EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma.

AIM: We assessed the diagnostic accuracy of epidermal growth factor receptor (EGFR) mutant-specific antibodies for detecting two common activating EGFR mutations. METHODS: Immunohistochemical expression of mutation-specific antibodies against EGFR exon 19 deletion E746-A750 ((c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg) were assessed in a cohort of 204 resected early stage node negative lung adenocarcinomas, and protein expression was compared with DNA analysis results from mass spectrometry analysis. RESULTS: Of seven cases with L858R point mutation, six were positive by immunohistochemistry (IHC). There were three false positive cases using L858R IHC (sensitivity 85.7%, specificity 98.5%, positive predictive value 66.7%, negative predictive value 99.5%). All seven E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. Four additional cases were positive for exon 19 IHC but negative by mutation analysis. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 98.0%, positive predictive value 63.6% and negative predictive value 100%. CONCLUSIONS: Mutant-specific EGFR IHC has good specificity and sensitivity for identifying targeted activating EGFR mutations. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases where limited tumour material is available, or in situations where molecular genetic analysis is not readily available.

CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion.

OBJECTIVE: Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs. METHODS: We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression. RESULTS: An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele. CONCLUSIONS: Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors.

Low yield in screening patients with sporadic motor neuron disease for Kennedy disease.

The diagnostic yield of testing for Kennedy disease in patients diagnosed with sporadic motor neuron disease (MND) is unclear. We measured the CAG repeat lengths in the androgen receptor gene of patients with progressive limb weakness who had either upper and lower motor signs (n = 130), or lower motor neuron signs alone (n = 30). Only one patient with a long history of lower motor weakness had a repeat length in the Kennedy disease range. Testing for Kennedy disease is unlikely to benefit MND patients with upper motor neuron signs or those with a short history of lower motor signs.

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.

Gene therapy: applications and progress towards the clinic.

Gene therapy was originally conceived as an approach to the treatment of genetic disease, to repair or replace a faulty gene. Subsequently, gene therapy clinical trials have been undertaken for a wide range of conditions, particularly cancer and AIDS. Overall, the results from gene therapy have been disappointing. The reasons include the following: (i) low gene transfer efficiencies and (ii) shortcomings in the identification and manipulation of appropriate target cells, including progenitor cell populations required for the maintenance of long-term effects. Today, the immense potential of gene therapy remains, but more basic research is required to improve technical aspects of this form of cellular therapy.

Warfarin for non-rheumatic atrial fibrillation: five year experience in a district general hospital.

OBJECTIVES: To assess the long term efficacy of and risks associated with computer aided oral anticoagulation for non-rheumatic atrial fibrillation (NRAF) in a district hospital setting. DESIGN: Retrospective, age stratified, event driven clinical database analysis. SETTING: District general hospital. PARTICIPANTS: 739 patients receiving warfarin for NRAF between 1996 and 2001. Patients were selected from an anticoagulation database through appropriate filter settings. MAIN OUTCOME MEASURES: Anticoagulation control (international normalised ratio (INR)) and hospitalisations for bleeding complications, thromboembolic events, and stroke. RESULTS: Over 1484 patient-years, computer assisted anticoagulation was uncontrolled in 38.3% of patients (INR < 2.0 or > 3.0). No significant differences in INR control were observed with respect to patient age (< 65, 65-75, and > 75 years), although to achieve adequate control of anticoagulation, the frequency of testing increased significantly with age. Annual risks of bleeding complications, thromboembolism, and stroke were 0.76%, 0.35%, and 0.84%, respectively. No significant differences in these events were observed between the three age groups studied. Patients who had thromboembolic events and haemorrhagic complications were significantly more likely to have been under-anticoagulated (INR < 2.0) and over-anticoagulated (INR > 3.0), respectively, at the time of their clinical event. CONCLUSIONS: Computerised long term oral anticoagulation for NRAF in a community setting of elderly and diverse patients is safe and effective. Anticoagulation control, bleeding events, thromboembolic episodes, and stroke rates are directly comparable with those reported in major clinical trials. The authors therefore support the strategy of rate control with long term oral anticoagulation for NRAF in general clinical practice.

