Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP.

Myotonic dystrophy, or dystrophia myotonica (DM), is an autosomal dominant multisystem disorder caused by the expansion of a CTG trinucleotide repeat in the 3' untranslated region of the DMPK protein kinase gene on chromosome 19q13.3 (refs 1-3). Although the DM mutation was identified more than five years ago, the pathogenic mechanisms underlying this most prevalent form of hereditary adult neuromuscular disease remain elusive. Previous work from our laboratory demonstrated that a DNase l-hypersensitive site located adjacent to the repeats on the wild-type allele is eliminated by repeat expansion, indicating that large CTG-repeat arrays may be associated with a local chromatin environment that represses gene expression. Here we report that the hypersensitive site contains an enhancer element that regulates transcription of the adjacent DMAHP homeobox gene. Analysis of DMAHP expression in the cells of DM patients with loss of the hypersensitive site revealed a two- to fourfold reduction in steady-state DMAHP transcript levels relative to wild-type controls. Allele-specific analysis of DMAHP expression showed that steady-state transcript levels from the expanded allele were greatly reduced in comparison to those from the wild-type allele. Together, these results demonstrate that CTG-repeat expansions can suppress local gene expression and implicate DMAHP in DM pathogenesis.

Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A.

Charcot-Marie-Tooth disease 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy, associated with a DNA duplication on chromosome 17p11.2. A related disorder in the mouse, trembler (Tr), maps to mouse chromosome 11 which has syntenic homology to human chromosome 17p. Recently, the peripheral myelin protein-22 (pmp-22) gene was identified as the likely Tr locus. We have constructed a partial yeast artificial chromosome contig spanning the CMT1A gene region and mapped the PMP-22 gene to the duplicated region. These observations further implicate PMP-22 as a candidate gene for CMT1A, and suggest that over-expression of this gene may be one mechanism that produces the CMT1A phenotype.

Rapid cloning of expanded trinucleotide repeat sequences from genomic DNA.

Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.

An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)

Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.

Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene.

P0, a major structural protein of peripheral myelin, is a homophilic adhesion molecule and maps to chromosome 1q22-q23, in the region of the locus for Charcot-Marie-Tooth neuropathy type 1B (CMT1B). We have investigated P0 as a candidate gene in two pedigrees with CMT1B and found point mutations which are completely linked with the disease (Z = 5.5, theta = 0). The mutations, glutamate substitution for lysine 96 or aspartate 90, are located in the extracellular domain, which plays a significant role in myelin membrane adhesion. Individuals with CMT1B are heterozygous for the normal allele and the mutant allele. Our results indicate that P0 is a gene responsible for CMT1B.

Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease.

To determine whether the presenilin 1 (PS1), presenilin 2 (PS2) and amyloid beta-protein precursor (APP) mutations linked to familial Alzheimer's disease (FAD) increase the extracellular concentration of amyloid beta-protein (A beta) ending at A beta 42(43) in vivo, we performed a blinded comparison of plasma A beta levels in carriers of these mutations and controls. A beta 1-42(43) was elevated in plasma from subjects with FAD-linked PS1 (P < 0.0001), PS2N1411 (P = 0.009), APPK670N,M671L (P < 0.0001), and APPV7171 (one subject) mutations. A beta ending at A beta 42(43) was also significantly elevated in fibroblast media from subjects with PS1 (P < 0.0001) or PS2 (P = 0.03) mutations. These findings indicate that the FAD-linked mutations may all cause Alzhelmer's disease by increasing the extracellular concentration of A beta 42(43), thereby fostering cerebral deposition of this highly amyloidogenic peptide.

A familial Alzheimer's disease locus on chromosome 1.

The Volga German kindreds are a group of seven related families with autosomal dominant early-onset Alzheimer's disease (AD). Linkage to known AD-related loci on chromosomes 21 and 14 has been excluded. Significant evidence for linkage to AD in these families was obtained with D1S479 and there was also positive evidence for linkage with other markers in the region. A 112-base pair allele of D1S479 co-segregated with the disease in five of seven families, which is consistent with a common genetic founder. This study demonstrates the presence of an AD locus on chromosome 1q31-42.

Absence of linkage of chromosome 21q21 markers to familial Alzheimer's disease.

