Hemodynamic evidence linking cognitive deficits in clinically isolated syndrome to regional brain inflammation.

BACKGROUND AND PURPOSE: To investigate the relation between hemodynamic measurements and memory function in patients with clinically isolated syndrome (CIS). METHODS: Forty CIS patients were administered tests of verbal short-term/working memory and passage learning. Using dynamic susceptibility contrast MRI cerebral blood volume (CBV), cerebral blood flow and mean transit time values were estimated in 20 cerebral regions of interest, placed in normal appearing white matter (NAWM) and normal appearing deep gray matter structures, bilaterally. RESULTS: CIS patients showed significantly impaired scores on working memory and secondary verbal memory that correlated inversely with elevated CBV values in the left frontal and periventricular NAWM, thalamus, right caudate and corpus callosum. CONCLUSIONS: Verbal memory in CIS correlates inversely with elevated CBV values of brain structures involved in memory. As these hemodynamic changes, detected in CIS, are indicative of inflammation, the observed cognitive disturbances may relate to widespread brain inflammatory processes that prevail in early multiple sclerosis.

Rising incidence of multiple sclerosis in females associated with urbanization.

OBJECTIVE: To design and perform a case-control study of multiple sclerosis (MS) in Crete, an island of 0.6 million people, that has experienced profound socioeconomic changes in recent decades. METHODS: All MS cases occurring on Crete from 1980 to 2008 were ascertained. To search for putative risk factors, a structured questionnaire of 71 variables was employed, with patients with MS (n = 657) being compared to random controls (n = 593) matched for age, gender, and current place of residence. RESULTS: MS incidence rose markedly on Crete over the past 3 decades. This increase was associated with a major shift in MS distribution among genders (1980: F/M = 0.9; 2008: F/M = 2.1), with females living in towns or having relocated at a young age from the countryside to urban centers being mainly affected. In rural Crete, MS showed lesser increases and gender preference. Of the major changes that accompanied urbanization, smoking among women with MS increased dramatically, while imported pasteurized cow milk virtually replaced fresh goat milk produced locally. Compared to controls, female patients with MS more often used contraceptives and were older at first childbirth. Besides smoking, alcohol drinking and vitamin intake was more common among female patients with MS. Also, the distribution of childhood diseases and chronic medical conditions differed significantly between patients with MS and controls. CONCLUSIONS: MS incidence rose markedly over 3 decades in a genetically stable population in tandem with a transition from rural to urban living, thus possibly implicating environmental factors introduced by urbanization.

Benign multiple sclerosis in Crete.

Our objective was to study multiple sclerosis on Crete, an island of 0.6 million inhabitants sharing a similar genetic background and the same environment. Case ascertainment was achieved using the MS Epidemiology Program Project of Crete. The diagnosis and classification of multiple sclerosis were made by established clinical and magnetic resonance imaging criteria, and disease evolution was assessed by periodic evaluations. Thorough clinical and laboratory evaluations were conducted; a detailed history, including a questionnaire of 36 items, was taken. Data obtained were analysed for possible interaction with disease prognosis. We identified 587 cases of multiple sclerosis (F:M = 1.6), >90% of which were of Cretan origin from both parental lines. Age at onset was 31.5 +/- 10.3 years (mean +/- SD) and disease duration 12.7 +/- 9.1 years. About 84.6% had relapsing remitting multiple sclerosis, 9.4% primary progressive multiple sclerosis and 6% clinically isolated syndrome. Nearly 40% of our multiple sclerosis patients with disease duration >10 years (mean = 16.2 +/- 5.3 years) remained with no or mild disability (Expanded Disability Status Scale [EDSS] < or =3). Also, about 30% of patients with relapsing remitting multiple sclerosis showed benign disease evolution (EDSS < or =3) more than 20 years (mean = 24.0 +/- 3.3) after onset. Factors predisposing to benign multiple sclerosis included younger age at onset, shorter disease duration and a lower number of relapses. We conclude that a substantial proportion of patients with multiple sclerosis from Crete follow a rather benign disease course, and this may relate to the genetic background of the population and/or to environmental factors.

Patients with horizontal gaze palsy and progressive scoliosis due to ROBO3 E319K mutation have both uncrossed and crossed central nervous system pathways and perform normally on neuropsychological testing.

