Prenatal Flashcards

Question

Describe the process of Haplotype-based NIPD NGS for X-linked & autosomal recessive disorders.

Answer 1

haplotype-based analysis used to determine the inheritance of parental mutations in a linkage type manner. This involves grouping SNPs into informative categories based on parental zygosity. Phasing of parental SNP haplotypes is achieved by comparing genomic DNA samples from the mother, father, and affected proband. Analysis of cfDNA sequencing data then enables the detection of the mutant or normal haplotype in the fetus

Answer 2

fetuses with sonographic abnormalities can reveal the underlying cause in a significant proportion of cases. It illustrated the power of whole-exome sequencing for identifying variants that potentially cause abnormal fetal development

Answer 3

The project's main objective is to answer questions regarding the use of exome sequencing as a diagnostic tool for invasively acquired samples when a structural anomaly is found on prenatal ultrasound examination

Answer 4

For fetus with multiple major structural abnormalities on ultrasound where an MDT considers a monogenic malformation disorder is likely

Answer 5

Major congenital abnormalities affect 1-1.5% of pregnancies, and chromosomal abnormalities account for 20-25% of these major fetal anomalies. Microarrays can provide higher resolution for detecting these abnormalities

Answer 6

For Abnormal ultrasound scan (USS) results and a normal QF-PCR result for the common aneuploidies (or other method for rapid aneuploidy screening)

Answer 7

Culture is not required prior to reporting, leading to a faster turnaround time and exclusion of culture artefacts Higher resolution and better detection of sub-microscopic abnormalities, resulting in a higher pickup rate

Answer 8

Requires a high quantity of high quality DNA Does not detect balanced rearrangements Lower level mosaicism may be missed Triploidy will be missed or mis-classified Difficulties in interpretation of Variants of uncertain clinical significance (VOUS

Answer 9

A gene within the deletion region on the homologous chromosome could be mutated (autosomal recessive condition) The CNV might contain an imprinted gene with a pathogenic effect only when inherited from a specific parent The CNV itself could be a risk factor with incomplete penetrance, requiring a specific genomic context Flashcard 9

Answer 10

Incidental findings generally not to be reported include: 15q13.1q13.3 duplications 15q11 BP1-BP2 duplications or deletions Xp22.31 (STS) duplications 16p13 duplications Heterozygous deletion of recessive genes that cannot be linked to the presenting phenotype Variants with no relation to the prenatal phenotype or no clinically actionable consequence

Answer 11

Results suggest that CMA is a robust, acceptable, and probably cost-effective diagnostic test and should replace karyotyping in care pathways when the indication is one or more structural anomalies or an isolated NT of ≥ 3.5 mm on ultrasound after a normal QF-PCR result

Answer 12

od communication is essential because many different results can be generated. There needs to be agreement on what to report to the clinician and the patient, especially regarding pathogenic CNVs inconsistent with ultrasound findings, the origin of CNVs, and how to handle unsolicited/incidental findings with implications for the fetus or parents

Answer 13

e percentage of total cell-free DNA in maternal plasma that comes from the placenta (cffDNA

Answer 14

Detectable from 4-5 weeks, reliably testable from 7-9 weeks

Answer 15

Low abundance Substantially outnumbered by cell-free maternal DNA (Nearly) indistinguishable from maternal cell-free DNA Need to prevent maternal genomic DNA release from blood cells

Answer 16

Present in very low numbers (~1 to 2 cells per ml) Potential for sampling bias Requires visualization of multiple cell markers and high throughput image processing Whole genome amplification issues (allele dropout/preferential amplification)

Answer 17

-Analyzing paternally inherited SNPs where maternal DNA is homozygous for the other allele -Or using universal fetal epigenetic markers, such as methylation patterns specific to the placenta (e.g., SERPINB5, RASSF1)

Answer 18

Many methods, like methylation-specific PCR, use bisulfite conversion which causes massive degradation of input DNA (up to 95%). This is problematic due to the already low levels of cell-free DNA

Answer 19

◦Fetal sex determination for sex-linked diseases ◦Diagnosis of fetal blood type (particularly RhD) ◦Detection of paternally inherited/de novo pathogenic variants for AD disorders ◦Testing for maternally inherited variants for AR/X-linked disorders using relative mutation dosage (RMD) or relative haplotype dosage (RHDO)

Answer 20

Pegnancies at risk of serious X-linked conditions (like Duchenne muscular dystrophy) and those at risk of congenital adrenal hyperplasia (CAH)

Answer 21

By detecting the presence of male Y chromosome DNA (e.g., SRY gene or DYS14) in maternal plasma using sensitive methods like Real-time PCR (RT-PCR). A female fetus is inferred by a negative result for the Y chromosome

Answer 22

Anti-D therapy can be selectively given only to RHD negative women carrying an RHD positive fetus, avoiding unnecessary treatment for those carrying RhD negative fetuses

Answer 23

Maternally inherited or de novo pathogenic variants. This is because these altered alleles are not present in the high background of maternal cfDNA

Answer 24

NGS NIPD panels (e.g., for FGFR3-related conditions, Retinoblastoma, Apert syndrome) or PCR-based assays ("bespoke NIPD") designed to detect the relevant varian

Answer 25

CAH is an autosomal recessive disorder. If a female fetus is detected, she can be treated with dexamethasone from 6-7 weeks gestation to prevent virilisation. If a male fetus is detected, this treatment is not required and can be avoided

