Vegetable and Fruit Intake Frequency and Mortality in Patients With and Without Chronic Kidney Disease: A Hospital-Based Cohort Study.

OBJECTIVE: Patients with advanced chronic kidney disease (CKD) are generally discouraged from consuming high amounts of vegetables and fruits given the potential risk of hyperkalemia. In the general population, however, lower vegetable and fruit intake is associated with higher mortality. This Japanese hospital-based prospective cohort study including non-CKD and CKD participants examined whether the frequency of vegetable and fruit intake is associated with mortality, and whether the presence of CKD modifies this association. METHODS: Participants were 2,006 patients who visited the outpatient department of a general hospital between June 2008 and December 2016 (55% men; mean age, 69 years). Among these participants, 902 (45%) and 131 (7%) were non-dialysis-dependent patients with CKD and hemodialysis patients, respectively. The frequency of vegetable and fruit intake was determined by a self-report questionnaire using an ordinal scale, "never or rarely," "sometimes," and "every day." Multivariable Cox proportional hazards analysis was conducted, adjusting for potential confounders. RESULTS: Vegetable and fruit intake frequency decreased with worsening CKD stage (P for trend < .001). Baseline serum potassium levels stratified by CKD stage were similar across the three vegetable and fruit intake frequency groups. During a median follow-up of 5.7 years, 561 participants died (47.1/1,000 person-years). Adjusted hazard ratios relative to the "every day" group were 1.25 (95% confidence interval, 1.04-1.52) and 1.60 (95% confidence interval, 1.23-2.08) for the "sometimes" and "never or rarely" groups, respectively, after adjusting for demographic factors, comorbidities, and CKD status. When stratified by CKD status, a similar, albeit non-significant, dose-dependent relationship was observed between vegetable and fruit intake frequency and all-cause mortality irrespective of CKD status, with no effect modification by CKD status (Pinteraction = .69). CONCLUSION: A lower frequency of vegetable and fruit intake is significantly associated with a higher risk of death regardless of CKD status.

Polypharmacy, chronic kidney disease, and incident fragility fracture: a prospective cohort study.

INTRODUCTION: Polypharmacy is associated with an increased risk of fracture in aging populations, but no study has accounted for the impact of kidney function on this association. This study aimed to examine the association between polypharmacy and incident fragility fracture based on chronic kidney disease (CKD) status. MATERIALS AND METHODS: Participants were 2023 patients (55% men; mean age, 69 years) of Sado General Hospital enrolled in the Project in Sado for Total Health (PROST) between June 2008 and December 2016. Among these, 65%, 28%, and 7% had non-CKD, non-dialysis-dependent CKD, and dialysis-dependent CKD, respectively. Multivariable Cox proportional hazards analysis was conducted with adjustments for potential confounders. RESULTS: Prevalences of polypharmacy (≥ 5 medications) and hyperpolypharmacy (≥ 10 medications) among participants were 43% and 9% for non-CKD, 62% and 23% for non-dialysis-dependent CKD, and 85% and 34% for dialysis-dependent CKD, respectively. During a median follow-up of 5.6 years, 256 fractures occurred. More medications were associated with a higher risk of fractures. Specifically, compared to participants without polypharmacy, adjusted hazard ratios were 1.32 (95% CI 0.96-1.79) and 1.99 (1.35-2.92) for those with polypharmacy and hyperpolypharmacy, respectively, after adjusting for osteoporosis risk factors, CKD status, and comorbidities. No effect modification by CKD status was observed (interaction P = 0.51). Population-attributable fractions of hyperpolypharmacy for fracture were 9.9% in the total cohort and 42.1% in dialysis-dependent CKD patients. CONCLUSION: Hyperpolypharmacy is associated with an increased risk of fragility fracture regardless of CKD status, and has a strong impact on incident fragility fractures in dialysis-dependent CKD patients.

Stroke incidence and chronic kidney disease: A hospital-based prospective cohort study.

