Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA

Abstract

Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families. (Blood. 2011;117(13): 3684-3691)

Intrauterine Diagnosis of Genodermatoses

“…Therefore, this approach is mostly useful for diagnosis of mutations that are inherited from the father and are absent in the mother's genome. To overcome this problem, an approach called the relative mutation dosage was developed [58,59]. This method includes accurate quantification of the relative dosage of the mutant and wild-type alleles in plasma.…”

Noninvasive detection of F8 int22h-related inversions and sequence variants in maternal plasma of hemophilia carriers

“…6 We have previously reported that it is feasible to perform NIPT for fetal genotype assessment by maternal plasma DNA analysis of F8 and F9 sequence variants by microfluidics digital PCR (dPCR). 7 The European Hematology Association Roadmap for European Hematology Research has identified development in NIPT for hemophilia as a research priority, in particular for severe and common sequence variants such as F8 int22h-related inversions. 8 The well-established hemophilia A and hemophilia B databases gather detailed descriptions of more than 2000 sequence variants occurring in the F8 gene and approximately 1000 sequence variants in the F9 gene, respectively.…”

“…This can potentially be resolved through a haplotypebased analysis examining mutation-linked polymorphic sites. [16][17][18] The protocol described by Tsui et al 7 for NIPT of hemophilia assesses the allelic imbalance from the proportion of the mutant (M) and normal/wild-type (N) allele in maternal plasma of pregnant hemophilia carriers. As for any NIPT-based protocol using a molecule counting strategy, the fetal DNA fraction governs the degree of such allelic imbalance, and thus requires precise molecular tools for its measurement.…”

“…19 The lower is the fetal DNA fraction in a particular sample, the higher is the number of plasma DNA molecules that would need to be counted. The chip-based microfluidics platform adopted by Tsui et al 7 has less flexibility in terms of scaling the number of digital reactions achieved. At this time, droplet dPCR (ddPCR) and massively parallel sequencing (MPS) technologies offer the highest sensitivity and precision for quantification of maternal plasma DNA.…”

Zirkulierende Nukleinsäuren – ein neues Universum in der laboratoriumsmedizinischen Diagnostik

“…Lo auf dem Gebiet der nicht-invasiven pränataler Testung (NIPT) gegeben, der schon 1999 erste Arbeiten zur pränatalen Untersuchung zirkulierender fetaler DNA im mütterlichen Plasma vorstellte [7][8][9]. In 2010 konnte er zeigen, dass sich das gesamte fetale Genom aus seiner im mütterlichen Plasma zirkulierenden DNA sequenzieren ließ [10] und in 2011 stellte seine Gruppe eine nicht-invasive pränatale Diagnostik der fetalen Hämophilie bei heterozygoten Schwangeren vor [11].…”