Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker.
BACKGROUND
The putative promoter of the holocarboxylase synthetase (HLCS) gene on chromosome 21 is hypermethylated in placental tissues and could be detected as a fetal-specific DNA marker in maternal plasma. Detection of fetal trisomy 21 (T21) has been demonstrated by an epigenetic-genetic chromosome dosage approach where the amount of hypermethylated HLCS in maternal plasma is normalized using a fetal genetic marker on the Y chromosome as a chromosome dosage reference marker. We explore if this method can be applied on both male and female fetuses with the use of a paternally-inherited fetal single nucleotide polymorphism (SNP) allele on a reference chromosome for chromosome dosage normalization.


METHODS
We quantified hypermethylated HLCS molecules using methylation-sensitive restriction endonuclease digestion followed by real-time or digital PCR analyses. For chromosome dosage analysis, we compared the amount of digestion-resistant HLCS to that of a SNP allele (rs6636, a C/G SNP) that the fetus has inherited from the father but absent in the pregnant mother.


RESULTS
Using a fetal-specific SNP allele on a reference chromosome, we analyzed 20 euploid and nine T21 placental tissue samples. All samples with the fetal-specific C allele were correctly classified. One sample from each of the euploid and T21 groups were misclassified when the fetal-specific G allele was used as the reference marker. We then analyzed 33 euploid and 14 T21 maternal plasma samples. All but one sample from each of the euploid and T21 groups were correctly classified using the fetal-specific C allele, while correct classification was achieved for all samples using the fetal-specific G allele as the reference marker.


CONCLUSIONS
As a proof-of-concept study, we have demonstrated that the epigenetic-genetic chromosome dosage approach can be applied to the prenatal diagnosis of trisomy 21 for both male and female fetuses.
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Special Administrative Region, China.
Yu K
YK
Tong
Rossa W K
RW
Chiu
Ranjit
R
Akolekar
Tak Y
TY
Leung
Tze K
TK
Lau
Kypros H
KH
Nicolaides
Y M Dennis
YM
Lo
D000199:Actins / Q000378:metabolism; D000483:Alleles; D019731:Carbon-Nitrogen Ligases / Q000235:genetics*; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D041322:Chromosomes, Human, Y / Q000235:genetics; D019175:DNA Methylation; D004314:Down Syndrome / Q000235:genetics*; D044127:Epigenesis, Genetic; D005260:Female; D018628:Gene Dosage; D005819:Genetic Markers / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods
D000199:Actins; D005819:Genetic Markers; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases
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