AIM: This prospective cohort study aimed to (i) examine stroke incidence and stroke subtypes by chronic kidney disease (CKD) stage, (ii) examine whether CKD patients with or without proteinuria have a high risk of stroke independent of traditional cardiovascular risk factors, and (iii) determine precise estimates of stroke risk by CKD stage while accounting for competing mortality risk. METHODS: Participants were 2023 patients enrolled in the Project in Sado for Total Health between June 2008 and December 2016 (55% men; mean age, 69 years), of whom 52% had CKD (stage 1-2, 10%; G3a, 48%; G3b, 17%; G4-5, 11% and G5D, 14%). RESULTS: During a median follow-up of 5.7 years, 157 participants developed stroke and 448 died without developing stroke. Most stroke cases were ischaemic among non-dialysis-dependent CKD participants, but the relative frequency of ischaemic stroke was near that of intracerebral haemorrhage among dialysis-dependent CKD participants. After adjustment, stage 1-2 (hazard ratio [HR], 2.97; 95% confidence interval [CI], 1.60-5.51) and stage G3-5 participants with proteinuria (HR, 2.50; 95% CI, 1.56-4.02), but not stage G3-5 participants without proteinuria (HR, 0.64; 95% CI, 0.38-1.08), had a higher stroke risk compared to non-CKD participants. In competing risk analyses, the association was attenuated but remained significant. CONCLUSION: Although the distribution of stroke subtypes differed, CKD participants with proteinuria and those with CKD stage 5D had a 2- and 4-times higher risk of stroke, respectively, than that of non-CKD participants, after accounting for competing mortality risk and traditional risk factors.

Immunological abnormalities in patients with early-onset ataxia with ocular motor apraxia and hypoalbuminemia.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is a neurodegenerative disorder caused by mutation in the aprataxin (APTX)-coding gene APTX, which is involved in DNA single-strand break repair (SSBR). The neurological abnormalities associated with EAOH are similar to those observed in patients with ataxia-telangiectasia. However, the immunological abnormalities in patients with EAOH have not been described. In this study, we report that EAOH patients have immunological abnormalities, including lymphopenia; decreased levels of CD4+ T-cells, CD8+ T-cells, and B-cells; hypogammaglobulinemia; low T-cell recombination excision circles and kappa-deleting element recombination circles; and oligoclonality of T-cell receptor ß-chain variable repertoire. These immunological abnormalities vary among the EAOH patients. Additionally, mild radiosensitivity in the lymphocytes obtained from the patients with EAOH was demonstrated. These findings suggested that the immunological abnormalities and mild radiosensitivity evident in patients with EAOH could be probably caused by the DNA repair defects.

Genetic Variations and Neuropathologic Features of Patients with PRKN Mutations.

BACKGROUND: Mutations in PRKN are the most common cause of autosomal recessive juvenile parkinsonism. The objective of this study was to investigate the association between genotype and pathology in patients with PRKN mutations. METHODS: We performed a sequence and copy number variation analysis of PRKN, mRNA transcripts, Parkin protein expression, and neuropathology in 8 autopsied patients. RESULTS: All the patients harbored biallelic PRKN mutations. Two patients were homozygous and heterozygous, respectively, for the missense mutation p.C431F. Seven patients had exon rearrangements, including 2 patients from a single family who harbored a homozygous deletion of exon 4, and 3 patients who carried a homozygous duplication of exons 6-7, a homozygous duplication of exons 10-11, and a heterozygous duplication of exons 2-4. In the other 2 patients, we found a compound heterozygous duplication of exon 2, deletion of exon 3, and a heterozygous duplication of exon 2. However, sequencing of cDNA prepared from mRNA revealed 2 different transcripts derived from triplication of exon 2 and deletion of exons 2-3 and from duplication of exons 2-4 and deletion of exons 3-4. Western blotting and immunohistochemistry revealed faint or no expression of Parkin in their brains. In the substantia nigra pars compacta, a subfield-specific pattern of neuronal loss and mild gliosis were evident. Lewy bodies were found in 3 patients. Peripheral sensory neuronopathy was a feature. CONCLUSIONS: Genomic and mRNA analysis is needed to identify the PRKN mutations. Variable mutations may result in no or little production of mature Parkin and the histopathologic features may be similar. © 2021 International Parkinson and Movement Disorder Society.

