The Genetic Screening Method Establishment And The Common Pathogenic Mutation Distribution Study On Familial Hypercholesterolemia Population From 16 Chinese Provinces

Objective:1.Homozygous Familial Hypercholesterolemia?Ho FH?is an extremely serious disorder of lipoprotein metabolism caused by genetic mutations,which can lead to early-onset coronary heart disease and life-threatening in children or adolescents.As far as the current research on heterozygous FH is concerned,the genetic background of Chinese FH patients is very different from other regions and races,but there is currently no large-scale systematic study of Chinese Ho FH patients.This study aimed to study and explore the clinical characteristics and genetic background of the only large sample Ho FH cohort in China.2.The genetic spectrum of familial hypercholesterolemia?FH?remains unclear,especially in northeastern China.Thus,the aim of this study was to clarify the FH genetic spectrum and identify specific characteristics of FH patients in this region.3.The early diagnosis of FH patients is crucial for the prolongation of survival and improvement of quality of life in this population,and the FH patients who have been diagnosed early and treated appropriately in China is extremely rare.Detection of suspected FH patients by cholesterol levels,followed by genetic testing will help China's economy to effectively screen and diagnose FH.The method of capturing enrichment or amplicon in the target region and then performing NGS is cost-effective,but as a screening method for FH,a disease with a low incidence rate,the cost is still too high and it is difficult to carry out in a wide range.According to the current status and the bottleneck of FH gene screening in China,this study intends to establish a two-stage FH gene detection method to reduce the cost of detection,and try to establish a feasible clinical laboratory FH genetic screening test method suitable for China's national conditions.Methods:1.A total of 63 FH-related individuals were enrolled in the study,including 48 Ho FH patients from 45 Ho FH families and 14 He FH patients from 7 He FH families,and 1 healthy Ho FH relative.All registered individuals are required to provide a complete medical record,including medical records previously seen in other hospitals.Serum samples were collected from patients and tested for blood lipid levels using the Hitachi 7600-020 Automated Analyzer?Japan Hitachi?in accordance with the daily testing procedures of the hospital's laboratory.Genomic DNA were extracted from whole blood samples and libraries were prepared using the Sure Select XT Custom 0.5-2.9Mb Kit library and sequenced using the Hiseq 4000 sequencer?illumina,USA?according to the official standard protocol for double-ended 150 bp sequencing.The study used ANNOVAR for annotation,and obtain information such as mutation location,variation type,and conservation prediction through various databases such as Db SNP,1000 Genome,Ex AC,CADD,and HGMD.Amino acid change information is obtained by gene transcription annotation databases?such as Concesus CDS,Ref Seq,Ensembl,and UCSC?.All mutation sites were analysed using in silico methods,such as SIFT,Polyphen2_HDIV,Polyphen2_HVAR,Mutation Taster,FATHMM,CADD,LRT,and gerp++gt2,for function and pathogenicity prediction.All suspected pathogenic sites were classified for pathogenicity according to the AMCG Guidelines?2015?.2.The family history,personal medical history,and lifestyle habits of two patients clinically diagnosed with homozygous FH were recorded,and DNA samples of the patients and their relatives were subjected to a newly designed next-generation sequencing panel using an Illumina Miseq platform.Detected variants were annotated and functionally predicted with in silico algorithms,and protein structures were modeled.3.According to the genetic information of hereditary lipid metabolism diseases,24 target genes were selected.A customized FH genetic screening panel was designed and optimized using Design Studio for testing the exons and UTR regions of these 24 genes.A total of 10 frozen blood samples were collected from FH patients who were recruited by Anzhen Hospital and tested with NGS sequencing for the mutation sites they were carrying and then verified by Sanger sequencing.Extracted whole blood genomic DNA was mixed at 1,2,4,6,8,10 samples at same concentrations,and the libraries was prepared using the previously mentioned