Molecular Detection Of Three Common Inborn Errors Of Metabolism In Newborn Screening

Newborn screening?NBS?is an important preventive measure in the field of public health.Inborn errors of metabolism?IEM?is an important part of the newborn screening program.Due to it s harm to the children's health,Early diagnosis,timely and correct treatment can largely avoid patient death,reduce disability and improve the prognosis effectively.However,most of IEM do not have typical features,and thus brings great difficulties to their diagnosis.The detection of causative gene mutations is an important method for the diagnosis of IEM diseases.This thesis aims to establish a rapid detection methods for methylmalonic acidemia?MMA?in the abnormal metabolism of organic acids,citrin deficiency?CD?in the abnormal metabolism of amino acid and primary carnitine deficiency?PCD?in the abnormal metabolism of fatty acid metabolism abnormalities.Real-time PCR was used to establish the detection methods.The first chapter starts with introduction of the clinical manifestations and classification of congenital metabolic abnormalities and their common detection methods.This is followed by description of the main purpose and standard of newborn screening,the current status of newborn screening in China and abroad.Finally,the main content and significance of this thesis are given.The second chapter focuses on the detection of high frequency mutations of MMACHC and MUT,the two main pathogenic genes of MMA,based on multi-color melting curve analysis technology.The established detection system of MMACHC mutations and MUT mutations could detect as low as 50 pg/reaction human genomic DNA?gDNA?.We detected 376 random samples and 2 of them was found positive and 9 were MMACHC mutation carriers.The mutation types were:c.609G>A?4/376?,c.658 660delAAG?3/376?,c.315C>G?1/376?,c.567dupT?1/376?,c.394C>T?1/376?,c.569T>G?1/376?and c.641 G>A?1/376?.Among them,c.569T>G and c.641 G>A were new mutations and had not been reported before.Also,we detected 376 samples for the MUT mutations,four mutation types,i.e.,c.1677-1G>A?5/376?,c.1280G>A?3/376?,c.2080C>T?1/376?and c.729-730insTT?1/376?.One sample was a compound mutations of a c.1677-1G>A and c.1280G>A.The third chapter describes the establishment a detection system for SLC25A13 high-frequence mutations associated with CD.The result showed that the analytical sensitivity of the SLC25A13 detection system was 50 pg human gDNA/reaction.The system was used to test 851 random samples,3 mutations were detected,i.e.,c.851₈54de1GTAT?7/851?,c.1638 1660dup?2/851?and IVS16ins3kb?2/851?.These results were concordant with Sanger sequencing.In chapter 4,we established a detection system for SLC22A5 mutations,which were associated with PCD.The analytical sensitivity was 50 pg human gDNA/reaction.Among 749 random samples,we detected one c.1400C>G homozygous mutant,two c.760C>T/c.1400C>G compound mutations,ten c.1400C>G carriers,one c.428C>T carrier and one c.752A>G carrier.The last one was a new mutation.In total,four mutation types were detected.The results fully agreed with Sanger sequencing.