Identification of the ataxin-1 interaction network and its impact on spinocerebellar ataxia type 1.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein. The pathogenic mechanism resulting in SCA1 is still unclear. Protein-protein interactions affect the function and stability of ataxin-1. METHODS: Wild-type and mutant ataxin-1 were expressed in HEK-293T cells. The levels of expression were assessed using real-time polymerase chain reaction (PCR) and Western blots. Co-immunoprecipitation was done in HEK-293T cells expressing exogenous wild-type and mutant ataxin-1 using anti-Flag antibody following by tandem affinity purification in order to study protein-protein interactions. The candidate interacting proteins were validated by immunoprecipitation. Chromatin immunoprecipitation and high-throughput sequencing and RNA immunoprecipitation and high-throughput sequencing were performed using HEK-293T cells expressing wild-type or mutant ataxin-1. RESULTS: In this study using HEK-293T cells, we found that wild-type ataxin-1 interacted with MCM2, GNAS, and TMEM206, while mutant ataxin-1 lost its interaction with MCM2, GNAS, and TMEM206. Two ataxin-1 binding targets containing the core GGAG or AAAT were identified in HEK-293T cells using ChIP-seq. Gene Ontology analysis of the top ataxin-1 binding genes identified SLC6A15, NTF3, KCNC3, and DNAJC6 as functional genes in neurons in vitro. Ataxin-1 also was identified as an RNA-binding protein in HEK-293T cells using RIP-seq, but the polyglutamine expansion in the ataxin-1 had no direct effects on the RNA-binding activity of ataxin-1. CONCLUSIONS: An expanded polyglutamine tract in ataxin-1 might interfere with protein-protein or protein-DNA interactions but had little effect on protein-RNA interactions. This study suggested that the dysfunction of protein-protein or protein-DNA interactions is involved in the pathogenesis of SCA1.

C1orf194 deficiency leads to incomplete early embryonic lethality and dominant intermediate Charcot-Marie-Tooth disease in a knockout mouse model.

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy and shows clinical and genetic heterogeneity. Mutations in C1orf194 encoding a Ca2+ regulator in neurons and Schwann cells have been reported previously by us to cause CMT disease. In here, we further investigated the function and pathogenic mechanism of C1or194 by generating C1orf194 knockout (KO) mice. Homozygous mutants of C1orf194 mice exhibited incomplete embryonic lethality, characterized by differentiation abnormalities and stillbirth on embryonic days 7.5-15.5. Heterozygous and surviving homozygous C1orf194 KO mice developed motor and sensory defects at the age of 4 months. Electrophysiologic recordings showed decreased compound muscle action potential and motor nerve conduction velocity in the sciatic nerve of C1orf194-deficient mice as a pathologic feature of dominant intermediate-type CMT. Transmission electron microscopy analysis revealed demyelination and axonal atrophy in the sciatic nerve as well as swelling and loss of mitochondrial matrix and other abnormalities in axons and Schwann cells. A histopathologic examination showed a loss of motor neurons in the anterior horn of the spinal cord and muscle atrophy. Shorter internodal length between nodes of Ranvier and Schmidt-Lanterman incisures was detected in the sciatic nerve of affected animals. These results indicate that C1orf194 KO mice can serve as an animal model of CMT with a severe dominant intermediate CMT phenotype that can be used to investigate the molecular mechanisms of the disease and evaluate the efficacy of therapeutic strategies.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

[Pathogenic Genes of A Hereditary Hemorrhagic Telangiectasia Pedigree].

OBJECTIVE: To identify the mutation of ENG and ALK1 genes in a hereditary hemorrhagic telangiectasia pedigree. METHODS: 14 exons of ENG gene and 9 exons of ALK1 gene in 11 menbers of this pedigree 4 generation were amplified by reverse transcription-polymerase chain reaction (RT-PCR), the PCR products were screened by direct sequencing. RESULTS: A nonsense mutation c.447G > A was found in exon 4 of ENG gen of the pedigreee, resulting in change of Trp 149 into Stop, while no gene mutation was found in ALK1 gene. CONCLUSION: The hereditary hemorrhagic telangiectasia in this pedigree is caused by the nonsense mutation c.447G > A in ENG gene.

[Sequence Analysis of the UGT1A1 Gene Untranslated Region in Chinese Han Population].

