Case Analysis Of Inherited Metabolic Diseases In Shandong Peninsula And Screening Of Hot Spot Pathogenic Gene Mutation Carriers

Part 1 Selection of high frequency pathogenic mutation and gene test in methylmalonic acidemiaObjectives: Methylmalonic acidemia is the most common organic acidemia in our country.The etiology of the disease is complex and mutiple gene mutation can cause the deficiency of methylmalonyl coenzyme-A mutase or metabolic disorder of its co-enzyme cobalamin which results in the onset of affected children.Biochemical phenotypes and gene spectrum vary from different countries and regions.As the incidence of methylmalonic acidemia in Shandong Peninsula is significantly higher than that in other regions,we aim to screen for carriers of methylmalonic acidemia in our area to identify the cause of high incidence of the disease.The main goal of this study are as follows: firstly,getting the gene spectrum of methylmalonic acidemia in our area through collectting patients with methylmalonic acidemia who had diagnosed by gene detection in our center compared with reported literature to select common genes and mutations in our region.The selected genes and mutations are used for the panel of carrier screening in Shandong Peninsula.Secondly,specific primers were designed according to selected mutations and PCR conditions were optimized.We applied a method of multiplex PCR combined with next generation sequencing for carrier screening.The aim of this paper is to establish and evaluate technical routes for gene detection and large-scale population screening of high frequency gene mutations suitable for the characteristics of the region,and to collect 4 positive samples for methylmalonic acidemia and 6 normal samples to identify scientific nature and accuracy of the gene test.Methods: According to 35 methylmalonic academia patients' spectrum and associated literature of our country to choose common genes and mutations in Shandong Peninsula.PC R primers were designed by primer z software according to choosed variants of MMACHC and MUT.4 cases of children with methylmalonic acidemia and 6 cases of normal subjects were collected for the verification coverage and accuracy of the test of screening for large-scale population carriers with methylmalonic acidemia in Shandong Peninsula.The library was conducted by two steps PCR: one for specific site amplification and another for adding barcode.We extracted DNA by centrifugal column.Purifing the DNA products of each step of PCR capture by magnetic beads.We evaluated the capture efficiency by Qubit and agarose gel electrophoresis.After mixing,diluting and quantifying the library,the enriched library sequence was read according to the standard procedure.The sequence results were analysed finally.Results: 35 children with methylmalonic acidemia were collected in our center include 31 for MMACHC and 4 for MUT.Combined with literature MMACHC gene and MUT gene were selected for screening.For MMACHC,10 mutations were detected and 8 of them were selected in combination with the literature which are c.609G>A,c.658₆60del AAG,c.80A>G,c.217C>T,c.567₅68ins T,c.482G>A,c.394C>T,c.315C>G.For MUT,5 mutations were detected as c.729₇30ins T,c.1106G>A,c.323G>A,c.2080C>T,c.1631G>A.10 mutations was selected in combination with the literature including c.729₇30ins T,c.1106G>A,c.323G>A,c.1280G>A,c.1631G>A,c.424A>G,c.1741C>T,c.914T>C,c.1677-1G>A,c.2080C>T.We designed specific primers based on the above mutations.During gene test,after two steps of PCR reaction,captured concentrations were 87.2 to 186 ng/?L which were higher than that of 60ng/?L with the average capture concentration of 160.14 ng/?L.The capture effect was satisfactory.Analysing data of sequence,4×coverage and 20×coverage are 100%.The data bases of all were from 23.02 to 32.75 Mb,with an average of 29.55 Mb.The depth of sequencing were from 12123.06 to 17205.88×,which were far more than 100 ×,indicating the accuracy of detection.In 10 samples,the detection of pathogenic mutations and normal samples were accurate,which were consistent with the information of pathogenic gene mutation.Conclusions: 1.The most common gene of methylmalonic acidemia in Shandong Peninsula was MMACHC followed by MUT.2.The most common mutation of MMACHC gene in Shandong Peninsula was c.609G>A,followed by c.658₆60del AAG.The study selected 8 relatively common mutations,which could cover about 90% of mutation on MMACHC gene.3.The mutation of MUT gene in Shandong Peninsula were scattered and there was no obvious advantage of high frequency mutation.The study selected 10 relatively common mutations,which could cover about 45% to 55% of mutations on MUT gene.4.According to the selected high frequency gene and mutation of methylmalonic acidemia,a multiplex PCR combined with next generation sequencing was developed to detect the related mutations.Ten samples were used for gene detection.According to the analysis of captured data and sequencing results,it is shown that the sequencing results of this experiment are satisfactory from the data of data quantity,coverage,and captured efficiency and so on.5.The detection method has the characteristics of high efficiency,accuracy,low cost,short experiment period and little result analysis workload.This method can be used to screen the carrier frequency of methylmalonic acidemia in large-scale population in Shandong Peninsula.Part 2 Carrier screening and analysis of high frequency pathogenic mutation of methylmalonic academia in Shandong PeninsulaObjectives: Methylmalonic acidemia is the most common organic acidemia in our country caused by the deficiency of methylmalonyl coenzyme-A mutase and metabolic disorder of its co-enzyme cobalamin.It accounts for 55-60% organic acidemias in China which is higher than other Asian countries?15-40%?.The incidence of methylmalonic acidemia was 