| BackgroundSpinocerebellar ataxias SCAs(Spinocerebellar ataxias SCAs)is a group of autosomal dominant hereditary neurodegenerative diseases with ataxia as the main clinical feature.The lesions mainly affect the spinal cord and cerebellum,but also affect the brain stem,thalamus,basal ganglia,cerebral cortex and peripheral nerves.SCA36(Spinocerebellar ataxias 36)is a relatively rare SCA subtype worldwide.The frequency of autosomal dominant ataxia in mainland China is about 1.6%.The main clinical symptoms are ataxia,hearing loss,dysarthria,motor neuron involvement,etc.The disease is caused by abnormal amplification of GGCCTG hexanucleotide in intron 1 of NOP56 gene.It is an exception that the repeat number is more than 15.Also,the expanded alleles usually contained between 650 and 2500 repeats.Currently,the Triplet repeat-primed Polymerase Chain Reaction(TP-PCR)screening combined with Southern blotting is commonly used to diagnose the disease.However,the above method has great limitations,such as heavy labor,radioactivity,and inability to describe the nucleotide sequence composition of the repeat expansion region.So,we tried to find new sequencing methods to solve these difficulties.Single molecule real time(SMRT)sequencing is a third-generation sequencing technology based on Pac Bio platform with a long-read length,high accuracy,uniform coverage,no PCR amplification and no GC preference.Because of the low incidence rate and limited diagnostic tools,it is important to report clinical features of SCA36 and explore the SMRT sequencing to diagnose the SCA36.ObjectiveIn this study,we collected and compiled a Han Chinese family pedigree with three generations of SCA36,and then described the clinical features,electrophysiology and imaging characteristics of the family.We applied SMRT technology to detect and analyze the base composition of repeat expansion sequences to explore the correlation between genotypes and phenotypes,and to conduct genetic counseling on SCA36 for family members with reproductive requirements.Further,we believe that these findings can deepen our knowledge of SCA36 and may lay the foundation for revealing the genetic mechanism,diagnosis,and treatment of this rare disease.Methods1.We collected the clinical features and draw the family pedigree of ataxia.Also,we performed the scales,pure tone audiometry and electrophysiological examinations of the family proband and other patients.The brain MRI and DTI image scanning were performed to measure and analyze by the pedigree members and the health control.FA values of multiple intracranial sites(cerebellum,midbrain,thalamus,bulbar medulla)of the family members.2.The peripheral blood was collected from the family members and DNA was extracted.The SCA type of the family was determined by TP-PCR.Also,we performed Whole Exome Sequencing(WES)of the proband and another family member,ruling out the possibility of other types of ataxic in the family.The SMRT sequencing was performed on the DNA of the proband and two patients in the family,and the resulting data were screened and assembled to obtain different kinds of reads.A detailed statistical analysis of reads across the repeat expansion region should be focused on,including the length of the d(GGCCTG)n,the repeat number of GGCCTG,the motif of composition and distribution.The correlation between genotype and phenotype of the family was explored based on the clinical symptoms of the patients.Results1.The ataxia feature of were found in 11 patients with SCA family.There were 7 in the second generation,with onset age ranging from 39 to 50 years.The clinical characteristics of this family are mainly late-onset ataxia,motor neuron injury,hearing loss,dysarthria,accompanied by mood disorders and sleep disorders.Also,The electrophysiology indicated the motor neuron injury,hearing loss,especially the high frequency threshold in patients.The brain MRI indicated cerebellar atrophy in patients.The DTI analysis manifested that FA values in the left upper and lower cerebellar feet were decreased compared with health controls.2.TP-PCR and capillary electrophoresis were performed on 8 individuals in the family,indicating abnormal amplification of NOP56 gene GGCCTG TP-PCR and capillary electrophoresis were performed on8 individuals in the family,indicating abnormal amplification of NOP56 gene GGCCTG hexanucleotide sequence of 7 individuals and TBP gene CAG sequence of 5 individuals.The abnormal amplification of NOP56 gene and TBP gene were 4 members.The number of CAG repeats in TBP gene associated with SCA17 was 43,with incomplete penetrance.WES was applied for the proband to rule out the SCA causing by the exons.We determined the family was SCA36,based on the clinical symptoms of the family and mode of inheritance not conform to the SCA17.3 Combining SMRT sequencing data with age of onset,course of disease and SARA(Scale for the Assessment and Rating of Ataxia)score,it suggested that the average length of full＿d GGCCTGn subreads and the average GGCCTG repeat number were negatively correlated with the age of onset,and positively correlated with the progression of disease.4.It suggested that SCA36 patients had interruptions in the repeat expansion region from the analysis of assembled reads,and the motifs varied around the core motif of GGCCTG.5.We selected the full-length schematics with the highest GGCCTG repeat number from each of the three patients for analysis based on the Practical Extraction and Report Language,which showed no obvious regularity in the interruptions.Conclusions1.This study suggested that the main clinical features of this pedigree are late-onset ataxia symptoms,dysarthria,hearing loss,affective and sleep disorders.In addition,sleep disorder symptoms for the first reported in this paper.2.We firstly applied the Pac Bio SMRT sequencing to the SCA36 patients.The results indicated that there’re interruptions in the repeat expansion region of NOP56 gene,but the motifs varied around the core motif of GGCCTG3.The SMRT sequencing technology has broad application prospects in similar dynamic mutational diseases. |
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