OBJECTIVE: To analyze the 5' and 3'-untranslated region sequences of the UGT1A1 gene in Chinese Han population and to find polymorphic variants within the untranslated region. METHODS: Genomic DNA was extracted from peripheral leukocytes in 220 healthy Han individuals. The 5' and 3'-untranslated region sequences of the UGT1A1 gene were amplified by polymerase chain reaction, and followed by DNA sequencing. RESULTS: Two polymorphic loci were identified in the 5'-untranslated region of the UGT1A1 gene with -64(G/C) and A(TA)6TAA/A(TA)7TAA in TATAA box region among Chinese Han population. Genotype frequencies were 98.4% (G) and 1.6% (C) in -64 locus of the UGT1A1 gene among the 220 individuals. The allele frequency of A(TA)6TAA and A(TA)7TAA within the promoter region was found to be 93.4% and 6.6%, respectively. Two polymorphic loci of 1813(C/T) and 1941(C/G) were detected in the 3'-untranslated region of the UGT1A1 gene, they showed a homozygous state at two loci with cosegregation pattern at 1813 and 1941 locus. The haplotype frequencies were 73.6% (CC/1813+CC/1941) and 26.4% (TT/1813+GG/1941) for 1813 and 1941 loci in the UGT1A1 gene. CONCLUTION: Cosegregation pattern, at 1813 and 1941 locus with homozygous state in the 3'-untranslated region of the UGT1A1 gene may be selected from the human genome among Chinese Han population. More studies should be focused on the mechanism of homozygous cosegregation.

[Mutation analysis of UGT1A1 gene in patients with unconjugated hyperbilirubinemia].

OBJECTIVE: To analyze potential mutations of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene in patients with unconjugated hyperbilirubinemia, and to explore the correlation between the mutations and total serum bilirubin levels. METHODS: Genomic DNA was extracted from peripheral blood samples of patients. Coding sequence and promoter region of the UGT1A1 gene were amplified. Mutations were identified through DNA sequencing. RESULTS: Mutations of the UGT1A1 gene were found in 46 out of 61 patients with unconjugated hyperbilirubinemia. Five types of mutations were detected, with a decreasing order of 211G>A, TA insertion in the TATAA promoter element, 686C>A, 1091C>T and 1352C>T. Compared with those carrying a single homozygous mutation or compound heterozygous mutations, total serum bilirubin was higher in those carrying a homozygous mutation in combination with other heterozygous mutations (P< 0.05). Based on the UGT1A1 gene mutations and level of total serum bilirubin, 44 patients were diagnosed with Gilbert syndrome, and 2 were diagnosed with Crigler-Najjar syndrome type 2. CONCLUSION: The level of total serum bilirubin is correlated with the number of UGT1A1 gene mutations as well as their heterozygous or homozygous status.

[Effect of BCL11A gene on transcription of γ-globin gene].

This study was aimed to explore the effect of BCL11A gene on transcription of γ-globin gene in K562 cells. B-cell lymphoma/leukemia 11A (BCL11A) gene was silenced by small interfering RNA (siRNA) expression vectors in K562 cells (human erythroblastic leukemia cell line). Gamma-globin mRNA level in K562 cells was determined by RT-PCR. Association between the BCL11A gene and γ-globin gene transcription was explored by comparison of mRNA levels. The results indicated that the silence rate of the BCL11A gene in K562 cells by 4 siRNA expression vectors was 49.7%, 55.4%, 78.2%, and 84.1%, respectively. The siRNA expression vector with 84.1% silence rate was transfected into K562 cells, transcription level of γ-globin mRNA in K562 cells transfected with siRNA expression vector increased 2.4 times as compared with control K562 cells. It is concluded that level of γ-globin mRNA increases when the BCL11A gene is silenced. It indicates that the BCL11A gene may be a negative regulator for γ-globin gene expression.

[Correlation between hemoglobin F levels and single nucleotide polymorphism at BCL11A gene rs11886868 locus in ß-thalassemia patients].

This study was aimed to analyze hemoglobin F (HbF) level and single nucleotide polymorphisms at rs11886868 locus of BCL11A gene in ß-thalassemia patients, and to explore correlation between them. 89 mild ß-thalassemia patients with known mutations were registered, and HbF levels were determined by capillary electrophoresis. Genomic DNA was extracted from peripheral leukocytes, fragment including rs11886868 locus in BCL11A gene was amplified by PCR, and polymorphism was determined by DNA sequencing. The results showed that 2 polymorphisms including C and T were found at rs11886868 locus in BCL11A gene among 89 mild ß-thalassemia patients. HbF levels in red blood cells were (4.47 ± 3.42)% and (2.79 ± 2.21)% for ß-thalassemia patients carrying C/C and C/T haplotypes, respectively. There was difference between 2 haplotype groups. It is concluded that the C and T polymorphisms are found at rs11886868 locus in the BCL11A gene for ß-thalassemia patients. C polymorphism may be related to high HbF expression in red blood cells.