self-designed Panel,sequenced using the Miseq sequencing platform,and the output data was annotated and analyzed using the Basespace program.The results obtained after the analysis were again verified using Sanger sequencing and then compared with the results obtained by Anzhen Hospital.Results:1.The Ho FH group included 21 males and 27 females with baseline LDL-C levels of 14.11 +/-3.39 mmol/L;He FH included 7 males and 7 females with baseline LDL-C levels of 6.56 +/-1.85 mmol/L.The 55 variant sites were identified as pathogenic variations of class 3 or class 4 or class 5.Among them,18 mutation sites were newly discovered mutation sites,APOB:exon26:c.T10927C:p.S3643P;LDLR: exon3:c.T310G:p.C104 G,exon5:c.G764T:p.C255 F,exon5:c.T727A:p.C243 S,exon17:c.G2487C:p.Q829 H,exon7:c.T973C:p.C325 R,exon8:c.T1129G:p.C377 G,exon10:c.C1427T:p.P476 L,exon10:c.T1532G:p.L511 X,exon3:c.G292A:p.G98 S,exon13:c.G1980C:p.Q660 H,exon8:c.A1178C:p.K393 T,exon4:c.338₃42del:p.E113 fs,exon5:c.720₇21del:p.E240 fs,exon13:c.1899 del C:p.R633 fs,exon11:c.1693₁696del:p.G565 fs,exon4:c.649₆60del:p.217₂20del;PCSK9: exon9:c.C1426T:p.R476 C,which have never been reported before.The 63 individuals enrolled were from 16 provinces,of which 7 LDLR gene variances appeared in multiple provinces,including p.W483 X was found in 5 provinces;p.H583 Y and p.A627 T were found in 4 provinces;c.C1216 A and c.1187-10G>A were found in 3 provinces;p.P685 L and p.C243 S were found in 2 provinces.Moreover,the geographical distribution of the provinces with the same mutation sites was relatively close.2.The patients' cholesterol levels were effectively reduced to 33.8% and 17.2% of the original level under conventional ezetimibe and statin treatment.Two pathogenic mutations,W483 X and the novel mutation W483 G,in low-density lipoprotein receptor?LDLR?were identified.Both patients were heterozygous for the respective mutations.Under a high cholesterol/carbohydrate diet,these mutations could trigger a severe FH phenotype,but both patients responded well to regular medical treatments and dietary control.The W483 X mutation results in a premature stop codon,leading to incomplete protein formation.Although the W483 G mutation results in translation of the complete protein with no apparent structural difference,it still led to a severe FH phenotype similar W483 X.3.The self-designed Panel covers 98% of the targeted area,with a total target length of 123,509 bp.After the sample was mixed and loaded onto the machine,the Q30 base percentage was 72.19-83.26,the average amplicon coverage was 563.4-1668.6,the number of detected SNV was 212-347,and the number of detected base insertions was 41-54.The number of deletions is 79-114.Validation of known mutations using Sanger sequencing revealed a 100% coincidence rate.When the average?per patient?coverage depth exceeds 200 times,the 100% coincidence rate can be achieved,while the result below 100 times is not reliable,and the coincidence rate is only 21.43%.Sample sites between 100 and 200 times have a coincidence rate of 70%-90%.Conclusion:1.This study was the first to describe the clinical features and gene detection of the large-scale Ho FH cohort from 16 provinces of China,which greatly expanded the genetic spectrum of Ho FH in China,confirmed the common pathogenic mutations of Ho FH in China and found China.The distribution of common pathogenic mutations in the Ho FH population is related to geographic location.These results have greatly helped the clinical understanding of patients with Ho FH in China and provided theoretical support for future laboratory screening and detection of FH and the development of future gene-targeted drugs.2.Identification of the novel W483 G mutation expands the genetic spectrum of FH.Both mutations cause a severe FH phenotype under certain conditions,suggesting that W483 is important for LDLR function,highlighting potential targets for genetic screening or drug development.3.Established a targeted NGS sequencing method of the 24 FH-related gene exon regions in this study can assist clinical diagnosis and differential diagnosis of FH-related diseases and assist in guiding the selection of clinical treatment options.4.The experimental procedure of the self-established method needs to be improved and optimized before being applied to the clinic.At the same time,it is necessary to increase the sample size to further study and verify the technical parameters of the method.