18.05.10 Application of NGS for prenatal diagnosis Flashcards Preview

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Flashcards in 18.05.10 Application of NGS for prenatal diagnosis Deck (16)

What is the incidence of chromosome abnormalities in live births?

1 in 160


What % of pregnancies have a foetal anomaly?



What are the existing prenatal testing strategies following the detection of foetal anomalies?

QF-PCR (or FISH) for rapid trisomy screening in parallel with cell culture and G-banding or array CGH.

Karyotyping required for QF-PCR +ve cases
aCGH for QF-PCR -ve cases


What % of foetal anomaly cases will go undiagnosed following current prenatal testing practices?



What sample type could be used for foetal genotyping using NGS?



What are the different possible applications of NGS in the prenatal setting?

1. Identify chromosome anueploidies and imbalances
2. For single gene disorders


What are some of the current limitations of using NGS in clinical practice for prenatal testing?

Turnaround times in time-critical cases
Amount of DNA available for testing (And confirmation)
Price, equipment, skilled staff etc.


What possibilities does NGS offer in the prenatal context?

Design panels that can screen for multiple mutations in a single assay

Analyse samples from different patients in a single run (reduced overall cost compare to single exon analysis).

use targeted cffDNA libraiires to improve sequence depth

be more suitable to the detection of de novo mutations or the diagnosis of diseases of unknown pathophysiology.


What are the different types of NGS technique that could be used in the prenatal setting?

1. Haplotype- based NGS
2. Relative haplotype dosage (RHDO)
3. Targets panel NGS
4. WES


Describe the study by Drury et al into the use of WES in cases with an abnormal ultrasound result and a normal result on karyotyping.

1. Recruited 24 cases from FMUs with NT >3.5mm or abnormality

2. Invasive test and parental bloods taken and consented.
Two cohorts 1) Proband only 2) Trios

3. Excess material used for molecular testing if karyotype normal.

4. Any relevant results were fed back at the end of pregnancy.


What were the results and conclusions from the Drury et al WES study?

Definitive diagnosis obtained in 5 out of 24 cases tested (21%)

Highly plausible diagnosis in one fetus

Highly suggestive of an autosomal recessive condition in two cases (8%)

Mutations detected unrelated to the sonograohic findings in two cases (8%)


1. Exome sequencing in fetuses with sonographic abnormalities can reveal the underlying cause in a significant proportion of cases

2. Inability to fully cover the exome hinders interpretation

3. Needs fast data interpretation pipeline and parental blood must be available upfront

4. Numbers too small to draw useful conclusions regarding the value of testing in fetuses with an isolated increased nuchal


Describe the study by Carss et al into the use of WES in cases with an abnormal ultrasound result and a normal result on karyotyping. Neonates and prenatal samples.

Proof-of-principle study of 30 fetuses and neonates with structural anomalies first identified by ultrasound.

llustrates the power of whole-exome sequencing for identifying variants (single-nucleotide variants (SNVs), indels and CNVs) that potentially cause abnormal fetal development.

Referrals from FMUs with structural abnormalities detected

DNA obtained from prenatal or neonatal samples

Exome seq using SureSelect + paired-end on the HiSeq

Variants divided into causual, likely causal (needs further work e.g. functional analysis), or unknown


What were the results from the Carss study?

Exome sequencing successful in 91% of cases (4 cases insufficient DNA)

The mean depth of coverage of the targeted coding regions was 103×.

A mean of 92.7% of bases in the targeted coding regions were covered by at least 10 reads

No parental phenotypic abnormalities were reported that might be related to the fetal abnormalities, suggesting dominant inheritance is unlikely.

Evaluated different classes of potentially pathogenic, rare coding variants under dominant de novo, recessive and X-linked modes of inheritance, through systematic manual curation of the existing literature, to classify variants into one of three categories: highly likely to be causal, possibly causal or unknown.


What were the conclusions from the Carss study?

Identified an average of one candidate gene with a de novo functional variant and five candidate genes with inherited functional variants per fetus.

Variants that are highly likely to be causal were noted in three cases (10%).

Variants that are possibly causal but require further confirmatory genetic and functional studies were noted in a further five cases (17%).

Exome sequencing in the prenatal setting is inevitable; the challenge will be to incorporate it into prenatal diagnostics in a responsible manner, with an ethical framework.


Give an overview of PAGE.

In 2013, the Wellcome Trust, alongside the Department of Health (England), awarded a Health Innovation Challenge Fund to the PAGE ‘consortium’ project.

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

1000 fetuses with structural abnormalities, along with maternal and paternal samples will undergo exome sequencing or whole gene sequencing using DNA extracted from CVS or AF.


What are the primary aims of PAGE?

1. elucidate the relative contribution of different forms of genetic variation to prenatal structural anomalies;

2. design cost-effective genome sequencing assays for improved prenatal diagnosis of structural anomalies;

3. catalyse the adoption by the NHS of prenatal diagnostic sequencing, through transfer of acquired know-how, rigorous health economic assessment and establishment of an ethical and social science framework for clinical implementation