[A novel mutation in ß-globin gene of a patient with ß-thalassemia].

This study was aimed to analyze the ß-globin gene mutations in a patient with ß-thalassemia minor. Genomic DNA was extracted from peripheral blood cells of the patient. The full-length DNA sequence coding for ß-globin was amplified by polymerase chain reaction, and the gene mutation was determined by DNA sequencing. The results indicated that a heterogeneous A→G mutation was found at position 129 in intron 1 of the ß-thalassemia minor patient. It is concluded that the IVS-I-129(A→G) mutation is a splicing site mutation leading to a splicing error in immature messenger RNA and a protein translation error for the ß-globin gene. Thus, the IVS-I-129(A→G) is a novel mutation.

[Polymorphism in BP1 binding site upstream of ß-globin gene in Chinese Han population].

This study was aimed to analyze the BP1 binding site sequence upstream of ß-globin gene in Chinese Han population, and to investigate polymorphism in the BP1 binding site upstream of ß-globin gene, so as to provide the basis for exploration of relation between polymorphisms in the BP1 binding site and ß-globin expression. Genomic DNA was extracted from peripheral leukocytes of 110 healthy individuals in Chinese Han population. Sequence of the BP1 binding site upstream of ß-globin gene was amplified by polymerase chain reaction, the polymorphic variation in the BP1 binding site was determined by DNA sequencing. The results indicated that 2 polymorphism loci were found in the BP1 binding site upstream of ß-globin gene, they were C/T at the -551 bp region and (AC)(n)(AT)(x)T(y) at the -530 bp region in Chinese Han population. Frequencies of C and T were 60.4% and 39.6% at position -551. Analysis of the (AC)(n)(AT)(x)T(y) polymorphism revealed 9 different genotypes: (AC)(2)(AT)(9)T(5), (AC)(2)(AT)(8)T(5), (AC)(2)(AT)(7)T(7), (AC)(3)(AT)(7)T(5), (AC)(2)(AT)(8)T(9), (AC)(3)(AT)(8)T(5), (AC)(2)(AT)(10)T(3), (AC)(2)(AT)(11)T(3), and (AC)(2)(AT)(7)T(5) at position -530. Frequencies of 9 (AC)(n)(AT)(x)T(y) polymorphisms were 33.2%, 29.1%, 24.1%, 5.4%, 3.2%, 1.8%, 1.4%, 0.9%, and 0.9% respectively. It is concluded that rich (AC)(n)(AT)(x)T(y) polymorphisms at the -530 bp region in the BP1 binding site upstream of ß-globin gene are found in Chinese Han population. (AC)(2)(AT)(9)T(5), (AC)(2)(AT)(8)T(5), and (AC)(2)(AT)(7)T(7) are 3 major polymorphisms among Chinese Han population, and (AC)(3)(AT)(8)T(5) is a novel polymorphism at the -530 bp region. More studies should be done to explore relation between (AC)(n)(AT)(x)T(y) polymorphisms and ß-globin expression.

[Analysis of GJB2 gene coding sequence in patients with nonsyndromic hearing loss].

OBJECTIVE: To analyze the coding sequence of GJB2 gene in six pedigrees with nonsyndromic hearing loss in order to find deafness-causing mutations in the GJB2 gene, and to explore the inherent pattern of deafness-causing mutations in the GJB2 gene. METHODS: Genomic DNA was extracted from peripheral blood for the probands and their family members. Coding sequence of the GJB2 gene was amplified by polymerase chain reaction, sequence variations were determined by DNA sequencing. Amplified fragments with overlapping peaks on sequencing chromatogram were sequenced by TA cloning in order to determine whether the mutations originated from the same allele. RESULTS: Mutations in the GJB2 gene were found in 4 out of the 6 pedigrees with nonsyndromic hearing loss. Four types of mutations were detected in the GJB2 gene, which were 235delC, 299-300delAT, 79G>A+341A>G, and 109G>A. Compound heterozygous polymorphisms 79G>A and 341A>G, and mutations 109G>A and 235delC had deafness-causing effects. CONCLUSION: Heterogeneous mutations of the GJB2 gene are frequently seen in patients with nonsyndromic hearing loss. Sometimes, polymorphisms may cause deafness when they are combined. Environmental factors and other genes may contribute to hearing loss caused by the GJB2 gene mutations.

[Single nucleotide polymorphisms of ß-globin gene in ß-thalassaemia patients].

