[Clinical and genetic analysis of a patient with Desminopathy manifesting initially with myalgia after lower limb activity].

OBJECTIVE: To explore the clinical characteristics and genetic variant of a patient with desminopathy manifesting with atypical symptoms. METHODS: A patient who was admitted to the Department of Neurology of Jing'an District Central Hospital on February 24, 2021 was selected as the study subject. Clinical data, laboratory tests, muscle pathology, muscle magnetic resonance imaging (MRI) and genetic testing of the patient were retrospectively analyzed. RESULTS: The patient had developed myalgia after lower limb activity, and gradually developed asymmetrical muscle weakness and atrophy of the lower limbs. Cardiac examination revealed atrioventricular block and decreased left ventricular diastolic function. Muscle MRI showed that semitendinosus, sartorius, gracilis, fibula, gastronemius and supinator muscles were selectively involved at the early stage. Muscle biopsy confirmed pathological changes of desmin positive myofibrils. Genetic testing revealed that the patient has harbored a c.1024A>G (p.n342d) missense variant in exon 6 of the DES gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic (PS4_moderate+PM2_supporting+PP3_moderate+PP1). CONCLUSION: Desmin disease has a great clinical heterogeneity. Postexercise myalgia of lower limbs is a rare clinical phenotype. For patients harboring the c.1024A>G (p.n342d) variant of the DES gene, in addition to semitendinosus and fibula, Cardiac involvement is relatively insidious and easy to be ignored in clinic. Timely muscle MRI, muscle biopsy and gene detection will help the early diagnosis of the disease.

Clinical features of NOTCH2NLC-related neuronal intranuclear inclusion disease.

BACKGROUND: Abnormal expanded GGC repeats within the NOTCH2HLC gene has been confirmed as the genetic mechanism for most Asian patients with neuronal intranuclear inclusion disease (NIID). This cross-sectional observational study aimed to characterise the clinical features of NOTCH2NLC-related NIID in China. METHODS: Patients with NOTCH2NLC-related NIID underwent an evaluation of clinical symptoms, a neuropsychological assessment, electrophysiological examination, MRI and skin biopsy. RESULTS: In the 247 patients with NOTCH2NLC-related NIID, 149 cases were sporadic, while 98 had a positive family history. The most common manifestations were paroxysmal symptoms (66.8%), autonomic dysfunction (64.0%), movement disorders (50.2%), cognitive impairment (49.4%) and muscle weakness (30.8%). Based on the initial presentation and main symptomology, NIID was divided into four subgroups: dementia dominant (n=94), movement disorder dominant (n=63), paroxysmal symptom dominant (n=61) and muscle weakness dominant (n=29). Clinical (42.7%) and subclinical (49.1%) peripheral neuropathies were common in all types. Typical diffusion-weighted imaging subcortical lace signs were more frequent in patients with dementia (93.9%) and paroxysmal symptoms types (94.9%) than in those with muscle weakness (50.0%) and movement disorders types (86.4%). GGC repeat sizes were negatively correlated with age of onset (r=-0.196, p<0.05), and in the muscle weakness-dominant type (median 155.00), the number of repeats was much higher than in the other three groups (p<0.05). In NIID pedigrees, significant genetic anticipation was observed (p<0.05) without repeat instability (p=0.454) during transmission. CONCLUSIONS: NIID is not rare; however, it is usually misdiagnosed as other diseases. Our results help to extend the known clinical spectrum of NOTCH2NLC-related NIID.

Classifying mild cognitive impairment and Alzheimer's disease by constructing a 14-gene diagnostic model.

