| BackgroundMaple syrup urine disease(MSUD)is a rare autosomal recessive inheritance disease.The mortality and disability rate is high and the clinical perniciousness is great.The general incidence rate in the world is 1:1 85000.Maple syrup urine disease is a metabolic disease of branched-chain amino acid(BCAA)caused by functional defects of the a-keto acid dehydrogenase complex(BCKDC)in the mitochondrial matrix of cell.BCKDC includes E1(divided into El a and E1?),E2,E3,BCKD kinase and BCKD phosphatase.Disfunction of each subunit of BCKDC will affect the catabiosis function.In addition,abnormal functions of the enzyme regulating BCKDC can also lead to MSUD.For example,abnormal PPMIK can also lead to MSUD.All the above abnormalities can give rise to accumulation of BCAA and ?-keto acid in higher concentration in blood,urine and cerebrospinal fluid.This hampers the energy metabolism of neuron which causes a series of neurological symptoms.At present,there are identified pathogenic genes including--BCKDHA,BCKDHB,DLD,DBT and PPMIK.According to the onset age,the progression of disease and the responsiveness to thiamine,MSUD can be divided into five types of classic,intermediate,intermittent,thiamine-responsive and E3 subunit deficiency.Classic type of MSUD accounts for majority of cases.The patients with classic MSUD exhibit neonatal onset of the disease and serious clinical manifestations including poor response,frequent convulsions,hypoglycemia and so on.The conditions of MSUD developed rapidly and postnatal death often occurred.Due to the lack of specificity of its clinical manifestations,it is easy to misdiagnose and delay treatment resulting in high mortality or high disability rate after survival.Therefore,early diagnosis,intervention and treatment to improve the survival rate and quality of life are urgent clinical issues to be solved.For a long time,study of gene function and pathogenesis on MSUD are limited by the research model.Induced pluripotent stem cells(iPSC)are acquired by reprogramming with features and functions of embryonic stem cells.iPSC coming from patient as research models of genetic diseases have been widely used in basic study.Because of its no ethical issues,with multiple differentiation potential,and carrying specific genes,they provide a revolutionary technique to study the pathogenetic mechanism of genetic diseases.On the basis of previous screening of genetic metabolic disease,we conducted detailed clinical phenotype,laboratory examination and genetic analysis on seven children with highly suspected MSUD,and found that 5 of 7 children had BCKDHB mutations and 2 of them had BCKDHA mutations.Of them,7 novel mutations were identified which had not been reported previously.In order to further explore the pathogenicity of the new mutations,we collected peripheral blood from a live child and induce into iPSC cells which were further identified and evaluated.Moreover the mutation site of the gene and defective protein expression were validated.The establishment of iPSC cell line from the child with MSUD provides a research model in vitro which laid a foundation for further investigation of MSUD pathogenesis,gene function,drug screening and possible treatment.Section 1 Clinical characteristics and gene mutation analysis of children with MSUDObjectiveTo analyze clinical characteristics,mutation types and mechanism of Chinese patients with maple syrup urine disease,and further summarize the pathogenesis characteristics and explore the relationship between phenotype and genotype of MSUD so as to save lives of children,improve the survival rate and the quality of life.Methods7 children with highly suspected MSUD were detected and clinical phenotype analysis,routine laboratory examination,gene testing and bioinformatics analysis were conducted.Specific methods were as follows.1.Clinical data:collecting the history of present disease,feeding history,personal history,family history,etc.Meanwhile,conducting detailed physical examination.2.Lab testing:Including blood routine test,biochemistry test,blood-gas analysis,cerebrospinal fluid routine examination,as well as blood amino acid and urine organic acid screening etc.3.Imageological examination:CMRI or cranial ultrasound examination.4.EEG examinations:amplitude integrated EEG.5.Molecular genetic examination5.1 High-throughput sequencing was performed on seven children using exome targeted capture combined with next generation sequencing.Exons and adjacent introns(50bp)of MSUD-related genes(BCKDHA,BCKDHB,DBT,DLD,PPMIK)were analyzed intensively.5.2 Bioinformatics analysis was