Quality standards for DNA sequence variation databases to improve clinical management under development in Australia

Despite the routine nature of comparing sequence variations identified during clinical testing to database records, few databases meet quality requirements for clinical diagnostics. To address this issue, The Royal College of Pathologists of Australasia (RCPA) in collaboration with the Human Genetics Society of Australasia (HGSA), and the Human Variome Project (HVP) is developing standards for DNA sequence variation databases intended for use in the Australian clinical environment. The outputs of this project will be promoted to other health systems and accreditation bodies by the Human Variome Project to support the development of similar frameworks in other jurisdictions.B. Bennetts, M. Caramins, A. Hsu, C. Lau, S. Mead, C.Meldrum, T.D. Smith, G. Suthers, G.R. Taylor, R.G.H. Cotton, V. Tyrrel

Cognitive Dysfunction in Huntington's Disease: Mechanisms and Therapeutic Strategies Beyond BDNF

One of the main focuses in Huntington's disease (HD) research, as well as in most of the neurodegenerative diseases, is the development of new therapeutic strategies, as currently there is no treatment to delay or prevent the progression of the disease. Neuronal dysfunction and neuronal death in HD are caused by a combination of interrelated pathogenic processes that lead to motor, cognitive and psychiatric symptoms. Understanding how mutant huntingtin impacts on a plethora of cellular functions could help to identify new molecular targets. Although HD has been classically classified as a neurodegenerative disease affecting voluntary movement, lately cognitive dysfunction is receiving increased attention as it is very invalidating for patients. Thus, an ambitious goal in HD research is to find altered molecular mechanisms that contribute to cognitive decline. In this review we have focused on those findings related to corticostriatal and hippocampal cognitive dysfunction in HD, as well as on the underlying molecular mechanisms, which constitute potential therapeutic targets. These include alterations in synaptic plasticity, transcriptional machinery, and neurotrophic and neurotransmitter signaling. This article is protected by copyright. All rights reserved

Deletion 13q Characterized by SNP Microarray Profiling of a Large Cohort of CLL Patients

The investigation of Chronic Lymphocytic Leukemia (CLL) by conventional cytogenetic analysis combined with fluorescence in-situ hybridisation (FISH) has been regarded as the gold standard. Detection and characterisation of non-random rearrangements is hindered in part by the limitation of cell culturing and the targeted approach of FISH testing. More recently the implementation of molecular karyotyping by high resolution Single Nucleotide Polymorphism (SNP) microarray platforms has augmented the investigation protocol of haematological malignancies in diagnostic pathology. Deletion 13q is reported in up to 50% of patients with CLL and has historically been reported in association with low risk disease. Molecular karyotyping has revealed the diversity and complexity of rearrangements within the del13q region. This has culminated in the classification of the 13q deletion into type I (exclusive of Rb1) and type II (inclusive of Rb1). However uncertainty exists with regard to the prognostic significance, particularly with respect to the large, type II deletion. To evaluate this we investigated the molecular karyotype of 500 CLL patients. We ascertained the incidence of del13q type I and type II, complexity of deletions, association with additional karyotypic changes and cnLOH chromosome 13. Our results show that there is no significant difference in the incidence or association with additional karyotypic changes, but type II deletion is associated with complex 13q deletions (p = 0.0001). Furthermore there is no significant difference with respect to cytogenetic progression in follow-up testing of 43 patients. This study highlights the benefit of SNP microarray analysis in characterising deletion anatomy and the need for re-evaluation of risk stratification of non-random rearrangements

Fine mapping studies of quantitative trait loci for baseline platelet count in mice and humans

Platelet count is a complex trait with a substantial genetic contribution. Platelets have a fundamental role in normal haemostasis, with both increases and decreases of the platelet count associated with significant mortality and morbidity. Despite the significant genetic component contributing to variation in baseline platelet count, the precise mechanisms remain incompletely understood. A detailed knowledge of these genetic and biochemical pathways could potentially lead to the development of new therapies for disorders of platelet number. The aim of this project has been to fine map quantitative trait loci previously identified in a murine F2 cross, of which two (Pltct1 and Pltct2) had been found to reach genome-wide statistical significance. Fine mapping was attempted by three independent approaches. First, a human association study for platelet count was performed, and a cross-species comparison conducted to identify regions where human associations and murine linkage peaks might coincide. Although this association study did not unequivocally identify syntenic regions associated with platelet count in humans where linkage was identified in the murine F2 cross, potentially suggestive associations were identified. Next, fine mapping of the murine regions was undertaken by QTL analysis in a murine F11 advanced intercross line. This approach successfully narrowed the support interval for pltct2. Although linkage at the pltct1 locus was also replicated, this failed to reach genome-wide significance in the F11 study. Next, an in-silico association analysis was undertaken where the methodology included the novel development of an underlying genetic similarity matrix index to correct for population stratification. The results from this in-silico association were then correlated with expression array data from parental samples and five genes were prioritised for more detailed analysis by resequencing. Two potential variants in these candidate genes were identified which merit further investigation for causal effects on platelet count.This work highlights the complexity of mapping genetic variants contributing to thrombopoiesis, despite the significant progresses which have been made at the technological, methodological and statistical levels in the field of complex trait mapping

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

Aims: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). Methods: The coding sequence and intron–exon boundaries of the cardiac β myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. Results: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. Conclusions: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions