Low-dose IL-2 induces CD56bright NK regulation of T cells via NKp44 and NKp46.

Low-dose interleukin (IL)-2 has shown clinical benefits in patients with autoimmune and inflammatory diseases. Both regulatory T cells (Tregs ) and natural killer (NK) cells are increased in response to low-dose IL-2 immunotherapy. The role of regulatory T cells in autoimmune diseases has been extensively studied; however, NK cells have not been as thoroughly explored. It has not been well reported whether the increase in NK cells is purely an epiphenomenon or carries actual benefits for patients with autoimmune diseases. We demonstrate that low-dose IL-2 expands the primary human CD56bright NK cells resulting in a contact-dependent cell cycle arrest of effector T cells (Teffs ) via retention of the cycle inhibitor p21. We further show that NK cells respond via IL-2R-ß, which has been shown to be significant for immunity by regulating T cell expansion. Moreover, we demonstrate that blocking NK receptors NKp44 and NKp46 but not NKp30 could abrogate the regulation of proliferation associated with low-dose IL-2. The increase in NK cells was also accompanied by an increase in Treg cells, which is dependent on the presence of CD56bright NK cells. These results not only heighten the importance of NK cells in low-dose IL-2 therapy but also identify key human NK targets, which may provide further insights into the therapeutic mechanisms of low-dose IL-2 in autoimmunity.

Bilateral Plantar Fibromatosis

Aim To describe an uncommon clinical finding and raise awareness of its manifestation and associated conditions. Methods This case describes a gentleman with bilateral plantar fibromatosis caused by type 2 Diabetes Mellitus and previous alcohol excess. Results Treatment options include physiotherapy, steroid and collagenase injection therapy. Surgical intervention can be considered for persistently symptomatic or recurrent cases. Discussion In conclusion, plantar fibromatosis is an under-recognised and disabling condition which should prompt intervention and optimisation of co-morbidities.

Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy.

Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Effect of anaesthetic technique on the natural killer cell anti-tumour activity of serum from women undergoing breast cancer surgery: a pilot study.

BACKGROUND: Animal models and retrospective clinical data suggest that certain anaesthetic techniques can attenuate immunosuppression and minimize metastasis after cancer surgery. Natural killer (NK) T cells are a critical component of the anti-tumour immune response. We investigated the effect of serum from women undergoing primary breast cancer surgery, randomized to propofol-paravertebral block (PPA) or sevoflurane-opioid (GA) anaesthetic techniques, on healthy human donor NK cell function and cytotoxicity against oestrogen and progesterone receptor-positive breast cancer cells (HCC1500). METHODS: Ten subjects who donated serum before operation and 24 h after operation in an ongoing randomized prospective trial (NCT 00418457) were randomly selected. Serum from PPA (n=5) and GA (n=5) subjects was co-cultured with HCC1500 and healthy primary NK cells. NK cell activating receptors (NKp30, NKp44, NKp46, 2b4, CD16, NKG2D), cytokine production, NK CD107a expression, and cytotoxicity towards HCC1500 were examined. RESULTS: Serum from PPA subjects did not alter normal NK marker expression or secretion of cytokines. Serum from GA subjects reduced NK cell activating receptor CD16 [from mean (sem), 82 (2)% to 50 (4)%, P=0.001], IL-10 [from 1700 (80) to 1200 (92) pg ml(-1), P=0.001], and IL-1ß [from 68 (12) to 19 (4) pg ml(-1), P=0.01]. An increase in NK cell CD107a [23 (2)% to 37(3)%, P=0.007] and apoptosis of HCC1500 [11 (1)% to 21 (2)%, P=0.0001] was observed with PPA serum, but not GA serum, treated NK cells. CONCLUSION: Serum from women with breast cancer undergoing surgical excision who were randomized to receive a PPA anaesthetic technique led to greater human donor NK cell cytotoxicity in vitro compared with serum from women who received GA. CLINICAL TRIAL REGISTRATION: NCT 041857.

Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis: in vitro evidence in cultured epithelial cells.

BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Increased expression of ER stress- and hypoxia-associated molecules in grey matter lesions in multiple sclerosis.

BACKGROUND: The endoplasmic reticulum (ER) stress pathway may play a role in the pathogenesis multiple sclerosis (MS), and while ER stress-associated molecules have been demonstrated in white matter (WM) lesions, these have not been analysed in grey matter (GM) demyelination. OBJECTIVE: The objective was to characterise the type and frequency of GM lesions and establish expression profiles of ER stress- and hypoxia-associated markers. METHODS: Sections from 16 MS cases and 12 non-MS controls were stained for ER stress molecules (BiP and CHOP) and hypoxia-associated D110 antigen. RESULTS: Of the GM lesions analysed, 24% were type 1 (continuous between GM and WM), 22% were type 2 (entirely within GM) and the majority (54%) were type 3 (extending from pia mater). Comparison of GM lesions, MS normal-appearing grey matter (NAGM) and non-MS control tissue showed that NAGM, type 1 and type 3 lesions all had significantly increased levels of CHOP compared to controls. According to morphological and dual-labelling criteria, the majority of CHOP-positive cells were microglia. Approximately 50% of GM lesions contained D110-positive cells. CONCLUSION: These data suggest that ER stress plays an important role in GM lesion development and may be critical in activation of microglia in pre-lesional NAGM. The high number of lesions containing D110-positive cells suggests a role for hypoxic-like insult in GM lesion development.

Graves' disease associated with HIV disease and late immune reconstitution inflammatory syndrome following the initiation of antiretroviral therapy.

SUMMARY: Both human immunodeficiency virus (HIV) and antiretroviral therapy (ART) are associated with endocrine dysfunction (1). The term 'immune reconstitution inflammatory syndrome' (IRIS) describes an array of inflammatory conditions that occur during the return of cell-mediated immunity following ART. Graves' disease (GD) occurs rarely as an IRIS following ART. In this study, we describe the case of a 40-year-old Brazilian female who was diagnosed with HIV following admission with cryptococcal meningitis and salmonellosis. At this time, she was also diagnosed with adrenal insufficiency. Her CD4 count at diagnosis was 17 cells/µL which rose to 256 cells/µL over the first 3 months of ART. Her HIV viral load, however, consistently remained detectable. When viral suppression was finally achieved 21 months post diagnosis, an incremental CD4 count of 407 cells/µL over the following 6 months ensued. Subsequently, she was diagnosed with a late IRIS to cryptococcus 32 months following initial ART treatment, which manifested as non-resolving lymphadenitis and resolved with high-dose steroids. Following the initiation of ART for 45 months, she developed symptomatic Graves' hyperthyroidism. At this time, her CD4 count had risen to 941 cells/µL. She has been rendered euthyroid on carbimazole. This case serves to remind us that GD can occur as an IRIS post ART and typically has a delayed presentation. LEARNING POINTS: Endocrinologists should be aware of the endocrine manifestations of HIV disease, in particular, thyroid pathology. Endocrinologists should be aware that IRIS can occur following the initiation of ART. Thyroid dysfunction can occur post ART of which Graves' disease (GD) is the most common thyroid manifestation. GD as a manifestation of ART-induced IRIS can have a delayed presentation. Infectious disease physicians should be aware of endocrine manifestations associated with HIV and ART.

Immunovirological correlates in human rabies treated with therapeutic coma.

A 37-year-old woman was admitted to hospital and over the next 5 days developed a progressive encephalitis. Nuchal skin biopsy, analyzed using a Rabies TaqMan(c) PCR, demonstrated rabies virus RNA. She had a history in keeping with exposure to rabies whilst in South Africa, but had not received pre- or post-exposure prophylaxis. She was treated with a therapeutic coma according to the "Milwaukee protocol," which failed to prevent the death of the patient. Rabies virus was isolated from CSF and saliva, and rabies antibody was demonstrated in serum (from day 11 onwards) and cerebrospinal fluid (day 13 onwards). She died on day-35 of hospitalization. Autopsy specimens demonstrated the presence of rabies antigen, viral RNA, and viable rabies virus in the central nervous system.

Investigation of killer cell immunoglobulin-like receptor gene diversity, KIR2DL1 and KIR2DS1.

Polymorphism in the alleles of the killer cell immunoglobulin-like receptor 2DL1 and 2DS1 genes has been investigated by the development of polymerase chain reaction-sequence-specific oligonucleotide probing systems. The methods have been applied to 77 Northern Irish families, establishing allele frequencies from the unrelated parents. Additionally, cell line DNA from individuals and CEPH families of the 13th International Histocompatibility Workshop panel were investigated. Eight of the reported KIR2DL1 alleles and only the KIR2DS1*002 allele were identified in the groups studied. Two individuals were positive for three alleles of KIR2DL1, and a putative variant of KIR2DL1*001 was observed. Results also indicated an inherited KIR2DL1/2DS1 splice variant present in a haplotype with several core loci absent, in two families.

Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis.

AIMS: In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell-to-cell spread in the central nervous system (CNS) has been assessed in vibratome-cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. METHODS: Recombinant MVs expressing EGFP were visualized at different levels in 200-microm vibratome-cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7-microm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). RESULTS: The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV-infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200-microm vibratome-cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in-depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. CONCLUSIONS: The use of self-tracing recombinant MVs, viewed in thick vibratome-cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.