BACKGROUND: Alzheimer's disease (AD) and mild cognitive impairment (MCI) are two neurodegenerative diseases. Most patients with MCI will develop AD. Early detection of AD and MCI is a crucial issue in terms of secondary prevention. Therefore, more diagnostic models need to be developed to distinguish AD patients from MCI patients. METHODS: In our research, the expression matrix and were screened from Gene Expression Omnibus (GEO) databases. A 14-gene diagnostic model was constructed with lasso logistic analysis. The efficiency and accuracy of diagnostic model have also been validated. In order to clarify the expression differences of 14 genes in health donor, AD and MCI, the blood samples of patients and healthy individuals were collected. The mRNA expression of the 14 genes in blood sample were detected. The SH-SY5Y cell injury model was constructed and biological function of POU2AF1 and ANKRD22 in SH-SY5Y have been proved. RESULTS: We obtained 16 genes which have an area under curve (AUC) ≥0.6. After that, a diagnostic model based on 14 genes was constructed. Validation in independent cohort showed that the diagnostic model has a good diagnostic efficiency. The expressions of 6 genes in AD patients were significantly lower than those in healthy individuals and MCI patients, while the expressions of 8 genes in AD patients were significantly higher than those in healthy individuals and MCI patients. In in vitro experiments, we found that two key genes POU2AF1 and ANKRD22 could regulate neuronal development by regulating cell viability and IL-6 expression. CONCLUSION: The diagnostic model established in this study has a good diagnose efficiency. Most of these genes in diagnostic model also showed diagnostic value in AD patients. This research also can help doctors make better diagnosis for the treatment and prevention of AD.

Subacute dermal toxicity study of bensulfuron-methyl in Sprague-Dawley rats.

Background: Bensulfuron-methyl has recently attracted attention given its widespread use as an herbicide in crops, especially its transdermal safety. However, no dermal toxicity study of this pesticide to mammals has been reported. The present study aims to investigate subacute dermal toxicity of bensulfuron-methyl following repeated doses exposure.Methods: Forty-eight Sprague-Dawley rats were randomly divided into four groups: a normal control group and bensulfuron-methyl groups of different concentrations (250, 500, 1000 mg/kg.bw/day). The rats were topically applied with the substance dermally for 6 h per day for 28 days. At the end of the experiment, all rats were monitored for any changes in their hematological, biochemical parameters, and pathological and histological sections.Results: There were no statistically significant differences (P ≥ 0.05) in the hematological parameters and biochemical parameters. The pathological histological results of rats in the control and the highest concentration group showed no significant abnormalities. The NOAEL of subacute dermal toxicity study was found to be 1000 mg/kg.bw/day in both female and male rats.Conclusion: The result indicated that bensulfuron-methyl is probably safe for humans as a pesticide.

Pinoresinol diglucoside alleviates ischemia/reperfusion-induced brain injury by modulating neuroinflammation and oxidative stress.

Brain ischemia/reperfusion (I/R) injury is a common pathological process after ischemic stroke. Pinoresinol diglucoside (PDG) has antioxidation and anti-inflammation activities. However, whether PDG ameliorates brain I/R injury is still unclear. In this study, middle cerebral artery occlusion (MCAO) model was established with male C57BL/6 mice, and the mice were treated with 5 and 10 mg/kg PDG via intravenous injection, respectively. The neurological deficit, infarct volume, and brain water content were then evaluated. HE staining and Nissl staining were used to analyze neuron injury. Besides, enzyme-linked immunosorbent assay and colorimetry assay were used to examine the level of inflammatory markers and oxidative stress markers, and Western blot was used to detect the expressions of p-p65, Nrf2, and HO-1. It was revealed that PDG could significantly alleviate the MCAO-induced neurological dysfunction of the mice and reduce the infarct volume, brain water content, and neuron injury. PDG treatment decreased the levels of TNF-α, IL-1ß, IL-6, NO, ROS, and MDA, and significantly increased the activities of SOD, GSH, and GSH-Px in the brain tissue of the mice. Additionally, PDG could repress the activation of p65 and promote Nrf2 and HO-1 expressions. In conclusion, PDG exerts anti-inflammatory and antioxidation effects via regulating the NF-κB pathway and Nrf2/HO-1 pathway, thereby reducing the I/R-induced brain injury of mice.

Simultaneous determination of TUG-891 and its metabolites in rat plasma using LC-HRMS with application to preclinical pharmacokinetic study.

