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Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia K.-W.G. Lam, P. Jiang, G.J.W. Liao, K.C. A. Chan, T.Y. Leung, R.W.K. Chiu, and Y.M.D. Lo October 2012 www.clinchem.org/cgi/content/article/58/10/1467.full © Copyright 2012 by the American Association for Clinical Chemistry
© Copyright 2009 by the American Association for Clinical Chemistry Introduction Prenatal diagnosis: Established part of obstetrics care Definitive fetal DNA testing typically involves invasive procedures (e.g., amniocentesis, chorionic villus sampling) with risk of fetal miscarriage Cell-free fetal DNA in maternal plasma: First reported in 1997 Only amounts to average of 10% of the total DNA Facilitates noninvasive prenatal diagnosis (NIPD) Early application: fetal sex determination for sex-linked diseases and congenital adrenal hyperplasia, rhesus D blood group testing
© Copyright 2009 by the American Association for Clinical Chemistry Introduction Massively parallel sequencing of maternal plasma DNA: Precise DNA measurement Allows NIPD of chromosomal aneuploidies (e.g., trisomy 21) Deep sequencing enabled fetal genetic and mutational analysis Targeted sequencing: To selectively capture and amplify DNA fragments in targeted regions from a DNA sample for sequencing Cost-effective for deep sequencing of the targeted regions
© Copyright 2009 by the American Association for Clinical Chemistry Introduction NIPD of β-thalassemia: An autosomal recessive monogenic disease causing anemia (HBB gene on chromosome 11) An affected fetus has inherited both the maternal and paternal mutations NIPD involves ascertaining the fetal inheritance of the maternal and paternal mutations in maternal plasma DNA
© Copyright 2009 by the American Association for Clinical Chemistry Question 1 What are the challenges to achieve NIPD of fetal β- thalassemia in maternal plasma compared with applications such as fetal sex determination?
© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods Samples: 2 families: parents were both carriers of β-thalassemia Blood specimens from 2 pregnant women and their husbands were collected in 1 st trimester Targeted sequencing of maternal plasma DNA: Maternal plasma DNA was extracted DNA molecules in HBB gene cluster were enriched for massively parallel sequencing
© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods Parental genetic information: Parental genomic DNA was extracted from buffy coat Parental haplotyping information of HBB gene cluster was interrogated by digital PCR Haplotype is a combination of alleles at adjacent loci on the chromosome that are transmitted together
© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. HBB mutations and pedigrees of β-thalassemic mutations in 2 families. (A), Targeted regions for enrichment and haplotyping. Filled and empty boxes represent HBB exons and introns, respectively. Three common mutations identified in the 2 studied families are marked with dotted lines. (B), The pedigree of β- thalassemic mutations in the first family. (C), The pedigree of β-thalassemic mutations in the second family. WT, wild-type allele. Materials and Methods
© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods Deduction of paternal fetal HBB inheritance: To detect the presence of paternally derived mutation in maternal plasma, if the paternal mutation differed from the maternal mutation Figure 2. Deduction of paternally derived mutation in HBB gene. M=mutant, W=wild-type
© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods Deduction of maternal fetal HBB inheritance: To detect the over-representation of mutation and adjacent alleles (as a haplotype) in plasma DNA using “relative haplotype dosage analysis” (RHDO analysis) Figure 3. Deduction of maternally derived mutation by RHDO analysis in HBB gene. M=mutant, W=wild-type
© Copyright 2009 by the American Association for Clinical Chemistry Question 2 What factors may affect the accuracy of RHDO analysis?
© Copyright 2009 by the American Association for Clinical Chemistry Main results NIPD of fetal mutational status in the 1 st family Paternal mutation (-CTTT deletion) was detected by deep sequencing (60/741 reads) Maternal haplotype carrying wild-type HBB gene was over-represented Conclusion: The fetus was a heterozygous carrier
© Copyright 2009 by the American Association for Clinical Chemistry Main results NIPD of fetal mutational status in the 2 nd family Paternal mutation (A→T at codon 17) was NOT detected by deep sequencing (0/826 reads) Maternal haplotype carrying maternal mutation (-CTTT deletion) was over-represented Conclusion: The fetus was a heterozygous carrier
© Copyright 2009 by the American Association for Clinical Chemistry Question 3 Ref: Lun FMF et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A 2008;105:19920–5. What are the pros and cons of targeted RHDO versus ‘simpler’ digital PCR-based approaches for relative mutation dosage analysis in NIPD of monogenic diseases?
© Copyright 2009 by the American Association for Clinical Chemistry Conclusions The combination of targeted sequencing and RHDO analysis is feasible for NIPD of β-thalassemia The concept could be generalized for other genetic disorders, thus expanding the application of plasma DNA-based NIPD
© Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us
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