Volume 20, Issue 8 (August 2022)                   IJRM 2022, 20(8): 613-626 | Back to browse issues page

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Mortazavipour M M, Mahdian R, Shahbazi S. The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review. IJRM 2022; 20 (8) :613-626
URL: http://ijrm.ir/article-1-2001-en.html
1- Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, ‎Tehran, Iran.
2- Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran.‎
3- Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, ‎Tehran, Iran. , sh.shahbazi@modares.ac.ir
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  1. Introduction
1- 1. Cell-free fetal DNA (cfDNA)
Cell-free DNA or cfDNA is released into the plasma following a variety of normal physiological functions such as the cell death programs of apoptosis and necrosis (1). The release of this double-stranded DNA is not limited to the plasma and may occur in other body liquids such as urine or cerebrospinal fluid. Since cfDNA has a fragmented structure, its half-life is very short (< 2.5 hr); as a result, it has a fast turnover with rapid plasma clearance (2). Increased levels of cfDNA are associated with a range of conditions from infections to inflammation. There is also evidence of elevated plasma cfDNA levels with increasing age. However, cfDNA has been most widely studied in cancer cases and has been successfully used for the evaluation of tumor progression (3). It is also applicable in other pathogenic cell proliferation conditions such as endometriosis (4). Currently, cfDNA molecular profiling techniques are employed by noninvasively identifying somatic mutations; thus precise monitoring of minimal residual disease in different cancers can help to provide appropriate clinical management (5).
Another promising application of cfDNA is in prenatal diagnosis. Lo et al. showed that maternal plasma is an unequal combination of fragmented fetal DNA molecules and a large amount of maternal cfDNA. With the new knowledge about cfDNA, prenatal diagnosis has entered a new era of progress (6). Before the availability of cffDNA, the first-line prenatal diagnosis in medical genetics centers included invasive methods such as chorionic villus sampling, amniocentesis, and cordocentesis. The risk of miscarriage and anxiety from invasive procedures should always be considered as they may discourage couples from undergoing prenatal diagnostic tests (7).
By identifying the characteristics of cffDNA, non-invasive prenatal diagnosis (NIPD) was able to be implemented in clinics (8). NIPD was first applied for the screening of chromosomal aberrations focusing on chromosomes 21, 18, and 13 aneuploidy and identification of sex chromosomes. It is now claimed that NIPD after the 10th wk of pregnancy has a detection rate of 99.4% and a false-positive rate of 0.16% for trisomy 21. For trisomy 18 the values are 96.6% and 0.05%; however, for trisomy 13 and monosomy X the detection rates are lower (86.4% and 89.5%, respectively) (9). NIPD screening of these major trisomies is based on deep sequencing techniques, although it requires confirmatory invasive testing due to the possibility of placental mosaicism. Microdeletions can also be detected using the chromosomal microarray technique. Both deep sequencing and microarray methods identify the areas of the genome with copy number variation. Detection of specific fetal mRNA expression is another way of screening for aneuploidies. Placenta-specific protein 4, located on chromosome 21, was one of the genes to be applied in trisomy 21 analysis. For detection of Edward's syndrome, the expression of the serpin peptidase inhibitor, clade B, membrane 2 gene, located on chromosome 18, has been employed (10). NIPD diagnostic applications were later expanded to Rh Blood Group D Antigen (RhD) evaluation and fetal sex determination (11). Ultimately, cffDNA has been proposed for NIPD of single-gene diseases. Early uses were focused on the detection of fetal de novo variants or paternally inherited gene mutations. Researchers are currently validating the diagnostic application of cffDNA for a variety of genetic diseases.
Here, we have reviewed the most relevant NIPD approaches that are available for common genetic disorders with an emphasis on single-gene diseases, including their advantages and levels of accuracy.
1-2. NIPD application in single-gene disorders
From the 7th-9th wk of gestation, the cffDNA makes up 10% of the circulating DNA in the mother's plasma which is sufficient for a variety of fetal molecular diagnoses. It should be noted that this quantity of DNA is still very low, which makes the implementation of NIPD for single-gene disorders very challenging. Also, since the X chromosome of a male fetus is similar to one of the mother's Xs and has the same haplotype, it can be difficult to discriminate between maternal and fetal genomic variants in X-linked diseases. Additionally, in autosomal recessive disorders, prenatal diagnosis becomes more complicated when the parents have the same mutation with the same haplotype (12). Therefore, various innovative methods have been introduced to perform genetic analysis of cffDNA samples. These methods are mainly based on allele discrimination assays or relative allele dosage analyses (Table I).

