Transcript Journal Club - Clinical Chemistry

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
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 1st 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
Materials and Methods
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 βthalassemic mutations in the first family. (C), The pedigree of β-thalassemic mutations in the second family.
WT, wild-type allele.
© 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 1st 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 2nd 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
 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?
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
S the
A 2008;105:19920–5.
Copyright
2009Uby
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
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