The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

Non-invasive prenatal testing for fetal inheritance of maternal ß-thalassaemia mutations using targeted sequencing and relative mutation dosage: a feasibility study.

OBJECTIVE: To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal ß-thalassaemia mutations in cell-free DNA. DESIGN: Feasibility study using samples collected in a prenatal clinic. SETTING: South East Asia. POPULATION: Couples where both partners were known to be carriers of one of four common ß-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with ß-thalassaemia. METHODS: 49 samples previously identified as having inherited a paternal ß-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele. MAIN OUTCOME MEASURES: Classification of the fetus as 'unaffected' (if the maternal wild-type allele was inherited) or 'affected' with ß-thalassaemia (if the maternal mutant allele was inherited). RESULTS: A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6-97.3%] and a specificity of 95.8% (95% CI 78.9-99.9%) for inheritance of maternal genotype. CONCLUSIONS: RMD for detection of inheritance of maternal ß-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders. TWEETABLE ABSTRACT: NIPT for ß-thalassaemia achieved using nested-PCR followed by relative mutation dosage.

Accuracy of Non-Invasive Prenatal Testing for Duchenne Muscular Dystrophy in Families at Risk: A Systematic Review.

BACKGROUND: Methodological advancements, such as relative haplotype and relative mutation dosage analyses, have enabled non-invasive prenatal diagnosis of autosomal recessive and X-linked diseases. Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by progressive proximal muscular dystrophy and a high mortality rate before the age of twenty. We aimed to systematically present obtainable data regarding a non-invasive prenatal diagnosis of DMD and provide a comprehensive resume on the topic. The emphasis was given to the comparison of different available protocols and molecular methods used for fetal inheritance deduction, as well as their correlation with prognostic accuracy. METHODS: We searched the Scopus and PubMed databases on 11 November 2022 and included articles reporting a non-invasive prenatal diagnosis of DMD in families at risk using relative dosage analysis methods. RESULTS: Of the 342 articles identified, 7 met the criteria. The reported accuracy of NIPT for DMD was 100% in all of the studies except one, which demonstrated an accuracy of 86.67%. The combined accuracy for studies applying indirect RHDO, direct RHDO, and RMD approaches were 94.74%, 100%, and 100%, respectively. Confirmatory results by invasive testing were available in all the cases. Regardless of the technological complexity and low prevalence of the disease that reduces the opportunity for systematic research, the presented work demonstrates substantial accuracy of NIPT for DMD. CONCLUSIONS: Attempts for its implementation into everyday clinical practice raise many ethical and social concerns. It is essential to provide detailed guidelines and arrange genetic counseling in order to ensure the proper indications for testing and obtain informed parental consent.

Noninvasive prenatal diagnosis of duchenne muscular dystrophy in five Chinese families based on relative mutation dosage approach.

BACKGROUND: Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach-cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. METHODS: Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. RESULTS: The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. CONCLUSION: This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types.