| ObjectiveInherited metabolic diseases(IMD)are a wide variety of diseases that are difficult to diagnose,as well as extremely harmful.Taking advantage of Tandem Mass Spectrometry(MS/MS),which is a kind of high-throughput technology,to detect multiple diseases through one experiment during newborn period,is an important means of prophylaxis and treatment for the inherited metabolic diseases.In this research,we adopt MS/MS assay for the IMD newborn screening and Next Generation Sequencing(NGS)for post-processing methods to confirm the gene diagnosis of children with IMD.We explored the clinical value of combined application of MS/MS assay and NGS in the screening as well as diagnosis of IMD,and also illuminated the features of native cases of IMD and the genetic characteristics of the pathogenic genes.Besides,we preliminarily investigated the influences of interference factors about the screening and diagnosis of IMD,to improve the level of quality control.Methods1.Taking 50844 newborns given birth in Changzhou Maternal and Children Healthcare Hospital from April,2015 to May,2018 as study objects,we started the research by using the standard process of IMD screening and diagnosis.We adopted non-derivatized MS/MS assay to test the concentration of 11 types of amino acids,31 types of acylcarnitines items and succinylacetone in the dried blood spots(DBS)specimens,we could screen out 27 types of IMD by using the assay.2.Using target gene sequencing,the gene panels contain 306 genes to detect 286 diseases,and performed genetic diagnosis in suspicious positive newborns.Meanwhile,we applied Sanger's sequencing technique to identify the mutation sites of pathogenic genes and find out the hereditary modes of their family.3.By analyzing clinical statistics thoroughly,we especially focused on four core quality control indexes: Positive rate of preliminary screening,Positive rate of screening,Positive rate of the called-back specimens and Positive predict value.Then,we discussed the influence of interference factors(gender,delivery modes,regions,seasons,birth weight,etc.)on the core quality control indexes,and tried to find measures to improve the quality of IMD detection.Results1.Among the 50844 newborn cases,650 positive cases were found after retesting of all the original DBS specimens.As a result,595 of them were successfully called back to recollect the DBS samples.We found that there were 54 cases remain positive after testing by MS/MS assay.According to the categories of the suspicious positive cases,different suspicious newborns received particular differential diagnosis and genetic diagnosis.And finally 19 cases were confirmed to be the IMD,involving 11 types in total.These cases consisted of 7 amino acid disorders,including 4 phenylketonuria(PKU),1 citrullinemia type ?(CTLN ?),1 isolated hypermethioninemia(MIM)and 1 Citrin deficiency;7 organic acid disorders,including 3 glutaric acidemia ?(GA-?),2 3-methylcrotonyl-coenzyme A carboxylase deficiency(MCCD)and 2methylmalonic acidemia(MMA);5 fatty acid disorders,including 2 short-chain acyl-Co A dehydrogenase deficiency(SCADD),1 primary carnitine deficiency(PCD),1 medium chain acyl-Co A dehydrogenase deficiency(MCAD),and 1 ?-ketothiolase deficiency(MIM).We established the follow-up files for all the 19 confirmed patients,all of them were under treatment according to the ?Clinical Inherited Metabolic Diseases?.2.The gene detection of this research consists of 10 types of diseases,including 3 amino acid disorders,3 organic acid disorders and 4 fatty acid disorders.All of the above diseases were autosomal recessive inherited diseases.Two mutation sites were found in the pathogenic genes in every one of the 15 cases who volunteered to accept NGS genetic analysis.Both of the mutation sites were heterozygous mutation.After querying the data base,we detected 29 life-threatening mutations,but there was still 1 mutation mode which was of unknown clinical significance.The detection rate was 96.7%.It was verified that the mutations were derived from its father and mother by analyzing its family mutation pedigree.3.The statistical analysis demonstrated that seasons and birth weight of the newborns might have effects on the core quality control indexes,especially on the fatty acid disorders(p <0.05),while gender,delivery modes and regions basically had no significant difference(p >0.05).Conclusion1.The native incidence of IMD is 1/2676,which was slightly higher than that reported in other regions.This should claim our attention.2.The application of NGS would contribute to revealing the genetic characteristics of diseases and finding the mutation sites of the pathogenic genes,while the Sanger's sequencing techniques could be conductive to confirm the hereditary modes of diseases.The combined application of these two techniques would help us to establish the gene mutation maps of the pathogenic genes.3.Quality control indexes of IMD might be related to newborn birth weight and seasons,but might have no relationship with gender or delivery modes.Eliminating interference factors would help to optimize quality control. |
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