Next-Generation Sequencing Identifies Pathogenesis And Diagnostic Strategy Of Intellectual Disability

Background: Intellectual disability(ID), also referred to as mental retardation(MR) or developmental delay(DD), is characterized by obvious limitations of cognitive and social adaptive functions and is diagnosed before 18 years old. DD is used for children younger than 5 years old. The score of intelligence quotient(IQ) can assess cognitive function. Patients with lower IQ(?70) are regarded as cognitive impairment. ID is serious social problem. The general population incidence of ID approaches 1%~3%. Male patients to female patients ratio is 1.4~1.6:1. Clinical manifestations of ID vary from cognitive limitations to multiple system disorders. The etiologies of ID are complex, including genetic functions and environments. Genetic factors are responsible for 2/3 ID patients, including chromosome abnormalities, gene mutations, and congenital diseases. Most researchers apply conventional techniques to ID etiology, including karyotype analysis, PCR-Sanger, fluorescence in situ hybridization(FISH) and multiplex ligation-dependent probe amplification(MLPA). However, more than half of cases remain undiagnosed. Neurological disorders, such as ID and Autism Spectrum Disorder(ASD), are related to copy number variants(CNVs). The methods of array comparative genomic hybridization(aCGH) and array-based single nucleotide polymorphisms(SNP array) are applied to find copy number variants(CNVs) in the human whole genome. However, genetic variants without CNVs cannot be found. Next generation sequencing rapidly develop in the medical field because its efficiency, specificity and sensitivity. It can detect point mutation, insertion and deletion(InDel). Some foreign researchers study the etiology by next generation sequencing. Point mutation plays an important role in ID. In recent years, there are several reports about single gene mutation. Nobody apply next- generation sequencing in large-scale etiology study of ID. Our innovative application of targets region gene enrichment and sequencing analyze the sequences of 384 ID genes, including point mutations, deletions, insertions, and common CNVs. It is difficult to diagnose ID because of complex causes and various symptoms. It is particularly urgent to develop an effective diagnostic strategy of ID.Objective: The aim of this study is to study causes and genetic mechanism and provide theoretical basis for genetic consultation, prenatal diagnosis, treatment and outcome. The research is arming at establishing simple and effective diagnosis process by etiology analysis of 160 ID patients and select right cytogenetic and molecular genetics methods according to clinical manifestations and associated symptoms.Methods: Non-genetic ID can be diagnosed by collecting detail medical history and taking both physical and laboratory examination. Regular genetic methods, including karyotype analysis, inherited metabolic screening, abnormal CGG copy number of FMR1 gene, and MLPA, were performed in unexplained ID patients. Next- generation sequencing and target region gene enrichment and sequencing were used to explore the etiology of 40 patients without clear diagnosis by sequence analysis. We analyzed the sequence data, including nucleotide polymorphisms(SNPs), nucleotide insertion, nucleotide deletion, and CNVs. Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA) were applied to verify these results and analyze origin.Results: Eighty-four ID patients were caused by non-enviromental factors in 160 patients. There were 2 Down's syndrome(2/84, 2.38%), 1 methylmalonic aciduria(1/84, 1.19%) and 7 subtelomeric disorders(7/84, 8.33%). We choose 40 undiagnosed patients and analyzed sequences of 384 ID genes by target region gene enrichment and sequencing. Sanger sequencing and MLPA were used to confirm the results. Two patients(2/40,5%) carried CNVs including 7q11.23 and 22q11.21 deletions. Twenty patients(20/40,50%) carried causative mutations, including 22 missense mutations, 2 splicing mutations, and 2 InDels. Positive rate was 55%. Twenty-three mutations were firstly reported. MutationTaster predicted these mutations were disease-causing. Diagnostic process was established by causes and genetic analysis to 160 cases and it can offer rapid diagnosis for more patients.Conclusions: Our results show de novo gene mutations are major causes of ID. Target region gene enrichment and sequencing is an effective and quick method and can be applied to study etiology and genetic mechanism. The results can help clinical diagnosis and offer scientific genetic counseling.