In this study, a simple and reliable LC-MS/MS method was first proposed for the simultaneous determination of TUG-891 and its metabolites TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat plasma. The analytes and fasiglifam (internal standard) were extracted from plasma samples with acetonitrile and separated using an Acquity BEH C18 column (1.7 µm, 2.1 × 50 mm) with water containing 0.05% ammonium hydroxide and acetonitrile containing 0.05% ammonium hydroxide as the mobile phase. A Q-Exactive Orbitrap mass spectrometer in full-scan mode was used for mass detection, and the data analysis was obtained using a mass extraction window of 5 ppm. The calibration curves exhibited excellent linearity (correlation coefficient > 0.9981) in the concentration range of 0.5-1000 ng/mL. The lower limit of quantification was 0.5 ng/mL for all analytes. The intra- and inter-day precision was less than 11.31%, and the accuracy ranged from -11.50 to 9.50%. The extraction recovery of the analytes from rat plasma was greater than 82.31%, and no obvious matrix effect was found. The established method was further applied to the pharmacokinetic study of TUG-891, TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat after a single dose of 5-mg/kg treatment of TUG-891. The results demonstrated that TUG-891 was rapidly metabolized into its metabolites and the systemic exposures of the metabolites were much higher than those of TUG-891.

Upregulation of the lncRNA MEG3 improves cognitive impairment, alleviates neuronal damage, and inhibits activation of astrocytes in hippocampus tissues in Alzheimer's disease through inactivating the PI3K/Akt signaling pathway.

OBJECTIVE: The purpose of this study was to elucidate the expression of the long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) in rats with Alzheimer's disease (AD) and to explore its potential mechanisms. METHODS: An AD rat model was induced by microinjection of Aß25-35 . On the first day after successful modeling, pcDNA3.1 plasmid and pcDNA3.1-MEG3 plasmid were continuously infused into the third ventricle through a micro-osmotic pump to interfere with the expression level of MEG3. The spatial learning ability and memory ability, the histopathological changes of hippocampus tissues, the ultrastructure of hippocampal neurons, astrocyte activation as well as the survival and apoptosis of hippocampal neurons in each group was observed. The expression of apoptosis, PI3/Akt signaling pathway-related proteins, glial fibrillary acidic protein, inflammatory factors, malondialdehyde, glutathione-peroxidase, and superoxide dismutase levels were determined. The deposition of amyloid beta (Aß) in the hippocampus of rats by was observed by Aß immunohistochemical staining. RESULTS: Downregulated MEG3 was detected in the tissues of AD rats. In addition, upregulation of MEG3 contributed to an improvement of spatial learning ability and memory ability, inhibited the pathological injury and its apoptosis of hippocampal neurons, decreased Aß positive expression, inhibited oxidative stress injury and inflammatory injury as well as the activated astrocytes in AD rats via inactivation of the PI3/Akt pathway. CONCLUSION: Our study highlights that upregulation of the lncRNA MEG3 improves cognitive impairment, alleviates neuronal damage, and inhibits activation of astrocytes in hippocampus tissues in AD through inhibiting the PI3K/Akt signaling pathway.

LncRNA SNHG12 as a potent autophagy inducer exerts neuroprotective effects against cerebral ischemia/reperfusion injury.

Long-non-coding RNA small nucleolar RNA host gene 12(SNHG12) was reported to be highly up-regulated in brain microvascular endothelium after cerebral ischemia. Autophagy has been shown to have protective effects against cerebral ischemic insults. However, molecular mechanisms of SNHG12 in regulating autophagy during cerebral ischemia/reperfusion (I/R) injury remain unclear. Here, we established middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice and adopted oxygen-glucose deprivation and reperfusion (OGD/R) SH-SY5Y cell model to mimic cerebral I/R injury in vitro. Triphenyltetrazolium chloride (TTC) staining was used to measure infarct size. Bederson and Longa score systems were used to evaluate neurological behavioral and defects, respectively. CCK-8, EdU staining, flow cytometry and Hoechst 33258 staining were performed to determine the biological function of SNHG12 on SH-SY5Y cell under OGD/R condition. The autophagy levels were determined by Western blotting and LC3B immunofluorescence. We found the expression of SNHG12 was up-regulated by cerebral I/R in mice andSH-SY5Y cell model after OGD/R. Up-regulated SNHG12 alleviated OGD/R-induced SH-SY5Y cell injury and induced autophagy activation, as indicated by an increased ratio of LC3 II/I and Beclin-1, decreased p62. On the contrary, down-regulation of SNHG12 exacerbated SH-SY5Y cell injury after OGD/R and inhibited autophagy. Furthermore, autophagy activator rapamycin or inhibitor 3-MA partially reversed the down-regulation or up-regulation of SNHG12 effect in OGD/R-inducedSH-SY5Y cell injury, respectively. Taken together, these findings suggest that SNHG12 as an autophagy inducer alleviates cerebral I/R injury, which might be a new therapeutic target of ischemic stroke.

