Validation of the new consensus criteria for the diagnosis of corticobasal degeneration.

BACKGROUND: Corticobasal degeneration (CBD) is a complex neurodegenerative disorder. Accurate diagnosis is increasingly important, with the advent of clinical trials of drugs aimed at modifying the underlying tau pathology. CBD often presents with a 'corticobasal syndrome' including impairments of movement and cognition. However, patients with similar corticobasal syndromes can have neurodegenerative pathologies that are not CBD. In addition, patients with CBD may present with aphasia or behavioural change. The clinical diversity of CBD and mimicry by non-CBD pathologies hinders accurate diagnosis. METHODS: We applied the new consensus criteria of Armstrong and colleagues et al 1 to a cohort of patients with detailed longitudinal clinical evaluation and neuropathology. RESULTS: In patients with pathologically confirmed CBD, accuracy of diagnosis was similar under the new and previous criteria: 9/19 (47%) met criteria for probable CBD at presentation, 13/19 (68%) at last clinical assessment. Patients with a corticobasal syndrome but without CBD pathology all (14/14) met the new diagnostic criteria of probable or possible CBD, demonstrating that the new criteria lacks the necessary specificity for an accurate ante mortem clinical diagnosis of CBD. None of the clinical features used in the new criteria were more common in the patients with CBD pathology (n=19) than without (n=14). CONCLUSIONS: The Armstrong criteria usefully broadens the recognised clinical phenotype of CBD but does not sufficiently improve the specificity of diagnosis to increase the power of clinical trials or targeted applications of tau-based disease-modifying therapies. Further work is required to show whether biomarkers could be more effective than clinical signs in the diagnosis of CBD.

The Syk-kinase inhibitor R406 impairs platelet activation and monocyte tissue factor expression triggered by heparin-PF4 complex directed antibodies.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4-heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. OBJECTIVES: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B-cell related hematological malignancies, on FcγRIIA-mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies-induced expression of TF and procoagulant activity of monocytic cells. RESULTS: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA-induced LAT phosphorylation and activation of phosphoinositide 3-kinase, Akt, phospholipase Cγ2 and p38 MAP-kinase. As a consequence, FcγRIIA-induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. CONCLUSION: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies-mediated platelet activation and monocyte procoagulant activity.

Clinical phenotypes in autopsy-confirmed Pick disease.

BACKGROUND: Neuropathology of frontotemporal lobar degeneration is variable and relationship between the pathology and the clinical presentation remains uncertain. Abnormal deposits of hyperphosphorylated and ubiquitinated tau protein are present in 30% of cases, which include the classic presentation of Pick disease with argyrophilic, intraneuronal inclusions known as Pick bodies. This study aimed to improve sensitivity of clinicopathologic relations in cases with neuropathologically confirmed Pick disease and to identify clinical symptoms and signs predictive of disease progression. METHODS: This was a retrospective analysis of 21 cases with a pathologic diagnosis of Pick disease and sufficient clinical information to establish early presenting clinical features from 2 specialist centers, representing 70% of all cases of Pick disease identified between 1998 and 2007 in these centers. RESULTS: At presentation, 13/21 cases (62%) were clinically diagnosed with behavioral variant frontotemporal dementia (bvFTD) and 8/21 (38%) with language variant frontotemporal dementia (lvFTD) including 2 with mixed syndromes. Patients with bvFTD died on average 5 years earlier than those with lvFTD (7 years vs 12 years after disease onset). Pathologically, fewer Pick bodies were present in the frontal and inferior temporal cortices of bvFTD than lvFTD cases. In contrast, both groups showed decreased neuronal density in the dentate gyrus with increasing disease duration. CONCLUSIONS: The pathologic course of the disease in FTLD cases with Pick bodies is not uniform and disease duration can be estimated based on early clinical features. These findings have relevance as treatment options, which are likely to be pathology specific, are developed.

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow.

BACKGROUND: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. OBJECTIVES: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. RESULTS: Videomicroscopy of DIOC-labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P-selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvß3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y-27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.

Atrophy patterns in histologic vs clinical groupings of frontotemporal lobar degeneration.

