| IntroductionBeta-thalassemia is a common autosomal recessive disorder caused by reduced (β+) or absent (β0) synthesis of the β-globin chains of the haemoglobin tetramer, located on the β-globin (HBB) gene cluster on chromosome11. In clinic, There are three conditions for β-thalassemia, beta thalassemia major, beta thalassemia trait and beta thalassemia intermedia.As so far, over200different mutations have been identified in the patients with β-thalassaemia. The phenotype of beta thalassemia was effected by the β0-thalassaemias and β+or β++-thalassaemias, the former was caused by no β-globin gene product, and the latter was the result of marked or mild reduction in the output of β-chains.In the Asia population, the five most common mutations were taking up92.5%of all the mutations in south of China, which was namely CD41-42(-TCTT),IVS-2-654(C>T),minus28(A>G), CD17and hemoglobin E.Prenatal diagnosis is an essential part of obstetric practice, and genetic diagnosis is currently offered to couples known to be carriers of β-thalassaemia. Prenatal diagnosis of fetal genetic status depends on the use of invasive diagnostic tests (chorionic villus sampling (CVS) and amniocentesis) to collect a sample of the baby’s genetic material. These procedures carry a small but significant risk of miscarriage of around1%. Non-invasive prenatal diagnosis (NIPD) using cell-free DNA (cfDNA) from maternal plasma provides an alternative means of prenatal diagnosis that does not carry any risk of miscarriage. Cell-free fetal DNA (cffDNA) constitutes approximately10%of cfDNA in maternal plasma, and can be used reliably as a source of fetal genetic material for NIPD from seven weeks gestation.NIPD for autosomal recessive is more complicated. First attempts involved the exclusion of the paternal mutation in autosomal recessive conditions where the parents were carriers of different mutations. If a paternally identified allele can be detected, there is a50%risk that the fetus will inherit the disorder, and therefore invasive testing would be recommended. Alternatively, in the absence of a paternal allele, the fetus would be a carrier at worst, and invasive testing would not be indicated. Since this study a number of publications have demonstrated that paternal exclusion is readily achieved However, in the50%of cases where the fetus has inherited the paternal allele an invasive test is still required. In addition this strategy can’t be performended on the cases where parents carried same mutations.Digital PCR (dPCR) is much more sensitive than qPCR, A quantitative approach to NIPT where both parents carry the same mutation known as Relative Mutation Dosage (RMD)was developed for β-thalassaemia and haemoglobin E (HbE) mutations using dPCR. Sequential Probability Ratio Testing (SPRT) is a statistical method used to determine whether the counts from the digital PCR are in allelic balance or whether there is an imbalance for the over-represented allele.There have been NGS analyses on cfDNA looking for a diagnosis of β-thalassaemia, based on the SNPs panel to find the imbalance allele in maternal blood. SNP is a biomarker in human genome, of which the informative information are varied from different rigion, race and need a database providing the most imformative SNPs. Although the millions of SNPs can be captured by in-solution capture method, it is too expensive to clinic practice.In this study, We are developing a next generation sequencing approach using targeted sequencing combined with RMD for the β-thalassaemia mutations on the Illumina MiSeq platform, focusing our initial efforts on the five most common mutation types.Materials and MethodsA total of144pregnant Chinese couples attending the prenatal diagnosis for beta thalassemia were recruited in Department of Obstetrics and Gynecology, National University of Singapore, Singapore and NanFang hospital,Southern medical university, China.124of them, couples carried the five most common mutations and received deeply analysis. The gestational weeks for prenangcy attending prental diagnosis were ranging from10.57-32.14weel, the average gestational week was20.29±0.36. Eighty-three of these couples carried different mutations, while41carried the same.10mL maternal peripheral blood were collected in two EDTA tubes before invasive puncture.2mL paternal peripheral blood were collected in EDTA tubes. Fetal specimens were collected as below:11-14weeks of gestation were obtained prior to chorionic villus sampling (CVS),14+1to24weeks of gestation were obtained to amniocentesis and in the third trimester, the