BACKGROUND: Horizontal gaze palsy and progressive scoliosis (HGPPS) is caused by mutations of the ROBO3 gene, which encodes a receptor associated with axonal guidance during development. Although there is evidence for uncrossed cuneatal and corticospinal tracts in HGPPS, it is unclear whether other central nervous system pathways are involved. OBJECTIVE: To study two patients with HGPPS homozygotic for the ROBO3 E319K mutation using a variety of neurophysiological and neuropsychological tests. METHODS: A battery of neuropsychological tests was applied to assess various cognitive and perceptual functions. The corticospinal, somatosensory and auditory pathways were evaluated using appropriate neurophysiological tests. To access motor pathways to the neck muscles, electromyographic recordings were obtained from the sternocleidomastoideus and splenius capitis muscle during active head rotation. RESULTS: Both patients performed normally on manual dexterity, complex sensory and visuospatial functions, reading and general intelligence tests. Motor evoked potentials in both patients showed uncrossed corticospinal tracts for the extremities, although in one patient, electromyography indicated pyramidal tract crossing for the neck muscles. Although somatosensory evoked potentials showed uncrossed somatosensory fibres subserving proprioception and light touch, right median nerve somatosensory evoked potential in one patient indicated a partial lemniscal crossing. Sympathetic skin response and blink reflex showed a midline crossing of the spinothalamic and quintothalamic tracts. Brain stem auditory evoked potentials indicated a lack of crossing in the level of the trapezoid body. CONCLUSIONS: Our patients with the ROBO3 E319Kappa mutation show normal perceptual and cognitive functions and have both crossed and uncrossed motor, sensory and auditory pathways.

Central nervous system involvement as the presenting manifestation of autoimmune rheumatic diseases: an observational study using the American College of Rheumatology nomenclature for neuropsychiatric lupus.

OBJECTIVE: We sought to describe CNS involvement as initial presentation of autoimmune rheumatic diseases using a standardized nomenclature. PATIENTS AND METHODS: A 6-year observational study (1999-2005) was conducted in the University Hospital of Heraklion Crete, a regional referral secondary/tertiary care academic center. Patients presenting with new neurological symptoms of acute/subacute onset underwent clinical and laboratory screening for systemic autoimmune disorders. The diagnosis of an autoimmune rheumatic disorder was based upon the American College of Rheumatology (ACR) classification criteria, whereas for primary antiphospholipid syndrome (PAPS) we used the Sapporo preliminary criteria. In order to describe the neurological syndromes we used the ACR nomenclature for neuropsychiatric lupus. RESULTS: During this period fourteen patients (ten females and four males) were recorded. Eight patients had systemic lupus erythematosus (SLE), four had primary APS and the remaining two had systemic vasculitis. Four out of the eight SLE patients had secondary APS. Two of them presented with movement disorder (chorea). The other two and all four patients with primary APS presented with cerebrovascular disease (CVD). These six patients comprised the 5.7% of young adults under < 45 years old with cerebrovascular accident admitted over the 6-year period. CONCLUSION: SLE and APS either primary or secondary to SLE were the most common underlying systemic autoimmune rheumatic diseases, in patients presenting with a neurological event of acute onset. Young adults (< 45 years old) with CVD should undertake screening for SLE/APS.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis.

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Regulation of human glutamate dehydrogenases: implications for glutamate, ammonia and energy metabolism in brain.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is probably involved in the metabolism of transmitter glutamate. The exact mechanisms that regulate glutamate fluxes through this pathway, however, have not been fully understood. In the human, GDH exists in heat-resistant and heat-labile isoforms, encoded by the GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) genes, respectively. These forms differ in their catalytic and allosteric properties. Kinetic studies showed that the K(m) value for glutamate for the nerve tissue GDH is within the range of glutamate levels in astrocytes (2.43 mM), whereas for the housekeeping enzyme, this value is significantly higher (7.64 mM; P < 0.01). The allosteric activators ADP (0.1-1.0 mM) and L-leucine (1.0-10.0 mM) induce a concentration-dependent enzyme stimulation that is proportionally greater for the nerve tissue-specific GDH (up to 1,600%) than for the housekeeping enzyme (up to 150%). When used together at lower concentrations, ADP (10-50 mM) and L-leucine (75-200 microM) act synergistically in stimulating GDH activity. GTP exerts a powerful inhibitory effect (IC(50) = 0.20 mM) on the housekeeping GDH; in contrast, the nerve tissue isoenzyme is resistant to GTP inhibition. Thus, although the housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Conditions associated with enhanced hydrolysis of ATP to ADP (e.g., intense glutamatergic transmission) are likely to activate nerve tissue-specific GDH leading to an increased glutamate flux through this pathway.

Differential expression of glutamate dehydrogenase in cultured neurons and astrocytes from mouse cerebellum and cerebral cortex.