OBJECTIVE: Predictable patterns of atrophy are associated with the clinical subtypes of frontotemporal dementia (FTD): behavioral variant (bvFTD), semantic dementia (SEMD), and progressive nonfluent aphasia (PNFA). Some studies of pathologic subtypes have also suggested specific atrophy patterns; however, results are inconsistent. Our aim was to test the hypothesis that clinical, but not pathologic, classification (FTD with ubiquitin inclusions [FTD-U] and FTD with tau inclusions [FTD-T]) is associated with predictable patterns of regional atrophy. METHODS: Magnetic resonance scans of nine FTD-U and six FTD-T patients (histologically confirmed) were compared with 25 controls using voxel-based morphometry (VBM). Analyses were conducted with the patient group classified according to histologic or clinical variant. Additionally, three Alzheimer pathology patients who had the syndrome of SEMD in life (FTD-A) were analyzed. RESULTS: The VBM studies in clinical variants confirmed established patterns of atrophy (SEMD, rostral temporal; bvFTD, mesial frontal; PNFA, left insula). FTD-U and FTD-T VBM results were very similar, showing severe atrophy in the temporal poles, mesial frontal lobe, and insulae. A conjunction analysis confirmed this similarity. Subgroup analysis found that SEMD associated with either FTD-T or FTD-U was associated with similar rostral temporal atrophy; however, FTD-A had a qualitatively different pattern of left hippocampal atrophy. CONCLUSIONS: While there is predictable atrophy for clinical variants of frontotemporal dementia (FTD), histologic FTD variants show no noticeable differences. Reports of specific atrophy profiles are likely the result of idiosyncrasies in small groups. Semantic dementia associated with Alzheimer pathology, however, presented a distinct atrophy pattern.

Alpha- and gamma-synuclein proteins are present in cerebrospinal fluid and are increased in aged subjects with neurodegenerative and vascular changes.

BACKGROUND: Disease-specific biomarkers should reflect a fundamental feature of neuropathology and be validated in neuropathologically confirmed cases. Several synaptic proteins have been described in cerebrospinal fluid (CSF) of patients with dementia. In Lewy body disease alpha-synuclein is incorporated within Lewy bodies and alpha-, beta- and gamma-synucleins in dystrophic neuritis. These pathological changes are expected to be seen in CSF. METHODS: A total of 25 CSF post-mortem samples (8 control and 17 subjects with dementia) were used to quantify alpha- and gamma-synucleins and IgG. RESULTS: We describe for the first time the presence of gamma-synuclein in CSF. There is an elevation of both alpha- and gamma-synucleins in CSF from elderly individuals with Alzheimer's disease, Lewy body disease (LBD) and vascular dementia (CVD), compared to normal controls. gamma-Synuclein showed a greater elevation in LBD, IgG in CVD. The elevation of alpha- and gamma-synucleins was seen from Braak stage III onwards and remained stable until Braak stage VI. These results were not influenced by age at death or post-mortem delay. CONCLUSIONS: The reported increases in alpha- and gamma-synucleins and IgG in the ventricular CSF of individuals with dementia are novel findings. They now need to be explored further using a greater number of cases in each subgroup, using lumbar CSF samples to determine their applicability and relevance to a clinical diagnostic setting. It needs to be established whether using these markers may help to discriminate LBD from other types of neurodegenerative and vascular dementias.

Loss of synaptophysin and synaptosomal-associated protein 25-kDa (SNAP-25) in elderly Down syndrome individuals.

AIMS: This study quantified the density of the synaptic proteins synaptophysin and synaptosomal-associated protein 25-kDa (SNAP-25) in brains from elderly Down's syndrome individuals. METHODS: Six areas (frontal, occipital, parietal and temporal lobes, cerebellum and hippocampus) of post mortem brains from elderly Down's syndrome (DS) individuals (with reported functional memory problems and pathologically established Alzheimer's disease) and elderly controls were studied. RESULTS: Collectively in the six brain areas studied, there were significantly lower levels of both synaptophysin and SNAP-25 immunostaining in the DS group compared with controls. The elderly control group displayed a significant decrease in the densities of synaptophysin and SNAP-25 as a function of age at death (AAD; P < or = 0.001), whereas the DS group only showed a significant decrease as a function of AAD for synaptophysin. Assessing synaptic density as a function of Braak stage (BST) revealed a significant decrease in synaptophysin density for both groups. SNAP-25 was only significantly decreased as a function of BST in the DS group. Synaptic protein density was also shown to vary according to gender. Thus, both DS and control female subjects had a higher synaptic density of SNAP-25 than their male counterparts. In contrast, male DS and control individuals had a significantly greater density of synaptophysin than females. CONCLUSIONS: These results indicate that elderly DS individuals have lower synaptic densities of both analysed proteins than cognitively intact elderly individuals. Although AAD and BST show varying significance to decreases in protein density for both DS and control groups, results suggest that gender differences also play a role in the type of synaptic protein lost in elderly DS individuals.

Focal cortical presentations of Alzheimer's disease.

