1. 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

2. © 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

3. © 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

4. © 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

5. © 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?

6. © 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

7. © 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

8. © 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

9. © 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

10. © 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

11. © Copyright 2009 by the American Association for Clinical Chemistry Question 2  What factors may affect the accuracy of RHDO analysis?

12. © 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

13. © 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

14. © 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?

15. © 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

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