Molecular Genetics Of Two Chinese Families With Spinocerebellar Ataxias

Objective1. To determinate the clinical features of all family members, which are from two Chinese families. We try to complete the pedigree diagrams and define the relationship between patients in the same family.2. We try to diagnose the disease and the subtype of the proband and other patients in their family and we try to know the severity and involvement of the disease. Our research can provide details about the disease for family members.3. We try to define pathogenic alleles in the two SCAs families and confirm the nature of family members carrying pathogenic alleles.4. We want to define where the pathogenic allele come from and provide more information about SCAs for our country.Content1. We try to determinate the clinical features of all family members of the two SCAs families, and complete the pedigree diagrams.2. We took the probands to take detailed neurologic examinations to acquire more information about the disease and finally clarify a diagnosis.3. We used the exons capture and the second generation sequencing technology to analyze the same mutated genes by DNA of peripheral vein blood in patients in two families.4. We used polymerase chain reaction(PCR), agarose gel electrophoresis and direct sequencing to study the mutated genes to acquire the disease-causing genes. At last, we try to study the pathogenic characteristics and molecular mechanism of the disease-causing genes.Methods1. The proband(Ⅲ-7) in the Gansu province family of familial diseases of the nervous system initially participated in MRI scans in LinXiaZhou people’s hospital of Gansu province. And a preliminary diagnosis is brain atrophy, especially in the cerebellum. According to the Harding standization, the proband(Ⅲ-7) and her younger brother(Ⅲ-5) were diagnosed as SCAs by clinical manifestations, imaging examinations and family history in the third hospital of Peking University.2. Our team members went to gansu weiyuan mountainous area for two times for the detailed investigation and family history of genetic research after we knew the family in gansu. We have got family members understanding and cooperation. Later on, we invited proband(Ⅲ-7) and her younger brother(Ⅲ-5) to the third hospital of Peking University to get professional clinic diagnose and at last they were diagnosed as SCAs.3. The proband(Ⅲ-9)in the Hebei-liaoning area family of SCAs was diagnosed as SCAs in neurology after the relevant inspections.Several common subtypes of SCAs were preliminary screening and ruled out one by one. At last,we think it is a new subtype of SCAs.4. We invited family members Ⅲ-5 and Ⅲ-11 to the third hospital of Peking University to get professional clinic diagnose after we knew the family in the Hebei-liaoning area and at last they were preliminarily diagnosed as SCAs.5. We have explained to the family members why and how we conduct the research and the detailed information about the causes and harm of the disease and the disease harm to their offspring. Then peripheral vein blood of part of the two families members was collected in 5 mL one person and placed in EDTA anticoagulation tubes and marked in tag number. All the blood samples are placed in- 80 ℃ cryogenic refrigerator in our laboratory. According to the requirements of exon sequencing for the samples, we send the prepared samples in the two families to BGI Tech to take the exons capture and the second generation sequencing. DNA samples of the members had extracted by standards are placed in- 80 ℃ cryogenic refrigerator in our laboratory for the following up tests. 6. We analyzed the exome sequencing results of the two families and got the same mutated genes in the family. Then we designed the primers of the same mutated genes to conduct PCR amplification tests. Amplification products were analyzed by agarose gel electrophoresis. Amplification products meeting the requirements of the direct sequencing samples were sent to Sangon biological engineering(Shanghai) co., LTD and the ones not meeting the requirements were required to redesign the primers. Then we can screen out the disease-causing genes for the two families and learn gene subtypes of the diseases. At last, we can analyze and research the pathogenic mechanism of the diseases and provide the basis for subsequent diagnosis before any symptoms are made in the family members.Results1. We obtained 84 point mutations and 8 insertion or loss mutations in the SCAs family of gansu through the comparison between the results of exome sequencing in BGI Tech of the two patients Ⅲ-5 andⅡ-8 and the reference of the human genome sequence(UCSC hg 19 version). Firstly, ATXN2 gene in the 8 insertion or loss mutations was conduct PCR amplification test combined with clinical diagnosis. Autosomal dominant heredity SCA2 has been diagnosed in this family caused by the dynamic nutation of CAG trinucleotide repeats, and 7 pathogenic allele carriers in this family were confirmed by genetic diagnosis.2. We obtained 15 point mutations in the SCAs family of Hebei-liaoning area through the comparison between the results of exome sequencing in BGI Tech of the two patients Ⅲ-5 and Ⅲ-11 and the reference of the human genome sequence(UCSC hg 19 version). Finally, we screen out five candidate genes(FERMT1,S100A12,SPATC1,SPTB,ZNF334)in which one may be the disease-causing gene but there is a need to follow-up to continue the research analysis.3. The CAG trinucleotide repeats of the pathogenic gene ATXN2 were amplified by polymerase chain reaction(PCR) in the 46 family members in the SCA2 family of gansu province. PCR products electrophoregram showed 2 strips of different sizes in both the 6 patients Ⅱ-8、Ⅲ-5、Ⅲ-7、Ⅲ-21、Ⅳ-8、Ⅳ-10 and 7 carriers of pathogenic allele Ⅲ-13、Ⅳ-1、Ⅳ-6、Ⅳ-19、Ⅴ-1、Ⅴ-3、Ⅴ-5. In theory for this site, each individual’s PCR products have two bands(a pair alleles). Normal individual’s CAG repeats in two alleles are same or small in gap, so we can not distinguish them in the electrophoregram. The normal allele of patients and carriers of the PCR products significantly smaller than abnormal allele, so we can distinguish them in the electrophoregram. According to the sequencing results, the repeated times of CAG trinucleotide are 40-46 in abnormal alleles of patients and carriers of pathogenic allele in the pathogenic gene ATXN2, while the number is 19-25 in other normal family members and the number is 13-31 in normal.Conclusions1. The patients in the two genetic SCAs families in our research have shown walking instability with taking things instability, gait ataxia, drinking water choking cough, mild dysarthria, finger-nose test and heel-knee-tibia test inexact,dysdiadochokinesia and dysmetria, etc. Head MRI(Magnetic Resonance Imaging) examination showed different degrees of brain atrophy and especially in the cerebellum atrophy. EMG revealed dystonia and tendon hyporeflexia. Clinical symptoms of patients in two families consistent with the typical symptoms of SCAs. But different patients in the same stage and in different stages of the disease in the same patient showed obvious different manifestations. Clinical and genetic heterogeneity are obvious.2. There is no obvious genetic anticipation between family members in the Gansu weiyuan SCAs family and there is no dysgnosia in family patients. This does not agree with some related reports.3. Finally, we screen out five candidate genes(FERMT1,S100A12,SPATC1,SPTB,ZNF334) in the Hebei-liaoning area SCAs family and in which one may be the disease-causing gene but there is a need to follow-up to continue the research analysis.4. SCAs have highly clinical and genetic heterogeneity, so the diagnosis and classification of SCAs can not just rely on clinical diagnosis. Besides, there are many overlapping symptoms between different subtypes, so the diagnosis and classification of SCAs must be combined with genetic diagnosis.