Introduction

The identification of cell-free fetal DNA (cffDNA) circulating in maternal plasma, which is present from early in pregnancy, has paved the way for the development of noninvasive prenatal diagnosis (NIPD), avoiding the small miscarriage risk associated with invasive prenatal diagnosis. The cffDNA is rapidly cleared from the maternal circulation after delivery, and because the whole fetal genome is represented, it offers scope for comprehensive prenatal diagnosis. NIPD for monogenic disorders is diagnostic, and results do not require confirmation on chorionic villi or amniocytes obtained by invasive testing as diagnosis is undertaken in pregnancies known to be at increased risk because of family history or sonographic findings and is targeted at a specific gene or panel of genes. This contrasts with noninvasive prenatal testing or screening (NIPT or NIPS) for aneuploidy, which is considered a very sensitive screening test requiring an invasive test to confirm a positive result ( Table 22.1 ). This is because cffDNA emanates from the placenta, and testing maternal plasma can detect confined placental mosaicism, and because the majority of cell-free DNA (cfDNA) in maternal plasma comes from the mother herself, NIPT can reveal unexpected maternal chromosomal rearrangements. Although maternal mosaicism can complicate NIPD for monogenic disorders, this can be taken into account by testing maternal genomic DNA (gDNA) in parallel with the cfDNA, but as far as we are aware, placental mosaicism for cell lines containing mutations found in single-gene disorders has not been reported.

Differences between NIPD and NIPT also apply to the timing of testing, largely because any cffDNA-based test is affected by the fetal fraction. The fetal fraction increases as pregnancy progresses, usually reaching a level that can be used for testing in straightforward applications such as the detection or exclusion of an allele not present in the maternal genome, for example, fetal sex determination, by 7 weeks’ gestation. However, for more complex applications that require accurate quantification (e.g., aneuploidy screening), accurate testing usually requires higher levels of cffDNA, which are attained around 10 to 12 weeks in most pregnancies. It is therefore critical that the gestational age is determined using ultrasonography before embarking on cfDNA testing. Furthermore, ultrasonography should be used to look for evidence of multiple pregnancies, which may complicate results. If an empty sac or ‘vanishing twin’ is identified, this may lead to false-positive results because the placenta continues to shed cffDNA in the absence of a fetal pole. NIPD is possible in multiple pregnancies, but in the absence of any abnormal ultrasound findings, it is not possible to tell which fetuses are affected, and invasive testing will be needed for definitive diagnosis. However, in dizygotic twins, it may be possible to analyse single nucleotide polymorphisms (SNPs) to measure variations in the fetal fraction between genomic regions to determine zygosity and analyse the fetal fragments for each fetus.

It is not possible to separate maternal and fetal cfDNA and, as described earlier, any test based on analysing cfDNA in maternal plasma will analyse both the maternal and fetal fractions, the maternal fraction being the most abundant. Consequently, the early clinical applications of NIPD focussed on the detection of paternal alleles or those arising de novo that were therefore not present in the mother (e.g., fetal sex determination, fetal RHD typing in RhD-negative mothers, paternally inherited single-gene disorders , or single-gene disorders arising de novo , such as achondroplasia ). NIPD can also be used in pregnancies at risk for autosomal recessive conditions if the parents are heterozygous for different mutations. In these cases, termed ‘paternal exclusion testing’, if the paternal allele is identified in maternal plasma, an invasive test is still required for definitive prenatal diagnosis to determine inheritance of the maternal allele and see whether the fetus is affected or not.

Many different laboratory approaches have been reported for NIPD, largely in research settings ( Table 22.2 ). New technologies, such as massively parallel sequencing (MPS), that allow accurate quantification of specific sequences has meant that tests are now being developed that take into account the presence of the maternal cfDNA, thus allowing diagnosis of autosomal recessive X-linked conditions.

TABLE 22.2

Studies Reporting Noninvasive Prenatal Diagnosis for Single-Gene Disorders Published Between January 2000 and August 2016

Adapted from Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn 33 :555–562, 2013.