To determine the frequency of Alzheimer's disease (AD) pathology in patients presenting with progressive focal cortical syndromes, notably posterior cortical atrophy (PCA), corticobasal syndrome (CBS), behavioural variant frontotemporal dementia (bvFTD), progressive non-fluent aphasia (PNFA) (or a mixed aphasia) and semantic dementia (SD); and to compare the age of onset, evolution and prognosis in patients with focal cortical presentations of AD versus more typical AD and those with non AD pathology. From a total of 200 patients with comprehensive prospective clinical and pathological data we selected 120 : 100 consecutive cases with focal cortical syndromes and 20 with clinically typical AD. Clinical files were reviewed blind to pathological diagnosis. Of the 100 patients with focal syndromes, 34 had AD as the primary pathological diagnosis with the following distribution across clinical subtypes: all 7 of the PCA (100%); 6 of 12 with CBS (50%); 2 of 28 with bvFTD (7.1%); 12 of 26 with PNFA (44.1%); 5 of 7 with mixed aphasia (71.4%) and 2 of 20 with SD (10%). Of 20 with clinically typical AD, 19 had pathological AD. Age at both onset and death was greater in the atypical AD cases than those with non-AD pathology, although survival was equivalent. AD is a much commoner cause of focal cortical syndromes than previously recognised, particularly in PCA, PNFA and CBS, but rarely causes SD or bvFTD. The focal syndrome may remain pure for many years. Patients with atypical AD tend to be older than those with non-AD pathology.

Sporadic malignant nerve sheath tumour of the oculomotor nerve.

Malignant peripheral nerve sheath tumours (MPNST) are exceedingly rare in an intracranial location. In this report clinical and pathological evidence for the diagnosis of a MPNST arising from of the oclumotor nerve is presented. To our knowledge this is the first such case reported in the medical literature.

Ubiquitin-positive inclusions and progression of pathology in frontotemporal dementia and motor neurone disease identifies a group with mainly early pathology.

Frontotemporal lobar degeneration (FTLD) with tau-negative, ubiquitin-positive inclusions has been a topic of major interest in recent years, with this group now accounting for the majority of tau-negative cases of frontotemporal degeneration. The severity of neurodegeneration in FTLD is dependent on the stage of disease and is substantial even in the earliest stages. Elucidating the pathogenesis of FTLD requires evaluation of changes during the earliest possible stage of disease. However, the long survival of most frontotemporal dementia cases means that cases with early neuropathology are not frequently encountered. Cases of FTLD with the shortest survival are those with coexisting motor neurone disease (FTLD + MND), making these the ideal group for studying early FTLD pathology. It is not clear, however, what the pathological contribution of MND is in these cases. This study evaluates the pathology of 20 cases of FTLD (11 with no clinical signs of MND and nine with FTLD + MND) as well as 10 cases of MND without dementia. Our findings indicate that the deposition of ubiquitin does not play a key role in the neurodegenerative process in FTLD, and that the severity of neurodegeneration in FTLD is similar in cases with and without clinical MND.

Cerebral neutrophil recruitment, histology, and outcome in acute ischemic stroke: an imaging-based study.

BACKGROUND AND PURPOSE: Evidence now exists for a pathogenic role for neutrophils in acute cerebral ischemia. We have studied the patterns and temporal profile of cerebral neutrophil recruitment to areas of acute ischemic stroke (IS) and have attempted to correlate this with neurological status and outcome. METHODS: Patients with cortical middle cerebral artery (MCA) IS were recruited within 24 hours of clinical onset. Neutrophil recruitment was studied using indium-111 (111In) troponolate-labeled neutrophils, planar imaging, and single-photon emission computed tomography (SPECT). Volume of brain infarction was calculated from concurrent computed tomography (CT). Hematoxylin and eosin sections were obtained postmortem (n=2). Outcome was measured using Barthel, Rankin, and National Institute of Health Stroke (NIHSS) scales. RESULTS: Fifteen patients were studied. Significant 111In-neutrophil recruitment to ipsilateral hemisphere, as measured by asymmetry index (AI), was demonstrated within 24 hours of onset in 9 patients; this response was heterogenous between patients and on repeated measurement attenuated over time. Histologically, recruitment was confirmed within intravascular, intramural, and intraparenchymal compartments. Interindividual heterogeneity in neutrophil response did not correlate with infarct volume or outcome. In an exploratory analysis, neutrophil accumulation appeared to correlate significantly with infarct expansion (Spearman rho=0.66; P=0.03, n=12). CONCLUSIONS: Neutrophils recruit to areas of ischemic brain within 24 hours of symptom onset. This recruitment attenuates over time and is confirmed histologically. While neutrophil accumulation may be associated with either the magnitude or the rate of infarct growth, these results require confirmation in future studies.

