Genetic And Clinical Analysis Of Usher Syndrome And Stargardt Disease In Patients Of Southwest China

Background：Hereditary retinal dystrophies （HRD） are a group of hereditary diseases that lead todebilitating visual impairment and in some instances to blindness. HRD are caused bymutations in more than250different genes. Usher syndrome （USH） is the most commonform of deaf–blindness and is characterized by the association of sensorineural hearing loss（SNHL）, visual impairment due to retinitis pigmentosa （RP） and variable vestibulardysfunction. There are three clinical subtypes, and USH1is the most severe form and and ischaracterised by prepubertal onset RP. Stargardt disease （STGD） is known as juvenilemacular degeneration. USH and STGD are two form of HRD that lead to progressiveirreversible loss of vision in childhood, and exhibit remarkable clinical and geneticheterogeneity. Molecular diagnosis is of great significance in revealing the molecularpathogenesis, aiding the prenatal and clinical diagnosis, and helping with the selection ofpatients for clinical stem cell-based therapy or gene therapy trials. However, moleculardiagnosis remains a challenge due to the high phenotypic and genetic heterogeneity in USHand STGD. Sanger sequencing of the individual gene one-by-one remains expensive andtime-consuming. Targeted exome sequencing （TES） based on next-generation sequencing（NGS） has recently been proven to be a powerful, robust, precise and cost-effective tool fordiscovering genetic mutations in large genomic regions. In the present study, we utilisedTES based on NGS to study the genetic defects of five USH and STGD families insouthwest China. We also identified genotype-phenotype correlations between themutations and the observed clinical symptoms.Methods：1. Collecting HRD genetic resources including written informed consent, completingHRD questionnaire, extracting genomic DNA from peripheral blood. Establishing thestandardized paper-based and electronic HRD genetic resources management system. 2. Clinical examinations2.1Comprehensive ophthalmic examinations included best-corrected visual acuity（BCVA） assessment, dilated fundus examination, colour fundus photography,autofluorescence （AF） imaging, optical coherence tomography （OCT）, and full-fieldelectroretinogram （FERG）, pattern electroretinography （PERG） and multifocalelectroretinogram （mfERG）.2.2. The USH patients were additionally subjected to audiologic assessments andvestibular function texts （caloric and rotary chair tests）.3. Molecular genetic methods3.1. Targeted region capture and next-generation sequencing: A sequence capture arraywas designed to capture the coded exons of the103HRD genes, including the12candidateUSH-related genes and5STGD-related genes.3.2. Bioinformatics analysis: SOAP aligner program was used to align the clean readsto the reference human genome. The single-nucleotide variants （SNVs） were firstlyidentified using the SOAPsnp program. Subsequently, we realigned the reads to thereference genome using BWA and identified the insertions or deletions （INDEls） using theGATK program. All SNVs and INDEls were determined using the NCBI database of singlenucleotide polymorphisms （dbSNP）, the HapMap project and the1000Genome Project.3.3. All novel variants were confirmed by polymerase chain reaction （PCR） and Sangersequencing.3.4. Assessment of novel mutations pathogenicity.Results：1. The RD genetic resources system was scientific, standard and reasonable.2. USH and STGD families’ phenotypic determination2.1. The patients in USH-Family1and USH-Family2showed typical and severeclinical features of USH1: delays in sitting independently and walking, profound SNHLfrom birth, and early onset of RP.2.2. The patients in USH-Family3, USH-Family4and USH-Family5exhibitedmoderate to severe SNHL, progressive night blindness and peripheral visual loss withoutvestibular dysfunction, which indicated diagnoses of USH22.3. The patients in different STGD families exhibited complex and diverse phenotypes. The phenotypes in STGD-Family1and Family4were mild: adult onset of adecrease in central vision （20Y）, better preserved BCVAs, type1or type2AF, and normalFERGs （ERG group1）. However, the phenotypes in the other three families were severve:early onset （3Y or6-7Y）, worse BCVAs （below o.1despite two probands being14and9years old, respectively） type2or type3AF; and ERG group3. OCT demonstrated the loss ofinner segment （IS）/outer segment （OS） conjunction partly or completely, which isconsistent with the abnormal AF area.3. Genetic analysis of USH and STGD3.1. The study utilised TES based on NGS technology to study the genetic defects offive Chinese USH families. Through systematic data analysis using an establishedbioinformatics pipeline and segregation analysis,10pathogenic mutations in the USHdisease genes were identified in the five USH families. Six of these mutations were novel:c.4398G>A and EX38-49del in MYO7A, c.988₉89delAT in USH1C, c.15104₁5105delCAand c.6875₆876insG in USH2A.3.2.9pathogenic mutations in the ABCA4gene were identified in the five STGDfamilies. The recurrent mutation c.6118C>T （p.Arg2040Ter） was deteced inSTGD-Familily3and Familily4. Six of these mutations were novel: c.6190G>A（p.Ala2064Thr）, c.1937+1G>A, c.2424C>G （p.Tyr808Ter）, c.1761-2A>G, c.371delG（p.Arg124LeufsX30） and large deletions EX37del.3.3. Sanger sequencing of the candidate mutations verified that these mutationscosegregated with the disease phenotypes in the respective families. All novel variationswere absent from ethnically matched control individuals.3.4. The two truncating mutations or one truncating mutation in combination with thelarge deletions in exons were found mostly to cause more severe disease than non-truncating mutations.Conclusion：1. We successfully established HRD genetic resources system. The standardized HRDsystem is of great importance in the large-scale molecular genetic study.2. It is very important to early and accurate diagnosis of USH1when the childrenexhibited congenital deaf and dumb, the delay in childhood motor development, and poorvision. 3. ERG together with AF and OCT is a powerful and robust non-invasive method.Consistent changes of their characteristics in the retinal function and structure are of greathelp to diagnosis and assessments of STGD.4. We proved that targeted NGS is an accurate and effective method for detectinggenetic mutations related to USH and STGD. Targeted exon sequencing of the103knownRD genes, including12USH-related genes and5STGD-related genes, was sufficient andclinically feasible for the comprehensive identification of genetic defects in USH and STGDpatients.5. We firstly reported6novel USH gene mutations and6novel ABCA4gene mutations.This study expanded the mutation spectrum of USH and STGD and revealed thegenotype-phenotype relationships of the novel USH and ABCA4mutations in southwestChina patients. The identification of pathogenic mutations is of great significance forelucidating the underlying pathophysiology of disaese, aiding the prenatal, early anddifferential diagnosis, and the selection of patients for clinical trials.