Application Of Molecular Genetics Techniques In Maple Syrup Urine Disease And Multiple Malformations

【Purpose】:The objective of this work was to combine target region sequencing in known genesand non-invasive prenatal detection based on trio-family information to investigate theunexplained cases in clinical practice, providing a prominent one-step method fornon-invasive prenatal testing of single-gene disorders.Toanalyze the molecular genetics change of multiple malformations using array-basedcomparative genomic hybridization, then investigate the value of this technique.【Methods】:1.we report a new strategy, combining discovering novel mutations in known genesand non-invasive prenatal testing, to diagnosis a Chinese family affected with Maplesyrup urine disease （MSUD）. Potential mutations of the proband and her parents wereanalyzed using a customized capture array （NimbleGen, Madison, USA） and IlluminaHiSeq2000sequencers.To verify the data obtained by targeted MPS, real-time PCR（7500Real-time PCR system） and Sanger sequencing （ABI3730） were performed forsamples of all family members.Maternal blood was collected at13thgestational weeksand the plasma was isolated by two-step centrifugations and used for the noninvasivelyprenatal testing. Parental haplotypes were constructed using genomic information of thetrio-family family trios, the fetal genotype had four candidate combinations that could be inherited from parents. Then, the probability of each mixed state of maternalgenotype and different fetal combinations was calculated in each SNP marker inmaternal plasma. Finally, according the linkage relationship obtained from parentalhaplotype, we established a Hidden Markov Model （HMM） with consideration of thestate of SNP markers and the parental recombination information. By this strategy, thefetal genotype and haplotype can be achieved in one step.Amniotic fluid （AF） frominvasive procedure at the19thgestational week at Chinese PLA General Hospital wasused for prenatal diagnostic genetic testing. After DNA extraction, real-time PCR andSanger sequencing for target variations were performed.With careful post-testcounseling, the couple finally decided to terminate the pregnancy. Muscular tissues ofthe aborted fetus were obtained and validated by real-time PCR and Sanger sequencing,and the results were in accordance with that of AF DNA.2.31cases of fetal multiple malformationsfound by ultrasound examinationbetweenFebruary2010andMay2013in the people’s liberation army general hospitalwere studied. The age ranged from20to37，the gestational week ranged from21to27weeks.Collecting the clinical information, the samples（amniotic fluid, umbilical cordblood）with fetal genetic material after informed consent were obtained.Chromosomekaryotype analysis and array-based comparative genomic hybridization were carried outon the samples.【Results】:1. c.392A>G in gene BCKDHA was detected by massively parallel sequencing. Sangersequencing validated the suspected novel missense mutation （c.392A>G） exists in theproband and her mother, consistent with that of the MPS results. we successfullyrevealed novel mutations in gene BCKDHA （Ex2₄dup and c.392A>G）in this Chinesefamily, and developed a prenatal haplotype-assisted method to noninvasively detect the genotypes of the fetus （transmitted from both parents）. With the makers of the probandand her parents, we recovered the parental haplotype of chromosome19in6,148locibased on Mendel’s laws. And using a sensitive Hidden Markov Model （HMM）, weidentified the parental transmitted alleles and recombination breakpoints in5,480loci ofmaternal plasma assisted with the parental haplotype information. In addition, aone-time recombination of chromosome19in the maternal and paternal allelerespectively was identified, and the breakpoints were both located in the short arm.Ourresults showed that in the causative gene, BCKDHA, the fetus inherited the same allelesas that of the proband, which indicated it was also a MSUD patient.2. All of31samples were successfully detected by G-band karyotype analysis andarray-based comparative genomic hybridization. Among karyotype analysis,4sampleswere cultured failure, one case had no nuclear clearvage.There were3normal and23abnormal, the abnormality rate was11.54%（3/26）,the abnormal result was47,xx,t(11;22（q23;q11）,+der（22）t（22;11）（q11;23）;47,XN,+13；47,XN,+18respectively。Among the aCGH analysis,4cases was normal,21cases were detected bymicrodeletion and microduplication with chromosomal polymorphism。6cases wereabnormal, the rate of abnormality was19.35%（6/31），including2cases of45XO,1case of18trisomy,2cases of13trisomy and one case of duplication on the long arm ofchromosome11with the size of18.21Mb and deletion on the long arm ofchromosome22with the size of3.16Mb.The accordant rate with karyotping was100%.【Conclusions】:1. It’s the first report of integration of target region sequencing and non-invasiveprenatal test into clinical practice.Our study proved this MPS-based strategy will be apromising workflow for single-gene disorders diagnose in the future. 2. Chromosomal abnormalities is the important reason of fetal multiple malformation,so it is very necessary of molecular genetics testing for the fetal structural abnormalitiesby ultrasound finding, especially multiple malformations, identifying the reason anddirect the next pregnancy.Array-based comparative genomic hybridization techniquecan assist the routine karyotyping with the advantage of high throughout and extensivecover.On one hand, it can identify chromosomal abnormalities which can’t be detectedby routine prenatal cytogenetic analysis because of culture failure and cell vigordefect,increasing the overall detection rate.; on the other hand,it can detect themicrodeletion and microduplication at submicrostructure, it is favorable forunderstanding the reson and providing the evidence of fetal malformation.3. The above methods were proved by testing the fetal tissues of induction of labor inmid-pregnancy.