Glutamate dehydrogenase (GDH) specific activities, kinetic properties and allosteric regulation were studied in extracts from cultured neurons and astrocytes prepared from mouse cerebral cortex and cerebellum. Considerable differences were observed in the specific activity of the enzyme among the different cell types with astrocytes expressing the highest GDH activity. This may reflect the functional importance of these cells in glutamate uptake and metabolism. Among the neurons, the glutamatergic cerebellar granule cells showed a GDH specific activity that was 60% higher (P < 0.01) than that of the GABAergic cerebral cortical neurons. Also, the K(m) for ammonia was 1.7-fold higher in the cortical neurons than in the other cell types. These findings may reflect a particular need for the glutamatergic granule cells to synthesize glutamate via the GDH pathway. No differences were observed among the different cell types with regard to the allosteric properties of GDH expressed by these cells.

Increased incidence of sporadic Creutzfeldt-Jakob disease on the island of Crete associated with a high rate of PRNP 129-methionine homozygosity in the local population.

Since the spring of 1997, when the Neurology Department of the University Hospital of Crete admitted its first patient, nine cases (eight neuropathologically confirmed and one probable) of sporadic Creutzfeldt-Jakob disease (sCJD) have been recorded. This represents an annual incidence five-fold higher than expected based on the island's population (0.54 million). Molecular analysis of the prion-protein gene (PRNP) showed no mutations in any of the seven CJD cases studied. Five patients (ages 64-88 years) were homozygous for methionine-129 of PRNP and showed the classic sCJD triad (subacute dementia, myoclonus, periodic electroencephalogram). Brains contained type 1 (unglycosylated 21.5 kDa band) protease-resistant prion protein (PrPres). Two patients (ages 56 and 57 years), both homozygous for valine-129, showed cerebellar ataxia and later dementia not associated with periodic electroencephalogram; brain PrPres was type 2. Genotyping of 205 Cretan controls showed that methionine-129 homozygosity, a susceptibility factor for sCJD, was significantly higher in this population than in other Caucasian populations (57.0% n = 205 vs. 41.5% n = 859, p < 0.0001). These data are the first to relate a high regional incidence rate for sCJD to the distribution of PRNP 129 genotypes in the local population; however, additional factors may be operational.

Managing Alzheimer's disease in primary care in Crete, Greece: room for improvement.

As Greece moves during the last two decades toward a national health care system, which gives emphasis to the development of a primary care system, many worry how to ensure that the quality of care is assessed. This is more apparent in the rural populations, in which health care is served to a large extent by physicians without formal training in general practice. This article explores the level of knowledge of primary care physicians in relation to Alzheimer's disease in geographically defined areas of Crete, Greece, in comparison with that of general practitioners in Ostergötland, Sweden, and in Iceland. It emphasizes the need for better education and training for primary care physicians in Crete in both the early diagnosis and management of Alzheimer's disease.

Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is associated with degeneration of the pigmented dopaminergic neurons located in the ventral mesencephalon. Although the mechanisms by which these neurons degenerate in PD are poorly understood, indirect evidence suggests involvement of glutamatergic mechanisms in the pathogenesis of this disorder. Glutamate, the major excitatory transmitter in the mammalian central nervous system, is known to be neurotoxic when present in excess at the synapses. Two major mechanisms protect neurons from glutamate-induced toxicity: (a) removal of synaptic glutamate via a high affinity uptake carried out by cytoplasmic membrane proteins known as excitatory amino acid transporters (EAAT); and (b) metabolism and recycling of glutamate by synaptic astrocytes via glutamine synthetase, an ATP-requiring reaction. However, when extra-cellular glutamate levels are high (0.5-1.0 mM), glutamate metabolism may be shifted toward the ATP-generating oxidative deamination (glutamate dehydrogenase)-TCA cycle pathway. We have cloned and characterized two human glutamate dehydrogenases (GDH), one of which is nerve tissue specific. This isoenzyme requires ADP for its activity and it may become functional when cellular energy charge is low. We have also cloned three human glutamate transporters. One of these (EAAT3) is neuron specific. In situ hybridization studies using human brain revealed that the pigmented dopaminergic neurons, which degenerate in PD, express EAAT3 at high levels. Primary nerve tissue cultures derived from rat ventral mesencephalon were established and studied for their ability to metabolize glutamate. Results showed that mature cultures expressing high levels of GDH activity were capable of rapidly utilizing glutamate added to the medium at high concentrations (1-1.2 mM). This was associated with little release of aspartate and alanine into the medium. In contrast, immature cultures expressing low GDH activity utilized glutamate at lower rates while releasing substantial amounts of aspartate and alanine into the medium. These data suggest that immature mesencephalic cells metabolize a substantial fraction of the glutamate they take up from the medium via the transamination pathway, compared to mature mesencephalic cultures. Immunocytochemical studies on these cultures revealed that dopaminergic neurons (identified by their tyrosine hydroxylase content) showed intense staining for GDH. Furthermore, inhibition of GDH expression by antisense oligonucleotides was toxic to cultured mesencephalic neurons, with dopaminergic neurons being affected at the early stages of this inhibition. Hence, the dense expression by dopaminergic neurons of proteins involved in the transport and metabolism of glutamate may serve particular biological needs intrinsic to these cells. Further studies are required to test whether these properties render these neurons vulnerable to excitotoxic mechanisms or to abnormalities of glutamate metabolism.

