Molecular Genetics Study On 56 Families Of Acute Intermittent Porphyria In China

Objective: Porphyria is a metabolic disease result from abnormal enzyme activity during heme synthesis.Acute intermittent porphyria(AIP)is caused by mutation of the gene encoding Porphobilinogen deaminase(also called Hydroxymethylbilance synthase).AIP is an autosomal dominant genetic disease which is rare in clinical practice and easily misdiagnosed and mistreated.At present,there is little research on it in China.Thisarticle aims to find out the characteristics of HMBS gene mutations in Chinese AIP patients,summarize the gene mutation rules,and explore its possible functional hazards.Method:The AIP patients who participated in the study were all from the porphyrin care center.Questionnaires were used to collect general patient data(including gender,age,history of marriage and childbirth,history of smoking and drinking,etc.),causes of morbidity,frequency of morbidity,etc.Venous blood was collected,and the second-generation sequencing and one-generation verification methods were used to find the gene mutation sites.The spatial structure prediction and subcellular localization of the proteins encoded by the four new mutations(c.597 dup C,c.673＿674del,c.653G>A,c.1045＿1046del AA)found in this study were performed.He La cells were transfected with wild-type and mutant HMBS plasmid containing FLAG tag.After transfection for 24 hours,immunoglobulin staining was used to detect the localization of mutant protein in cells.The amino acid sequences of HMBS wild type and each mutant were input into SWISS-Mode website for homology modeling,and PDB-Viewer software was used to analyze and compare the tertiary structure changes of the mutant proteins.At the same time,reference to the relevant literature for functional prediction of partial mutations were reviewed.Result:General informations: In this study,there were 48 females and 8males.The age of onset fluctuated between 17 and 38 years old.The common causes of women was menstruation,followed by fatigue,mood swings,etc.Men were the first to suffer from fatigue.Gene detection results: A total of 56 AIP families were included in the study.Genetic mutations revealed 35 gene mutations,14 of which were new mutations not reported in the literature before.Among the 35 gene mutations,14 were missense mutations(40%),8 were splicing mutations(23%),9 were frameshift mutations(26%),and 4 were nonsense mutations(11%).In this research,c.673C>T(R225X)(13%),occurring in 7 families,c.517C>T(R173W)(11%),occurring in 6 families,c.973C>T(p.R325X),(5%),occurred in 3 families,c.267-1G>C,(5%),occurred in 3 families,c.76C>T,c.77G>A,c.331G>A,c.445C>T,c.806C>G,occur in two families,c.92C>T,c.88-1 G>C,c.160+3G>T,c.181G>A,c.229T>G et al,these 25 mutations occurred in one family.The most frequent occurrences of mutations were exon 12(9 gene mutations accounted for 31%)> Exon 10(4gene mutations accounted for11%)>3,5,7,15 exons,introns 4 and 14(two gene mutations occur)> exons 8 and 14,introns 6,7 and 12(one gene mutation occurs).Subcellular localization: None of the four new mutations affects the subcellular localization of proteins.After transfection of the plasmid,most HMBS was expressed in the cytoplasm,and the wild type was relatively evenly distributed in the cytoplasm,while the mutant HMBS was prone to form obvious aggregation in the cytoplasm.Spatial structure prediction results: This study found no significant changes in the spatial structure of the protein encoded by c.653G>A(P.G218E).c.597 dup C caused amino acid coding to change to position 200,and terminates at the 8th amino acid after the change,c.673＿674del caused amino acid coding to start change at position 225,and terminates early at 232,both Mutants lack more amino acids and therefore had a greater impact on enzyme structure.The new mutation c.1045＿1046del A A resulted in a change in amino acid coding to position 349 and premature termination at position 358,which lacked fewer amino acids and therefore had a relatively little effect on the spatial structure of the enzyme.Conclusion:The most common exons in this study were No.12(31%)> No.10(19%),and exon 7 coexisted with other exons in the third place,which was basically consistent with foreign countries.c.517C>T is the second commonmutant genotype in this study and the most common mutation in many countries.The difference is that c.673C>T is the most common mutant genotype in this study but has not been reported as the most common mutant genotype in foreign countries.