Troponin I, laboratory issues, and clinical outcomes in a district general hospital: crossover study with "traditional" markers of myocardial infarction in a total of 1990 patients.

AIMS: Review of the clinical outcomes and practical issues of replacing traditional cardiac enzymes with troponin I (cTnI) in a district general hospital. METHODS: Crossover study of three sequential three month stages during which serial cardiac enzymes were replaced with a single cTnI measurement available at three set times within 24 hours for the duration of the second three month stage. The study was carried out in a 630 bed district general hospital with 1990 admissions of suspected cardiac ischaemia over the study period as a whole. Account was taken of seasonal factors. RESULTS: The introduction of troponin was associated with 8.5% more patients with non-ischaemic heart disease (IHD) being discharged on the day after admission, saving approximately 107 bed days each year. Approximately 50% more patients were diagnosed with myocardial infarction during the cTnI stage. There was no increase in readmission within one month or early death with cTnI. Approximately 3% false positive and 1.5% false negative cTnI results were recorded. All false positive cTnI results were coding errors or attributable to known assay interference effects. All false negatives were potentially explained by sample timing factors. The lack of standardisation in troponin assay services impacts clinically. CONCLUSION: Younger patients without IHD were discharged earlier during the cTnI stage in apparent safety. Blood sample timing needs to be verified when cTnI is used as an adjunct to early discharge. There were no unexplained false positives or negatives. Standardisation related issues arose.

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.

Interaction of normal and expanded CAG repeat sizes influences age at onset of Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the HD gene on chromosome 4p16.3. Past studies have shown that the size of expanded CAG repeat is inversely associated with age at onset (AO) of HD. It is not known whether the normal Huntington allele size influences the relation between the expanded repeat and AO of HD. Data collected from two independent cohorts were used to test the hypothesis that the unexpanded CAG repeat interacts with the expanded CAG repeat to influence AO of HD. In the New England Huntington Disease Center Without Walls (NEHD) cohort of 221 HD affected persons and in the HD-MAPS cohort of 533 HD affected persons, we found evidence supporting an interaction between the expanded and unexpanded CAG repeat sizes which influences AO of HD (P = 0.08 and 0.07, respectively). The association was statistically significant when both cohorts were combined (P = 0.012). The estimated heritability of the AO residual was 0.56 after adjustment for normal and expanded repeats and their interaction. An analysis of tertiles of repeats sizes revealed that the effect of the normal allele is seen among persons with large HD repeat sizes (47-83). These findings suggest that an increase in the size of the normal repeat may mitigate the expression of the disease among HD affected persons with large expanded CAG repeats.

Frequency of cytochrome P450 3A4 variant genotype in transplant population and lack of association with cyclosporin clearance.

OBJECTIVE: Cytochrome P450 3A4 (CYP3A4) plays a vital role in the oxidative metabolism of many xenobiotics. Some recent reports have provided circumstantial evidence in support of an association between a genetic polymorphism (A-->G) in the 5'-flanking region (-290) of CYP3A4 and altered enzyme activity. We sought to determine whether genotyping patients for CYP3A4-G could assist with the dose optimisation of drugs metabolised by this system. METHODS: Normal subjects and renal-transplant patients receiving cyclosporin for immune modulation were genotyped for the CYP3A4-G variant. A surrogate for cyclosporin clearance was estimated from the ratio of the cyclosporin dose, normalised for body weight and the corresponding trough concentration. The association between genotype and clearance was examined in patients who received twice-daily doses of cyclosporin and who were not on concurrent medication known to modify CYP3A4 function. RESULTS: The allelic frequencies of the CYP3A4-G variant were estimated to be 2.6% and 3% in transplant patients and normal subjects, respectively. The median cyclosporin pseudo-clearance of transplant patients with wild-type CYP3A4 was 0.90 l/h/kg (range: 0.35-3.8 l/h/kg; n = 86), whereas the corresponding value for the five patients heterozygotic for the CYP3A4-G variant was 0.71 l/h/kg (range 0.35-0.91 l/h/kg). The distribution of the pseudo-clearance according to genotype was not found to be significant according to a Fisher's exact test (P = 0.15). CONCLUSION: Genotyping for the CYP3A4-G polymorphism is unlikely to assist cyclosporin dose selection in transplant patients.

