Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function.

Due to the importance of 4R tau in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in iPSC-derived neurons, which express very low levels of 4R tau. To address this problem we have developed a panel of isogenic iPSC lines carrying the MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation. Transcriptomic and functional analyses of S305 mutant neurons revealed shared disruption in glutamate signaling and synaptic maturity, but divergent effects on mitochondrial bioenergetics. In iPSC-astrocytes, S305 mutations induced lysosomal disruption and inflammation and exacerbated internalization of exogenous tau that may be a precursor to the glial pathologies observed in many tauopathies. In conclusion, we present a novel panel of human iPSC lines that express unprecedented levels of 4R tau in neurons and astrocytes. These lines recapitulate previously characterized tauopathy-relevant phenotypes, but also highlight functional differences between the wild type 4R and mutant 4R proteins. We also highlight the functional importance of MAPT expression in astrocytes. These lines will be highly beneficial to tauopathy researchers enabling a more complete understanding of the pathogenic mechanisms underlying 4R tauopathies across different cell types.

SMAD transcription factors are altered in cell models of HD and regulate HTT expression.

Transcriptional dysregulation is observable in multiple animal and cell models of Huntington's disease, as well as in human blood and post-mortem caudate. This contributes to HD pathogenesis, although the exact mechanism by which this occurs is unknown. We therefore utilised a dynamic model in order to determine the differential effect of growth factor stimulation on gene expression, to highlight potential alterations in kinase signalling pathways that may be in part responsible for the transcriptional dysregulation observed in HD, and which may reveal new therapeutic targets. We demonstrate that cells expressing mutant huntingtin have a dysregulated transcriptional response to epidermal growth factor stimulation, and identify the transforming growth factor-beta pathway as a novel signalling pathway of interest that may regulate the expression of the Huntingtin (HTT) gene itself. The dysregulation of HTT expression may contribute to the altered transcriptional phenotype observed in HD.

Exceptions to the rule: case studies in the prediction of pathogenicity for genetic variants in hereditary cancer genes.

Based on current consensus guidelines and standard practice, many genetic variants detected in clinical testing are classified as disease causing based on their predicted impact on the normal expression or function of the gene in the absence of additional data. However, our laboratory has identified a subset of such variants in hereditary cancer genes for which compelling contradictory evidence emerged after the initial evaluation following the first observation of the variant. Three representative examples of variants in BRCA1, BRCA2 and MSH2 that are predicted to disrupt splicing, prematurely truncate the protein, or remove the start codon were evaluated for pathogenicity by analyzing clinical data with multiple classification algorithms. Available clinical data for all three variants contradicts the expected pathogenic classification. These variants illustrate potential pitfalls associated with standard approaches to variant classification as well as the challenges associated with monitoring data, updating classifications, and reporting potentially contradictory interpretations to the clinicians responsible for translating test outcomes to appropriate clinical action. It is important to address these challenges now as the model for clinical testing moves toward the use of large multi-gene panels and whole exome/genome analysis, which will dramatically increase the number of genetic variants identified.

A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes.

Genetic testing has the potential to guide the prevention and treatment of disease in a variety of settings, and recent technical advances have greatly increased our ability to acquire large amounts of genetic data. The interpretation of this data remains challenging, as the clinical significance of genetic variation detected in the laboratory is not always clear. Although regulatory agencies and professional societies provide some guidance regarding the classification, reporting, and long-term follow-up of variants, few protocols for the implementation of these guidelines have been described. Because the primary aim of clinical testing is to provide results to inform medical management, a variant classification program that offers timely, accurate, confident and cost-effective interpretation of variants should be an integral component of the laboratory process. Here we describe the components of our laboratory's current variant classification program (VCP), based on 20 years of experience and over one million samples tested, using the BRCA1/2 genes as a model. Our VCP has lowered the percentage of tests in which one or more BRCA1/2 variants of uncertain significance (VUSs) are detected to 2.1% in the absence of a pathogenic mutation, demonstrating how the coordinated application of resources toward classification and reclassification significantly impacts the clinical utility of testing.

Gene expression and behaviour in mouse models of HD.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, resulting in expansion of the CAG repeat in exon 1 of the HTT gene. The resulting mutant huntingtin protein has been implicated in the disruption of a variety of cellular functions, including transcription. Mouse models of HD have been central to the development of our understanding of gene expression changes in this disease, and are now beginning to elucidate the relationship between gene expression and behaviour. Here, we review current mouse models of HD and their characterisation in terms of gene expression. In addition, we look at how this can inform behaviours observed in mouse models of disease. The relationship between gene expression and behaviour in mouse models of HD is important, as this will further our knowledge of disease progression and its underlying molecular events, highlight new treatment targets, and potentially provide new biomarkers for therapeutic trials.