Answer 26

RMD is a method for testing maternally inherited variants in AR/X-linked disorders where the fetal material is present in the background of maternal cfDNA containing the maternal variant. It involves determining the relative amounts of mutant and normal alleles in the maternal plasma *An affected fetus (homozygous mutant) would contribute only mutant alleles, leading to more mutant alleles than expected *An unaffected fetus (homozygous wildtype) would contribute only wildtype alleles, leading to more wildtype alleles than expected The shift in allele ratios is proportional to the fetal fraction, which must be high enough and accurately determined

Answer 27

Does not require paternal or proband samples

Answer 28

Technically challenging as accurate counting of mutant and wild-type alleles is required Requires techniques like digital PCR or targeted NGS with high read depth and unique molecular identifiers High depth NGS is expensive It is targeted, only looking at a single mutation site, making it less statistically robust than RHDO

Answer 29

RHDO is a linkage-based method that uses thousands of SNPs flanking the disease gene to determine maternal and paternal inheritance *The method used is capture-based targeted NGS *It is in diagnostic use for SMA, DMD, and CF

Answer 30

Proband or unaffected sibling sample Paternal sample Known parental carrier status *For X-linked RHDO, a paternal sample is not essential, but a closely-related unaffected/affected male is needed

Answer 31

Statistically robust as it uses many different loci Follow-up invasive testing is not necessary if a clear result is obtained (provided fetal fraction is high enough) Can be used for mutation types/genes not amenable to direct NGS Workup is not required prior to testing as it is a generic approach Possibility of multiplexing the assay

Answer 32

Risk of double recombination Requires proband or unaffected sibling sample and paternal sample Expensive as it needs NGS depth and multiple samples Consanguinity may lead to issues if insufficient informative SNPs are available Can only be used for singleton pregnancies

Answer 33

For paternal inheritance (using a KS test), RHDO uses SNPs that are homozygous in the maternal genotype and heterozygous in the paternal genotype (category 3 SNPs). If the paternal haplotype is present, approximately 5% of cfDNA (half of cffDNA) will contain the SNP present only in the paternal genotyp

Answer 34

for maternal inheritance, RHDO uses SNPs that are heterozygous in the maternal genotype and homozygous in the paternal genotype (category 4 SNPs). These are divided into α-SNPs (mutant maternal allele matches paternal allele) or β-SNPs (normal maternal allele matches paternal allele). An over-representation of either the mutant (α-Mat) or normal (β-Mat) maternal allele can be used to infer the fetal genotype

Answer 35

Whole genome screening for multiple genetic diseases (including common microdeletion syndromes) from maternal blood Non-invasive exome screening to detect paternally inherited alleles and de novo pathogenic variants Applying a universal haplotyping approach to extend RHDO to many more disorders ‘Proband-free’ RHDO using methods for direct parental haplotyping from cell-free DNA, potentially using targeted locus amplification or linked read sequencin

Answer 36

PHG – The Foundation for Genomics and Public Health (UK Expert Working Group) RAPID Project (Reliable Accurate Prenatal non Invasive Diagnostic) - a 5-year UK national programme

Answer 37

Reduced number of invasive tests Safer for the fetus and mother (almost no risk of infection or physical trauma, unlike invasive tests which have ~1% miscarriage risk) Less expertise required (a blood test vs. invasive procedure) Can be offered earlier than invasive testing, allowing parents more time to decide about the pregnancy *Less traumatic for the parents * Improved quality and management of antenatal care (e.g., better targeting of anti-D therapy)

Answer 38

in multiple pregnancies, it is not possible to differentiate between the fetuses cffDNA may not be present in equal proportions from all fetuses in multiple pregnancies Lower fetal fraction in women with a high BMI increases the chance of an inconclusive result Potential for incorrect results (false negatives due to low cffDNA or low fetal fraction; false positives due to technical issues like contamination or clinical abnormalities like a vanishing twin

Answer 39

Risk of false negative results leading to lack of treatment or birth of an affected child Risk of false positive results leading to termination of healthy pregnancies or unnecessary distress Availability of non-clinical applications (e.g., social sex selection, prenatal paternity testing) directly to consumers over the internet, raising questions about regulation of direct public access

Answer 40

By amplifying highly polymorphic short tandemly repeated DNA fragments (STRs) found in non-coding regions. These STRs are used due to their polymorphic nature and tendency to mutate

Answer 41

To detect maternal cell contamination (MCC). MCC cases are expected to produce a characteristic pattern with extra alleles or skewed ratios between peaks for all chromosomes. Running the maternal sample allows for comparison of traces

Answer 42

Multiple polymorphic X markers (though not 100% reliable), non-polymorphic markers such as AMELX/Y (Amelogenin), SRY, and non-polymorphic markers to determine X chromosome dosage compared to an autosome. Use of TAF9 on chromosome 3 and the X chromosome can help

Answer 43

It can also detect specific microdeletions, and cryptic or subtle duplications. It's particularly helpful when analysing complex structural rearrangements and marker chromosome

Answer 44

Due to confined placental mosaicism

Answer 45

: The limit for detection of mosaicism by QF-PCR is approximately 5-20%. However, mosaicism with an almost equal ratio (e.g. 50%) of 45,X and 47,XXX cells cannot be detected.

Prenatal Flashcards

(72 cards)