Masami Shizuka

Combination assay of CSF Tau, Aβ1-40 and Aβ1-42(43) as a biochemical marker of Alzheimer's disease

Cerebrospinal fluid samples from a total of 157 subjects consisting of 55 patients with sporadic Alzheimers disease (AD), 34 normal controls, 23 patients with non-AD dementia, and 45 with other neurological diseases were examined by ELISA of tau, Aβ1-40, and Aβ1-42(43). The AD group had a significantly higher level of tau than the normal control group (P<0.001), and the diagnostic sensitivity was 31% and specificity was 94%. CSF Aβ1-40 levels did not show any significant differences. Although the level of Aβ1-42(43) was decreased significantly in the AD group compared to the control group (P<0.005), the overlap of Aβ1-42(43) levels among all groups meant that none of the AD samples exceeded the cut-off value, the mean 2SD of normal control subjects. Reduction of Aβ1-42(43) levels in AD resulted in a significant increase in the ratio of Aβ1-40 to Aβ1-42(43) (Aβ ratio) as an improved marker. The diagnostic sensitivity and specificity of Aβ ratio were 51% and 82% respectively. The three indexes, using the tau level and Aβ ratio (tau or Aβ ratio, deviation score and tau×Aβ ratio), showed better sensitivity (58%, 67%, 69%) and specificity (82%, 86%, 88%) than previously reported methods. Combination assay for CSF tau, Aβ1-40 and Aβ1-42(43) in CSF is a biological marker of AD and may be useful to biochemically monitor subjects under treatment.

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis

Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. Mutations in the CHAC gene on 9q21 were recently found to cause chorea-acanthocytosis. CHAC encodes a large, novel protein with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in CHAC were screened for mutations by denaturing high-performance liquid chromatography in 43 probands with ChAc. We identified 57 different mutations, 54 of which have not previously been reported, in 39 probands. The novel mutations comprise 15 nonsense, 22 insertion/deletion, 15 splice-site and two missense mutations and are distributed throughout the CHAC gene. Three mutations were found in multiple families within this or our previous study. The preponderance of mutations that are predicted to cause absence of gene product is consistent with the recessive inheritance of this disease. The high proportion of splice-site mutations found is probably a reflection of the large number of exons that comprise the CHAC gene. The CHAC protein product, chorein, appears to have a certain tolerance to amino-acid substitutions since only two out of nine substitutions described here appear to be pathogenic.

Molecular and clinical analyses of spinocerebellar ataxia type 8 in Japan

Objective: To clarify the molecular and clinical features of the newly identified spinocerebellar ataxia type 8 (SCA8). Methods: We analyzed the CTG repeat region of the SCA8 gene in a series of Japanese patients with cerebellar ataxia. We also investigated the frequency of the CTG repeat length in Japanese normal elderly subjects older than age 79. Morphometric measurements on the cerebral MRI were compared between patients with SCA8 and SCA6. Results: The number of the combined CTA/CTG repeats of six affected SCA8 alleles was 106.3 ± 24.4 (mean ± SD) ranging from 89 to 155 and that of normal elderly subjects was 24.3 ± 4.4 (n = 104 alleles) ranging from 15 to 34. The mean age at onset of the SCA8 cases was 53.8 ± 19.7 years, with a range from 20 to 73 years. One father and daughter from an SCA8 family showed remarkable paternal anticipation. The number increase from father to daughter was +16 CTG repeats, with a 31-year acceleration of onset. The six identified SCA8 patients were clinically characterized by high frequencies of incoordination of trunk and limbs, ataxic dysarthria, impaired smooth pursuit, and horizontal nystagmus, and the MRI showed significant atrophy of the cerebellar vermis and hemispheres compared with that of normal controls. There was no significant difference between SCA8 and SCA6 on the morphometric MRI study. Conclusions: The CTG repeat expansions in the SCA8 alleles were much greater than the range of repeats in normal elderly subjects. The SCA8 phenotype manifested by cerebellar symptoms and atrophy corresponded to features of the autosomal dominant cerebellar ataxia type III (ADCA III).