Novel gene mutations in patients with left ventricular noncompaction or Barth syndrome.

BACKGROUND: Mutations in the gene G4.5 result in a wide spectrum of severe infantile cardiomyopathic phenotypes, including isolated left ventricular noncompaction (LVNC), as well as Barth syndrome (BTHS) with dilated cardiomyopathy (DCM). The purpose of this study was to investigate patients with LVNC or BTHS for mutations in G4.5 or other novel genes. METHODS AND RESULTS: DNA was isolated from 2 families and 3 individuals with isolated LVNC or LVNC with congenital heart disease (CHD), as well as 4 families with BTHS associated with LVNC or DCM, and screened for mutations by single-strand DNA conformation polymorphism analysis and DNA sequencing. In 1 family with LVNC and CHD, a C-->T mutation was identified at nucleotide 362 of alpha-dystrobrevin, changing a proline to leucine (P121L). Mutations in G4.5 were identified in 2 families with isolated LVNC: a missense mutation in exon 4 (C118R) in 1 and a splice donor mutation (IVS10+2T-->A) in intron 10 in the other. In a family with cardiomyopathies ranging from BTHS or fatal infantile cardiomyopathy to asymptomatic DCM, a splice acceptor mutation in exon 2 of G4.5 (398-2 A-->G) was identified, and a 1-bp deletion in exon 2 of G4.5, resulting in a stop codon after amino acid 41, was identified in a sporadic case of BTHS. CONCLUSIONS: These data demonstrate genetic heterogeneity in LVNC, with mutation of a novel gene, alpha-dystrobrevin, identified in LVNC associated with CHD. In addition, these results confirm that mutations in G4.5 result in a wide phenotypic spectrum of cardiomyopathies.

Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human delta-sarcoglycan gene, a member of the dystrophin-associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in delta-sarcoglycan staining. No skeletal muscle disease occurred in any of these patients. These data suggest that delta-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our "final common pathway" hypothesis that DCM is a cytoskeletalopathy.

Construction of a high-resolution physical map of the chromosome 10q22-q23 dilated cardiomyopathy locus and analysis of candidate genes.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality and a leading cause of cardiac transplantation worldwide. Multiple loci and three genes encoding cardiac actin, desmin, and lamin A/C have been described for autosomal dominant DCM. Using recombination analysis, we have narrowed the 10q21-q23 locus to a region of approximately 4.1 cM. In addition, we have constructed a BAC contig, composed of 199 clones, which was used to develop a high-resolution physical map that contains the DCM critical region (approximately 3.9 Mb long). Seven genes, including ANX11, PPIF, DLG5, RPC155, RPS24, SFTPA1, and KCNMA1, have been mapped to the region of interest. RPC155, RPS24, SFTPA1, and KCNMA1 were excluded from further analysis based on their known functions and tissue-specific expression patterns. Mutational analysis of ANX11, DLG5, and PPIF revealed no disease-associated mutations. Multiple ESTs have also been mapped to the critical region.

The "final common pathway" hypothesis and inherited cardiovascular disease. The role of cytoskeletal proteins in dilated cardiomyopathy.

The genetic basis of a number of inherited cardiovascular diseases has been elucidated over the last few years, including the long QT syndromes, hypertrophic cardiomyopathy and dilated cardiomyopathy. While genetic heterogeneity has been demonstrated in most of these diseases, a pattern has emerged, specifically that genes encoding proteins with similar functions or involved in the same pathway are responsible for a particular disease or syndrome. Based on this observation we proposed the "final common pathway" hypothesis. In the case of the arrhythmogenic disorders, the long QT syndromes and Brugada syndrome, mutations have been described in a number of ion channel proteins, including cardiac potassium (KVLQT1, HERG and minK) and sodium (SCN5A) channels. Thus, using the "final common pathway" hypothesis we have proposed these diseases to be "ion channelopathies". Hypertrophic cardiomyopathy appears to be a disease of the sarcomere ("sarcomyopathy") since all the disease-causing mutations have been identified in the gene encoding many of the sarcomeric proteins, including beta-myosin heavy chain, alpha-tropomyosin, troponin I and troponin T, as well as in actin, close to the beta-myosin heavy chain binding site. The genes responsible for familial dilated cardiomyopathy have been less well characterized. For X-linked dilated cardiomyopathy, mutations in the dystrophin and G4.5 genes have been reported. In addition, mutations in actin (close to the dystrophin binding domain) and desmin, a component of the intermediate filaments, have been reported. However, the genes at a further 6 loci associated with autosomal dominant dilated cardiomyopathy (associated with conduction disease in 2 cases) remain unidentified. Due to the mutations in dystrophin, actin and desmin, we have proposed that dilated cardiomyopathy is a "cytoskeletalopathy", and we are currently investigating the involvement of these genes in patients.