Alzheimer's disease is the most common form of dementia among the elderly population. Although the etiology is unknown, inheritance plays a role in the pathogenesis of the disease. Recent work indicates that an autosomal dominant gene for Alzheimer's disease is located on chromosome 21 at band q21. In the present study of a group of autopsy-documented kindreds, no evidence for linkage was found between familial Alzheimer's disease (FAD) and chromosome 21q21 markers (D21S1/D21S72 and the amyloid beta gene). Linkage to the D21S1/D21S72 locus was excluded at recombination fractions (theta) up to 0.17. Linkage to the amyloid gene was excluded at theta = 0.10. Apparent recombinants were noted in two families for the amyloid gene and in five families for the D21S1/D21S72 locus. These data indicate that FAD is genetically heterogeneous.

Genetic linkage evidence for a familial Alzheimer's disease locus on chromosome 14.

Linkage analysis was used to search the genome for chromosomal regions harboring familial Alzheimer's disease genes. Markers on chromosome 14 gave highly significant positive lod scores in early-onset non-Volga German kindreds; a Zmax of 9.15 (theta = 0.01) was obtained with the marker D14S43 at 14q24.3. One early-onset family yielded a lod score of 4.89 (theta = 0.0). When no assumptions were made about age-dependent penetrance, significant results were still obtained (Zmax = 5.94, theta = 0.0), despite the loss of power to detect linkage under these conditions. Results for the Volga German families were either negative or nonsignificant for markers in this region. Thus, evidence indicates a familial Alzheimer's disease locus on chromosome 14.

Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.

Tau proteins aggregate as cytoplasmic inclusions in a number of neurodegenerative diseases, including Alzheimer's disease and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Over 10 exonic and intronic mutations in the tau gene have been identified in about 20 FTDP-17 families. Analyses of soluble and insoluble tau proteins from brains of FTDP-17 patients indicated that different pathogenic mutations differentially altered distinct biochemical properties and stoichiometry of brain tau isoforms. Functional assays of recombinant tau proteins with different FTDP-17 missense mutations implicated all but one of these mutations in disease pathogenesis by reducing the ability of tau to bind microtubules and promote microtubule assembly.

A novel progranulin mutation associated with variable clinical presentation and tau, TDP43 and alpha-synuclein pathology.

Mutations in the progranulin (GRN) gene have recently been reported as a cause of the frontotemporal dementia (FTD) syndrome. We performed a clinical, neuropathological and molecular genetic study of two families with FTD and the same novel mutation in GRN. Age of onset ranged from 35 to 75 years and all individuals progressed to a severe dementia syndrome with a mean disease duration of approximately 6-10 years. Variable clinical presentations included language impairment, behaviour change or parkinsonism. Seven total autopsies in the families (five in Family 1, two in Family 2) showed gross and microscopic evidence of neuronal loss in the neocortex, striatum, hippocampus and substantia nigra. All cases with material available for immunohistochemistry had cytoplasmic and intranuclear ubiquitin positive, tau negative inclusions that stained best with an antibody to the TDP43 protein. In addition, all but one had evidence of distinctive tau pathology. Two cases in Family 1 also had alpha-synuclein (SNCA) pathology, one with diffuse neocortical inclusions and neurites and unusual striatal cytoplasmic inclusions. Affected persons in both families had the same mutation in GRN (c.709-2A>G). A minigene construct showed that this mutation alters splicing of exon 7 and results in reduced mRNA message in brain. A single GRN mutation in these two families was associated with variable clinical presentations consistent with the FTD syndrome. All cases had ubiquitin/TDP43 immuno-positive inclusions and most had additional tau pathology. Two cases had SNCA pathology. These findings suggest a link between mutations in GRN and aggregation of tau, TDP43 and SNCA.

Variants regulating ZBTB4 are associated with age-at-onset of Alzheimer's disease.