performed.NextGene V2.3.4 software was applied to compare the sequencing data with UCSC hg 19 human reference genome sequence and coverage and sequencing quality in the target region were evaluated.The mutation was filtered according to strict screening criteria and the mutation information was searched through HGMD,Clin Var and OMIM databases.UCSC,Ensembl database and Clustx were used for protein conservative prediction.Mutation taster,PolyPhen2 and other bioinformatics software predicted the pathogenicity of variation;Swiss-PDB Viewer software predicted the tertiary structure of protein,etc.The detected potentially pathogenic mutations in the children and their parents were verified.Point mutations and small insertion deletion mutations were verified by Sanger sequencing;large deletion and duplication were verified by real-time quantitative PCR.6.Analysis of the relationship between the clinical phenotype and genotype of the children:According to the clinical manifestations and disease progression of the 7 children,the MSUD was clinical classified and compared with genotype.Results1.Clinical results:Among 7 cases,5 were male and 2 were female.The onset age was less than 10 days.The main manifestations included irritability,poor response,feeding-difficulty,abnormal muscular tension and seizures.The time of diagnosis ranged from 4-5 hours to 22 days after birth.Among 7 cases,case 1 and 2 showed hypermyotonia while the rest showed hypomyotonia.Csae 1,4,5,6,7 appeared convulsions and case 1,3,4 with varying degree of fever.Case 3,4,5,7 presented with acid-base imbalance including respiratory acidosis,respiratory alkalosis and metabolic acidosis.Case 6 was unable to maintain respiration and required mechanical ventilation.The disease progressed rapidly.Case 1,3,4,5,6 died on 17,33,10,18,22 days respectively after birth.Case 7 was still under clinical observation and case 2 survived for a long time with obvious neurological development retardation.2.Laboratory examination2.1 Blood routine test:Case 2 was found to be complicated with severe anemia at the age of 40 days.After comprehensive treatment of MSUD,hemoglobin level rose to 120g/L at the age of 15 months.Case 5 had mild anemia while the rest of the children had no anemia.2.2 Biochemical indicators detection:Case 2 showed low blood glucose monitoring and the result was 2.8mmol/L while the others children ranged from 3.2 to 3.9mmol/L.Blood NH3 detection was that case 2,3,4,5 slightly increased while case 7 progressively increased.The blood NH3 concentration of case 1 and 6 was within the normal range.The results of glutamine transpeptidase obviously increased in 95.14-178U/L except for the normal case 3.The serum amylase decreased except for the normal case 3.The results showed that serum lacteal dehydrogenase and hydroxybutyric acid dehydrogenase increased to varying degrees.Except for case 1,the level of serum myoenzyme increased between 224-330U/L.2.3 Blood-gas analysis:Case 3 showed mild respoiratory acidosis and case 4 presented mild respiratory alkalosis while metabolic acidosis and respiratory alkalosis were seen in case 5 and 7.Case 1,6 were normal results of blood-gas analysis and case 2 did not performed this test.2.4 CSF examination:Case 1,4,5,6,7 with convulsion had CSF examination which showed normal glucose.CSF protein was higher than normal while the cells number was less than 10×106/L and negative bacteria test.2.5 Blood amino acid test:Except that the valine level of case 2 was within the normal range,the valine of the other children ranged from 300.01 to 744.48umol/L;Leucine plus isoleucine fluctuated from 605.91 to 3249.18umol/L while Leucine plus isoleucine/phenylalanine at 16.91-100.19umol/L.2.6 Urine organic acid detection:Most of children showed a significant increase in branched ketone acid such as 2-hydroxyisovalerate increased from 181.2 to 446.76.The level of 2-ketone-isovaleric acid was between 101.92 to 207.97;The level of 2-ketone-3-methylvaleric acid ranged from 58.1 to 442.98;The level of 2-ketoisocaproic acid ranged from 138.2 to 679.11.3.Imaging examination results:Cranial magnetic resonance showed that widespread abnormal signals of metabolic encephalopathy were consistent with those of bilateral pons,midbrain,medulla oblingata,vermis cerebelli,thalamus,corona radiata,centrum semiovale,bilateral globus pallidum,endocyst and so on.The cranial MRI results of case 6 and 7 manifested abnormal symmetrical signal shadow in bilateral basal ganglia region,brain stem and cerebellum.It was finally confirmed as metabolic encephalopathy imaging.Patient 1 bedside