Upregulation of miR-496 decreases cerebral ischemia/reperfusion injury by negatively regulating BCL2L14.

Neurological functions were seriously impaired by cerebral ischemia-reperfusion (I/R) injury following ischemic stroke and its molecular mechanism is still not fully understood. MicroRNA-496 (miR-496) has been reported to be deregulated in several diseases but it still remains unknown about the function of miR-496 in cerebral I/R injury. Here, Middle cerebral artery occlusion/reperfusion (MCAO/R) was performed to induce cerebral I/R injury in rats. Then, neurological deficits were assessed by Bederson and Longa score system. The 2,3,5-triphenyltetrazolium chloride (TTC) and terminal deoxynucleotidyltransferase UTP nick end labeling (TUNEL) staining were used to evaluate the rat brain pathology. The oxygen-glucose deprivation and reperfusion (OGD/R) induced in vitro I/R injury was constructed in SH-SY5Y cells. The expression of miR-496 was determined using Real-time PCR. Bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the target gene of miR-496. CCK-8, EdU staining, flow cytometry and Hoechst 33258 staining were respectively utilized to measure cell viability, proliferation and apoptosis. The results showed the expression of miR-496 was found to be down-regulated by cerebral I/R in rats and OGD/R-inducedSH-SY5Y cell model. MiR-496 overexpression alleviated the OGD/R-induced injury in SH-SY5Y cells. In addition, pro-apoptosis factor Bcl-2-like protein 14 (BCL2L14) was predicted and verified as the direct target of miR-496 and suppressed by miR-496. Furthermore, BCL2L14 knockdown exhibited similar effects similar to those of miR-496 overexpression, and the restored BCL2L14expression reversed the protective effects of miR-496 on SH-SY5Y cells. In conclusions, our results suggest that miR-496 alleviates cerebral I/R injury possibly via inhibiting BCL2L14 expression.

Toward Understanding Non-coding RNA Roles in Intracranial Aneurysms and Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a common and frequently life-threatening cerebrovascular disease, which is mostly related with a ruptured intracranial aneurysm. Its complications include rebleeding, early brain injury, cerebral vasospasm, delayed cerebral ischemia, chronic hydrocephalus, and also non neurological problems. Non-coding RNAs (ncRNAs), comprising of microRNAs (miRNAs), small interfering RNAs (siRNAs) and long non-coding RNAs (lncRNAs), play an important role in intracranial aneurysms and SAH. Here, we review the non-coding RNAs expression profile and their related mechanisms in intracranial aneurysms and SAH. Moreover, we suggest that these non-coding RNAs function as novel molecular biomarkers to predict intracranial aneurysms and SAH, and may yield new therapies after SAH in the future.

miRNA profiling in autism spectrum disorder in China.

Autism spectrum disorder (ASD) is a clinically complex and heterogeneous disorder. It is characterized by impaired social abilities, disordered language, isolated areas of interest, and repetitive behaviors. Evidence suggested that the neuropathology of ASD is widely distributed, involving epigenetic regulation in the brain. MiRNAs are a group of endogenous non-coding RNAs that play a critical role in neurodevelopment, neuroplasticity, and other fundamental neurobiological processes. To study miRNA profiling in Autism spectrum disorder in China, we performed miRNA microarray followed quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here, we describe detailed methods and analysis on these microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE67979.