The identification of novel genetic modifiers of age-at-onset (AAO) of Alzheimer's disease (AD) could advance our understanding of AD and provide novel therapeutic targets. A previous genome scan for modifiers of AAO among families affected by early-onset AD caused by the PSEN2 N141I variant identified 2 loci with significant evidence for linkage: 1q23.3 and 17p13.2. Here, we describe the fine-mapping of these 2 linkage regions, and test for replication in 6 independent datasets. By fine-mapping these linkage signals in a single large family, we reduced the linkage regions to 11% their original size and nominated 54 candidate variants. Among the 11 variants associated with AAO of AD in a larger sample of Germans from Russia, the strongest evidence implicated promoter variants influencing NCSTN on 1q23.3 and ZBTB4 on 17p13.2. The association between ZBTB4 and AAO of AD was replicated by multiple variants in independent, trans-ethnic datasets. Our results show association between AAO of AD and both ZBTB4 and NCSTN. ZBTB4 is a transcriptional repressor that regulates the cell cycle, including the apoptotic response to amyloid beta, while NCSTN is part of the gamma secretase complex, known to influence amyloid beta production. These genes therefore suggest important roles for amyloid beta and cell cycle pathways in AAO of AD.

Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.

Gerstmann-Sträussler-Scheinker disease (GSS), a cerebello-pyramidal syndrome associated with dementia and caused by mutations in the prion protein gene (PRNP), is phenotypically heterogeneous. The molecular mechanisms responsible for such heterogeneity are unknown. Since we hypothesize that prion protein (PrP) heterogeneity may be associated with clinico-pathologic heterogeneity, the aim of this study was to analyze PrP in several GSS variants. Among the pathologic phenotypes of GSS, we recognize those without and with marked spongiform degeneration. In the latter (i.e. a subset of GSS P102L patients) we observed 3 major proteinase-K resistant PrP (PrPres) isoforms of ca. 21-30 kDa, similar to those seen in Creutzfeldt-Jakob disease. In contrast, the 21-30 kDa isoforms were not prominent in GSS variants without spongiform changes, including GSS A117V, GSS D202N, GSS Q212P, GSS Q217R, and 2 cases of GSS P102L. This suggests that spongiform changes in GSS are related to the presence of high levels of these distinct 21-30 kDa isoforms. Variable amounts of smaller, distinct PrPres isoforms of ca. 7-15 kDa were seen in all GSS variants. This suggests that GSS is characterized by the presence PrP isoforms that can be partially cleaved to low molecular weight PrPres peptides.

Frontotemporal dementia and Parkinsonism linked to chromosome 17: a new group of tauopathies.

Frontotemporal dementia is a neurological disorder characterised by personality changes, deterioration of memory and executive functions as well as stereotypical behaviour. Sometimes a Parkinsonian syndrome is prominent. Several cases of frontotemporal dementia are hereditary and recently families have been identified where the disease is linked to chromosome 17q21-22. Although, there is clinical and neuropathological variability among and within families, they all consistently present a symptomathology that has led investigators to name the disease "Frontotemporal Dementia and Parkinsonism linked to chromosome 17." Neuropathologically, these patients present with atrophy of frontal and temporal cortex as well as of basal ganglia and substantia nigra. In the majority of cases these features are accompanied by neuronal loss, gliosis and microtubule-associated protein tau deposits which can be present in both neurones and glial cells. The distribution, structural and biochemical characteristics of the tau deposits differentiate them from those present in Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy and Pick's disease. No beta-amyloid deposits are present. The clinical and neuropathological features of the disease in these families suggest that Frontotemporal Dementia and Parkinsonism linked to chromosome 17 is a distinct disorder. The presence of abundant tau deposits in the majority of these families define this disorder as a new tauopathy.

The effect of apolipoprotein E genotype on expression of an autosomal dominant schizophreniform disorder with progressive dementia and neurofibrillary tangles.

The apolipoprotein E (APOE) epsilon 4 allele is associated with an increased and the epsilon 2 allele a decreased risk for Alzheimer's disease (AD). It has been hypothesized that these risks are mediated by differential effects of the APOE alleles on the cytoskeletal degeneration, which results in neurofibrillary tangle (NFT) formation. It has also been suggested that APOE alleles differentially affect the beta amyloid accumulation. We examined APOE genotypes and their effects on age of onset in a family with an autosomal dominant "neurofibrillary tangle only" dementia. This disorder is manifested by schizophreniform psychosis followed by progressive dementia and neuropathologically by prominent AD-like neurofibrillary tangles without neuritic plaques. The only affected epsilon 4 heterozygote in this family did not demonstrate accelerated disease onset. In contrast, the affected epsilon 2 heterozygote had the latest age of onset of any affected family member. The two other epsilon 2 heterozygotes remained unaffected at an age much greater than the mean age of onset for the disease. These results are consistent with a protective effect of the epsilon 2 allele in a hereditary neuropsychiatric disorder with prominent NFT formation.

