9. NIPD - ffDNA and RNA in maternal serum & applications

Question 1

Why are intact fetal cells not used for NIPD?

Answer 1

Present in small numbers & may persist in maternal circulation for years –> risk of false positive in subsequent pregnancies

cffDNA cleared from circulation after delivery

Question 2

How is the presence of cffDNA confirmed?

Answer 2

Panel of SNPs

Look at SNPs where mother is homozygous - presence of paternally inherited allele quantified

Question 3

Why is CPM not a consideration for NIPD?

Answer 3

CPM not reported for monogenic disorders, unlike aneuploidies

Question 4

Why is an ultrasound recommended in advance of NIPD?

Answer 4

1. To exclude the possibility of a vanishing twin which could obscure the genotype of the surviving fetus
2. To date the pregnancy as abundance of cffDNA increases with gestational age

Question 5

Why is fetal sex determination carried out by NIPD?

Answer 5

For pregnancies at risk of sex-linked disease, e.g. DMD, ALD

Question 6

How is fetal sex determined? What is the outcome for an X-linked disorder?

Answer 6

Detection of SRY by qPCR

Multiple replicates over 2 different extractions

Male fetus –> invasive test for condition
Female fetus –> no further testing

Question 7

Why is CAH tested for by NIPD? What is the outcome?

Answer 7

Females with CAH show virilisation - develop characteristics associated with male hormones (androgens).

Female fetuses treated with dexamethasome from 6 weeks

Question 8

In what scenarios can NIPD be used for AD disorders?

Why?

Answer 8

1.If variant is paternal inherited

2. If variant was de novo in previous offspring (risk of germline mosaicism)

cffDNA will contain variant not present in high background of maternal cfDNA

Question 9

What is the limitation for CNVs in NIPD?

Answer 9

Size of cffDNA fragments (approx. 300bp)

Question 10

How are AD disorders tested for by NIPD?

Answer 10

1. Targeted NGS NIPD panel, e.g. for FGFR3-related disorders
2. Bespoke PCR-based method - required DNA from parent/proband in advance

Question 11

What are the 3 ways that AR disorders can be tested for by NIPD?

Answer 11

1. Paternal exclusion testing (fetus is at most a carrier if paternal variant is absent)
2. Relative mutation dosage
3. Relative haplotype dosage

Question 12

How does RMD work?

What is it used for?

Answer 12

Determine relative quantities of mutant and wildtype alleles

AR disorders when both parents have same variant

Question 13

RMD - what does it mean if there are equal amounts of mutant and WT alleles?

Answer 13

Carrier mother and carrier fetus

Question 14

RMD - what does it mean if there are increased levels of mutant alleles?

And increased levels of WT alleles

Answer 14

Increased levels of mutant alleles = homozygous affected fetus

Increased levels of WT alleles = unaffected fetus

Question 15

What method is used for RMD and why?

Answer 15

Usually ddPCR or NGS with v high read depth

Question 16

What are the advantages & disadvantages of RMD over RHDO?

Answer 16

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

Question 17

Why must the fetal fraction be accurately determined for RMD?

Answer 17

Shift in allele ratios is proportional to fetal fraction

Question 18

How does RHDO work?

Answer 18

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

Question 19

What are the advantages of RHDO over RMD?

Answer 19

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

Question 20

What is the key to RMD and RHDO?

Answer 20

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

Question 21

What is the phenotype of achondroplasia?

Answer 21

Normal limb length in trimesters 1 & 2, shortening in 3
Macrocephaly
Frontal bossing
Short fingers
Polyhydramnios

Question 22

What is the phenotype of thanatophoric dysplasia?

Answer 22

Short limbs from first trimester
Short ribs & small chest
Frontal bossing
Short fingers
Polyhydramnios

Question 23

How are FGFR3 variants tested for by NIPD?

Answer 23

NGS panel targeted to 31 known mutations

Question 24

What is the common mutation associated with achondroplasia?

Answer 24

Gly380Arg accounts for 99% of mutations

Question 25

What are the benefits of NIPD for FGFR3-related skeletal dysplasias?

Answer 25

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

Question 26

What do mutations in FGFR2 cause?

Answer 26

Apert syndrome & Crouzon syndrome