The significance of measles virus antigen and genome distribution in the CNS in SSPE for mechanisms of viral spread and demyelination.

The distribution of measles virus (MV) antigen and genomic RNA in the central nervous system (CNS) in 10 cases of subacute sclerosing panencephalitis (SSPE) was determined using optimized immunocytochemistry and in situ hybridization techniques. As in previous reports neurons and oligodendrocytes were found to be the most frequently infected cells. It was confirmed that MV infection in neuronal processes was predominantly dendritic but there was also some evidence suggestive of occasional axonal involvement, a finding confirmed by electron microscopy. In addition MV genomic RNA was detected in neuronal processes, in some cases in the absence of demonstrable MV antigen. The relationship between myelin destruction and oligodendrocytic infection suggested that the demyelination may be solely the result of virus infection. A possible correlation between viral distribution and form and the clinical duration of disease was examined. Viral antigen and genome were equally abundant in the cerebral cortex in most short duration cases (<6 months). However, in two of these cases viral RNA but not antigen was detected in the spinal cord. In long duration cases (>36 months) viral RNA was abundant in all areas of the CNS examined, frequently in the absence of demonstrable antigen. These findings may suggest viral spread in a cephalo-caudal direction, probably by transneuronal spread.

Leukaemia inhibitory factor mRNA is expressed in the brains of patients with subacute sclerosing panencephalitis.

We have examined the in situ transcription of leukaemia inhibitory factor (LIF) in brain tissue from 3 cases of subacute sclerosing panencephalitis (SSPE) and in 2 non-neurological control brains. This has been compared with expression of interleukin 2 (IL-2), interleukin 6 (IL-6) and tumour necrosis factor beta (TNF beta) in the same tissues. All of the cytokines in the study were expressed in cells in the inflammatory infiltrate as well as in glial cells. LIF mRNA was also found to be expressed in neurons, in foci where these cells were also virally infected. No hybridization was found with any of the probes in areas of SSPE brain, which were negative for measles virus RNA or in the non-neurological control cases, although expression was demonstrated in the latter by use of reverse transcription-polymerase chain reaction (RT-PCR). Differentiated, cultured human neuronal cells were also positive, by RT-PCR, for LIF. This is the first demonstration of LIF expression in human brain and the results suggest that this cytokine is up-regulated, in several cell types, including neurons, following virus infection.

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Transcriptional activation of the glutathione S-transferase pi gene in human ureteric and bladder carcinomas.

Using a cDNA clone derived from the rat Glutathione S-Transferase P (GST-P) gene, we have investigated homologous transcripts in a range of normal and malignant human tissues. A major transcript, of 0.75 Kb, representing human GST-pi mRNA, was present in total RNA purified from peripheral white blood cells, bladder, ureteric and prostate tissues. In total RNA derived from histopathologically benign/normal tissues, peripheral leucocytes and from hyperplastic and malignant prostates, levels of the mature transcript varied by less than 1.5-fold (n = 70). In contrast, levels of transcript were significantly elevated (up to 16-fold) in total RNA derived from bladder and ureteric carcinomas, with the highest levels of elevation approaching those previously found only for the GST-P gene in experimentally induced rodent hepatocellular carcinomas. A wide range of stage and grade of tumour has been investigated (pT1 to pT4; G1 to G3, respectively), suggesting that the transcriptional activation of the GST-pi gene is a characteristic common to many bladder/ureteric carcinomas in man.

Analysis of transcripts homologous to acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase induced in rat liver by methylclofenapate.

Methylclofenapate is a potent peroxisome proliferating agent and liver carcinogen in rats. Animals exposed to daily oral doses (2.5 mg/kg body wt.) for a 21-day period were studied to determine the levels of mRNA homologous to peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in total liver RNA. Northern blotting revealed transcripts of approximately 3.8 and 3.3 kilobases (kb), homologous to acyl-CoA oxidase and the bifunctional enzyme, respectively. Levels of these transcripts began to rise at approximately 4 h after the initial dose of the agent, and reached maximum induction (35- and 60-fold, respectively, in excess of control levels) at 2-8 days after the start of the study. The kinetics of induction for acyl-CoA oxidase mRNA resembled those of palmitoyl-CoA oxidase activity, and the induction of mRNA preceded the expression of enzyme activity, further supporting a transcriptional control model of induction of the peroxisomal enzymes. The levels of mRNA induction for the peroxisomal enzymes were higher in the present study than those reported elsewhere for single doses of peroxisome proliferating agents and probably reflect the increased tissue levels achievable in long term carcinogenesis studies.

Salivary insulin-like growth factor-I originates from local synthesis.

