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Clinical applications

Non-invasive prenatal diagnosis (NIPD) can be offered to women whose pregnancy is at risk of a specific monogenic condition, or to determine the sex of the fetus in the context of an X-linked condition.

Before NIPD can be undertaken, a suitable assay has to be available. For some conditions, an assay may have already been designed; for others, it may need to be created in a bespoke manner.

NIPD assays designed for specific conditions

Currently, genetic conditions for which NIPD assays have been developed include:

  • Duchenne and Becker muscular dystrophy;
  • spinal muscular atrophy;
  • congenital adrenal hyperplasia (female fetuses only);
  • cystic fibrosis;
  • heritable retinoblastoma; and
  • selected FGFR2– and FGFR3-related conditions, such as Crouzon syndrome with acanthosis nigricans, Apert syndrome and FGFR3-related skeletal dysplasia.

Bespoke NIPD assays

For pregnancies at risk of other genetic disorders, it may be possible to design a bespoke NIPD assay for paternal exclusion testing. This form of NIPD may be used if a pregnancy is at risk of a paternally inherited dominant condition.

This method may also be used if the risk is of an autosomal recessive condition and both parents are carriers of different pathogenic variants. In such cases, paternal exclusion testing can confirm or exclude the presence of the paternal variant in the fetus. However, if the fetus is found to have the paternal variant, further invasive testing is required to determine whether the fetus has also inherited the maternal variant.

Non-invasive testing for a maternally inherited variant is technically difficult. This is because the cell-free DNA (cfDNA) tested is a mixture of maternal and fetal DNA fragments, and if the variant of interest is detected it is difficult to tell whether it is just present in the mother, or in both the mother and the fetus. This means that bespoke NIPD for maternally inherited autosomal dominant or recessive variants is not yet available in the NHS.

For a small number of conditions, however, (in particular, spinal muscular atrophy) special assays have been developed that are able to detect maternally inherited single nucleotide polymorphism (SNP) combinations (haplotypes) linked to the variant or normal maternal alleles (see below). The conditions for which this is available are listed in the National Genomic Test Directory.

It is important to note that pre-pregnancy workup is required for bespoke assays, both to confirm that it is possible to design an assay and to make sure NIPD can be performed quickly during pregnancy.

Non-invasive prenatal sexing

NIPD can also be used to non-invasively determine the sex of the fetus. This non-invasive prenatal sexing can help inform the management of pregnancies at risk of severe sex-linked conditions (for example, Duchenne muscular dystrophy).

It may also be used to avoid unnecessary treatment in male fetuses in suspected cases of congenital adrenal hyperplasia, or in cases in which the fetal genitalia are ambiguous on ultrasound.

How does it work?

NIPD is performed on a sample of maternal blood. This contains a mixture of maternal DNA and placental DNA called cell-free DNA (cfDNA). In virtually all cases, the placental DNA will be representative of the fetal genome in terms of the inheritance of small variants (such as single nucleotide variants or small insertions and/or deletions).

There are two forms of NIPD currently in use in the NHS, which are outlined below.

PCR-based testing, including paternal exclusion testing

This form of NIPD is used for paternal exclusion testing (including bespoke assays). This is also the method used for non-invasive prenatal sexing.

  • CfDNA is extracted from a maternal blood sample.
  • CfDNA is used as a template in a PCR reaction with primers targeting the variant of interest (or in the case of non-invasive prenatal sexing, the Y-chromosome).
  • The PCR products are then used for targeted next-generation sequencing (NGS). If the variant is present, a small proportion of the NGS reads covering the location of the variant of interest will show the variant, indicating that it is present in the fetus. If all the reads are normal, this indicates that the fetus has not inherited the variant.
  • A separate assay is usually used to determine how much of the cfDNA came from the fetus (the fetal fraction). This is important to make sure that the result is not a false negative due to insufficient fetal DNA being present.

Relative haplotype dosage

This form of NIPD is used for some autosomal recessive conditions, for example spinal muscular atrophy. In contrast to the paternal exclusion testing above, haplotype dosage testing gives information on the inheritance of both the maternal and the paternal alleles. It can also be used regardless of the variant in the targeted gene, as the variant itself is not directly tested for.

  • CfDNA is extracted from a maternal blood sample.
  • DNA is also extracted from blood samples from both parents and from a previous affected or unaffected child.
  • The cfDNA and the familial DNA samples are used for NGS using a targeted panel. This targets the chromosomal region surrounding the gene of interest.
  • During analysis of the NGS data, genotyping of more than 1,000 SNPs in the chromosomal region surrounding the gene of interest is performed. The pattern of inheritance of SNPs in the previous child is used to determine which SNP combinations (haplotypes) are linked to the variant or normal maternal alleles, and to the variant or normal paternal alleles.
  • This information is then used to interpret the NGS SNP data from the cfDNA and identify which gene copies (variant or normal, maternal or paternal) the fetus has inherited.
  • The NGS SNP data are also used to determine how much of the cfDNA came from the fetus (the fetal fraction).

NIPD vs NIPT

NIPD is closely related to a similar method known as non-invasive prenatal testing (NIPT). In both methods, a maternal blood sample is taken to detect genetic conditions in the fetus. The difference lies in the fact that only NIPD is considered a diagnostic test, with no follow-up invasive testing required. NIPT for aneuploidies is regarded as a screening test, meaning that follow-up invasive diagnostic testing is required for confirmation.

Advantages and limitations of NIPD

Advantages

  • There is no need to take a sample from inside the uterus (in contrast with invasive prenatal diagnosis using amniotic fluid or chorionic villus sampling). This removes the small risk of miscarriage associated with invasive procedures.
  • It can be performed from as early as eight to nine weeks’ gestation (dependent on the condition to be tested for).
  • It is diagnostic: no confirmatory invasive testing is required (except in autosomal recessive conditions where the presence of the paternal variant is detected through paternal exclusion testing, and the presence or absence of the maternal variant in the fetus needs to be determined).

Limitations

  • It is only available for a limited range of genetic conditions.
  • Pre-pregnancy work-up is usually required for confirmation that NIPD is possible and to allow timely delivery in pregnancy.
  • It may not be possible in multiple pregnancies (such as twins or triplets). In these cases, you should contact the laboratory.
  • It may not be possible if the parents are consanguineous, or if the mother has had an organ transplant.
  • Some forms of NIPD (for example, relative haplotype dosage testing) require samples from both parents and from a previous (unaffected or affected) child.
  • In relative haplotype dosage testing, there is a small risk of an inconclusive result due to a recombination event (the DNA has swapped between chromosomes, uncoupling the condition-causing variant from the haplotype surrounding it), as well as a small risk of a misdiagnosis due to a double recombination event.

Practicalities

  • Pregnant patient blood samples are required in cell-stabilising blood tubes (Streck or PAXgene).
  • For fetal sexing from seven to nine weeks’ gestation, two 10ml samples are required, taken one week apart. At nine-plus weeks’ gestation, for fetal sexing and other NIPD two 10ml samples may be taken at the same time.
  • Control samples for comparison may include paternal blood (5ml EDTA), proband or confirmed non-carrier child (1ml EDTA or DNA sample).
  • Testing must be arranged in advance through your local clinical genetics department or fetal medicine unit.
  • Target reporting time from receipt of sample is 21 days, with 42 days for pre-pregnancy test work up.

Resources

For clinicians

Tagged: Technologies

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  • Last reviewed: 02/09/2022
  • Next review due: 02/09/2024
  • Authors: Dr Julia van Campen
  • Reviewers: Professor Barbara Jennings, Dr Siobhan Simpson