cord blood were obtained.Plasma was separated from lOmL of maternal blood by centrifugation at1,600g for10min, The supernatant was transferred to clean polypropylene tubes, then microcentrifuged at16,000g for10min. The plasma was carefully transferred to2-mL Lo-Bind tuebs(Eppendorf).Plasma and buffy coat were stored at-80℃immediately until further processing. Plasma DNA was extracted from2mL plasma with QIAamp Circulating Nuclei Acid Kit (Qigen),with a Qiagen vacuum manifold and in accordance with the manufacturer’s instructions.The gender identification and fetal fraction is detected by ZFX/ZFY and INDEL panel.The male fetus is identified as proportion of ZFY>4%and female fetus showing ZFY%<1%. The unclassified result is obtained by ZFY%being larger than1%but less than4%. A multiplex panel of primers was designed for45indels with a minor allele frequency>0.25. The fetal fraction is quantified by the ratio of counts for heterozygous alleles.The informative SNPs must be more than2for each sample and the cutoff for ratio is from4%to50%.The paternal mutation exclusion in maternal blood can be detected directly for cases where parents carried different mutations. While, maternal haplotype analysis is to detect the imbalance allele in maternal plasma and the results diagnosed by SPRT.All the measurement data were displayed by M±SD. Statistically significant between samples were tested by both sides. The comparison between two library construction methods and fetal fraction quantified methods used paried student test. To evaluate the accurary of paternal mutation exclusion method and maternal haplotype analysis, McNemar test was used for statistical analysis.With a=0.05level of significance test. All statistics were analyzed using SPSS19.0statistical software.ResultsThe fetal gender was identified by ZFX/ZFY, where rsults from two methods that are long primer and short primer targeted sequencing. In124cases,52cases were identified as male by both two methods,47cases were identified as female,7cases was shown unclassified results. The accuracy of diagnosis for gender identification was87.90%(109/124),83.87%(104/109) separately with long primer and short primer method. Comparison to these methods, the difference between the two methods was no significate, P>0.05(P=0.255)The fetal fraction was detected by IN/DEL method, the Mean of fetal fraction ws11.834+7.706with male samples. Comparison to ZFX/ZFY results, the IN/DEL method provid a higher percentage of cell free fetal DNA in maternal blood (P<0.05). In124cases, there were average11INDEL SNPs proving informative data for each sample.In83cases where parents carried different mutations, the sensitivity for paternal exclusion is100%95%CI (92.4%-100%) and specificity is91.67%,95%CI(78.17%-97.13%). Relative mutation dosage results for all cases where couples carried different mutations and couples carried same mutations. In36cases where parents carried different mutations, the maternal mutation was excluded,47cases was along with maternal mutations. The overall sensitivity for maternal exclusion in couples with different mutations was84.78%(95%CI:71.78,92.43%) and specificity was78.38%,95%CI (62.8,88.61%). In the other41cases where parents carried the same mutation,32cases with correct results after analyzing by RMD, the accurary of the method was75.61%.DiscussionThe informative INDEL SNPs were performed on quantification of fetal fraction in maternal blood. The average numbers of informative INDEL for each sample was11, which was helpful to quantify not only the fetal fraction in male samples but also the fetal fraction in female samples.In our stuy, the5most common mutations were targeted by overlapping PCR amplicons. The diagnosis strategy was to exclude the inheritance of paternal mutation, occuring in50%of cases. This is a relatively straightforward procedure, since we are looking for the presence or absence of the mutation and this should be quite apparent from the sequencing data. In our study, the negtive predictive value was100%, indicating that all the samples with non-inheritance of paternal mutations were truth. The maternal mutation detecting used by RMD and SPRT, the accurary was lower than the accurary of paternal mutation exclusion strategy., which would caused by lower fetal fraction; mismatch or errors happend during the PCR amplification process; in addition, the error was lead to sequencing bias.Next step,It is important to increase the yield of RMD and SPRT and find the causes leading to the error results. |
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