1.2.1. New diagnostic approaches: Relative mutation dosage (RMD) and relative haplotype dosage (RHD)
RMD analysis is mainly based on the droplet digital polymerase chain reaction (PCR), a technique that is comparable to conventional PCR while including quantification features of real-time PCR. The main technical concept of this method is diluting DNA samples as low as one copy of the genome per micro-unit droplet (13). The micro-units could contain one or no copy of the template DNA. Micro-unit droplets are prepared using water-in-oil emulsion or micro-channel chips (27). This level of dilution accounts for the increased sensitivity and specificity of the PCR reaction by reducing competition effects of target DNA molecules (11). Each digital PCR micro-unit contains 6 nL of reagent mixture which is the minimum volume required for successful PCR amplification. PCR reactions are performed in a real-time PCR apparatus where the amplification of each variant on the target DNA can be detected by a particular fluorophore (28). Recently, researchers have started implementing this assay for NIPD of single-gene disorders. The interpretation of the results depends on the inheritance pattern of the disorder, which is explained in more detail below.
RHD is a next generation sequencing-based NIPD technique, involving maternal and paternal whole-genome sequencing followed by the analysis of cffDNA. The characteristics of the affected fetus are determined by estimating the proportion of mutated to normal alleles. The advantage of RHD over RMD is that with RHD it is possible to examine areas where direct PCR is not possible due to pseudogenes, rearrangements, and complex mutations. However, the problem with RHD is that it requires samples of the proband and his/her siblings. Other limitations of RHD are the high costs of whole exome sequencing and the complexities of the associated bioinformatics data analysis (29).
To overcome these challenges, targeted locus amplification (TLA) of genomic regions around mutations that harbor informative single nucleotide polymorphisms (SNPs) has been considered. These linked SNPs that are inheritable from one parent have known SNP haplotypes (16). The main purpose of this method is to determine the paternal or maternal origin of the allele. Therefore, it is a successful strategy to find maternal mutations in the fetus which has always been challenging (30). Following targeted sequencing of the desired regions of the maternal and paternal genome, parental haplotypes are mapped to relevant haplotype data obtained from cffDNA (31). TLA has the ability to analyze multiple genomic regions, which is an advantage for the simultaneous detection of multiple single-gene disorders. It will not only reduce costs but also help in cases where the first pregnancy is examined and there is no previous patient sample available. TLA can also be used in trinucleotide repeat expansions that are challenging to be investigated by other methods (29).
1-3. Sex determination approaches and X-linked recessive inheritance
Mutations in the genes located on the X chromosome can lead to an X-linked disorder that is predominantly exhibited in male fetuses, while females are either carriers or unaffected. In this regard, sex determination using cffDNA can prevent unnecessary invasive tests such as chorionic villus sampling in female fetuses (Figure 1). While NIPD sex determination has reliable results between the 7th-12th wk of gestation, conventional ultrasound often is unable to detect the fetus’ sex earlier than the 13th wk of gestation (18). The presence of Y-specific chromosomal sequences in maternal plasma logically indicates male-bearing pregnancies (32). Based on this, studies have been conducted to determine the reliable Y-chromosomal target sequences for non-invasive sex determination (33-35). The real-time PCR assay has been widely applied to detect Y-chromosomal sequences including SRY, DYS14, DAZ, AMYLY, and PAP genes. The results of a study have indicated a sensitivity of up to 100% at the 8th wk of gestation (36).

However, complementary molecular tests should be performed to determine whether a male fetus has inherited the maternal causative mutation. These tests should be able to effectively differentiate the fetal mutation in the presence of a large amount of maternal DNA harboring the same allele. In this case, highly sensitive and specific techniques such as RMD and RHD have been implemented for clinically validated assays. In a previous study, RMD analysis was able to detect maternal mutations causing X-linked abnormalities in male fetuses

The inheritance of X-linked causative mutations in an at-risk pregnancy (XnXm × XnY) could result in either an affected fetus (XmY) or a normal fetus (XnY). Thus, the maternal plasma may consist of maternal XnXm mixed with either XnY (normal fetus) or XmY (affected fetus) genotypes. To determine the fetus genotype, maternal plasma is diluted and transferred into micro-units as a water-in-oil emulsion or micro-channel chips. Statistically, each micro-unit may contain either one or no copy of the template DNA. Each micro-unit contains all the essential reagents for a PCR reaction which is performed in a real-time PCR instrument. 2 distinct TaqMan probes are designed for normal and mutant alleles which are labeled with different dyes such as VIC and FAM. Amplification signals are emitted if a micro-unit contains template DNA. The emitted signals are collected and an amplitude diagram of each dye is drawn for every micro-unit. The mutant allele ratio (M) is calculated by using the formula M = m / m + n (Figure 2).