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody.

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP.

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with approximately 40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05-0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550-1,300%) than of the housekeeping enzyme (by 120-150%). Magnesium chloride (1-2 mM) inhibited the nonactivated housekeeping GDH (by 45-64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05-0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.

Stability of the Huntington disease (CAG)n repeat in a late onset form occuring on the Island of Crete.

Huntington disease (HD) is an autosomal-dominant disorder of mid-life onset characterized by chorea, dementia, and oculomotor disturbances. Anticipation is commonly seen in HD families, particularly when the disease is inherited through the father. The disorder is associated with an expanded (CAG)n repeat in the IT15 gene that is unstable and tends to increase in size during meiotic transmissions, particularly of paternal origin. We have detected an unusual form of HD on the island of Crete which has distinctly different characteristics. Data from eight families encompassing 48 HD patients, showed a median age at onset 15-20 years later than that for HD occurring worldwide. There is no juvenile cases and no anticipation. DNA analysis in 12 HD patients showed expansion of the (CAG)n repeat the size of which was identical among members of each family or varied by only one unit. The elongated DNA segment was passed stably or contracted during both paternal and maternal transmissions thus indicating that unique molecular mechanisms may be operational in this form of HD.

X-linked adrenoleukodystrophy presenting as neurologically pure familial spastic paraparesis.

The familial spastic paraplegias (FSPs) are heterogeneous neurologic disorders that are known to occur clinically as "pure" or "complicated" forms. Although some of the complicated FSPs have been linked to specific metabolic defects, the pure forms of this disorder remain idiopathic and are considered to be primary system degenerations. We report the case of a 28-year-old man who has evidenced a neurologically pure spastic paraparesis since age twenty-five. Consistent with this diagnosis were the findings of magnetic resonance imaging, which revealed atrophy of the thoracic spinal cord without evidence for white matter disease in the cerebrum, cerebellum, or brainstem. His 32-year-old brother has also evidenced progressive spastic paraparesis since age 30, but his case is confounded by a severe head injury at age 24 that caused a skull fracture and a focal demyelinating lesion of the right frontal lobe. Both patients have had hypogonadism, requiring treatment with testosterone, since age 20. Measurement of plasma levels of very long-chain fatty acids (VLCFA) revealed that both brothers had concentrations diagnostic of adrenoleukodystrophy; their mother had plasma VLCFA levels in the heterozygous range. We conclude that neurologically pure FSP can be an early manifestation of adrenoleukodystrophy and that levels of plasma VLCFA should be determined for all cases of FSP in which X-linked inheritance appears tenable. These considerations may have bearing on the ongoing linkage studies for these disorders.

Linkage disequilibrium between the expanded (CAG)n repeat and an allele of the adjacent (CCG)n repeat in Huntington's disease patients of Greek origin.

Huntington's disease (HD) is associated with an expanded unstable (CAG)n repeat in the IT15 gene. This repeat was investigated in 44 HD patients and 59 of their relatives at risk who were members of 29 unrelated families from various parts of Greece. Abnormal elongation of the (CAG)n repeat ranging from 39 to 95 trinucleotide units was found in all but one of the 44 HD patients tested with 70% of these patients showing 42-47 repeats. The size of the expanded sequence correlated inversely with the age at disease onset (r = 0.77, p < 0.00001, n = 43). In a single sporadic case, de novo expansion of the (CAG)n repeat was detected. Twenty-four of 59 asymptomatic family members at risk showed expansion of the (CAG)n repeat in the HD range (39-56 trinucleotide units) while three had intermediate alleles (36-37 repeats). Evaluation of the adjacent polymorphic (CCG)n repeat showed a strong linkage disequilibrium between the 7-unit (CCG)n repeat allele and the HD mutation, with 51% of normal and 93% of HD chromosomes showing this allele (chi 2 = 15.55, p < 0.0001, n - 260). These data on HD patients of Greek origin are consistent with the thesis that the (CAG)n expansion is the primary gene defect of the disease and that this mutation occurred primarily on chromosomes with the (CCG)7 repeat haplotype.