Introduction:
Due to the specificity of inheritance patterns and the interference of maternal DNA, noninvasive prenatal diagnosis of recessive monogenic diseases is relatively lagging behind in cell free fetal DNA research. This study explored the efficiency of noninvasive prenatal diagnosis for monogenic disorders (NIPD-M) based on relative haplotype dosage (RHDO) and Bayes Factor (BF) for pregnant women of high-risk recessive genetic disease in the first trimester.

 

Methods:
A total of 206 cases of high-risk recessive genetic disease pregnant women and their family samples were collected at the First Affiliated Hospital of Zhengzhou University, between September 2022 and November 2023. The cell free DNA (cfDNA) was extracted from the pregnant woman's plasma and the genomic DNA was extracted from their family blood samples. The designed capture panel covered 10 genes (DMD, SMN1, PAH, MMACHC, MMUT, F8, F9, SLC26A4, GJB2, CYP21A2). Enriching targeting genes and informative SNP sites in adjacent regions, the family haplotype was constructed. The fetal genotype was determined based on the dose change of the informative SNPs in cfDNA combined with the BF algorithm. The CBS algorithm was used to exclude the interference of recombination events. All NIPD-M results were validated by invasive prenatal diagnosis or newborn genetic testing.

 

Results:
Among the 206 recruited families, the average age of the pregnant women was 31 years old, the average gestational age of blood collection was 9 weeks, and the average fetal fraction (FF) was 6.15%. Among them, there were 44 cases of Duchenne muscular dystrophy, 12 cases of hemophilia A, and 8 cases of hemophilia B with high risk of the X-chromosome recessive genetic disease, and 35 cases of phenylketonuria, 35 cases of spinal muscular atrophy, 49 cases of methylmalonic acidemia, 13 cases of non-syndromic hearing loss, and 10 cases of congenital adrenal hyperplasia with high risk of autosomal recessive genetic disease.

202 (98%) pregnant women had family samples collected in the first trimester (7-13⁺⁶ weeks), and 4 pregnant women chose to undergo NIPD-M in the second and third trimesters for not available for invasive prenatal diagnosis, including 2 cases after cervical cerclage for threatened abortion, 1 case with signs of uterine infection, and 1 case with bleeding tendency due to abnormal coagulation function. 190 families obtained the NIPD-M results and the success rate was 92.23% (190/206). The main reason for the failure of NIPD-M in 16 families was insufficient number of informative SNP sites. NIPD-M failed in 11 pregnant women due to insufficient number or imbalance distribution of information SNPs, inability to judge recombination events and unclear BF, 4 cases failed due to the recombination breaking point near pathogenic mutation, and 1 case failed due to spontaneous embryo termination and FF declined. All the families that passed the quality control obtained NIPD-M complete reports within 7 days.

Among the 206 pregnant women, 120 cases underwent amniocentesis, 80 cases underwent chorionic villus puncture, 4 cases underwent neonatal verification after birth due to puncture contraindications, and 2 cases underwent abortion due to embryo arrest. Compared with validation testing, the NIPD-M results are consistent with the invasive prenatal diagnosis and newborn genetic testing, showing its accuracy is 100%.

 

Conclusion:
This study confirmed that NIPD-M is an early, safe, accurate and rapid method, which provides valuable experience for clinical consultation. Haplotype construction and BF analysis are reliable and feasible in the real clinical environment of recessive monogenic diseases diagnosis, and can be used as the preferred method for contraindication of puncture and early diagnosis.