Apolipoprotein E4 accelerates dementia and increases cerebrospinal fluid tau levels in Alzheimer's disease

To examine the effect of apolipoprotein E (ApoE) 4 on the progression of Alzheimers disease (AD), the clinical course of 33 AD patients (17 cases with ApoE epsilon4 and 16 cases without ApoE epsilon4) was evaluated with the mini-mental state examination (MMSE) and cerebrospinal fluid (CSF) biological markers. The decline of MMSE scores to zero was shortened in the ApoE4 group. During a mean follow-up of 20 months, a significant increase of CSF tau levels was observed in the ApoE4 group. A lower level of CSF A beta1-42(43) was found in both the ApoE4 and non-ApoE4 groups than in age-matched normal controls. The ApoE epsilon4 allele accelerates the progression of dementia and increases the levels of CSF tau in AD patients.

Cerebrospinal fluid tau in dementia disorders: a large scale multicenter study by a Japanese study group

A large scale multicenter study of cerebrospinal fluid (CSF) tau levels was conducted to determine the cut-off value, sensitivity and specificity for clinical usage as a biomarker of Alzheimers disease (AD). Its use for early and differential diagnosis and the factors that increase CSF tau levels were also examined. CSF samples from a total of 1,031 subjects including 366 patients with AD, 168 patients with non-Alzheimer type dementia (NA), 316 patients with non-dementia neurological diseases (ND) and 181 normal controls (NC) were measured using ELISA for tau. The cut-off value of tau, 375 pg/ml, showed 59.1% sensitivity and 89.5% specificity for diagnosis of AD compared with the other groups. The tau levels were increased from the early to late stages of AD. Elevation of CSF tau in the non-tauopathy and tauopathy dementia groups, chronic and acute damage to the cerebrum, and meningeal disturbance were other factors that required attention for clinical practice. Measurement of CSF tau was useful as a biomarker for early and differential diagnosis of AD.

The levels of cerebrospinal fluid Aβ40 and Aβ42(43) are regulated age-dependently

Decreased levels of cerebrospinal fluid (CSF) Abeta42 is a diagnostic marker of Alzheimers disease. To clarify the biological basis of this marker, the physiological alterations of CSF Abeta40 and Abeta42 by aging were studied. CSF samples from 92 normal subjects between 8 and 89 years old were measured using a specific ELISA for Abeta40 and Abeta42(43). High concentrations of Abeta40 and Abeta42(43) in the young group, under 29 years old, changed to be at low concentrations in the adult group between 30 and 59 years old. Subsequently, the levels increased again with age. Third order regression analysis showed a significant correlation between the levels of Abeta40 and age (Y = - 169 X(3) + 3.1X(2)- 0.02X + 4135; P < 0.034) and between the levels of Abeta42(43) and age (Y = - 46 X(3) + 0.9 X(2)- 0.005X + 992; P < 0.005). The levels of CSF Abeta40 and Abeta42(43) were physiologically regulated to show a U-shaped natural course in normal aging. These findings suggested that the physiological increase of Abeta42(43) over 59 years of age is selectively inhibited in Alzheimers disease.

Accumulation of NACP/alpha-synuclein in Lewy body disease and multiple system atrophy

OBJECTIVES NACP/α-synuclein is an aetiological gene product in familial Parkinsons disease. To clarify the pathological role of NACP/α-synuclein in sporadic Parkinsons disease and other related disorders including diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA), paraffin sections were examined immunocytochemically using anti-NACP/α-synuclein antibodies. METHODS A total of 58 necropsied brains, from seven patients with Parkinsons disease, five with DLBD, six with MSA, 12 with Alzheimers disease, one with Downs syndrome, one with amyotrophic lateral sclerosis (ALS), three with ALS and dementia, one with Huntingtons disease, two with progressive supranuclear palsy (PSP), one with Picks disease, one with myotonic dystrophy, and three with late cerebellar cortical atrophy (LCCA), and 15 elderly normal controls were examined. RESULTS In addition to immunoreactive Lewy bodies, widespread accumulation of NACP/α-synuclein was found in neurons and astrocytes from the brainstem and basal ganglia to the cerebral cortices in Parkinsons disease/DLBD. NACP/α-synuclein accumulates in oligodendrocytes from the spinal cord, the brain stem to the cerebellar white matter, and inferior olivary neurons in MSA. These widespread accumulations were not seen in other types of dementia or spinocerebellar ataxia. CONCLUSION Completely different types of NACP/α-synuclein accumulation in Parkinsons disease/DLBD and MSA suggest that accumulation is a major step in the pathological cascade of both diseases and provides novel strategies for the development of therapies.