Low serum 25-hydroxyvitamin D increases cognitive impairment in elderly people.

It has been reported that many elderly people have low serum levels of 25-hydroxyvitamin D [25(OH)D] and that serum 25(OH)D levels may have a relationship with cognitive function. The aim of this study was to examine the relationship between serum 25(OH)D levels and cognitive function in a Japanese population. This cross-sectional study was performed as a part of the Project in Sado for Total Health (PROST). The PROST study evaluated cognitive state and serum vitamin D level from June 2011 to November 2013 for 740 patients (431 men and 309 women). The Mini-Mental State Examination-Japanese version (MMSE-J) and serum 25(OH)D level measurements were used as assessment tools. Cognitive impairment was defined using MMSE-J ≤ 23 as a cutoff. Multiple logistic regression analyses were performed to calculate the odds ratios (ORs) for low MMSE-J scores. The average subject age was 68.1 years, the average MMSE- J score was 25.9, and the average 25(OH)D level was 24.6 ng/mL. Significant ORs for cognitive impairment were observed for both high age and low serum 25(OH)D. The adjusted OR for the lowest versus highest serum 25(OH)D quartiles was 2.70 (95% confidence interval 1.38-5.28, P = 0.0110). Low serum 25(OH)D levels were independently associated with a higher prevalence of cognitive impairment.

Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder. In motor neurons of ALS, TAR DNA binding protein-43 (TDP-43), a nuclear protein encoded by TARDBP, is absent from the nucleus and forms cytoplasmic inclusions. TDP-43 auto-regulates the amount by regulating the TARDBP mRNA, which has three polyadenylation signals (PASs) and three additional alternative introns within the last exon. However, it is still unclear how the autoregulatory mechanism works and how the status of autoregulation in ALS motor neurons without nuclear TDP-43 is. Here we show that TDP-43 inhibits the selection of the most proximal PAS and induces splicing of multiple alternative introns in TARDBP mRNA to decrease the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. When TDP-43 is depleted, the TARDBP mRNA uses the most proximal PAS and is increased in the cytoplasm. Finally, we have demonstrated that in ALS motor neurons-especially neurons with mislocalized TDP-43-the amount of TARDBP mRNA is increased in the cytoplasm. Our observations indicate that nuclear TDP-43 contributes to the autoregulation and suggests that the absence of nuclear TDP-43 induces an abnormal autoregulation and increases the amount of TARDBP mRNA. The vicious cycle might accelerate the disease progression of ALS.

Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis.

Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Role of the p.E66Q variant of GLA in the progression of chronic kidney disease.

BACKGROUND: The p.E66Q variant of the α-galactosidase A gene (GLA) is frequently found during screening for Fabry disease in dialysis patients in Japan. However, recent reports suggest that the p.E66Q variant is not a disease-causing mutation but is a risk factor for cerebral small-vessel occlusion. To evaluate the role of the p.E66Q in the progression of renal diseases, we performed a genetic association study in patients with chronic kidney disease (CKD). METHODS: In this study, we enrolled 1651 chronic hemodialysis and 941 non-dialysis patients who attended medical institutions in the Niigata Prefecture, Japan. The frequency of the p.E66Q allele was compared between hemodialysis and non-dialysis patients, with data from a previously published study of Japanese male newborns. In addition, we compared estimated glomerular filtration rates (eGFR) in the presence or absence of the p.E66Q variant in non-dialysis patients. RESULTS: Of the 2233 alleles in hemodialysis and 1447 alleles in non-dialysis patients, 21 and nine harbored p.E66Q, respectively. However, p.E66Q allele frequencies did not differ between the two patient groups (0.90 versus 0.62 %, P = 0.35), and no significant difference in p.E66Q allele frequency was observed between male hemodialysis patients and the general Japanese population (0.52 versus 0.63 %, P = 0.67). Moreover, eGFR did not significantly differ between non-dialysis patients with the p.E66Q variant and patients with the wild-type allele (65.5 ± 10.7 versus 62.7 ± 16.6 mL/min/1.73 m(2), P = 0.69). CONCLUSION: This study indicated that the p.E66Q variant of GLA does not affect the progression of CKD.