Evidence for etiologic heterogeneity in Alzheimer's disease.

We briefly describe the evidence for etiologic heterogeneity in Alzheimer's disease. Several different genetic and environmental factors may act separately or in combination to produce the AD phenotype. This potential heterogeneity presents special challenges to those attempting to identify specific genetic influences in the pathogenesis of AD.

Intracellular calcium response is reduced in CD4+ lymphocytes in Alzheimer's disease and in older persons with Down's syndrome.

Abnormalities in intracellular free calcium ([Ca2+]i) regulation are likely to play a role in brain aging and have been described in cells from patients with Alzheimer's disease (AD). [Ca2+]i acts as a second messenger in transmembrane signaling and regulates diverse functions in many cell types. Therefore, abnormalities in [Ca2+]i response may have far-ranging effects. Using flow cytometric assay for [Ca2+]i, we examined whether mitogen-induced increases in [Ca2+]i are abnormal in CD4+ T-lymphocytes from patients with familial AD (FAD), other AD, and Down's syndrome (DS) compared to age-matched controls. We observed that the peak [Ca2+]i responses were significantly decreased in CD4+ cells from 6 FAD patients (59% of control), 34 other AD patients (69% of age-matched control), and 6 older persons with DS (> 25 years old, 47% of control), after stimulation with 10 micrograms/ml anti-CD3 monoclonal antibody (mAb). The number of CD3 receptors on T lymphocytes of the AD patients was not decreased. In contrast, lymphocytes from subjects with FAD, other AD and older DS patients had no decrease in response to phytohemagglutinin (30 micrograms/ml). CD3 and related classes of membrane receptors are present on many cells of the central nervous system. Therefore, receptor signaling defects via this receptor in T lymphocytes of AD patients may be relevant to the central nervous system pathology seen in AD and DS.

Neuropathological findings in patients with clinical diagnoses of probable Alzheimer's disease.

To assess prospectively the accuracy of standard antemortem clinical diagnostic criteria for Alzheimer's disease, post-mortem examinations were performed on 25 patients who had met DSM-III criteria for primary degenerative dementia and National Institute of Neurological and Communicative Disorders and Stroke criteria for probable Alzheimer's disease. Seventeen patients (68%) met neuropathological criteria for Alzheimer's disease. Two presenile-onset patients had diffuse neocortical senile plaques of insufficient number for definite Alzheimer's disease. Six patients had non-Alzheimer's disease diagnoses. Five of these six had presenile-onset dementia. These results suggest caution in the antemortem diagnosis of Alzheimer's disease in presenile-onset dementia.

Familial aggregation of psychotic symptoms in Huntington's disease.

OBJECTIVE: The mutation responsible for Huntington's disease is an elongated and unstable trinucleotide (CAG) repeat on the short arm of chromosome 4. Psychotic symptoms are more common in patients with Huntington's disease than in the general population. This study explored the relationship of psychosis in Huntington's disease patients with the number of CAG repeats and family history of psychosis. METHOD: Forty-four patients with Huntington's disease, 22 with and 22 without psychotic symptoms, were recruited from two university-affiliated medical genetics clinics in Seattle and Vancouver, B.C. Psychiatric assessments of the subjects were made through chart review, and diagnoses were validated by structured interviews in a subset of patients. The demographic and clinical characteristics of the psychotic and nonpsychotic patients were compared. RESULTS: The two groups did not differ in demographic and clinical characteristics, except that subjects with psychosis were significantly more likely than nonpsychotic subjects to have a first-degree relative with psychosis. In eight of nine families in which Huntington's disease probands with psychosis had a first-degree relative with psychosis, the relative's psychosis co-occurred with Huntington's disease. In the Huntington's disease probands with psychosis, the onset of psychosis correlated with the onset of the neurological symptoms of Huntington's disease, and the age at onset of psychosis was lower in probands with a higher number of CAG repeats. CONCLUSIONS: Patients with Huntington's disease and psychotic symptoms may have a familial predisposition to develop psychosis. This finding suggests that other genetic factors may influence susceptibility to a particular phenotype precipitated by CAG expansion in the Huntington's disease gene.