Huntington's disease: neurological assessment of potential gene carriers presenting for predictive DNA testing.

One hundred and fifty-six potential gene carriers who were 50% 'at risk' of inheriting the Huntington's disease (HD) mutation, and who presented for predictive testing, underwent neurological assessment before their gene status had been determined. The association between pre-gene result symptoms and minimal neurological signs (insufficient for diagnosis in their own right) and subsequent gene status was determined. Of these, 38% tested positive for the HD mutation. Fifty-one individuals had minor neurological signs. After exclusions, 61% of gene-positive patients had minor neurological signs, whereas only 8% testing gene negative had signs. Minimal chorea observed in the toes and feet with the subject supine, and the patient being stressed by a mental task carried 96% specificity and 86% positive predictive value for gene-positive status. Neurological symptoms did not distinguish gene status, but behavioural and cognitive symptoms were more often reported by the gene-positive group. Although an 'at-risk' individual may receive a gene-positive result, neurological examination remains the most accessible, reliable and cost effective means of determining clinical disease onset.

Dobutamine magnetic resonance imaging as a predictor of myocardial functional recovery after revascularisation.

OBJECTIVE: To assess the use of dobutamine magnetic resonance imaging (MRI) as a preoperative predictor of myocardial functional recovery after revascularisation, comparing wall motion and radial wall thickening analyses by observer and semi-automated edge detection. PATIENTS: 25 men with multivessel coronary disease and resting wall motion abnormalities were studied with preoperative rest and stress MRI. MAIN OUTCOME MEASURES: Observer analysis for radial wall thickening was compared with a normal range, while wall motion analysis used a standard four point scale. Semi-automated analysis was performed using an edge detection algorithm. Segments displaying either improved or worsened thickening or motion with dobutamine were considered viable. Postoperative rest images were performed 3-6 months after coronary artery bypass grafting (CABG) for comparison. RESULTS: For observer analysis the values for sensitivity and specificity were 50% and 72% for wall motion, with respective values of 50% and 68% for thickening. With semi-automated edge detection the figures for motion were 60% and 73%, with corresponding values of 79% and 58% for thickening. Combining thickening and motion for the semi-automated method to describe any change in segmental function yielded a sensitivity of 71% and specificity of 70%. CONCLUSIONS: Dobutamine MRI is a reasonably good predictor of myocardial functional recovery after CABG. The use of semi-automated edge detection analysis improved results.

Polymorphisms in the CAG repeat--a source of error in Huntington disease DNA testing.

Five of 400 patients (1.3%), referred for Huntington disease DNA testing, demonstrated a single allele on CAG alone, but two alleles when the CAG + CCG repeats were measured. The PCR assay failed to detect one allele in the CAG alone assay because of single-base silent polymorphisms in the penultimate or the last CAG repeat. The region around and within the CAG repeat sequence in the Huntington disease gene is a hot-spot for DNA polymorphisms, which can occur in up to 1% of subjects tested for Huntington disease. These polymorphisms may interfere with amplification by PCR, and so have the potential to produce a diagnostic error.

DNA testing for haemochromatosis: diagnostic, predictive and screening implications.

Since 1996, the identification of the HFE gene has enabled DNA testing for hereditary haemochromatosis (HH). The range of DNA testing available includes: (1) diagnostic, (2) predictive (also called presymptomatic testing) and (3) screening. Access to DNA testing has been facilitated by an Australian Medicare rebate, the first available for genetic disorders. Despite the availability of HFE DNA testing in HH, it remains necessary to interpret results in the context of the clinical picture. Traditional markers based on phenotype (transferrin ferritinsaturation, and liver biopsy) are still required in some circumstances. We report our experience with HFE DNA testing using a semi-automated approach, which allows multiplexing for the two common mutations (C282Y and H63D). Screening a cohort of beta-thalassaemia major and sickle cell anaemia patients of predominantly Mediterranean origin showed that these individuals do not have the common C282Y mutation. This excluded C282Y as a factor in the pathogenesis of iron overload in these haemoglobinopathies. It also showed that the C282Y mutation is of limited value when investigating HH in certain ethnic groups. An Australian family studied illustrated the relative contribution of C282Y and H63D in iron overload. A recently reported third mutation (S65C) in the HFE gene was detected in a low frequency in the populations tested.