Minor splicing pathway is not minor any more: implications for the pathogenesis of motor neuron diseases.

To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate-rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.

UCP2 polymorphisms, daily step count, and number of teeth associated with all-cause mortality risk in Sado City: A hospital-based cohort study.

Objective: Uncoupling protein 2 (UCP2) is an ion/anion transporter in the mitochondrial inner membrane that plays a crucial role in immune response, regulation of oxidative stress, and cellular metabolism. UCP2 polymorphisms are linked to chronic inflammation, obesity, diabetes, heart disease, exercise efficiency, and longevity. Daily step count and number of teeth are modifiable factors that reduce mortality risk, although the role of UCP2 in this mechanism is unknown. This study aimed to assess the possible effects of UCP2 polymorphisms on the association between daily step count and number of teeth with all-cause mortality. Methods: This study was conducted as a cohort project involving adult Japanese outpatients at Sado General Hospital (PROST). The final number of participants was 875 (mean age: 69 y). All-cause mortality during thirteen years (from June 2008 to August 2021) was recorded. The functional UCP2 genotypes rs659366 and rs660339 were identified using the Japonica Array®. Survival analyses were performed using multivariate Cox proportional hazard models. Results: There were 161 deaths (mean observation period: 113 months). Age, sex, daily step count, and the number of teeth were significantly associated with mortality. In females, UCP2 polymorphisms were associated with mortality independent of other factors (rs659366 GA compared to GG + AA; HR = 2.033, p = 0.019, rs660339 C T compared to CC + TT; HR = 1.911, p = 0.029). Multivariate models, with and without UCP2 genotypes, yielded similar results. The interaction terms between UCP2 genotype and daily step count or number of teeth were not significantly associated with mortality. Conclusion: The effects of UCP2 polymorphisms on the association between daily step count or the number of teeth and all-cause mortality were not statistically significant. In females, UCP2 polymorphisms were significantly associated with all-cause mortality. Our findings confirmed the importance of physical activity and oral health and suggested a role of UCP2 in mortality risk independently with those factors.

Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

Cerebral small-vessel disease protein HTRA1 controls the amount of TGF-ß1 via cleavage of proTGF-ß1.

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-ß (TGF-ß) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-ß family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-ß1 signaling triggered by proTGF-ß1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-ß1 in the endoplasmic reticulum (ER), and cleaved proTGF-ß1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-ß1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-ß1, specifically, the intracellular cleavage of proTGF-ß1 in the ER.

Sporadic ALS with compound heterozygous mutations in the SQSTM1 gene.

Accumulating evidence suggests that heterozygous mutations in the SQSTM1 gene, which encodes p62 protein, are associated with amyotrophic lateral sclerosis (ALS). Here, we report a Japanese patient with sporadic, late-onset ALS who harbored compound heterozygous SQSTM1 mutations (p.[Val90Met];[Val153Ile]). Autopsy examination revealed that although TDP-43 pathology was rather widespread, the selective occurrence of p62-positive/TDP-43-negative cytoplasmic inclusions in the lower motor neurons (LMNs) was a characteristic feature. No Bunina bodies were found. Ultrastructurally, p62-positive cytoplasmic inclusions observed in the spinal anterior horn cells were composed of aggregates of ribosome-like granules and intermingled bundles of filamentous structures. Another feature of interest was concomitant Lewy body pathology. The occurrence of distinct p62 pathology in the LMNs in this patient indicates the pathogenic role of SQSTM1 mutations in the development of a subset of ALS.

Japanese amyotrophic lateral sclerosis patients with GGGGCC hexanucleotide repeat expansion in C9ORF72.