Rapidly reversible dementia in cerebral amyloid inflammatory vasculopathy.

This report discusses a biopsy proven case of cerebral amyloid angiopathy, with additional prominent vascular inflammatory features, characterized by a rapidly progressive dementia and leukoencephalopathy, where the clinical and radiological abnormalities resolved rapidly with minimal therapeutic intervention. We propose the term cerebral amyloid inflammatory vasculopathy (CAIV) to describe this condition.

Survival in frontotemporal dementia.

OBJECTIVES: To establish survival in patients with pathologically confirmed frontotemporal dementia (FTD) and to determine whether clinical or pathologic subtype affects prognosis. METHODS: The authors reviewed the presenting clinical features of 61 patients with dementia and pathologically confirmed FTD studied in Sydney (n = 31) and Cambridge (n = 30) over a 10-year period. Data were available on time of symptom onset, diagnosis, institutionalization, and death. Cases were classified pathologically as tau-positive and tau-negative. RESULTS: Of the 61 patients with FTD, 26 presented with frontal variant (fvFTD), 9 with semantic dementia, 8 with progressive nonfluent aphasia (PNFA), 9 with associated motor neuron disease (FTD-MND), and 9 with corticobasal degeneration features. There was no difference between the groups in age at symptom onset (overall mean 58.5 +/- 7.8 years), but at diagnosis the PNFA (68.3 +/- 2.7) group was significantly older than the fvFTD (59.9 +/- 7.4) and FTD-MND (57.7 +/- 7.9) groups. The median survival from symptom onset and from diagnosis was 6 +/- 1.1 years (95% CI) for fvFTD and 3 +/- 0.4 years for FTD-MND. Survival across subgroups was equivalent except for the FTD-MND group, which had significantly shorter survival. Cases with tau-positive pathology had an older age at onset and a significantly better prognosis: median survival 9.0 +/- 0.9 years vs 5.0 +/- 1.1 years. CONCLUSIONS: FTD is a malignant disorder with limited life expectancy. FTD-MND has the shortest duration both before and after diagnosis. Tau-positivity is associated with a more slowly progressive form of FTD.

Identification and characterization of the promoter region of the GRM3 gene.

We have recently described the genomic organisation of the human metabotropic glutamate receptor 3 (GRM3) gene. The putative promoter region is characterised by the presence of a CCAAT and Sp1 site and the absence of a TATA box. Using a reporter gene assay, now we describe the functional activity of GRM3 promoter by transient transfection in both human neuroblastoma and astroglioma cell lines. Deletion of the CCAAT box and Sp1 site resulted in a pronounced reduction of reporter gene expression in both cell types, which indicates that these elements to correspond to the core promoter region. Moreover, we found that the genomic sequence 140 bp upstream of the first transcription initiation site appears to contain regulatory promoter elements for a preferential transcription of the gene in neuroblastoma cells. We also provide evidence that the genomic sequence spanning exon I, corresponding to the GRM3 5'-untranslated region, contains a negative regulatory element that represses gene transcription.

Activated astrocytes in areas of kainate-induced neuronal injury upregulate the expression of the metabotropic glutamate receptors 2/3 and 5.

All forms of brain injury induce activation of astrocytes, although different types of injury induce different astrocytic responses. Activated astrocytes are characterised by hypertrophy, proliferation and increased expression of glial fibrillary acidic protein (GFAP). However, neither the process by which astrocytes become reactive nor the consequences are well understood. Recently, the application of specific growth factors to primary astrocytic cultures was shown to regulate dramatically the level of expression of the metabotropic glutamate receptors (mGluR) 5 and 3. In the present study, we have used an intracerebroventricular injection of a subconvulsive dose of kainic acid to produce a lesion of CA3a pyramidal neurones in the mouse hippocampus and to investigate whether mGluR expression was altered in reactive astrocytes in vivo. Immunohistochemical analysis showed strong mGluR5 and mGluR2/3 immunoreactivity in glial cells within the area of neuronal loss possessing the morphological feature of activated astrocytes. Double labelling with GFAP confirmed the expression of mGluRs by reactive astrocytes. The mechanical injury produced by the needle insertion in the cerebral cortex also produced enhanced expression of mGluR5 and mGluR2/3 in activated astrocytes proximal to the area of neuronal injury. Our finding of an increased mGluR expression in reactive astrocytes in vivo suggests that transcriptional regulation by specific growth factors on mGluRs is a phenomenon extendible to specific circumstances in vivo and not limited to in vitro models. Identification of the mechanisms of this adaptive plasticity will be central in the understanding of the events leading to neuronal survival and/or death.

