Noninvasive prenatal diagnosis of hemophilia A by a haplotype-based approach using cell

Abstract of BGI prenatal test

Aim: We aimed to demonstrate noninvasive prenatal diagnosis (NIPD) of hemophilia A (HA) using a haplotype-based approach. Methods: Two families at risk for HA were recruited for this study. First, maternal haplotypes associated with pathogenic variants were constructed using the genotypes of the mothers and probands. Then, fetal haplotypes were deduced using a maternal haplotype-assisted hidden Markov model. Finally, the NIPD results were further confirmed by invasive prenatal diagnosis. Results: Two fetal genotypes were successfully inferred, with one normal fetus and one carrier fetus. The NIPD results were confirmed by invasive prenatal diagnosis, with a 100% consistency rate. Conclusion: Our test has been shown to be accurate and reliable. With further validation in a large patient cohort, this haplotype-based approach could be feasible for the NIPD of HA and other X-linked single-gene disorders.

SUMMARY POINTS

We propose a haplotype-based noninvasive prenatal diagnosis approach for hemophilia A using a small target region. The genomic DNA of the parents and proband and cell-free DNA of maternal plasma are analyzed by targeted sequencing and maternal pathogenic haplotypes deduced using the genotypes of mother and proband. Then, fetal haplotypes were inferred using a hidden Markov model and Viterbi algorithm.

factor VIIIhaplotypehemophilia Anoninvasive prenatal diagnosistargeted sequencing

Hemophilia A (HA) is an X-linked recessive bleeding disorder affecting approximately 1/5000 male live births [1]. HA is caused by a pathogenic variant in the F8 gene, resulting in a qualitative or quantitative deficiency in factor VIII (FVIII). More than 2000 sequence variants in F8 have been collected in the HA databases [2]. The F8 intron 22 inversion (Inv22) is found in approximately half the patients with severe HA [3]. Without prophylactic treatment, patients with severe HA suffer from spontaneous or traumatic bleeding in several organs. Intracranial hemorrhage can occur after even mild head trauma and lead to death. Replacement therapy with FVIII concentrate is the most common approach used in clinical practice at present. However, the ability to manage bleeding episodes is greatly impaired in patients who develop alloimmune antibodies (inhibitors) [4]. Because of the severity of the disease, pregnant carriers of severe HA can benefit from prenatal diagnosis.

However, invasive prenatal diagnosis (IPD) approaches such as chorionic villus sampling and amniocentesis pose a small risk of miscarriage and infection [5]. The discovery of cell-free DNA (cfDNA) in maternal plasma makes noninvasive prenatal diagnosis (NIPD) possible, which carries no risk to the pregnant woman or her fetus [6]. The NIPD available in the clinic at present focuses on detecting de novo or paternally inherited variants [7]. The high percentage of maternal cfDNA present interferes with direct observation of a fetal allele inherited from the mother, which presents significant technical challenges for NIPD of recessive disorders [8]. Few studies have reported NIPD of HA using fetal cfDNA. Tsui et al. first developed a NIPD method for the detection of F8 sequence variants using digital PCR and relative mutation dosage analysis [9]. Hudecova et al. tried NIPD for the detection of F8 Inv22 using target region sequencing and relative haplotype dosage analysis and the detection of sequence variants using droplet digital PCR and relative mutation dosage analysis [2].