BACKGROUND: A GGGGCC hexanucleotide repeat expansion in C9ORF72 occurs on a chromosome 9p21 locus that is linked with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in white populations. The diseases resulting from this expansion are referred to as 'c9FTD/ALS'. It has been suggested that c9FTD/ALS arose from a single founder. However, the existence of c9FTD/ALS in non-white populations has not been evaluated. RESULTS: We found two index familial ALS (FALS) patients with c9FTD/ALS in the Japanese population. The frequency of c9FTD/ALS was 3.4% (2/58 cases) in FALS. No patients with sporadic ALS (n=110) or control individuals (n=180) had the expansion. Neuropathological findings of an autopsy case were indistinguishable from those of white patients. Although the frequency of risk alleles identified in white subjects is low in Japanese, one patient had all 20 risk alleles and the other had all but one. The estimated haplotype indicated that the repeat expansion in these patients was located on the chromosome with the risk haplotype identified in white subjects. CONCLUSIONS: C9ORF72 repeat expansions were present in a Japanese cohort of ALS patients, but they were rare. Intriguingly, Japanese patients appear to carry the same risk haplotype identified in white populations.

Cataract Surgery and Chronic Kidney Disease: A Hospital-based Prospective Cohort Study.

Objective Cataract and chronic kidney disease (CKD) occur with increasing frequency with age and share common risk factors including smoking, diabetes, and hypertension. We evaluated the risk of incident cataract surgery in patients with non-dialysis-dependent CKD and dialysis-dependent CKD compared to non-CKD patients, while taking into account the competing risk of death. Methods The participants included 1,839 patients from Sado General Hospital enrolled in the Project in Sado for Total Health (PROST) between June 2008 and December 2016 (54% men; mean age, 69 years). Among these patients, 50%, 44%, and 6% had non-CKD, non-dialysis-dependent CKD, and dialysis-dependent CKD, respectively. Results During a median follow-up of 5.6 years (interquartile range, 4.7-7.1), 193 participants underwent cataract surgery [18.7 (95% confidence interval (CI), 16.2 - 21.5)/1,000 person-years] and 425 participants died without undergoing cataract surgery [41.0 (95% CI, 37.4 - 45.2)/1,000 person-years]. The cumulative incidence of cataract surgery was the highest in the dialysis-dependent CKD group, followed by the non-dialysis-dependent CKD and non-CKD groups (log-rank p=0.002). After adjusting for potential confounding factors, the dialysis-dependent CKD group (hazard ratio (HR) 2.48; 95% CI 1.43-4.31), but not the non-dialysis-dependent CKD group (HR, 1.01; 95% CI 0.74-1.38), had a higher risk of cataract surgery than the non-CKD group. However, this association was no longer significant according to a competing risk analysis (sub-hazard ratio, 1.67; 95% CI 0.93-3.03). Conclusion Dialysis-dependent CKD patients were found to have an increased risk of cataract surgery; however, the association was attenuated and no longer significant when death was considered a competing risk.

Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease.

BACKGROUND: The genetic cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which is characterized by ischemic, nonhypertensive, cerebral small-vessel disease with associated alopecia and spondylosis, is unclear. METHODS: In five families with CARASIL, we carried out linkage analysis, fine mapping of the region implicated in the disease, and sequence analysis of a candidate gene. We also conducted functional analysis of wild-type and mutant gene products and measured the signaling by members of the transforming growth factor beta (TGF-beta) family and gene and protein expression in the small arteries in the cerebrum of two patients with CARASIL. RESULTS: We found linkage of the disease to the 2.4-Mb region on chromosome 10q, which contains the HtrA serine protease 1 (HTRA1) gene. HTRA1 is a serine protease that represses signaling by TGF-beta family members. Sequence analysis revealed two nonsense mutations and two missense mutations in HTRA1. The missense mutations and one of the nonsense mutations resulted in protein products that had comparatively low levels of protease activity and did not repress signaling by the TGF-beta family. The other nonsense mutation resulted in the loss of HTRA1 protein by nonsense-mediated decay of messenger RNA. Immunohistochemical analysis of the cerebral small arteries in affected persons showed increased expression of the extra domain-A region of fibronectin and versican in the thickened tunica intima and of TGF-beta1 in the tunica media. CONCLUSIONS: CARASIL is associated with mutations in the HTRA1 gene. Our findings indicate a link between repressed inhibition of signaling by the TGF-beta family and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

Genotype-phenotype correlations in early onset ataxia with ocular motor apraxia and hypoalbuminaemia.

