Non-invasive prenatal diagnosis (NIPD)

Non-invasive prenatal diagnosis (NIPD) uses the mother’s blood rather than the fetal genetic material when making prenatal diagnosis of genetic conditions.

NIPD for sex determination

NIPD for fetal sex determination can be undertaken from 7 weeks gestation for pregnancies at a high genetic risk.

Common referral indications are pregnancies at risk of serious X-linked conditions or congenital adrenal hyperplasia, or where there are ultrasound findings such as ambiguous genitalia.

Some common examples of the application of this technique in the clinic include:

• Fetal sexing in pregnant women who are carriers for an X-linked disorder such as Duchenne Muscular Dystrophy (DMD) or haemophilia. In such cases, early fetal sex determination can reduce the invasive diagnostic testing rate by around 50% (as female fetuses may not require further testing).
• Fetal sexing for couples where both parents are carriers of congenital adrenal hyperplasia (CAH). Early identification of a male fetus can avoid unnecessary fetal exposure to steroids as only female fetuses require steroid treatment to avoid virilisation.

For fetal sex determination, ultrasound scanning must first be undertaken to confirm the gestation, and to identify multiple pregnancies or empty sacs from early twin demise, which could confound the results.

Once the cfDNA has been extracted, it is analysed using real time quantitative PCR methods to look for Y chromosome markers, indicating a male pregnancy.

Absence of Y signal indicates a female pregnancy.

NIPD for monogenic disorders

NIPD can be used for some monogenic disorders to exclude either paternal or de novo alleles (i.e. alleles that are not present in the mother). The techniques can be used in both dominant conditions, or recessive conditions where the parents are carriers of different mutations.

NIPD has been worked up for certain monogenic disorders such as skeletal dysplasias, congenital adrenal hyperplasia and cystic fibrosis, but bespoke tests can also be designed for other single gene disorders, especially in the context of testing for a subsequent pregnancy for a mutation previously identified by invasive prenatal or postnatal testing.

A) NIPD for dominant conditions – the exemplar of skeletal dysplasia

NIPD has been approved for prenatal diagnosis of FGFR3-related skeletal dysplasia (achondroplasia and thanatophoric dysplasia) in the NHS since 2012.

Achondroplasia is the most common short stature syndrome, presenting in the 3rd trimester. It shows autosomal dominant inheritance, although often arises de Novo.

A single mutation is responsible for 98% of cases. cfDNA testing can, therefore, be undertaken using a mutation-specific PCR-based restriction enzyme digest. NIPD allows confirmation of a diagnosis made on ultrasound, excluding other more severe dysplasias without the risk of preterm labour.

Thanatophoric dysplasia (TD) is the most common lethal skeletal dysplasia. It arises de novo and may be caused by at least 13 different mutations in FGFR3.

NIPD enables early diagnosis, allowing the option of surgical termination if requested (which would not be possible later in the pregnancy), and allowing accurate diagnosis in twin pregnancies without putting an unaffected twin at risk.

Initially, NIPD for TD was also developed using restriction enzyme digest PCR to identify the most common mutations, however since it can be caused by such a variety of possible mutations, less costly and time-consuming testing using a next-generation sequencing gene panel was developed, which is now being used for all FGFR3-related skeletal dysplasias.

NIPD can also be used in subsequent pregnancies following a pregnancy affected by an apparently de novo mutation, to exclude the possibility of recurrence resulting from germline mosaicism.

B) NIPD for recessive conditions – the exemplar of cystic fibrosis

NIPD can be used in recessive conditions where couples are known to be carriers of different mutations.

In this case, testing is done to exclude the presence of the paternal mutation, which means that the fetus is either a carrier (of only the maternal mutation), or completely unaffected.

If a fetus is shown to have the paternal allele, then they might be either a carrier of only the paternal mutation, or affected, and invasive testing will be required to distinguish this. Nevertheless, paternal mutation exclusion allows for a reduction in invasive testing.

In the case of cystic fibrosis, NIPD can currently be used where the couple are each known to be carriers of different mutations, and the paternal mutation is one of the 10 most common mutations covered by the NGS panel used for analysis.

NIPD for cystic fibrosis has been approved under these circumstances in the NHS since 2014.

Future directions

Current research in NIPD is now focusing on how to extend its use in order to detect a mutation in the fetal DNA against the background of the same mutation in the mother.

These approaches depend on precise measurement of relative mutation dosage (RMD). If the mother is heterozygous for the location of interest, then the presence or absence of the same mutation in the fetus will affect the expected extent of allelic imbalance between mutant and wild-type DNA in the total cfDNA (i.e. maternal and fetal cell-free DNA combined).

But the concentration of cfDNA varies between samples so this approach requires a precise quantification of the cfDNA by measuring a fetal allele not present in the maternal genome. This approach is extremely technically demanding and at present is only in the research phase.

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