Genomic organization and chromosomal localization of the human Coxsackievirus B-adenovirus receptor gene.

Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children. Many studies have implicated the enteroviruses and, particularly, the Coxsackievirus-B family as etiologic agents of the acquired forms of these diseases. However, we have shown the group-C adenoviruses to be as commonly detected as enteroviruses in the myocardium of children and adults with these diseases. It has remained something of a conundrum why two such divergent virus families cause these diseases. The recent description of the common human Coxsackievirus B-adenovirus receptor (CAR) offers at least a partial explanation. In order to characterize the CAR gene, we screened a bacterial artificial chromosomal (BAC) library (RPCI11) using a polymerase chain reaction (PCR) product derived from the 3' end of the CAR cDNA sequence. This identified 13 BACs that were further characterized by PCR amplification of seven contiguous regions of the entire cDNA sequence. Eleven of the BACs were determined to encode pseudogenes while the other two BACs (131J5 and 246M1) encoded the presumed functional gene. PCR amplification of a monochromosomal hybrid panel indicated the presence of pseudogenes on chromosomes 15, 18, and 21 while the functional gene is encoded on chromosome 21. Fluorescence in situ hybridization analysis indicated that the gene is located at 21q11.2. DNA sequencing of BACs 131J5 and 246M1 revealed the presence of seven exons. The DNA sequences have been determined for each exon-intron boundary, and putative promoter sequences and transcription initiation sites identified. No consensus polyadenylation signal was identified.

Genomic characterization of the human peptidyl-prolyl-cis-trans-isomerase, mitochondrial precursor gene: assessment of its role in familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common cause of morbidity and mortality, with >30% of cases being inherited. In one family with autosomal dominant familial dilated cardiomyopathy (FDCM), we localized the gene to the region of 10q21-10q23 and have performed candidate positional gene cloning. The peptidyl-prolyl-cis-trans-isomerase, mitochondrial precursor (PPIF: previously known as cyclophilin 3) is a protein that is part of the mitochondrial permeability transition pore, the activation of which is involved in the induction of necrotic and apoptotic cell death. Since it is encoded by a gene located within this FDCM critical region, PPIF was considered a potential candidate gene for FDCM. In order to screen patient genomes for evidence of disease-associated mutations, the genomic organization of this gene was determined. BAC libraries were screened by PCR, using primers designed from the published cDNA sequence, and positive clones were identified. This enabled the gene to be further localized to between the CEPH markers D10S1777 and D10S201. The DNA from a BAC clone was digested and subcloned into pUC18. Following identification of a subclone by whole-cell PCR, the gene was characterized by DNA sequencing; five introns were identified, and the sequences of the intron-exon boundaries were characterized. Additionally, 450 bp of DNA sequence upstream of the published cDNA were obtained and a potential transcription initiation site and promoter sequence were identified. DNA analysis of the entire PPIF coding region (including the intron-exon boundaries) of two affected and one unaffected family member revealed no mutations, therefore excluding this gene as the cause of FDCM in this family.

Gene mapping of familial autosomal dominant dilated cardiomyopathy to chromosome 10q21-23.

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial disorder, accounting for 60% of all cardiomyopathies. In 20-30% of cases, familial inheritance can be demonstrated; an autosomal dominant transmission is the usual type of inheritance pattern identified. Previously, genetic heterogeneity was demonstrated in familial autosomal dominant dilated cardiomyopathy (FDCM). Gene localization to chromosome 1 (1p1-1q1 and 1q32), chromosome 3 (3p25-3p22), and chromosome 9 (9q13-9q22) has recently been identified. We report one family with 26 members (12 affected) with familial autosomal dominant dilated cardiomyopathy in which linkage to chromosome 10 at the 10q21-q23 locus is identified. Using short tandem repeat polymorphism (STR) markers with heterozygosity > 70%, 169 markers (50% of the genome) were used before linkage was found to markers D10S605 and D10S201 with a pairwise LOD score = 3.91, theta = 0, penetrance = 100% for both markers. Linkage to 1p1-1q1, 1q32, 3p25-3p22, and 9q13-9q22 was excluded. We conclude that a new locus for pure autosomal dominant FDCM exists, and that this gene is localized to a 9 cM region of 10q21-10q23. The search for the disease causing gene and the responsible mutation(s) is ongoing.