Molecular analysis of a de novo mutation for spinocerebellar ataxia type 6 and (CAG)n repeat units in normal elder controls

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant spinocerebellar degenerative disease caused by CAG repeat expansions in the human alpha1A voltage-dependent calcium channel subunit gene (CACNL1A4). We analyzed 15 SCA6 patients in 14 unrelated Japanese families and 52 healthy Japanese aged over 74 years. Sequence analysis was performed to determine the correct number of CAG repeats. The expanded CAG repeat number was 23.6+/-2.1 (mean+/-S.D., n=15) with a range of 20-29, and the shortest expanded allele was 20 repeats. Moreover, the analysis of normal subjects revealed that the CAG repeat number of normal alleles was 12.3+/-1.9 (n=104) with a range of 7-18. We concluded that the normal range of CAG repeats in the CACNL1A4 gene is 18 or less, and that the disease range is 20 or more. Of 15 SCA6 patients, three sporadic cases were observed. In one male patient with 26 CAG repeats, the CAG repeat numbers of his parents were within normal range. His expanded allele was considered to be caused by an expansion of a normal allele from his mother (14 or 17 repeats). This is the first SCA6 case which was genetically proven to occur due to a de novo mechanism, suggesting that larger CAG repeats of normal alleles in the CACNL1A4 gene may be unstable and result in full expansion.

Mitotic and meiotic stability of the CAG repeat in the X‐linked spinal and bulbar muscular atrophy gene

X‐linked spinal and bulbar muscular atrophy (SBMA) occurs due to an expansion of the trinucleotide repeat (CAG)n in the androgen receptor gene. Anticipation is relatively rare in SBMA in contrast to spinocerebellar ataxia type 1 (SCA1), and dentatorubral and pallidoluysian atrophy (DRPLA) which show obvious paternal anticipation. The differences in the CAG repeat number were compared among sperm, leukocytes and skeletal muscles of SBMA patients. In SBMA, the sperm of most patients and the skeletal muscle of all patients showed the same repeat number as their leukocytes, whereas the increase in the repeat number from leukocytes to sperm was evident in SCA1 and DRPLA patients. The higher mosaicism level in sperm compared with leukocytes was common in SBMA, SCA1 and DRPLA, and the level of sperm was lower in SBMA than in SCA1 and DRPLA. Thus, spermatogenesis was suggested to be strongly associated with paternal anticipation. The mosaicism level was smaller in SBMA than in other (CAG)n expanded disorders, and smallest in the SBMA carrier females. These findings demonstrate that the CAG repeat in SBMA is relatively stable in mitotic and meiotic processes, and there is a possibility that the lower mosaicism level of the carrier females compared with the SBMA patients is associated with X‐linked recessive inheritance.

Analysis of spinocerebellar ataxia type 2 gene and haplotype analysis: (CCG)1- 2 polymorphism and contribution to founder effect

Spinocerebellar ataxia type 2 is a familial spinocerebellar ataxia with autosomal dominant inheritance. The gene responsible was recently cloned and this disorder was found to be the result of a CAG expansion in its open reading frame. We analysed 13 SCA2 patients in seven unrelated families in Gunma Prefecture, Japan. In four of the seven families, we detected CCG or CCGCCG interruptions in only the expanded alleles. Cosegregation of these polymorphisms with SCA2 patients was established within each family. Together with the results of haplotype analyses, we considered that at least two founders were present in our area and that these (CCG)1-2polymorphisms may make analysis of founder effects easier. By sequencing analysis we found that although the number of the long CAG repeat varied in each subclone of expanded alleles, these polymorphisms did not change their configuration. This finding suggests that CCG or CCGCCG sequences are stable when surrounded by the long CAG repeat and a single CAG. Moreover, the presence of these polymorphisms may lead to miscounting the repeat size by conventional estimation using a size marker such as an M13 sequencing ladder. Therefore we should consider these polymorphisms and accurately determine the repeat size by sequencing.