Genetic association of an LBP-1c/CP2/LSF gene polymorphism with late onset Alzheimer's disease.

OBJECTIVES: The only locus unequivocally associated with late onset Alzheimer's disease (AD) risk is APOE. However, this locus accounts for less than half the genetic variance. A recent study suggested that the A allele of the 3'UTR biallelic polymorphism in the LBP-1c/CP2/LSF gene was associated with reduced AD risk. Samples were diagnosed predominantly by clinical rather than pathological criteria. We have sought to replicate this finding in a series of necropsy confirmed, late onset AD cases and non-demented controls. METHODS: The 3'UTR polymorphism in the LBP-1c/CP2/LSF gene was typed in 216 necropsy confirmed AD cases and 301 non-demented controls aged >73 years. RESULTS: We found different LBP-1c/CP2/LSF allele distributions in our AD cases and controls (p=0.048); the A allele was associated with reduced AD risk. The allele and genotype frequencies observed in our cases and controls were similar to those previously reported. No significant effects emerged when the data were adjusted for age, sex, or apoE epsilon4 carrier status. CONCLUSIONS: Our data support LBP-1c/CP2/LSF as a candidate gene/risk factor for AD and provide justification for future studies to investigate the role of this gene in Alzheimer's disease.

Cortical degeneration associated with phonologic and semantic language impairments in AD.

OBJECTIVE: To compare the pattern of cortical degeneration associated with different language deficits in cases of AD. METHODS: Cases for detailed neuropathologic analysis (Patients 1 and 2) were selected because of their detailed clinical and neuropsychological assessments of language dysfunction in AD. Patient 1 had severe phonologic impairment with relatively preserved semantic aspects of language. Patient 2 had severe semantic language impairment with relatively preserved phonologic skills. The tissue volume of cortical regions associated with speech and language function was measured using standardized three-dimensional techniques. Neuronal areal fraction was also measured from histologic tissue samples. The degree of volume atrophy and neuronal loss was calculated in comparison to control measures (n = 10 men and 11 women). Measurements more than 2 SD from controls were considered abnormal. RESULTS: Both AD cases had significant degeneration of the superior temporal gyrus and area 37. Cortical language regions affected only in Patient 1 included the anterior and posterior insula and part of Broca's area. In contrast, Patient 2 had a greater degree of degeneration in the temporal gyri and their white matter connections with the hippocampal/entorhinal complex. CONCLUSIONS: Variable patterns of neurodegeneration underlie the clinical differences observed in patients with AD. Disconnection within the temporal lobe appears associated with semantic language difficulties, whereas disconnection of the anterior and posterior language areas appears associated with phonologic and grammatical impairment.

The BACE gene: genomic structure and candidate gene study in late-onset Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterized by beta-amyloid plaques and neurofibrillary tangles. Studies of autosomal dominant early-onset AD mutations suggest that beta-amyloid overproduction is sufficient to cause AD. Recently, the BACE gene, which encodes beta-secretase, the rate limiting enzyme in beta-amyloid formation, has been identified. Since this gene is a strong candidate gene for late-onset AD because of its function, we have characterized its genomic organization and identified two polymorphisms. Neither of these polymorphisms were associated with AD risk in genetic association studies comparing autopsy-confirmed late-onset AD cases and age-matched non-demented controls. Thus, we find no evidence that this locus influences risk for late-onset AD.

HLA class I, II & III genes in confirmed late-onset Alzheimer's disease.

We first examined all the then known alleles (1997) at the HLA-A, B, Bw, C, DRB1, 3, 4 and 5, and DQB1 loci in 55 late-onset (>65y) AD cases and 73 elderly controls from Oxford. We found an association of HLA-B7 with late-onset AD (odds ratio = 3.1, corrected P = 0.04) that was limited to apolipoprotein E epsilon4-negative subjects (odds ratio = 5.1, corrected P = 0.005). We then studied linkages with Class III genes and, finally, we sought to replicate our HLA-B7 result in cohorts from Montreal and Nottingham. Altogether, we used 299 histopathologically confirmed cases of late-onset AD and 175 controls. Our initial, clear finding was not replicated in Montreal and Nottingham, however. We also failed to support any other previously reported association of AD with an HLA gene. Though we cannot exclude distinct linkages in different cohorts as an explanation of the conflicting results of HLA/AD studies, we conclude that there is no compelling evidence of a strong, direct association between late-onset AD and any HLA Class I or II allele.