NIPT Or Non-invasive prenatal testing for aneuploidy

Non-invasive prenatal testing (NIPT) for aneuploidy is rapidly becoming the first line screening test for trisomies 13, 18 and 21 in the first trimester, with more and more hospitals offering the test as part of their routine prenatal care.

Three main methods are used in NIPT. Each is associated with specific advantages and disadvantages and we will discuss each in turn.

Massively Parallel Shotgun Sequencing (MPSS)

This approach was described by Fan et al. in 2008 (PNAS, 1056(16):266-71) and uses next-generation sequencing technologies to generate reads representing chromosome regions across the genome.

If there is trisomy of a particular chromosome, this would be detectable by a relatively greater number of reads mapping back to that chromosome region on the reference genome. However, this relative increase might be very small indeed.

For instance (as reported by the NIPT diagnostic laboratory at the South West Thames Regional Genetics Service) the reads representing chromosome 21 in a normal pregnancy typically might constitute 1.36% of the total cfDNA (remember, the total cfDNA is maternal and fetal cell free DNA).

However, in a pregnancy affected with trisomy 21, the total contribution of reads mapping to chromosome 21 from both mother and fetus might increase to just 1.42%. That is only a 0.06% difference!

Therefore, to distinguish such small differences in the amount chromosomal DNA found, an incredibly accurate counting and sorting method is required.

A disadvantage of MPSS is that, depending upon the underpinning NGS technology, the method can be costly with only a relatively small proportion of the generated reads being analysed.

Targeted multiple parallel sequencing (MPS)

There are various different forms of targeted MPS. However, they are all underpinned by the same concept - amplifying and quantifying chromosome-specific sequences.

One form of targeted MPS is DANSR (Digital Analysis of Selected Regions). DANSR uses oligonucleotide probes that hybridise to target sequences.

The principle is a bit like that behind MLPA in that it is the probes that are amplified rather than the target sequence. However, in contrast to MLPA, DANSR used three rather than two probes.

The number of each of the amplified probes reflects the amount of target sequence. Therefore, where a trisomy is present, there will be a relatively greater number of amplified oligonucleotides.

An advantage of targeted MPS is that costs are reduced because fewer sequences are generated. However, the targeted sequencing introduces biases and some believe reduces accuracy.

SNP detection

SNP detection is very different from the above two methods as, rather than analysing cfDNA as a whole, it differentiates between maternal and fetal cfDNA. In the commercially available kits, about 20,000 SNPs are utilised.

First of all, the maternal SNP genotype is determined through analysis of the buffy coat (the buffy coat is a layer of blood, mostly containing leukocytes and platelets and therefore not containing cfDNA, obtained through the centrifugation of a whole blood sample).

The plasma is then SNP genotyped which represents both fetal and maternal SNPs. Computationally, the two SNP results are compared and individual fetal and maternal SNP profiles are calculated.

Fetal fraction is first calculated. For instance, if mum is AA and fetus is AB for a given SNP locus, if there is 1000 of AA to 100 AB, then the fetal fraction is 10%.

Then, in order to determine copy number for each interrogated chromosome locus, the maternal and fetal genotypes are compared and evaluated to see whether there is more or less than expected.

Accuracy of NIPT

NIPT has been shown to have high sensitivity and specificity (>99%) for trisomy 21 detection. These figures are currently slightly lower for the other trisomies and for sex chromosome imbalance detection.

It is important to remember that these tests should be considered an advanced screening test rather than a diagnostic test. Sensitivity (likelihood of detecting a trisomy if present) is not the same as positive predictive value (PPV; likelihood of a positive result being a true positive), and the PPV for these tests are rather lower than the sensitivities.

Thus any positive result must be confirmed by invasive testing before any management decisions are made for the pregnancy.

The negative predictive value (NPV; likelihood of a negative result being a true negative) is high for these tests, so in the context of normal ultrasound findings a negative test is reassuring, and confirmation with an invasive test is not usually done.

However, in the context of abnormal findings on ultrasound, a negative NIPT should not be considered reassuring as other underlying chromosomal abnormalities have not been excluded and further invasive testing should be considered.

clinical situations is cell-free fetal DNA analysis not possible for detection of a maternally inherited single gene mutation.

NIPD cannot distinguish the presence or absence of a maternally inherited mutation in the fetus. This is because the mother carries the same mutation, so it is present in both the fetal and maternal cell free DNA fractions in the maternal plasma. Researchers are working on developing techniques which analyse the dosage of a given mutation in the maternal blood, but this is technically highly demanding and not currently developed for clinical use.

Twin pregnancy is not likely to cause an inconclusive result when performing NIPT for aneuploidy.
NIPT can be performed in twin pregnancies and is usually informative. If the aneuploidy screening comes back negative then this applies to both fetuses, and in the absence of any other indications, invasive testing can be avoided. If NIPT shows a possible aneuploidy then it could be difficult to know whether one of both foetuses is affected, and invasive testing is indicated.

Cell free DNA (cfDNA)

Cell free DNA (cfDNA) analysis is revolutionising prenatal genetic testing. Rather than subjecting women to invasive tests with associated discomfort and increased risk of miscarriage, analysis of cfDNA (through non-invasive prenatal testing, NIPT and non-invasive prenatal diagnosis, NIPD) is offering pregnant women an earlier, safer alternative.

Because of the close contact between maternal and fetal circulations in utero, a small amount of cell-free fetal DNA (not encased in a cell or nucleus) enters the maternal blood stream.

It originates from the trophoblast (placenta). This can be detected and analysed by taking a blood sample from the mother. Fetal DNA makes up around 10-20% of the total circulating cell free DNA in the maternal plasma. The cell free DNA in the maternal plasma comprises both maternal and fetal cell free DNA fractions, and distinguishing one from the other is one of the main challenges of cell free DNA analysis.

The presence of cell-free fetal DNA in maternal plasma and serum was first described by Lo and colleagues in the Lancet back in 1997.

Lo also spoke about how the arrival of whole genome sequencing brought about groundbreaking developments in the field of prenatal diagnosis at the PHG Foundation 15th anniversary conference, ‘Translating genomics: making science work for health’ in 2012.

The earliest stage in gestation that cell-free fetal DNA can be detected 4-5 weeks gestation. How soon after delivery do the cell-free fetal DNA levels fall. It is cleared from the maternal circulation within 30 minutes of delivery.

NIPD refers to the use of cfDNA analysis to diagnose monogenic disorders or to determine fetal sex or rhesus status. It does not require an invasive test for confirmation of the result.

NIPT refers to the use of cfDNA analysis as an advanced screening test for aneuploidy. Whilst this provides an accurate screening test for aneuploidy from 10 weeks’ gestation, positive results require confirmation by invasive testing.

Karyotype methods and applications

A karyotype describes all the chromosomes of a cell, usually visualised as a systemised arrangement of chromosome pairs, in descending order of size.

It is used to analyze the number and structure of all the chromosomes and provides a low-resolution genome-wide screen for chromosomal abnormalities.

An image of a normal male karyotype with 23 pairs of chromosomes

Figure 1: A normal male karyotype (note the X and Y sex chromosomes)
Original image from the National Institutes of Health and used under a public domain license.

When clinical cytogenetic analysis started in the 1960s, solid staining was used, which meant that the different chromosomes were only recognizable by size and shape.

In the 1970s the visualisation of chromosomes improved due to the development of specific stains and enzyme digestion techniques, giving the chromosomes reproducible banding patterns. Different techniques were utilised by different countries.

For instance, France used Q-banding whereas the UK used a combination of enzyme digestion with trypsin and G-banding (see below). Using these techniques, each chromosome pair could be visualised with a unique pattern, resembling a bar code.

This not only helped cytogeneticists to recognise the different chromosomes, but also allowed them to see when parts of the chromosome were rearranged, deleted or duplicated. The most widely used stain is the Giemsa stain, giving the familiar G-banding appearance shown in Figure 1.