It is assumed that a heterozygote non-pregnant woman has an M proportion of 0.5 (16).             M values > 0.5 indicate an affected fetus whereas non-affected samples display M values < 0.5. The M and fetal DNA fraction data are analyzed by the sequential probability ratio test. The interpretation of RMD is rather convenient as it needs specific primers and probes for every particular mutation (30).
RHD can be considered as an alternative approach to detect maternal mutations in male fetuses. As shown in figure 3, haplotype-I is considered as normal haplotype and haplotype-II as the mutated one. The genomic data obtained from maternal plasma cfDNA samples are collected and analyzed in parallel to corresponding genomic data of the parents. The relative quantity of haplotype-I and haplotype-II are determined and if the haplotype-I amount is greater than that of haplotype-II, the fetus might be considered normal, whereas a higher relative quantity of haplotype-II indicates that the fetus might be affected.

1-5. Autosomal dominant disorders
In autosomal dominant inheritance, detecting mutations of fetal origin in maternal plasma indicates an affected fetus. As shown in figure 4, there are 2 different approaches for the implementation of NIPD in maternally or paternally inherited autosomal dominant disorders. The detection/exclusion of paternal mutations in maternal plasma determines the status of the fetus in at-risk pregnancies (37). So far, various procedures have been developed for the detection/exclusion of paternal mutations in maternal plasma (Table I). In contrast, the differentiation of fetal alleles is very challenging for maternally inherited mutations. To overcome this limitation, RMD and RHD methods have been successfully applied. In both methods, an equal quantity of mutated and wild-type alleles represents an affected fetus.
However, these methods are not applicable in some autosomal dominant disorders such as Huntington's disease and myotonic dystrophy. These diseases are mainly caused by dynamic mutations classified as trinucleotide repeat expansions. Given that the fragmented fetal DNA in plasma measures about 150 bp, the detection of paternal allele expansions may need alternative methods. For example, the trinucleotide expansions of myotonic dystrophy and Friedreich’s ataxia consist of more than 50 and 200 repeats, respectively (5). To this end, quantitative fluorescent PCR (19), TLA (31), and PCR plus fragment analysis (12) have been used for the detection of paternal expansions in maternal plasma. TLA is appropriate for pregnancies in which either paternal or maternal repeats are identical or paternal repeats are much longer than their maternal counterparts (20). Nevertheless, to improve the analysis accuracy, these approaches may be designed exclusively for particular inheritance patterns.

Given the cost and complexity of the management of beta-thalassemia, prenatal diagnosis is recommended to at-risk families (41, 42). Some countries such as Italy, Greece, Cyprus, and Iran have developed screening programs for carrier detection and prenatal diagnosis (40). More than 200 distinct mutations in the HBB gene involving single base mutations, small and large deletion, and aberrant splicing mutations can lead to the clinical features of the disorder (41).

Considering the autosomal recessive inheritance pattern of this disease, NIPD for beta-thalassemia can be implemented according to the parent's mutation types (43). RMD and RHD approaches have been recommended for parents that share similar mutations (22, 44, 45).
Furthermore, detection/exclusion strategies can identify a paternal mutation in maternal plasma in compound heterozygote cases (46, 47). Investigation of 75 beta-thalassemia carriers for 2 common mutations (Cd39 and IVSI.110) using full COLD-PCR showed promising results in a study conducted in Italy (39). We also successfully developed a COLD-PCR-based method to detect the paternal beta-thalassemia IVS-II-1 (G>A) mutation in maternal cell-free DNA (48). Furthermore, another study showed that fast temperature-gradient COLD-PCR was able to identify 2 paternal SNPs in maternal plasma samples (38). The studies which have reported the results of NIPD of beta-thalassemia as an archetype of single-gene disorders are summarized in table II.

2. Conclusion
Recent advances in NIPD for the identification of single-gene diseases have provided promising prospects in prenatal diagnosis. We can expect that in the coming decade, a significant number of single-gene disorders will be detected by NIPD methods. However, ethical issues are among the concerns that should be addressed before the widespread implementation of advanced NIPD techniques. Ultimately, population-specific genome databases are highly desired for their high specificity in the detection of mutations within the context of inter-population variation.

Conflict of Interest
The authors declare that there is no conflict of interest.

Type of Study: Review Article | Subject: Reproductive Genetics

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