This study was aimed to analyze the ß-globin gene sequence and single nucleotide polymorphisms of the ß-globin gene in ß-thalassaemia patients from Shenzhen area, and to explore linkage relationships between ß-globin gene mutations and single nucleotide polymorphisms. Genomic DNA was extracted from peripheral leukocytes in 125 ß-thalassaemia patients from Shenzhen population. ß-globin gene was amplified by polymerase chain reaction, mutations and single nucleotide polymorphisms in the ß-globin gene were determined by DNA sequencing. The results indicated 10 types of mutation and 12 single nucleotide polymorphism loci were found in the ß-globin gene of 114 ß-thalassaemia patients. Linkage disequilibrium between mutations and single nucleotide polymorphisms was found in 6 loci including 6 haplotypes among these single nucleotide polymorphism loci in the ß-globin gene. It is concluded that a number of single nucleotide polymorphisms is found in the ß-globin gene, where an average of one single nucleotide polymorphism every 230 bp there is. Linkage disequilibrium occurs between ß-thalassaemia mutations and some haplotypes in single nucleotide polymorphism loci. This study may be helpful to gene diagnosis for ß-thalassaemia patients.

[Different splice of the calpain 3 gene in human skeletal muscle tissue and white blood cells].

OBJECTIVE: To investigate the splice variants of the calpain 3 gene existing in human skeletal muscle tissue and white blood cells, and to explore the feasibility of gene diagnosis using CAPN3 mRNA extracted from peripheral leukocytes. METHODS: Total RNA was extracted from peripheral blood and skeletal muscle tissue in healthy individuals. CAPN3 cDNAs were determined by reverse transcriptase polymerase chain reaction and DNA sequencing. CAPN3 cDNAs from peripheral leukocytes were compared with sequences obtained from skeletal muscle tissue. RESULTS: RT-PCR and DNA sequencing showed that the CAPN3 cDNAs comprised 24 exons in human skeletal muscle tissue, while the number of exons was 23 in white blood cells. Exon 15 was spliced out in human white blood cells. CONCLUSION: Splice variants exist in human skeletal muscle tissue and white blood cells. Gene diagnosis may omit the mutations of exon 15 using mRNA extracted from peripheral leukocytes. These findings suggest that mutation analysis of the CAPN3 cDNA should use skeletal muscle tissue as materials instead of peripheral blood.

[Changes of serum creatine kinase levels in children with Duchenne muscular dystrophy].

OBJECTIVE: Duchenne muscular dystrophy (DMD) usually occurs prior to 3 years old. The value of serum creatine kinase changes with clinical progression and age in patients with DMD. This study aimed to investigate the regularity in the changes of serum creatine kinase activities in children with DMD. METHODS: Peripheral blood samples were obtained from 40 children with DMD (ranged from 3-14 years). Serum creatine kinase levels were assayed by kinetic UV test. RESULTS: Serum creatine kinase level in the 40 DMD patients (ranged from 2 595- 45 495 U/L) was remarkably higher than the reference value (35-174 U/L). The highest serum creatine kinase level (average: 27750-31173 U/L) was found in 3-5 years old patients. Afterwards, serum creatine kinase level decreased with clinical progression and age, with a yearly average rate of decline was 8.7%. CONCLUSIONS: Serum creatine kinase level reaches a peak between 3 and 5 years old and then reduces with increasing age in children with DMD. The characteristic changes of serum creatine kinase are suspected to reflect the rate of muscle decay.

Polymorphism of the D4Z4 locus associated with facioscapulohumeral muscular dystrophy 1A in Shanghai population.

OBJECTIVE: To investigate the D4Z4 repeats on chromosome 4q35 in normal individuals in Shanghai and analysis the polymorphism of the D4Z4 locus. METHODS: The length of D4Z4 repeats on chromosome 4q35 in 191 normal individuals in Shanghai was determined by pulsed-field gel electrophoresis and Southern blotting after double digestion with Eco RI and Bln I. The number of short D4Z4 repeats was counted after partial digestion with Kpn I. RESULTS: Among 191 normal individuals in Shanghai, seventeen showed the size of D4Z4 fragments ranged from 22 to 34 kb, i.e. 8.9% of individuals had fewer numbers of D4Z4 repeats. Of these 17 individuals, sixteen showed the short D4Z4 fragment on chromosome 4q35, and one low D4Z4 fragment was correlated to 4q35--> 10q26 translocation. CONCLUSION: The frequency of individuals having fewer numbers of D4Z4 repeats on chromosome 4q35 in Shanghai population is higher than that in Caucasian population although the short D4Z4 fragment on chromosome 4q35 is associated with facioscapulohumeral muscular dystrophy. These findings suggest that other factors may also contribute to facioscapulohumeral muscular dystrophy.