelium of all vessels. The hemizygous cell line and eight endothelial cell lines originating from the umbilical cords of normal newborns were grown for more than 10 passages. They had a short generation time that allowed us to get sufficient cells for qualitative and quantitative investigations of alpha-galactosidase. The enzyme in normal endothelial cells had a similar thermostability and isoelectric focusing pattern as that in fibroblasts, but the activity was essentially higher in endothelial cells. The hemizygous endothelial cells were deficient in alpha-galactosidase A. It is concluded that endothelial cell lines are an important alternative to fibroblasts for in vitro studies of the lysosomal storage diseases.

ConA-mediated binding and uptake of purified alpha-galactosidase A in Fabry fibroblasts.

In most human tissues there are at least two different alpha-galactosidases, A and B. The former is deficient in patients hemizygous for Fabry disease. We have isolated it from human placenta and found that it was labile even at culture conditions, but was stabilized after binding to concanavalin A (conA). The alpha-galactosidase activity was markedly increased in Fabry fibroblasts when these were treated with conA and exposed to alpha-galA at 37 degrees C. The maximum activity was obtained after 1/2-2 h of incubation and was maintained for at least 4 h. The binding and uptake of conA into Fabry cells was followed by microscopical studies of fluorescein-labelled conA. We assume that alpha-galA is taken up by endocytosis of the enzyme-conA complex.

alpha-Galactosidase isozymes in normal individuals, and in Fabry hemizygotes and heterozygotes.

A method for staining of alpha-galactosidase with the synthetic substrate alpha-naphthyl-alpha-galactopyranoside after isoelectric focusing on gel slabs has been devised. Depending on the method used for cell extraction, at least seven isozymes could be detected in cell extracts of cultured fibroblasts from normal individuals. Thermal treatment revealed that both heat-stable and heat-labile isozymes occur in normal fibroblasts. The heat-labile isozymes were not detected in cells from Fabry hemizygotes and thus truly reflect products of the alpha-gal A locus. Three heat-stable isozymes observed in normal individuals were also found in Fabry heterozygotes and hemizygotes and are presumably determined by the alpha-gal B locus. The remaining isozymes were stained very weakly in the hemizygotes and were heat-stable. The relation of these isozymes to the A or B locus is uncertain. After treatment with neuraminidase the alpha-gal A isozymes could not be detected and one of the alpha-gal B isozymes appeared broader. The isozyme pattern observed in heterozygotes was almost identical to the normal one.

Fabry's disease.

Fifteen hemizygotes and 30 heterozygotes have been diagnosed since our investigations of Fabry's disease were started 10 years ago. They belong mainly to three Danish families. Genetic counseling and prenatal diagnoses have been performed, and in vitro studies of cultured fibroblasts and endothelial cells have been made with special reference to enzyme therapy.