Milestones in the Human Genome Project: genesis to postgenome.

The Human Genome Project (HGP) will change medicine and medical research irrevocably. The obvious gains in genetic knowledge from the HGP, together with the advances which will flow into bioinformatics, biotechnology and the potential for novel therapeutic agents, will ensure that the financial investment in the HGP is repaid many times over. The HGP's costs in terms of ethical and social issues remain to be determined, but it is to be hoped that these will not detract from the scientific and medical achievements. How did such an endeavour start, and what path did it follow?

An online locus-specific mutation database for familial hypertrophic cardiomyopathy.

The aim of this locus-specific mutation database was to provide an online resource that contains summarised and updated information on familial hypertrophic cardiomyopathy (FHC)-associated mutations and related data, for researchers and clinicians. It also serves as a means of publishing previously unpublished data, which could be of value in understanding genotype/phenotype correlations. There are 123 FHC-associated mutations catalogued along with ancillary information. By implementing the cgi/http method, remote users can query the database via the HTML interface on the Web browser and obtain data of relevance to them. The online service is available on http://www.angis.org.au/Databases/Heart.

Risk stratification after acute myocardial infarction by Doppler stroke distance measurement.

OBJECTIVE: To establish the value of Doppler stroke distance measurement as a predictor of mortality risk following acute myocardial infarction. DESIGN: Follow up study. SETTING: Coronary care unit of a teaching and district general hospital. SUBJECTS: 378 patients (mean age 61 years) with acute myocardial infarction followed up for a mean of five years (range 2-7 years); 299 (79%) patients received thrombolysis. MAIN OUTCOME MEASURES: Stroke distance (the systolic velocity integral of blood flow in the aortic arch (percentage of age predicted normal value)); presence or absence of left ventricular failure on the admission chest radiograph; the codified admission ECG; death during follow up. RESULTS: Mean (SD) stroke distance was 81 (19)% and five year survival 76%. For patients with stroke distance > 100% (n = 60), 82-100% (n = 134), 63-81% (n = 122), and < 63% (n = 62), the one month mortality rates were 0%, 1.5%, 4%, and 18%, respectively; the corresponding estimates for mortality at five years were 17%, 19%, 24%, and 43%. Survival was independently related to age (p < 0.0001), stroke distance (p < 0.0001), and chest radiograph appearance (p = 0.002), but not to ECG codes (p = 0.31) or receipt of thrombolysis (p = 0.60). The areas under receiver operator characteristic plots for stroke distance measurements were 82%, 76%, 71%, and 65% for deaths within one month, six months, one year, and two years, respectively. CONCLUSIONS: The bedside measurement of stroke distance stratifies mortality risk after acute myocardial infarction. The predictive ability of this simple measure of left ventricular systolic function compares well with published accounts of the more complex measurement of ejection fraction.

Determination of normal regional left ventricular function from cine-MR images using a semi-automated edge detection method.

A semi-automated edge detection method for the delineation of the endo- and epicardial borders of the left ventricle from cine MR images has been developed. The feasibility of this was demonstrated by processing end diastolic and end systolic ECG-gated images of four short axis images in 10 healthy subjects. The first derivative method combined with a 2D weighted polynomial fitting procedure was used to determine the endo- and epicardial borders, which then allowed determination of the wall motion, wall thickening, and ejection fraction, of the left ventricle. The results show that the end-systolic radial wall motion varies from (32+/-8)% to (76+/-12)%, and wall thickening from (0.60+/-0.46) cm to (1.26+/-0.50) cm. An average ejection fraction of (69+/-6)% was found which agrees well with literature values. The method described, for the delineation of the borders, reduces considerably the long and tedious operator time inherent in manual measurement and greatly increases the reproducibility of the measurements.