Mir-664 promotes osteosarcoma cells proliferation via downregulating of FOXO4.

BACKGROUND: Uncontrol cell growth and proliferation is acknowledged to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation. Aberrant expression of microRNA play essential roles in cancer development, leads to cell proliferation, growth and survival, and promotes the development of various human tumors, including osteosarcoma. Elucidating the molecular mechanism of this abnormality in osteosarcoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy. METHODS: The expression of miR-664 in osteosarcoma cell lines and osteosarcoma tissues was examined using real-time PCR. The effects of miR-664 on osteosarcoma cell proliferation were evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, colony formation and Anchorage-independent growth ability assay. The effect of miR-664 on FOXO4 was determine by luciferase assays and western blot assay. RESULTS: The expression of miR-664 was markedly upregulated in osteosarcoma cell lines and tissues, and upregulation of miR-664 enhanced, whereas downregulation of miR-664 inhibited the proliferation of osteosarcoma cells in vivo. Furthermore, using bioinformatics and biological approaches, we showed that miR-664 directly targeted and suppressed the expression of tumor suppressors FOXO4. CONCLUSIONS: Our findings suggest that miR-664 functions as an oncogene miRNA and has an important role in promoting human osteosarcoma cell proliferation by suppressing FOXO4 expression. These data suggests that miR-664 may represent a novel therapeutic target of microRNA-mediated suppression of cell proliferation in osteosarcoma.

Sodium valproate alleviates neurodegeneration in SCA3/MJD via suppressing apoptosis and rescuing the hypoacetylation levels of histone H3 and H4.

Spinocerebellar ataxia type 3 (SCA3) also known as Machado-Joseph Disease (MJD), is one of nine polyglutamine (polyQ) diseases caused by a CAG-trinucelotide repeat expansion within the coding sequence of the ATXN3 gene. There are no disease-modifying treatments for polyQ diseases. Recent studies suggest that an imbalance in histone acetylation may be a key process leading to transcriptional dysregulation in polyQ diseases. Because of this possible imbalance, the application of histone deacetylase (HDAC) inhibitors may be feasible for the treatment of polyQ diseases. To further explore the therapeutic potential of HDAC inhibitors, we constructed two independent preclinical trials with valproic acid (VPA), a promising therapeutic HDAC inhibitor, in both Drosophila and cell SCA3 models. We demonstrated that prolonged use of VPA at specific dose partly prevented eye depigmentation, alleviated climbing disability, and extended the average lifespan of SCA3/MJD transgenic Drosophila. We found that VPA could both increase the acetylation levels of histone H3 and histone H4 and reduce the early apoptotic rate of cells without inhibiting the aggregation of mutant ataxin-3 proteins in MJDtr-Q68- expressing cells. These results collectively support the premise that VPA is a promising therapeutic agent for the treatment of SCA3 and other polyQ diseases.

[Polynucleotide repeat expansion of nine spinocerebellar ataxia subtypes and dentatorubral-pallidoluysian atrophy in healthy Chinese Han population].

OBJECTIVE: To assist the establishment of platform and provide the reference standard for mutation detection in spinocerebellar ataxia (SCA) subtypes 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) in Chinese Han population. METHODS: The nucleotide repeat numbers of the 9 SCA subtypes and DRPLA were detected using fluorescence-PCR and capillary gel electrophoresis technique in 300 healthy Chinese Han individuals. RESULTS: Among the 300 healthy controls, the range of the CAG trinucleotide repeat number was 17 to 35 in SCA1, 14-28 in SCA2, 13-41 in SCA3/MJD, 4-16 in SCA6, 5-17 in SCA7, 5-21 in SCA12, 23-41 in SCA17, and 12-33 in DRPLA; and the range of CTA/CTG trinucleotide repeat number on SCA8 locus was 12-43 and the range of ATTCT pentanucleotide repeat number on SCA10 locus was 9-32. Of which, the 12 CTA/CTG repeats of SCA8, 9 ATTCT repeats of SCA10, 23 CAG repeats of SCA17 were the shortest normal repeat number, while the 41 CAG repeats of SCA3/MJD, 32 CAG repeats of SCA10 were the largest normal number that have not been reported. CONCLUSION: The normal ranges of polynucleotide repeats of different subtypes of SCA vary with geographical areas and ethnicities. It might be associated with the genetic and ethnic backgrounds. This is the first normal reference standard of polynucleotide repeat number of these ten SCA subtypes in Chinese Han.

