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 beta 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.

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.

Genetically confirmed clinical Huntington's disease with no observable cell loss.

Huntington's disease (HD) results from neurodegeneration of the neostriatum. The mutation on chromosome 4 is an expansion in a triplet repeat (CAG)(n) located within the IT15 gene. Only six patients have been reported with clinical features of HD in association with limited neuropathology. Of these, only one has had the diagnosis confirmed by genetic (DNA) testing. We describe a patient with the clinical phenotype and genetically confirmed HD but unexpected limited neuropathology. The patient was seen because of aggressive behaviour and memory problems of two years duration. The differential diagnosis included HD although there was no family history. DNA testing was positive for the HD mutation. Clinical follow up three months later confirmed classic features of HD. Progression of the disease was rapid with death three years later. Neuropathology revealed a largely intact neostriatum with bilateral ischaemic damage and cell loss in the external globus pallidus. Such pathology alone could explain the clinical features of HD. This is only the second report of genetically confirmed clinically manifest HD with little evidence of HD neuropathology. There are several unusual features which could not have been predicted by the clinical picture, in particular the progressive course of bilateral ischaemic changes restricted to the external globus pallidus. The potential to miss other HD cases at post-mortem examination, and the implications of this for family members, are discussed.