Condition	Method	Total Samples	Results	Reference
Autosomal Dominant Conditions
Achondroplasia	Restriction digest	1	1 affected	Saito et al. (2000)
	Restriction digest	1	1 affected	Li et al. (2004)
	MALDI-TOF MS	2	2 affected	Li et al. (2007)
	PCR-RED	6	4 affected 2 unaffected	Chitty et al. (2011)
	QF-PCR	2	1 affected 1 unaffected	Lim et al. (2011)
	NGS		PCR-RED: 14/14 affected MPS: 8/9 affected	Chitty et al. (2015)
Apert syndrome	Allele-specific real-time PCR	1	1 affected	Au et al. (2011)
	PCR-RED	2	1 affected 1 unaffected	Raymond et al. (2010)
Crouzon syndrome	PCR-RED	1	1 unaffected; recurrence excluded	Raymond et al. (2010)
Huntington disease	QF-PCR	1	1 unaffected	Gonzalez-Gonzalez et al. (2003)
	QF-PCR	4	2/3 affected 1/1 unaffected	Bustamante-Aragones et al. (2008)
	QF-PCR	1	1 unaffected	Gonzalez-Gonzalez et al. (2008)
Myotonic dystrophy	Nested PCR	1	1 affected	Amicucci et al. (2000)
Thanatophoric dysplasia types 1 and 2	PCR-RED	4	3 affected 1 recurrence excluded at 12 wk	Chitty et al. (2013)
	MPS		PCR-RED: 9/11 affected MPS: 9/9 affected	Chitty et al. (2015)
Torsion dystonia	RT-PCR	2	2 affected	Meaney and Norbury (2009)
Autosomal recessive: parents carrying different mutations
Congenital adrenal hyperplasia	Fluorescent SNPs	1	1 unaffected	Chiu et al. (2002)
Craniosynostosis	COLD-PCR	1	1 affected	Galbiati et al. (2014)
Cystic fibrosis	PCR-RFLP	1	1 affected	Gonzalez-Gonzalez et al. (2002)
	SnaPshot	3	2 affected 1 unaffected	Bustamante-Aragones et al. (2008)
	NGS panel with 10 common mutations	4	4 correctly classified: 2 inherited paternal mutations	Hill et al. (2015)
Haemoglobin E	Nested PCR and restriction digestion	5	3 affected 2 unaffected	Fucharoen et al. (2003)
	Seminested and nested real-time PCR for three different mutations	39 beta(E) 12 beta(17) 9 beta(41/42)	All correctly classified 26 affected/carrier beta(E) 6 affected/carrier beta(17) 5 affected/carrier beta(41/42)	Tungwiwat et al. (2007)
HB Lepore	Allele-specific PCR	1	1 unaffected	Lazaros et al. (2006)
Leber congenital amaurosis	Denaturing HPLC	1	1 affected	Bustamante-Aragones et al. (2008)
Propionic acidaemia	SnaPshot; melt curve analysis.	1	1 affected (positive using both methods)	Bustamante-Aragones et al. (2008)
α-Thalassaemia	Real-time nested PCR	13	8 carriers, 1 HbH, 2 HbBarts, 2 no mutation	Tungwiwat et al. (2006)
	QF-PCR	30	10/30 paternal allele correctly excluded	Ho et al. (2010)
β-Thalassaemia	COLD-PCR	35	10/21 affected Cd39 11/21 unaffected 12/14 affected IVSI-110 2/14 unaffected	Galbiati et al. (2011)
	AS-PCR for SNPs	2	1 affected 1 unaffected	Papasavva et al. (2006)
	APEX	7	3 inherited paternal mutations 3 unaffected 1 incorrect	Papasavva et al. (2008)
	Genome-wide MPS and SPRT analysis	1	1 carrier	Lo et al. (2010)
	Fetal DNA enrichment and real-time PCR	10	10 paternal mutations correctly identified	Ramezanzadeh et al. (2016)
Autosomal recessive: parents carrying the same mutations
Congenital adrenal hyperplasia	Targeted MPS and haplotype analysis	14	14 affected	New et al. (2014)
	Targeted MPS and haplotype analysis – hidden Markov model	1	1 unaffected	Ma et al. (2014)
Cystic fibrosis	Single-cell short tandem repeat genotyping	32	32 correctly classified (7 affected)	Mouawia et al. (2012)
Maple syrup urine disease	Targeted MPS and haplotype analysis	1	1 correctly classified	You et al. (2014)
Methylmalonic academia	Relative mutation dosage with digital droplet PCR and parental SNP analysis	1	1 correctly classified	Gu et al. ( 2014 )
Sickle cell disorder	Relative mutation dosage using digital RT-PCR	65	52 correctly classified 7 incorrectly classified 5 unclassified	Barrett et al. (2012)
	PAP	1	1 negative for linked paternal SNP allele	Phylipsen et al. (2012)
Spinal muscular atrophy	Single-cell short tandem repeat genotyping	31	31 correctly classified (7 affected)	Mouawia et al. (2012)
	Targeted MPS and haplotype analysis	5	5 correctly classified	Chen et al. (2016)
α-Thalassaemia	Real-time quantitative PCR	158	61/62 affected Sensitivity: 98.4% False-positive rate: 20.8%	Sirichotiyakul et al. (2012)
	Allele-specific real-time PCR	67	33/67 correctly classified unaffected	Yan et al. (2011)
β-Thalassaemia	Relative mutation dosage using digital PCR	10	5 correctly classified 1 incorrect 4 unclassified	Lun et al. (2008)
	Targeted MPS and relative haplotype dosage	2	2 correctly classified as carriers	Lam et al. (2012)
	(PAP)	13	Paternal SNP allele detected in maternal plasma in 13 cases	Phylipsen et al. (2012)
	Targeted MPS and haplotype analysis	10	NIPD possible in 8/10 cases	Papasavva et al. (2013)
	High-resolution melting analysis	50	25/27 correctly classified as affected 19/23 correctly classified as unaffected	Zafari et al. (2016)
X-linked
Haemophilia A and B	Relative mutation dosage using digital PCR	7	3/3 affected haemophilia A 4/4 affected haemophilia B	Tsui et al. (2011)
Retinitis pigmentosa (X-linked)	Sequencing	1	1 mutation on paternal allele detected	Bustamante-Aragones et al. (2006)
DMD and Becker muscular dystrophy	Targeted MPS and haplotype analysis	9	2/2 affected DMD 7/7 unaffected	Parks et al. (2016)

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