Researchers have developed a faster, less expensive, reliable method for non-invasive prenatal diagnosis (NIPD) of hemophilia A.
Future, larger studies are required to confirm the utility of the method, which could be used for the NIPD of hemophilia A and type B, as well as other X-linked disorders caused by mutations in a single gene, the researchers said.
The study, “Noninvasive prenatal diagnosis of hemophilia A by a haplotype-based approach using cell-free fetal DNA,” was published in the journal BioTechniques.
Hemophilia A and B are X-linked disorders, meaning they are caused by mutations in genes located on the X chromosome, one of the two sex chromosomes that play a part in determining gender.
Since men only have one X chromosome, those who inherit a mutated F8 gene will develop the disease, while in women — who have two X chromosomes — a healthy F8 gene copy typically partly compensates for a mutated one.
Women with a mutated copy of the F8 gene are called carriers, and usually produce normal amounts of FVIII. To develop hemophilia A, a baby girl will have to inherit two copies of the F8 mutation, one from each parent; this is why hemophilia A is rarer in women.
Prenatal testing is important to determine whether the unborn baby of a pregnant hemophilia A carrier will develop the disease.
The discovery of circulating cell-free fetal DNA (cffDNA) in maternal blood has led to the development of various NIPD methods, avoiding the 1% chance for miscarriage associated with invasive sampling methods.
In pregnant women, cffDNA are DNA fragments generated mainly from natural death of specific placental cells. Fetal fraction, or the percentage of cffDNA in a maternal blood sample, is generally at an average of 13% (ranging from 3% to 30%).
While cffDNA has the potential to revolutionize prenatal diagnosis, there are some limitations in its analysis, and few studies have reported its use in NIPD methods for hemophilia A.
Researchers in China developed a new method of NIPD based on cffDNA that is faster and less expensive than those previously reported.
Their method relies on statistical models and on the genetic analysis of a small DNA region comprising the F8 gene, two sections surrounding the gene (which include 445 common variants) and a chromosome Y-specific region (to help determine the baby’s gender).
According to the researchers, this region is significantly smaller than a previously reported region used for the same purpose, resulting in a faster and less expensive approach.
The researchers validated their method in two families affected by hemophilia A. Two pregnant women and their respective brothers carried a common F8 mutation of severe hemophilia A — Inv22 — with the brothers having the disease and the women being classified as carriers.
Blood samples of the pregnant women, their affected brothers, and their unaffected spouses were analyzed for that predefined DNA region to build the women’s genetic signature surrounding each F8 gene copy (the region containing either the healthy or the mutated gene). These signatures, or haplotypes, were compared with that of the fetus to determine which of the gene copies was inherited from the mother, without directly looking at the F8 mutation.
Results showed that one of the babies was a boy (positive detection of the chromosome Y-specific region) and had inherited the healthy F8 gene copy from his mother. The other baby was a girl, and the presence of a signature of the mutated F8 gene was detected, suggesting that she was a carrier.
Genetic analysis of the amniotic fluid of the two pregnant women confirmed the NIPD results, validating the new method.
“Our research demonstrated the feasibility of haplotype-based NIPD for [hemophilia A] using sequencing of a small target region,” the researchers wrote.
The new approach allows the simultaneous analysis of DNA from up to five families, and costs about $250. Invasive prenatal diagnosis methods cost about $1,000.
Larger studies are required to confirm the method’s value before it can be implemented in the clinic, the researchers said.
“As the sequencing cost continues to fall, we hypothesize that this haplotype-based approach could be feasible for NIPD of [hemophilia] and other X-linked single-gene disorders in the future,” they said.
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