Clinical Application Of Next-generation Sequencing Technology In Diagnosis Of Hereditary Ataxia

BackgroundHereditary ataxia(HA)is a large group of hereditary nervous system degenerative diseases with high clinical and genetic heterogeneity,high morbidity and mortality,accounting for approximately 10%?15%of CNS hereditary diseases.HA is classified as autosomal dominant cerebellar ataxia(ADCA),autosomal recessive cerebellar ataxia(ARCA),X-linked hereditary ataxia and ataxia with mitochondrial disorders.Prevalence of ADCA is about 1/100,000-5/100,000 in the general population,the incidence of ARCA is about 3/100,000 in the general population.In China,ADCA are the mostly reported in the literature,of which spinocerebellar ataxia type 3(SCA3)is the most common,accounting for 60%-70%of SCA,however,other hereditary ataxia were reported less.Friedreich ataxia is the most common recessive ataxia in the world,but in our country,there is no friedreich ataxia confirmed by genetic testing.ADCA often has obvious family history,early onset phenomenon,combined with genetic testing,not easily be wrongly diagnosed,missed diagnosis,but ARCA,X-linked hereditary ataxia and mitochondrial syndrome with ataxia often show no family history,is difficult to diagnose.There are not many reports about ARCA,X-linked hereditary ataxia and mitochondrial syndrome with ataxia at present,and there is no epidemiological data in our country.However,China has a large population base,the number of patients should be not small.With the development of genetic testing techniques,more and more cases are reported and some HA can be treated with diet and medication,such as coenzyme Q10 deficiency,ataxia with vitamin E defriciency,refsum disease,cerebrotendinous xanthomatosis and so on.Therefore,a precise molecular diagnosis is very important.Prior to the advent of next-generation sequencing,genetic diagnosis of ataxia relied on capillary electrophoresis fragment analysis,TA cloning sequencing,and direct sequencing to detect polyglutamine amplification of SCA,FA and a few other types of ataxia.In 2005 Roche developed Genome Sequencer 20 System,the first high-throughput genomic sequencing system which based on pyrosequencing principle,with the first NGS sequencer to appear,followed by Illumina's NGS system,Genome Analyzer,and ABI developed The SOLiD system,and the BGISEQ-500 sequencing platform from BGI.NGS technology has developed rapidly in these ten years and is widely used in the diagnosis of hereditary diseases.With the development of pathology,biochemistry and imaging techniques,the molecular diagnosis of HA has gradually become feasible.However,there are many genetic ways and pathogenic genes in HA.Therefore,how to select genetic testing methods and detect genes is the key step.Next generation sequencing technology can detect many kinds of genes at the same time,but it also brings a large number of mutation data,How to interpret these data also directly affects the molecular diagnosis.In order to set standards and guidelines for the interpretation of sequence variants,in 2013,the American College of Medical Genetics and Genomics combined with the Association for Molecular Pathology(AMP),the American Association of Pathologists College of American Pathologists,(CAP)set up a working group to revisit and revise standards and guidelines for interpretation of sequence variants.The new guidelines recommend the use of specific standard terminology-"pathogenic," "likely pathogenic,""uncertain significance," "likely benign," and "benign" to describe Mendelian disease-related sequence variants.In addition,the new guidelines also describe how to classify variants into five-level classification based on criteria using typical types of variant evidence such as population data,computational data,functional data,segregation data).The publication of this guideline regulates the interpretation of sequence variants.The major genetic testing companies present the genetic test results and classify the mutation according to the standards and guidelines.Starting from January 2013,department of Neurology,Nanfang Hospital conducted genetic tests on patients with ataxia to explore the application of next-generation sequencing in the diagnosis of hereditary ataxia.ObjectiveThe purpose of this study is to:1.Establish a standardized genetic ataxia diagnosis process;2.Provide genetic analysis to patients with clinically suspected "ataxia" and genetic counseling to the family;3.Explore how to select suitable genetic testing methods and detect appropriate genes for ataxia patients;4.To analyze the relationship between clinical phenotype and genotype;5.To explore how to further interpret the variant of uncertain significance(VUS)in the next-generation sequencing test reports.MethodFrom January 2013 to December 2016,31 patients with clinical diagnosis of hereditary ataxia in Department of Neurology,Nanfang Hospital,Southern Medical University were enrolled in this study.According to the patient's medical history,physical signs,biochemical tests,physiological,pathological and radiological findings,we obtained the patient's clinical phenotype.The first step is excludes non-hereditary ataxias,including degenerative ataxias(eg,multiple system atrophy,primary late-onset cerebellar ataxia)and acquired ataxia(such as ataxia due to stroke,poisoning,immune-mediated,infection,trauma,cancer,etc.);The second step is to get complete family history;Thus,we can speculate probable disease classification,genetic patterns,involved genes.According to the different type of sequence variants,we choose different genetic testing techniques for genetic testing.Genetic test report will be further analyzed as following:1)Patient with the negative results will be considered for changing the genetic testing methods or to expand the scope of genetic testing.2)The variant of uncertain significance(VUS)needs to be further verified by the genotype-clinical phenotype match,protein function prediction and pedigree segregation,even cell function study or animal experiment,to upgrade or downgrade the VUS,and finally provide molecular diagnosis and genetic counseling.Results19 of 31 patients with suspected clinical diagnosis of hereditary ataxia in this study were confirmed by genetic testing,the age of onset range from 8 to 54 years old,of whom spinocerebellar ataxia type 3(SCA3)was the most common,9 patient,3 patient were diagnosed with episodic ataxia,2 cases were diagnosed with spinocerebellar ataxia type 2(SCA2),2 cases were diagnosed with sialidosis type I,1 case was diagnosed with ataxia-oculomotor apraxia-2(AOA2),1 case was diagnosed with spinocerebellar ataxia type 14(SC A14),and a rare case was diagnosed with Mohr-Tranebjaerg syndrome.Five variants of uncertain significance were identified in the diagnosed patients.Another 7 cases were identified with uncertain significance variation,5 cases were negative.Conclusions1.We had established a genetic testing service platform to standardize the hereditary ataxia genetic diagnosis process.2.HA positive diagnosis rate reached up to 61.3%,in which,SCA3 accounted for 47.4%.Therefore,capillary electrophoresis analysis was the first choice of patients with family history and early onset.This study diagnosed other diseases and pathogenic genes that are not traditional hereditary ataxia and enriched the clinical phenotype and genotype of ataxia.3.Among all the detection methods,the usage frequency of next generation sequencing was the highest,accounting for 52.9%,the capillary electrophoresis is 35.3%.4.Next-generation sequencing technology can detect multiple genes at the same time.With many variants of uncertain significance were identified,it is important to further interpret the significance of the cause of the disease.5.Expanding sample size,multicenter studies,and data sharing will help the interpretation of uncertain significance variants.