Biomedical subjects
Publications and source records attributed to N T Potter.
We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.
BACKGROUND: The Ataxia Molecular Diagnostics Testing Group was established to generate quantitative proficiency and outcomes data regarding molecular testing for the autosomal dominant cerebellar ataxias (spinocerebellar ataxia types 1 [SCA-1] through -3, -6, and -7, and dentatorubral-pallidoluysian atrophy) in North America. METHODS AND RESULTS: Twenty-four North American laboratories that offered diagnostic testing for one or more ataxia genes were initially identified through GeneTests (Children's Health Care System, Seattle, WA). Eighteen laboratories agreed to participate in the study, which consisted of completing a technical survey, clinical survey, and molecular proficiency test. One laboratory returned the completed surveys but did not perform the proficiency testing. Ten of 18 laboratories (56%) provided data on test volumes, and these laboratories collectively performed 2,240 tests; approximately 5% of the tests yielded a positive result (i.e., identification of a pathological trinucleotide (CAG) repeat expansion). In proficiency testing, 100% of the laboratories correctly genotyped all samples, and 93% of the laboratories were within 1 SD of the mean for sizing normal alleles (one repeat unit or less). Ninety percent of the laboratories were within 1 SD for sizing expanded alleles. CONCLUSIONS: Proficiency testing showed little difference between laboratories with respect to allele sizing. However, additional phenotype/genotype correlations are necessary to define CAG repeat-length descriptors for SCA-1, SCA-2, and SCA-7 alleles of intermediate size.
Compound heterozygosity at the Friedreich's ataxia locus accounts for approximately 2% of molecularly confirmed cases. Genotype-phenotype correlation in this subgroup of patients reveals a spectrum of clinical variability. This report describes the clinical and molecular findings in a 6-year-old patient with Friedreich's ataxia who carried a pathologic GAA expansion of approximately 1,000 repeats on one allele and a novel initiation codon point mutation (3G-->A) on the other.
A rapid, simple, and reliable gender determination of human DNA samples was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Detection sensitivity reached 0.01 ng or less for DNA samples.
Genotyping of the dentatorubral-pallidoluysian atrophy (DRPLA) locus in six patient samples, representing four normal individuals and two DRPLA patients, was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). DRPLA is a dominantly inherited neurodegenerative disorder associated with the expansion of an unstable trinucleotide (CAG) repeat. The accurate determination of repeat length utilizing MALDI supports the use of this methodology for the analysis of genes containing unstable CAG trinucleotide repeats.
MAB21L1, originally termed CAGR1, is the human homologue of the C. elegans cell fate determining gene mab21. MAB21L1, mapped to 13q13, contains a highly polymorphic 5' untranslated CAG repeat that normally ranges from six to 31 triplets in length. A pedigree has been previously reported in which the repeat length is expanded to 45-50 triplets and is transmitted unstably between generations; the expansion did not correlate to a clinical phenotype but did exhibit somatic mosaicism. We now report a second pedigree with an expanded and unstably transmitted MAB21L1 CAG repeat of similar length. The expansion is not clearly associated with a clinical phenotype, though the complexity of the pedigree renders any conclusion concerning phenotype-genotype relationships speculative. The expansion did not result in decreased expression of MAB21L1 protein. The length, C-G rich composition, somatic mosaicism, and unstable transmission of the expanded CAG repeat in MAB21L1 resemble the premutations observed in other genes, such as FMR1 and MDPK, in which longer expanded repeats are associated with a clinical phenotype. This raises the possibility that longer expansions in the MAB21L1 repeat may also be associated with a clinical phenotype.
The discovery and characterization of thousands of genes involved in human disorders has the potential for great benefit in patient diagnosis and treatment. A combined approach using molecular genetic testing for some disorders as an adjunct to the clinical evaluation of children with developmental or behavioral features is rapidly occupying a more prominent role in the diagnostic evaluation. In some instances, molecular testing might confirm a genetic etiology in a child initially referred with a DSM-IV diagnosis, but molecular testing might be of limited use in other instances. This review presents advances in the diagnosis of inherited disorders affecting the pediatric population, with an emphasis on those with a developmental/behavioral component. We examine several relatively common disorders in detail (and less common disorders in a more cursory fashion) to elucidate the strengths and weaknesses of molecular diagnosis in clinical practice.
