A Novel Mutation In ATRX Causes Alpha-Thalassemia X-Linked Intellectual Disability Syndrome In A Han Chinese Family

Background and Objective:Alpha-thalassemia X-linked intellectual disability syndrome（ATR-X syndrome）is a rare X-linked dominant disorder characterized by intellectual disability,developmental delay,characteristic facies,seizures,gastrointestinal dysfunction and genital anomalies.Additionally,some patients haveα-thalassemia.ATR-X syndrome is caused by mutations in the ATRX gene,which is located on Xq21.1,containing 37 exons and spanning approximately 280kb.The encoded ATRX protein contains an ATPase/helicase domain,belonging to the SWI/SNF family of chromatin remodeling proteins.It participating in a wide range of biological functions:transcriptional regulation,DNA repair and chromosome segregation.ATRX protein is a heterochromatin interacting protein.The proteins interacting with ATRX include MeCP2,HP1 and Daxx.The abnormal expression of ATRX gene affects the protein interaction and leads to intellectual disability.One hundred and twenty-seven disease-causing mutations have been reported,including non-sense mutations,deletions,insertionsand splicing variants.Here,we investigated a Han Chinese family with ATR-X syndrome and detected a novel alternative splicing variant through whole-exome sequencing（WES）.We studied the function and potential pathogenic mechanism of the mutant gene and found that the novel mutation resulted in haploinsufficiency of the ATRX gene,which caused ATR-X syndrome.Materials and methods:a pedigree with ATR-X syndrome was collected,with two generations.The proband was a boy aged 5 years and 8 months old.The clinical manifestations were consistent with the characteristics of ATR-X syndrome.Methods:Clinical examination and gene function analysis were performed on family members.The clinical test included blood routine,hemoglobin electrophoresis,sex hormone and growth hormone.Function analysis experiment included WES,Sanger validation,reverse transcription and real-time fluorescence quantitative PCR analysis,product of real-time fluorescence quantitative PCR sequencing and Western blot.Bioinformatics Analysis:Mutation Taster was used to predict the genetic mutation effects,and the analysis of 5’ splice site score was estimated with MaxEntScan.Changes in amino acid sequences caused by nucleotide mutations were estimated with Mutalyzer.The threedimensional structures of wild type and mutant ATRX protein were predicted with I-TASSER.Results:The proband presented with severe intellectual disability,developmental delay,characteristic facies and cryptorchidism.The clinical results showed that the proband had decreased mean corpuscular volume and testosterone.A novel hemizygous duplication mutation in the ATRX gene in a splice site between exons 3 and 4,NM₀00489:c.189+1dupG,was identified with WES in the proband.Sanger sequencing confirmed that the mutation was inherited from his mother,who carried a heterozygous mutation,while his father was not affected.The level of mRNA expression showed that ATRX gene transcription decreased due to the mutation（P<0.05）.Sanger sequencing with the Quantitative Real-Time PCR product containing a G base inserted between bases 189 and 190.Bioinformatics analysis indicated that the splicing region where the mutation was located is highly conserved and the variant was damaging,producing a truncated protein due to the premature translation of a stop codon.Conclusion:A novel mutation in ATRX was found in this pedigree and was confirmed to be pathogenic through functional studies.Our research expanded the spectrum of ATRX gene mutations,providing a precise diagnosis and a basis for genetic counseling.