Non-Invasive Prenatal Diagnosis of Sickle Cell Disease in Spain

Background:

Non-Invasive Prenatal Testing (NIPT) allows obtaining genetic information about the fetus by studying the circulating cell-free fetal DNA in the maternal plasma. During pregnancy, the fetus releases DNA into the maternal bloodstream, making it possible to access the genetic information of the fetus through the mother's peripheral blood. This procedure enables prenatal diagnoses without the need for invasive obstetric techniques such as amniocentesis or chorionic villus sampling, which may pose risks to the fetus during sample collection.

Sickle cell disease is a recessive monogenic disease. NIPT for these diseases, in cases where both parents carry the same mutation, does not follow the principle of presence or absence of the mutation. Instead, the diagnosis must be based on the relative mutation dosage (RMD), which refers to whether there is a balance or imbalance between the mutated allele and the normal allele, with fetal DNA causing the deviation.

Objective:

We present the results of a non-invasive prenatal diagnostic method for sickle cell disease using digital PCR (dPCR) in Spain.

Methods:

The population of interest consisted of pregnant women who were heterozygous for sickle cell disease. Cell-free DNA was extracted from their plasma and subjected to dPCR with probes labeled with different fluorophores for the normal allele (VIC) and the mutated allele (FAM). This allows for the quantification of both alleles, thus obtaining the RMD of the mutation. The RMD was translated into a z-score using non-pregnant heterozygous women as the reference population. In this way, balance/imbalance ranges were established, allowing a specific genotype to be assigned to the fetus.

Using the mean and standard deviation of the reference population, the z-score values for each pregnant woman were calculated, and cutoff points were established to achieve 99% confidence at +2.6 and -2.6, and +1.5 and -1.5. If the z-score value was between +1.5 and -1.5, it was considered allelic equilibrium, indicating that the fetus should be heterozygous. If the value was below -2.6 or above +2.6, it was considered allelic imbalance, indicating that the fetus should be homozygous for the normal allele or homozygous for the mutated allele, respectively. Finally, an intermediate zone was maintained where the result is considered ambiguous, and the process needs to be repeated

Results:

The study included the results of 25 pregnant women who were heterozygous for sickle cell disease. The results we obtained were 11 cases of pregnancies with a z-score lower than -2.6, which we considered as fetuses homozygous for the wild-type allele, 13 cases with a z-score between -1.5 and +1.5, which we considered as heterozygous fetuses, and one case where the z-score fell into the intermediate zone, making it impossible to determine the fetal genotype.

All results were verified after birth.

Conclusion:

Determining the fetal genotype in cases where both parents are carriers of the same recessive hereditary disease has been a significant challenge due to the inability to separate maternal DNA from fetal DNA.

The fetal DNA present in the maternal blood contributes to the RMD, allowing for the study of the fetal genotype without resorting to invasive methods. This has significant implications for the prenatal diagnosis of sickle cell disease.

The results obtained have been as expected. In healthy fetuses (βA/βA), an allelic imbalance was observed with an increased load of the normal allele, and in fetuses with the sickle cell trait (βA/βS), allelic equilibrium was maintained. Although we did not have any cases of fetuses affected by the disease (βS/βS), we would expect an allelic imbalance shifted towards the mutated allele.

This study has allowed us to determine the fetal genotype from maternal blood, avoiding invasive methods.

Funding: This work has been possible thanks to the financial support granted by the scholarship awarded by the Spanish Erythropathology Group of the Spanish Society of Hematology and Hemotherapy in 2022.