T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.
Utilizing an affinity-purified antiserum directed against the carboxyl terminal region of atrophin-1/drplap (residues 1170-1185), we have examined the expression and distribution of the protein in a variety of neuronal and non-neuronal tissues. Immunohistochemical analyses of gelatin-embedded sections of monkey brain demonstrated a wide-spread distribution of the protein throughout the cerebrum and cerebellum. Labeling was primarily cytoplasmic within neuronal cell bodies and dendrites. Prominently staining regions included layers II, III, V, and VI of cerebral cortex, CA1-4 of the hippocampus, caudate nucleus, putamen, globus pallidus, amygdala, thalamus, red nucleus, pons, Purkinje cells, and deep cerebellar nuclei. Immunoblot analysis of extracts of frontal cortex from a wide variety of mammalian species (human, monkey, rabbit, rat, mouse, and bovine) detected a 190 kDa band in each extract. No cross-reactive material of similar molecular weight was detected in an extract of avian (chicken) central nervous system (CNS) tissue. Furthermore, in the rat, expression of the protein was predominantly neuronal in origin as immunoblot analyses of non-neuronal tissue extracts detected little or no 190 kDa protein. Collectively these investigations suggest a ubiquitous expression of atrophin-1/drplap in mammalian CNS tissue and provide initial immunochemical data for the study of the neuroanatomic and perhaps, phylogenetic relationships between mammalian and non-mammalian forms of the protein.
CAGR1 is a recently characterised polymorphic trinucleotide repeat localised to 13q13, which has been suggested as a possible candidate gene for neurological disorders that manifest genetic anticipation. To provide evidence in support of this hypothesis, a large number of chromosomes (n = 928) from patients with a wide variety of neurological diseases were screened for evidence of repeat expansion and meiotic instability. One person with a CAGR1 repeat number of 50 was identified (normal range 9-29). Subsequent molecular analyses of CAGR1 repeat number in additional family members showed meiotic instability of a (CAG)45 allele through three generations. While CAGR1 repeat number did not correlate with a readily discernible phenotype in this family, the finding of meiotic stability and mendelian inheritance of normal CAG alleles and meiotic instability of larger repeats fulfil several criteria thought essential for pathologically relevant mutations of this type. Thus, these data strengthen the hypothesis for a role of CAGR1 in the development of an as yet molecularly uncharacterised human neurological disease.
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Pooled polyclonal rabbit anti-rat myelin and mouse anti-human proteolipid protein (PLP) antisera were screened against a panel of PLP synthetic peptides spanning residues 178-238 of the protein. Cross-reactivity against one determinant defined by PLP(200-219) was particularly prominent in both the anti-myelin and anti-PLP antisera and was chosen for further study. Competitive inhibition studies, utilizing a panel of overlapping synthetic peptides, demonstrated that the C-terminal portion of PLP(200-219), specifically residues comprising PLP(200-217), was important for antibody recognition of this region. Immunohistochemical analyses with an affinity-purified rabbit anti-PLP(200-219) antiserum demonstrated antibody cross-reactivity with PLP in both paraffin- and gelatin-embedded brain sections and immunocytochemical staining of mouse oligodendrocyte-enriched cultures demonstrated antibody binding with native PLP in situ. Staining of living non-permeabilized cells localized binding to the extracellular face of the myelin membrane. Collectively, these data argue for the presence of an immunodominant B-cell determinant defined by PLP residues 200-219. Furthermore, the structural conformation of this determinant in native PLP can be mimicked by the synthetic peptide, resulting in the generation of an antibody reagent that has considerable utility for immunohistochemical and immunocytochemical investigations of PLP expression and localization within the central nervous system myelin membrane.
Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder associated with the expansion of a CAG repeat at chromosome band 12p13. Epidemiological studies have demonstrated an increased prevalence of DRPLA in Japan, although several DRPLA kindreds of non-Japanese ancestry have been identified. To define the molecular basis for this geographic variation in prevalence, we have analyzed haplotypes around the repeat in several different ethnic groups. Two intragenic biallelic polymorphisms distinguished three haplotypes, each of which formed a predominant haplotype found in the three major racial populations. All the expanded repeats of Japanese and Caucasian patients studied were associated with a particular haplotype, which otherwise was associated with longer repeats commonly found in Asians. Our results support a multi-step model for repeat expansion, and suggest that expanded DRPLA repeats may have evolved from an ancient chromosomal haplotype of Asian origin. We also propose that a combination of a highly polymorphic microsatellite marker with relatively stable biallelic markers in a range of PCR amplification is a powerful tool for studies on human genome diversity, which may reveal the ancient human migration and the formation of ethnic groups.
Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare neurodegenerative disorder characterised by variability in both age of onset and clinical features. Despite the recent identification of the CAG expansion mutation in DRPLA, the number of molecularly confirmed cases remains small. Given its rarity and prominent phenotypic heterogeneity, some care needs to be exercised in the interpretation and dissemination of test results derived from direct gene testing for the DRPLA specific expansion mutation.
Herein we describe the molecular and clinical findings in a North American Caucasian family with dentatorubral-pallidoluysian atrophy (DRPLA). These patients all presented with an autosomal dominant neurodegenerative disorder characterized by a variable combination of clinical symptoms including seizures, ataxia, dementia, choreiform movements, mental retardation, and psychiatric disease. Neuroradiologic findings in the index case revealed deep subcortical white matter changes on magnetic resonance imaging. Prior to referral, the family carried a diagnosis of Huntington's disease (HD). Subsequent direct molecular testing for HD failed to identify the HD expansion mutation in affected individuals. Molecular testing for DRPLA, however, demonstrated the presence of the recently characterized DRPLA expansion mutation in all affected individuals. The size of the expansion correlated with the age of onset of clinical symptoms. As DRPLA has rarely been reported in North American and European populations, the molecular confirmation of DRPLA in this family provides support for the hypothesis that DRPLA may not be as geographically restricted as once thought.
ALS is associated with the P2 blood group phenotype. Molecular evidence now shows the gene encoding this antigen to be on the long arm of human chromosome 22 near the newly discovered gene for heavy neurofilament (NF-H). Since an ALS-type condition can be generated in transgenic mice expressing the human NF-H gene, and since the gene for the CNTF-related cytokine leukemia inhibitory factor (LIF) is located adjacent to this gene, it is hypothesized that a defect on the chromosome 22 band region q12 is involved in the pathogenesis of sporadic ALS.
SJL/J mice were immunized with human PLP as well as encephalitogenic PLP peptides 139-151 and 178-191 and the resulting antibody responses examined for immunochemical specificity employing a panel of 17 synthetic PLP peptide ligands. All animals had demonstrable circulating titers of antibodies early in the humoral immune response to their respective encephalitogens, however, there was no clear qualitative correlation between antibody responses and the induction of EAE. In the majority of PLP immunized animals, determinant-specific antibody populations, including those against encephalitogenic centers, were not detectable in the presence of an anti-PLP antibody response. Antiencephalitogenic peptide antibodies were present in both 139-151 and 178-191 immunized animals regardless of clinical/histologic status. Neither group produced cross-reactive anti-PLP antibodies as detected by ELISA. In animals immunized with peptide 139-151, only anti-139-151 antibody specificities were noted. In contrast, all animals immunized with peptide 178-191 had an antibody population cross-reactive with three other PLP peptides: 97-110, 209-217, and 215-228. As humoral immune responses can be demonstrated against PLP-specific encephalitogenic epitopes, the significance of these B cell responses should be considered in the context of their potential role in the development, modulation, and/or potentiation of EAE.
We report the direct molecular detection of the fragile X genotype in 111 individuals from 17 families with a total of 31 cases of fragile X syndrome. Comparison of our molecular data with our previous cytogenetic and linkage data from these same families indicates the effectiveness of the direct molecular analysis. We have been able to assign a genotype unambiguously in 100% of the persons tested, and in all cases the molecular data correlated with the cytogenetic or linkage findings or both. Two of the three families presented in this study represent inheritance of this gene through normal transmitting males, and the third is strongly suggestive of this mode of inheritance. Our data show that the direct molecular approach will be of great utility for confirmation of the diagnosis and for the detection of female carriers and normal transmitting males who are at high risk for having affected children or grandchildren.