Spinocerebellar ataxia type 11 in the Chinese Han population.

The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative diseases. Researchers have recently found that SCA type 11 (SCA11) is associated with mutations in the TTBK2 gene. In our previous work, we performed mutation detection in SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy gene in Chinese SCA patients, but the genes responsible for approximately 40% of our patients have not yet been identified. To investigate the frequency of SCA11 in Chinese SCA patients, we examined the TTBK2 gene in 68 unrelated probands diagnosed with dominantly inherited ataxia using the denaturing high-performance liquid chromatography method. All analyzed samples displayed the normal elution profile, which denoted that no disease-related mutation was identified. We provided the evidence that SCA11 is a rare form of ataxia in China.

[Studies on the CAG repeat expansion in patients with hereditary spinocerebellar ataxia from Chinese Han].

OBJECTIVE: To investigate the CAG trinucleotide repeat expansion in spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, 12, and 17 from Chinese Han. METHODS: The pathological CAG triplet repeat expansions of the SCA1, SCA2, SCA3/Machado-Joseph disease (MJD), SCA6, SCA7, SCA12 and SCA17 genes were analyzed in a cohort of 559 Mainland Chinese patients affected by spinocerebellar ataxia, including 363 probands from families with autosomal dominant SCA and 196 sporadic cases. Polymerase chain reaction, agarose gel electrophoresis, recombinant DNA technology by T-vector cloning and direct sequencing were performed to detect the CAG-repeat number of abnormal allele. RESULTS: Among the 559 SCA patients, twenty-three were positive for SCA1, the ranges of expanded CAG repeats were from 39 to 60 (mean:51.09+/-4.88); thirty-two were positive for SCA2, the ranges of expanded CAG repeats were from 36 to 51 (mean:40.34+/-4.40); three hundred and five were positive for SCA3/MJD, the ranges of expanded CAG repeats were from 49 to 86 (mean:73.84+/-5.07); nine were positive for SCA6, the ranges of expanded CAG repeats were from 23 to 29 (mean:25.56+/-1.94); twenty-seven were positive for SCA7, the ranges of expanded CAG repeats were from 38 to 71(mean:58.22+/-10.90); three were positive for SCA12, the ranges of expanded CAG repeats were from 51 to 52 (mean:51.33+/-0.58); and finally, two were positive for SCA17, the range of expanded CAG repeats were from 53 to 55 (mean:54.00+/-1.41). CONCLUSION: The 39 CAG repeats of SCA1, 49 CAG repeats of SCA3 and 51 CAG repeats of SCA12 are all the shortest known causative expanded alleles, while the 86 CAG repeats of SCA3/MJD is the largest full expanded allele that has never been reported. Furthermore, it is the first report of SCA17 subtype in Mainland Chinese and first research that established the abnormal reference standard of CAG repeat number of different subtypes of SCA in Chinese Han.

[Study on the single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene in Chinese patients with spinocerebellar ataxia].

OBJECTIVE: To study the single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene in spinocerebellar ataxia (SCA) patients in China. METHODS: The single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene was detected by PCR, digested with EcoN I, separated on 8% polyacrylamide gel in 68 probands of autosomal dominant SCA families and 119 sporadic SCA patients, who had been excluded CAG/CAA repeat expansion at the SCA1, 2, 3, 6, 7, 17 and dentatorubral-pallidolluysian atrophy (DRPLA) loci. The results were confirmed in four patients by direct sequencing. RESULTS: The single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene was not identified in authors' cohort. CONCLUSION: The mutation of c.-16C to T of the PURATROPHIN-1 gene might be rare in SCA patients in China.