Prenatal Diagnosis of Congenital Factor XIII Deficiency Using Cell-Free Fetal DNA (cffDNA)


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Background
Cell-free fetal DNA (cffDNA) was rst described by Lo et al. in 1997. Using polymerase chain reaction (PCR) for the Y chromosome they found that fetal DNA circulates in the plasma of women carrying a male fetus (1). The main source of this cffDNA is trophoblastic breakdown (2). It is a low fraction of free DNA in the maternal plasma, reported to be approximately 10-20% in the last weeks of pregnancy. The amount of cffDNA increases with gestation. The average fetal DNA is higher than 10% in the 10th week, increasing 0.1% weekly between the 10th and 20th weeks. After the 21st week it increases more rapidly, 1% weekly (3). Extracting cffDNA requires great precision and a sensitive and speci c method for isolation. The amount of cffDNA depends on maternal health, body weight, aneuploidies, and twin pregnancy (2). Immediately after childbirth, fetal DNA is eliminated from the maternal plasma (4)(5)(6).
In comparison to invasive methods such as amniocentesis and chorionic villus sampling (CVS), which carry ~ 1% risk of miscarriage, noninvasive prenatal diagnosis (NIPD) using cffDNA has no risk for the mother or the fetus (2,7,8). In addition, its early screening helps to provide better clinical management (3). The only issue regarding NIPD is the low amount and small size of cffDNAs (9). These fetal DNAs are fragmented and smaller than 313 bp, while maternal DNAs are more than 400 bp in size (2). For this reason, NIPD requires a sensitive process to provide su cient cffDNA for analysis. Nonetheless, NIPD using cffDNA has been used to determine fetal sex, fetal rhesus D (RhD) genotyping, aneuploidies and inherited monogenic disorders (8,10,11). Congenital factor XIII de ciency (FXIIID), with an estimated incidence of 1 per 2 million, is a rare coagulation factor de ciency (RCFD) transmitted in an autosomal recessive manner (12,13). The disorder presents with a variety of clinical manifestations, including delayed wound healing, umbilical cord bleeding (UCB), central nervous system (CNS) bleeding, and recurrent miscarriage (14,15). Although UCB is the most common clinical feature, CNS bleeding is the main cause of morbidity and mortality (15,16). Iran has the highest prevalence of the disease worldwide (17)(18)(19)(20). The most effective programs to reduce this rate are PND and premarital counseling.
PND based on invasive techniques such as chorionic villus sampling (CVS), used to test for FXIIID in Iran, carry the risk of miscarriage. Therefore, it is suggested that NIPD, based on cffDNA analyses and obtained through a simple peripheral blood sample, be used for these cases. The aim of this study was to investigate the e ciency and accuracy of NIPD using cffDNA to nd potential cases of FXIIID.

Study population and sample collection
This study was conducted on 7 pregnant women with heterozygous FXIIID and a positive family history of severe (homozygote) FXIIID. It was approved by the Medical Ethics Committee of Iran University of Medical Sciences.
The pregnant women were in their rst trimester (8-13 weeks). Maternal blood samples were collected in ethylenediamine tetra acetic acid (EDTA) anticoagulant. Within 2 hours of collection, plasma was separated from the maternal blood by centrifuge for 10 minutes at 1600 g followed by further centrifuge of the supernatant for 10 minutes at 28000 g. The nal supernatant was kept at -20˚C for the next processes.

DNA extraction and molecular studies
The cffDNA extraction was conducted using the QIAamp Mini Elute ccfDNA kit (Qiagen, Germany). Maternal DNA contamination was assessed through ApoB3'VNTR gene (21,22). DNA sequencing was performed on some PCR products in order to con rm the results. Prenatal and postnatal results were compared to determine the accuracy of this method.

Results
Among 7 fetuses, four (57%) were heterozygotes, while three (43%) were unaffected. The rst (#1) had positive parental consanguinity ( rst degree) and one sibling with FXIIID. The mother had a history of hematoma. The fetus was found to be heterozygote. The PND and NIPD were offered to another woman in the rst trimester whose two children had severe FXIIID. This fetus had second-degree parental consanguinity. Moreover, there was a history of death due to UCB in the family. The mother also experienced recurrent epistaxis. Heterozygote state of the fetus (#2) was detected. The third case (#3), with parents of rst-degree consanguinity and one sibling with FXIIID, was diagnosed as a heterozygote of FXIIID. Its mother suffered from recurrent easy bruising. The fourth case (#4) had rst-degree parental consanguinity and one sibling with FXIIID. Its mother was asymptomatic. The PCR-RFLP on cffDNA showed a heterozygote state for this case, which was con rmed by sequencing. The three remaining cases (#5, 6, and 7) were diagnosed as unaffected after PCR-RFLP and sequencing on cffDNA. Following their births, the molecular study on fetal DNA samples con rmed the reported results: 4 cases (#1, 2, 3, and 4) were heterozygote and the rests were unaffected (Table 1). Table 1 The results of the molecular study of 7 fetuses with a positive family history of severe factor XIIID  (25). In our study, one subject had UCB-related death in his family. As with previous studies, all our cases had parental consanguinity and a family history of FXIIID.
Due to the rising prevalence of FXIIID, and related morbidity and mortality, the most important life-saving and preventive procedure is PND. The invasive method through CVS is used in Iran, which presents some risks for both mother and fetus. The rst study on PND of FXIIID, published in 1996, determined that HUMF13A01 STR can be easily applied for PND of FXIIID (28). Another study, performed on a pregnant woman whose rst son was affected with FXIIID, showed the fetus to be unaffected (29). Yet another proved the factor assay test as a PND method for FXIIID. That subject's FXIII level was shown to be below 5%; consequently the parents terminated the pregnancy (30). PND for FXIIID, using invasive methods such as amniocentesis and CVS, has been reported in previous studies (31,32). The only report of PND for FXIIID in Iran was published by Naderi et al. in 2016 on the CVS samples of 8 fetuses. They concluded PND is a substantial way of reducing morbidity and mortality related to the disease, particularly in newborns (33). Similarly, in the current study, we investigated PND for FXIIID but using cffDNA to assess NIPD.
Previous studies also have used cffDNA for fetal sex determination, RhD genotyping, aneuploidies, preeclampsia, and detection of inherited monogenic disorders (34,35). The Y chromosome is helpful for differentiation between fetal DNA and maternal DNA. Moreover, the cffDNA test is considered a screening test for 13, 18, and 21 trisomy (4,36,37). The studies concluded that the test could be used as NIPD for β thalassemia with high accuracy. For molecular analysis, the studies used dPCR, NGS, and qPCR, which are able to make use of a small amount of cffDNA (8).
According to our results, the molecular analysis via PCR-RFLP on cffDNA for mentioned mutations yielded the same results as molecular analysis on neonate DNA in all 7 cases. The results of Sanger sequencing on cffDNA con rmed the molecular results.

Conclusion
In conclusion, our study demonstrated that this non-invasive test may be used productively instead of invasive methods, such as CVS, which carry risks for mother and fetus. Moreover, we suggest assessing PND by cffDNA for determining the presence of other severe congenital disorders.