A Study Of Mutation Spectrum And Clinical Features In Patients With Autosomal Recessive Cerebellar Ataxia And Related Disorders

Objective: Autosomal recessive cerebellar ataxia(ARCA)is a large group of neurodegenerative disorders with a prevalence of 3-5 per 100,000 in the general population.ARCA is characterized by a marked genetic heterogeneity.Currently,more than 200 genes associated with ARCA have been identified,and more than 90 are ARCAcausing gene which have cerebellar ataxia as the main symptom.Various ARCA gene mutations ranging from point mutations,insertion,deletion to complex rearrangements have been identified.Although many causative genes have been uncovered in recent years,genetic diagnosis is still missing for approximately 50% of ARCA patients.Besides,ARCA also presents with obvious clinical heterogeneity,many cases were genetically diagnosed with non-ARCA disorders but showed a typical phenotype of recessive inherited ataxia have been successively reported,such as hereditary spastic paraplegia,Charcot-Marie-Tooth disease,or some rare syndromes,which further increases the difficulty in the diagnosis.With the enlargement of gene pleiotropy,ambiguous genotype-phenotype relationship,and diversified mutation types,this disorder is easily misdiagnosed or missed in clinical practice.As such,in order to implement a precision medicine plan for ARCA,it’s necessary to refine the genetic screening strategy and make a valid differential diagnosis.Moreover,due to the low incidence,few studies have reported the genetic or clinical characteristic of ARCA patients in the Chinese population.Therefore,in this study,dynamic mutation screening of common spinocerebellar ataxia type(SCA)was firstly performed in patients with clinically proposed ARCA.Then,whole exon sequencing(WES)combined copy number variation(CNV)analysis were used to investigate the causative mutations for an accurate diagnoses purpose.Genotypic and phenotypic characteristics of patients with definite diagnosis were analyzed and summarized to provide the clinician with a greater understanding of this disease.Methods:1.After screened the dynamic mutations of SCA,54 Chinese index patients with unexplained autosomal recessive or sporadic ataxia were investigated by WES and CNV calling with Exome Depth.The sanger sequencing was applied to identify the detected candidate mutations and perform pedigree verification.Likely causal CNV predictions were validated by CNVseq.Suspicious uniparental disomy were further proved by Affymetrix Cyto Scan? Dx Assay.The interpretation and classification of variants was performed based on the American College of Medical Genetics and Genomics standards.Finally,the mutation spectrum of ARCA causative genes was summarized by review related literature.2.Detailed clinical analysis was conducted for ARCA patients with clear genetic diagnosis,and phenotypic characteristics were summarized in combination with literature.3.Six Chinese PLA2G6-associated neurodegeneration(PLAN)families were clinically examined in detail and whole-exome sequencing was performed in the probands.Haplotype analysis was performed in those families with PLA2G6 c.991G>T mutation using 23 single nucleotide polymorphism markers.Furthermore,all previously reported PLA2G6 mutations and patients in Chinese were reviewed to summarize the genetic and clinical features of PLAN.4.We enrolled a consanguineous Chinese family initially diagnosed as "hereditary ataxia" which had normal parents but 2 children(a pair of siblings)had cerebellar atrophy and other complex phenotypes,and performed clinically examination in detail and WES in the family.Minigene assay were performed in splicing analysis.All previously reported ERCC6 splicing mutations and cases were reviewed to summarize the genetic and clinical features of ERCC6-related Cockayne Syndrome(CS).Results:1.Thirty-eight mutations including 29 novel ones were identified in 25 out of the 54 patients,providing a 46.3% positive molecular diagnostic rate.Ten different genes were involved,of which four most common genes were SACS,SYNE1,ADCK3 and SETX,which accounted for 76.0%(19/25)of the positive cases.The de novo microdeletion in SACS was reported for the first time in China and the uniparental disomy of ADCK3 was reported for the first time worldwide.2.We summarized the clinical characteristics and described some atypical symptoms of25 probands who had confirmed ARCA genetic diagnosis.In addition,this study reported the first autosomal recessive spastic ataxia 2,patient in China.3.All the six probands were compound heterozygote for PLA2G6 variants,including four novel likely pathogenic mutations(c.967G>A,c.1450G>T,c.1631T>C,and c.1915 del G)and five known pathogenic mutations.We found that up to 66.67%(4/6)patients had c.991G>T mutation and the haplotype analysis revealed that those patients shared a haplotype of 717 kb,which confirmed that the high frequency mutation was due to the founder effect.Haplotype analyses.The frequency of psychiatric features,cognitive and myoclonus in Chinese patients with PLA2G6-related PD were significant different from the European.4.A novel homozygous splice site mutation(c.543+4A>T)in ERCC6 were identified in both affected siblings.Functional studies showed that c.543+4A>T led to splicing defect,thus both siblings were consequently diagnosed with CS.The symptoms of them were less severe and slowly progressed compared to the most reported CS patients,and the psychiatric symptoms were firstly reported in patients with ERCC6 mutations.Genotypephenotype correlation analysis indicated that affected individuals with splicing mutations were more likely to exhibit a milder form of CS(type Ⅲ)than non-splicing mutations.Conclusion: We established a genetic diagnosis screening system for ARCA patients.Genotype frequency distribution of ARCA in China is depicted.Our findings expand the genetic and phenotypic spectrum of ARCA and related diseases(such as PLAN and CS),demonstrate the high efficiency and reliability of WES combined with CNV analysis in the diagnosis of suspected ARCA,and emphasize the importance of complete bioinformatics analysis of WES data and comprehensive evaluation of clinical information for accurate diagnosis.