Insulin-like growth factor-I (IGF-I) is a GH-dependent growth factor found in its highest concentrations in plasma. It is also measurable in saliva. The origins of salivary IGF-I concentrations were studied. Intracardial administration of Sprague-Dawley rats with 125I-labelled IGF-I and subsequent analysis of plasma and saliva samples by exclusion gel chromatography and SDS-PAGE, followed by autoradiography, demonstrated the apparent inability of IGF-I to cross from the plasma pool through to saliva. 125I-Labelled IGF-I was not chromatographed immediately before injection, resulting in administration of free iodide along with the iodinated peptide. This free iodide was demonstrable in saliva, indicating that movement of substances from plasma to saliva was measurable using the levels of 125I activity administered. Free iodide in saliva was not contributed to by 125I-labelled IGF-I degradation since 125I-labelled IGF-I was shown to be stable in saliva over 24 h. These data indicated that IGF-I in saliva is produced locally. Identification of a 4.7 kb IGF-I mRNA transcript in rat parotid salivary gland was consistent with IGF-I synthesis within that tissue.

Use of immunocytochemistry and biotinylated in situ hybridisation for detecting measles virus in central nervous system tissue.

Optimised immunocytochemical (ICC) and in situ hybridisation (ISH) protocols for long term, formalin fixed, central nervous system tissue infected with measles virus were developed. The effectiveness of 10 proteases for the enzymatic unmasking of formalin fixed antigen and nucleic acid was investigated. Protease VIII gave maximal signal generation with optimal tissue preservation and no background staining for both techniques. The use of a microwave oven as an additional pre-hybridisation step for RNA-RNA in situ hybridisation produced a significant increase in the number of cells labelled for genomic RNA. The ability to show the presence of antigen and nucleic acid in long term, formalin fixed tissue facilitates the use of stored necropsy material available in pathology departments for ICC and ISH investigations.

Comparison of reporter molecules for viral in situ hybridization.

A number of streptavidin-linked reporter molecules at the endpoint of a five-step detection protocol for viral in situ hybridization using biotinylated probes were examined. DNA-DNA and RNA-RNA model systems were used. Streptavidin linked to either peroxidase or fluorescein was found to be optimal in terms of sensitivity and resolution within individual cells. All other reporter molecules labelled similar numbers of cells with low background reaction. However, streptavidin-5 nm gold followed by silver enhancement gave very high background staining making interpretation of positive signals very difficult.

Typical Friedreich's ataxia without GAA expansions and GAA expansion without typical Friedreich's ataxia.

We clinically assessed and performed polymerase chain reaction analysis for the GAA trinucleotide repeat expansion in 103 patients from 73 families in Ireland, with a prior clinical diagnosis of Friedreich's ataxia (FA) or an unclassified progressive ataxic syndrome. The patients were classified as "typical" or "atypical" FA according to Harding's mandatory clinical diagnostic criteria. All patients underwent blood glucose analysis, and electrocardiography and echocardiography was performed in 99 and 101 patients, respectively. Mutation screening for expanded CAG trinucleotide repeats, associated with spinocerebellar ataxia (SCA) 1, 2, 3 and 6 was performed in 86 patients overall, including all GAA negative patients. Forty-nine of 56 typical patients and 13 of 47 atypical patients were either homozygous or heterozygous for the GAA expansion. Seven patients with a typical FA phenotype were negative for the GAA expansion. Although one of these patients had vitamin E deficiency, and two had raised alpha-fetoprotein levels, three other GAA negative patients with a typical FA phenotype had no other identifiable cause for their ataxia, once again raising the possibility of locus heterogeneity in FA. It is also possible that these patients have two point mutations in the X25 gene, or that they have another ataxic syndrome mimicking the FA phenotype. Two families who were homozygous for the GAA expansion exhibited intrafamilial phenotypic variability. Only one GAA negative patient had the SCA 3 mutation, and this was the only patient in the study with a possible autosomal dominant inheritance pattern. In the homozygous GAA population typical patients had significantly more repeats on the smaller allele than atypical patients, and there was an inverse relationship between the number of repeats on the smaller allele and the age at presentation. There was also an inverse relationship between the repeat size on both the larger and the smaller of the two alleles and the age at becoming wheelchair bound. There was no significant relationship between repeat size and the other indices of disease severity, including the presence or absence of diabetes or cardiomyopathy. This is the first large study of an Irish population with progressive ataxia that has shown a similar phenotype/genotype relationship to studies of FA in other European and non-European populations. The relatively low sensitivity and specificity of Harding's clinical diagnostic criteria must be appreciated when clinically assessing patients with a progressive ataxic syndrome. Although molecular genetic analysis now plays an essential role in diagnosis and classification, patients with a typical FA phenotype without any identifiable cause for their ataxia exist.