Identification Of Pathogenic Gene Of Progressive Myoclonic Ataxia

Objectives:To capture and verify the pathogenic genes in two families with progressive myoclonic ataxia.The discovery of pathogenic genes provides a new basis for the pathogenesis of the disease and the study of molecular genetics.Methods:(1)Collected data: Collected information from two families with progressive myoclonic ataxia.Collected general information of family patients,age of onset,clinical manifestations,related auxiliary examination results such as electroencephalogram(EEG),brain Magnetic resonance imaging(MRI),etc.We used EDTA anticoagulant tube to take 5ml blood sample,and put them in-80?refrigerator to be stored for test.Normal people need to be collected general information and taken blood samples.At the same time,we need to draw a family tree.(2)Whole exome sequencing: We performed DNA extraction and completed DNA quality assessment after blood sample collection.Then we selected the DNA from two patient who have an early onset age,typical clinical manifestation and severe illness from family 1.Performed a series of operations on them which included DNA Purification and Shearing,End Repair,Adenlylate 3' Ends,Adapter Ligation,Size Selection,PCR Amplification,Library Hybridization with Exome Array,Library Wash and Purification,PCR Amplification,Sequencing.Then the high flux sequencing was carried out with Illumina hiseq2000.First of all,the quality of the obtained data information was evaluated,Then performed reference sequence alignment analysis,SNV/In Del detection,SNV/In Del annotation.Compared all SNV/In Del site with the newly released population database,functional database, disease database and other known information,and evaluated the frequency of variation,functional characteristics,conservatism,pathogenicity,etc of these SNV/In Del site,Searched for candidate pathogenic genes.(3)Sanger sequencing verification: First of all,to proceed the self-validation of the patients who completed the whole exome group sequencing,and excluded the false positive sites.Secondly,the family co separation validation was carried out in PMA family 1.Considering that the pedigree may be an autosomal dominant genetic imperfection,the exclusion criteria are as follows: mutation sites not found in patients' DNA.An external validation is then performed.Another PMA family that has been collected was also tested for the same pathogenic gene,to determine whether it has the same pathogenic gene.Finally,the human genome database was used as contrst data to determine whether the mutation was carried in normal population.Results:(1)PMA pedigree 1 has 67 members,including 34 males and 33 females.16 patients have clinical phenotypes,9 males,7 females,And 51 patients without clinical phenotype.There are 35 members in PMA family 2,including 16 males and19 females.5 patients have clinical phenotypes,including 2 males and 3 females,and30 patients with no clinical phenotype.(2)The onset age of most patients in pedigree 1 was ranged from 10 to 20 years old,the average onset age was 15 years old,the minimum and the maximum onset age was 4 and 24 years old respectively.The mean onset age of patients in generation II,III,IV was 21.25,15 and 12.25 years old respectively.The onset age of most patients in family 2 was also between 10 and 20 years old,the average onset age was14 years,the minimum onset age was 10 years,and the maximum onset age was 20 years old.In the first generation,the onset age of one patient was 18 years old,the average age of onset was 14 years for all 3 patients in generation II,and 11 years old for one patient in the third generation.(3)Clinical manifestations include progressive exacerbation of myoclonus, ataxia,no cognitive impairment,no seizures.The auxiliary examination showed that the blood,urine routine,liver and kidney function were basically normal.ECG and X ray chest radiography were normal.Electromyography showed no characteristic changes in all muscles.MRI showed no abnormal signal in brain parenchyma,and part of cerebral sulcus widened slightly.Video-EEG showed that the low-amplitude spike waves and slow-spike waves can be seen in the bilateral frontal lobes during sleep.After sequencing,89162 mutation sites were obtained.26 candidate mutation sites were obtained by further screening with various databases and prediction software which include TACC2,OR8D4,SLC8A3,C16orf3,MARCH10,MROH7,OSBP2,WDR60,SGCE,PABPC1,CWF19L1,TAS2R43,INSC,ANO5,KLRF2,TAS2R43,CCDC168,IL34,FOXK2,LTBP2,JSRP1,LRRC3 C,CYP4B1,ZGPAT,TENM2,PABPC1.(4)Sanger sequencing of patients with whole exome sequence confirmed the existence of this mutation in the SGCE gene.Sanger sequencing was performed on11 patients with blood samples and 18 normal phenotypic members in family 1.And the results showed that all patients carried the same mutation of SGCE gene,15 members of the normal phenotype family did not have the same mutation of SGCE gene,and 3 members of them have the same mutation of SGCE gene,which is consistent with the family co separation.The sanger sequencing of another PMA family showed that none of the 10 DNA samples have the same mutation in SGCE gene.In the end,no SGCE gene mutation was found in the human genome.Conclusion:(1)Two PMA families are autosomal dominant inheritance,both of which were prematurely inherited,and family 1 is not fully explicit.(2)Pathogenicity gene mutation of PMA pedigree 1 is SGCE.For the first time,the SGCE mutation was found to be a PMA pathogenic gene.(3)SGCE gene mutation was not found in PMA pedigree 2,and the pathogenetic gene is not clear yet,which is considered to be caused by genetic heterogeneity.