1 in 3,220 to 3,920 in Shandong Province which was significantly higher than that of 1 in 50,000 to 250,000 in the global level.These suggest methylmalonic acidemia is prevalence in our area.Since most of the disease is autosomal recessive genetic disease,this paper speculates that the high incidence of the disease is related to the high carrier frequency of methylmalonic acidemia mutation in population in Shandong Peninsula.The purpose of this study was to investigate the carrier station of high frequency methylmalonic academia mutations in normal newborns in Shandong Peninsula,to provide the first molecular epidemiological data in this area,to explore the relationship between the high incidence of the disease and carrying frequency and provide a theoretical basis for explaining the high incidence of disease.In addition,parts of patients do not have effective treatment which gets high mortality and poor prognosis.Though others are effective for vitamin b12,progressive brain injury and even sudden death can be caused by delayed diagnosis and treatment.Implementing carrier screening not only evaluate reproductive risk,guide reproductive decision and reducing the incidence of disease,but also improve the prognosis in neonatal screening.The purpose of this study also is to find suitable and effective measures of methylmalonic academia carrier screening for prevention and control of methylmalonic academia in this region.Methods: Normal newborns that screened for newborn screening in Shandong Peninsula from 2016 to 2018 were collected.The screening specimens were chose as 13 mm in diameter dry blood filter paper.DNA was extracted from dry Blood Filtration Paper by Magnetic beads.The gene tests were detected by multiplex PCR combined with next generation sequencing and libraries were conducted by two steps PCR: one for specific site amplification and another for adding barcode.We Purified captured DNA products of each step of PCR by magnetic beads and evaluated the capture efficiency by Qubit assay and agarose gel electrophoresis.After mixing,diluting and quantifying the library,the enriched library sequences were read according to the standard procedure if the library was qualified.The sequence results were analysed finally.Statistical software SPSS20 was used for statistical analysis.The results were analyzed and the information of positive variants was counted.Results: A total of 5,006 newborns were screened for high frequency mutation of methylmalonic academia.The main gene of methylmalonic acidemia was screened that MUT was for isolated methylmalonic academia and MMACHC was for combined methylmalonic aciduria and homocystinuria.18 mutations were detected,of which 8 were MMACHC gene and 10 were MUT gene.The detected mutations of MMACHC include c.609G>A,c.658₆60del AAG,c.80A>G,c.217C>T,c.567₅68ins T,c.482G>A,c.394C>T,c.315C>G.The detected mutation of MUT include c.729₇30ins T,c.1106G>A,c.323G>A,c.1280G>A,c.1631G>A,c.424A>G,c.1741C>T,c.914T>C,c.1677-1G>A,c.2080C>T.The quality of carrier screening were as follows: Raw data bases were 50.33±96.78 Mb.Clean data bases were 47.52±92.33 Mb.Of all 18 mutaions can be tested as fraction of target covered with at least 4X,10 x and 20 x were100%.Average sequencing depth on target of all samples varied from 106× to 57825×,average on 11096.70±7220.92×.Of the 18 screening variants,125 out of 5006 carried pathogenic mutations.One hotspot mutation was carried per person,and no one carried two or more mutations.109 people were carrier of MMACHC accounting for 87.2% and 16 people were carrier of MUT accounting for 12.8%.In these 125 individuals 14 mutations were covered and no one carried other 4 mutations.All high frequency mutations of MMACHC were detected and 6 variants were detected on MUT include c.1106G>A,c.323G>A,c.1677-1G>A,c.2080C>T,c.1631G>A and c.914T>C.The overall carrier rate of high frequency mutations in MMACHC carrier screening was 1 in 46.c.609G>A was the most common mutation which accounting for 38.53% of all,followed by c.482G>A and c.658₆60del AAG,which accounting for 16.51% and 12.84% of all respectively.The overall frequency of high frequency mutations in MUT gene screening was 1 in 313.The carrier rate of c.1106 G >A,c.323 G> A,c.1677-1G >A,c.2080 C >T,c.1631G>A and c.914T>C were 0.14%,0.08%,0.04%,0.02%,0.02% and 0.02% respectively.c.1106G>A?c.323G>A were relatively common.Conclusions: 1.Among 5,006 normal newborns in Shandong Peninsula in this study,125 people were carrier of one of 18 hotspot variants and the carrier frequency was 1 in 40.05?2.5%?.Cbl C genotype accounted for 87.2% and MUT accounted for 12.8% in total.2.The hotspot variants of MMACHC screening were carried in 109 individuals and the carrier rate of cbl C hotspot mutations was 1 / 46.Mutations carried by 109 individuals covered all 8 high frequency mutations which we selected.c.609G>A was the most common mutation which accounting for 38.53% of al,followed by c.482G>A and c.658₆60del AAG,which accounting for 16.51% and 12.84% of all respectively.Since only 90% of the MMACHC mutations were screened,the overall carrier frequency of cbl C was estimated to be 1 in 41.16 individuals carried hotspot variants of MUT and the carrier rate was 1 in 313.16 people carrier 6 of mutations which were c.1106G>A,c.323G>A,c.1677-1G>A,c.2080C>T,c.1631G>A and c.914T>C.c.1106G>A?c.323G>A were relatively common.Since only 45% to 55% of the MUT mutations were screened,the overall carrier frequency of it was estimated to be 1 in 156.4.The carrier frequency of high frequency mutation of methylmalonic acidemia in Shandong Peninsula was high,which was the molecular biological basis leading to the high incidence of methylmalonic acidemia in this area.5.Carrier screening for methylmalonic acidemia should be carried out step by step in our region to reduce the incidence of the disease.