9. NIPD - ffDNA and RNA in maternal serum & applications Flashcards
Why are intact fetal cells not used for NIPD?
Present in small numbers & may persist in maternal circulation for years –> risk of false positive in subsequent pregnancies
cffDNA cleared from circulation after delivery
How is the presence of cffDNA confirmed?
Panel of SNPs
Look at SNPs where mother is homozygous - presence of paternally inherited allele quantified
Why is CPM not a consideration for NIPD?
CPM not reported for monogenic disorders, unlike aneuploidies
Why is an ultrasound recommended in advance of NIPD?
- To exclude the possibility of a vanishing twin which could obscure the genotype of the surviving fetus
- To date the pregnancy as abundance of cffDNA increases with gestational age
Why is fetal sex determination carried out by NIPD?
For pregnancies at risk of sex-linked disease, e.g. DMD, ALD
How is fetal sex determined? What is the outcome for an X-linked disorder?
Detection of SRY by qPCR
Multiple replicates over 2 different extractions
Male fetus –> invasive test for condition
Female fetus –> no further testing
Why is CAH tested for by NIPD? What is the outcome?
Females with CAH show virilisation - develop characteristics associated with male hormones (androgens).
Female fetuses treated with dexamethasome from 6 weeks
In what scenarios can NIPD be used for AD disorders?
Why?
1.If variant is paternal inherited
- If variant was de novo in previous offspring (risk of germline mosaicism)
cffDNA will contain variant not present in high background of maternal cfDNA
What is the limitation for CNVs in NIPD?
Size of cffDNA fragments (approx. 300bp)
How are AD disorders tested for by NIPD?
- Targeted NGS NIPD panel, e.g. for FGFR3-related disorders
- Bespoke PCR-based method - required DNA from parent/proband in advance
What are the 3 ways that AR disorders can be tested for by NIPD?
- Paternal exclusion testing (fetus is at most a carrier if paternal variant is absent)
- Relative mutation dosage
- Relative haplotype dosage
How does RMD work?
What is it used for?
Determine relative quantities of mutant and wildtype alleles
AR disorders when both parents have same variant
RMD - what does it mean if there are equal amounts of mutant and WT alleles?
Carrier mother and carrier fetus
RMD - what does it mean if there are increased levels of mutant alleles?
And increased levels of WT alleles
Increased levels of mutant alleles = homozygous affected fetus
Increased levels of WT alleles = unaffected fetus
What method is used for RMD and why?
Usually ddPCR or NGS with v high read depth
What are the advantages & disadvantages of RMD over RHDO?
Advantages:
- Doesn’t require familial haplotype info, therefore only single maternal sample required
Disadvantages:
- Difficult to apply to pseudogenes/complex variants
- Less statistically robust than RHDO for low fetal fractions
Why must the fetal fraction be accurately determined for RMD?
Shift in allele ratios is proportional to fetal fraction
How does RHDO work?
Multiple SNPs around variant of interest are sequenced by NGS for parents, affected child and cffDNA
Determine high risk and low risk haplotypes
Determines allelic ratios between 2 alleles in maternal plasma - deduce whether fetus has inherited high risk or low risk hapolotype
What are the advantages of RHDO over RMD?
More statistically robust
Can be used for more complex variants not amenable to direct NGS/ddPCR:
- complex rearrangements e.g. F8 inversion
- pseudogenes e.g. CYP21A1, SMN1
What is the key to RMD and RHDO?
Ability to detect low levels of fetal variant that are also present in high background of maternal mutant cfDNA
Assess relative quantities of mutant and WT alleles
NGS with very high coverage or ddPCR
What is the phenotype of achondroplasia?
Normal limb length in trimesters 1 & 2, shortening in 3
Macrocephaly
Frontal bossing
Short fingers
Polyhydramnios
What is the phenotype of thanatophoric dysplasia?
Short limbs from first trimester
Short ribs & small chest
Frontal bossing
Short fingers
Polyhydramnios
How are FGFR3 variants tested for by NIPD?
NGS panel targeted to 31 known mutations
What is the common mutation associated with achondroplasia?
Gly380Arg accounts for 99% of mutations
What are the benefits of NIPD for FGFR3-related skeletal dysplasias?
Confirmation of sonographic diagnosis without risk of preterm labour
Allows parental counselling and preparation for delivery
Allows for a surgical termination if requested
Provides accurate diagnosis in twins without putting the normal twin at risk
What do mutations in FGFR2 cause?
Apert syndrome & Crouzon syndrome