cranial displayed subependymal saccular dark areas and bilateral parenchyma echogenicity was slightly enhanced.4.EEG and other examination results:Case 6 showed abnormal amplitude of the whole electroencephalogram.The waveform was continuous and voltage amplitude was normal.The sleep cycle was poor and there were scattered sharp waves,spikes,fast wave and one time epileptiform discharge.5.Genetics examination results:Case 1,2,3,4,6 of 7 children had BCKDHB gene mutation including 4 complex heterozygous mutations and 1 homozygous mutation.Two patients(case 5 and 7)had compound heterozygous mutations of BCKDHA gene.No other gene mutations were detected.Seven of them were new mutations that have not been reported including c.863G>A and exon5-9 gross deletion were BCKDHA mutations while c.523T>C?c.659delA?c.550delT?c.665A>C?exon 1-7 gross deletion were BCKDHB mutations.6.Analysis of the relationship between clinical phenotype and genotype in children:All children were caused by mutations of BCKDHB and BCKDHA gene,but there was no corresponding relationship between clinical phenotype and genotype.Case 1,3,4,5,6 were classic type MSUD with poor prognosis and died from 17 to 33 days after birth.Case 2 was intermediate type.The onset of case 7 was consistent with classic type,but the clinical progress was similar to thiamine-responsive which was still under clinical observation.ConclusionsBCKDHB and BCKDHA gene may be the main pathogenic genes of MSUD in our province.c.659delA,a new mutation of BCKDHB may be a mutation hotspot for children of MSUD.Screening of amino acids in blood and organic acids in urine is conducive for rapid screening of MSUD which facilitates timely clinical treatment for MSUD and discover new mutant gene.Section 2 Establishment of iPSC cell line from Chinese patient with MSUDObjectiveTo establish iPSC cell line from a child with MSUD for further research on MSUD pathogenesis,gene function,drug screening.Methods1.iPSC cell induction:Peripheral blood mononuclear cells(PBMCs)were isolated from the seventh case of MSUD.Electroporation was performed with targeting vectors mixed with PBMCs.The PBMCs were planted onto 12 wells coated with feeder cells.On the day after transfection,ReproTeSR medium was used to induce clones.Two weeks later,iPSC colonies were manually picked up on Matrigel in mTeSR1 medium,maintained at 37? and 5%C02 in a humidified atmosphere.2.iPSC cells line identification:iPSC cells were identified with typical pluripotency-related cell markers by immunofluorescence and endogenous pluripotent genes OCT4,SOX2,NANOG were detected by qRT-PCR.The formation of embryonic body was performed to verify the differentiation potential iPSC cells.Expression levels of representative markers of three germ layers were detected by qRT-PCR,including the ectodermal markers PAX6,NR2F2,the mesodermal markers RUNX1,HAND1,and the endodermal markers SOX 17,GATA4.G-band chromosome analysis of iPSC cells at passage 15 after was analyzed to determine whether the karyotype was normal.3.Verification of gene mutation:Sanger sequencing was used to verify gene mutation on 3130XL DNA Analyzer.Western blot was performed to analyze the expression of proteins compared with normal control.Results1.iPSC cell induction:Peripheral blood cells from children with non-integrated reprogramming technique showed typical morphology of human embryonic stem cell.2.Identification of iPSC cells line:Immunofluorescence results showed that iPSC cells could express multiple pluripotency markers such as TRA-1-81?TRA-1-60,SSEA4,OCT3/4 and NANOG The expression of three endogenous genes OCT4,SOX2,NANOG was confirmed by qRT-PCR.iPSC were differentiated into a typical embryoid structure and the expression of specific markers SOX 17,GATA4,RUNX1,HAND1,PAX6 and NR2F2 of three germ layers could be detected.The chromosome karyotype of iPSC cells was identified which showed normal karyotype.PCR results showed that iPSC had no exogenous gene expression.3.Verification of gene mutation:Sanger sequencing verified that iPSC from child had the same c.632C>T and c.1280-1282delTGG compound heterozygous mutations in BCKDHA gene as that of the child.The results of western blot showed that the expression of BCKDH-Ela in the iPSC from the child was significantly lower compared with control group(P<0.01).Conclusions iPSC cell lines were established from the MSUD child carrying c.632C>T and c.1280-1282delTGG compound heterozygous mutations.It would be a useful tool for investigating pathogenesis,gene function and screening drug of MSUD. |
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