Early onset ataxia with ocular motor apraxia and hypoalbuminaemia/ataxia-oculomotor apraxia 1 is a recessively inherited ataxia caused by mutations in the aprataxin gene. We previously reported that patients with frameshift mutations exhibit a more severe phenotype than those with missense mutations. However, reports on genotype-phenotype correlation in early onset ataxia with ocular motor apraxia and hypoalbuminaemia are controversial. To clarify this issue, we studied 58 patients from 39 Japanese families, including 40 patients homozygous for c.689_690insT and nine patients homozygous or compound heterozygous for p.Pro206Leu or p.Val263Gly mutations who were compared with regard to clinical phenotype. We performed Kaplan-Meier analysis and log-rank tests for the ages of onset of gait disturbance and the inability to walk without assistance. The cumulative rate of gait disturbance was lower among patients with p.Pro206Leu or p.Val263Gly mutations than among those homozygous for the c.689_690insT mutation (P=0.001). The cumulative rate of inability to walk without assistance was higher in patients homozygous for the c.689_690insT mutation than in those with p.Pro206Leu or p.Val263Gly mutations (P=0.004). Using a Cox proportional hazards model, we found that the homozygous c.689_690insT mutation was associated with an increased risk for onset of gait disturbance (adjusted hazard ratio: 6.60) and for the inability to walk without assistance (adjusted hazard ratio: 2.99). All patients homozygous for the c.689_690insT mutation presented ocular motor apraxia at <15 years of age. Approximately half the patients homozygous for the c.689_690insT mutation developed cognitive impairment. In contrast, in the patients with p.Pro206Leu or p.Val263Gly mutations, only ∼50% of the patients exhibited ocular motor apraxia and they never developed cognitive impairment. The stepwise multivariate regression analysis using sex, age and the number of c.689_690insT alleles as independent variables revealed that the number of c.689_690insT alleles was independently and negatively correlated with median motor nerve conduction velocities, ulnar motor nerve conduction velocities and values of serum albumin. In the patient with c.[689_690insT]+[840delT], p.[Pro206Leu]+[Pro206Leu] and p.[Pro206Leu]+[Val263Gly] mutations, aprataxin proteins were not detected by an antibody to the N-terminus of aprataxin. Furthermore Pro206Leu and Val263Gly aprataxin proteins are unstable. However, the amount of the 689_690insT aprataxin messenger RNA was also decreased, resulting in more dramatic reduction in the amount of aprataxin protein from the c.689_690insT allele. In conclusion, patients with early onset ataxia with ocular motor apraxia and hypoalbuminaemia homozygous for the c.689_690insT mutation show a more severe phenotype than those with a p.Pro206Leu or p.Val263Gly mutation.

Successful twin pregnancy in a patient with parkin-associated autosomal recessive juvenile parkinsonism.

BACKGROUND: Pregnancy in patients with Parkinson disease is a rare occurrence. To the best of our knowledge, the effect of pregnancy as well as treatment in genetically confirmed autosomal recessive juvenile parkinsonism (ARJP) has never been reported. Here, we report the first case of pregnancy in a patient with ARJP associated with a parkin gene mutation, ARJP/PARK2. CASE PRESENTATION: A 27-year-old woman with ARJP/PARK2 was diagnosed as having a spontaneous dichorionic/diamniotic twin pregnancy. Exacerbation of motor disability was noted between ovulation and menstruation before pregnancy as well as during late pregnancy, suggesting that her parkinsonism might have been influenced by fluctuations in the levels of endogenous sex hormones. During the organogenesis period, she was only treated with levodopa/carbidopa, although she continued to receive inpatient hospital care for assistance in the activities of daily living. After the organogenesis period, she was administered sufficient amounts of antiparkinsonian drugs. She delivered healthy male twins, and psychomotor development of both the babies was normal at the age of 2 years. CONCLUSION: Pregnancy may worsen the symptoms of ARJP/PARK2, although appropriate treatments with antiparkinsonian drugs and adequate assistance in the activities of daily living might enable successful pregnancy and birth of healthy children.