How is a karyotype done?

Karyotyping can be done from any sample from which you can obtain nucleated cells which retain the potential to divide, most commonly:

Blood
Bone marrow
Skin
Prenatal samples (such as CVS or amniotic fluid)

Cells are cultured in an environment which includes stimulants for cell division, then arrested at metaphase and harvested for analysis. Cell culture can take anything from 3 days (blood/bone marrow) to 7-14 days (skin, prenatal samples).

This requirement to culture the cells increases the turnaround time, but is unavoidable.

Once harvested, the cells are mounted on slides, banded (enzyme treated and stained), then viewed under a light microscope (x1000).

How is a karyotype analysed?

The cytogeneticist counts the chromosomes, pairs the homologues and compares the banding pattern in each set of homologues to identify any differences. They are aided by software which allows them to ‘cut and paste’ the chromosomes so that they can be aligned for comparison, as shown in Figure 1.

Many attempts have been made to develop computer software to replace the job of the trained cytogeneticist but it has not been possible to replace the pattern-recognition skills of the human eye.

Even in the best hands however, the limit of resolution of a karyotype is around 5 Mb, and in many applications karyotyping has been superseded by the use of higher-resolution techniques such as array-CGH.

What sorts of abnormalities can a karyotype identify?

Karyotyping is able to identify numerical abnormalities in the chromosomes (also known as aneuploidy), where a chromosome is missing or present in one or more extra copies. Examples of this include trisomy of chromosomes 13, 18 or 21, or sex chromosome abnormalities such as Turner syndrome (45,X) or Klinefelter syndrome (47,XXY).

Karyotyping can also identify structural abnormalities in the chromosomes including rearrangements (such as translocations, insertions or inversions), deletions and duplications.

Chromosome deletions can be detected on karyotype down to a resolution of around 5 Mb, but some are very subtle and require a trained eye.

The karyotype shown in Figure 2 shows a 22q11.2 deletion associated with velocardiofacial (or DiGeorge) syndrome. Would you have spotted it?

Image of a Karyotype showing 22q11.2 deletion

Figure 2: Karyotype showing 22q11.2 deletion
© St George’s, University of London

How are karyotypes reported?

A clinical karyotype report will include a description of the cytogenetic findings, described using standardized nomenclature and displayed using a cartoon representation of the chromosome and its banding pattern, called an ideogram (Figure 3).

Image of an ideogram representation of chromosome 13

Figure 3: Ideogram representation of chromosome 13 Original image from Human chromosome ideograms from NCBI’s Genome Decoration Page

There may also be a description of any additional tests undertaken to confirm the results, a literature review with any suspected associations with phenotype, a description of any follow-up studies required and, where appropriate, recurrence risks will be given.

What is karyotyping used for currently?

In many contexts karyotyping has been superseded by the use of array CGH, which can also pick up numerical and structural abnormalities in the chromosomes, and at much higher resolution, however karyotyping is still in use in certain clinical situations.

One such situation is in the investigation of infertility or recurrent miscarriage, where a balanced rearrangement is suspected. A balanced translocation describes a rearrangement of chromosomal material, but with no overall gain or loss of genetic material.

The balanced translocation carrier is usually unaffected themselves, however problems may arise when they try to have children as the balanced translocation can affect the way that the chromosomes divide during meiosis, and imbalance can then occur in the offspring.

In this context, karyotyping has a distinct advantage over array CGH in that it is able to detect balanced rearrangements, which array CGH is not.

This is because a karyotype provides a visual representation of the chromosomes, unlike array CGH, which provides quantitative information on the amount of chromosomal material present, but not its location.

Question 1

4. Study the image below which includes a chromosome deletion associated with a recognised developmental disorder. Compare each of the chromosomes to its pair to see whether you can recognise any difference in banding pattern which might be indicative of there being a chromosome deletion or duplication. What syndrome do you think this patient has?

(Click to expand)

Smith Magenis (17p11.2)

Williams syndrome (7q11.23)

Velocardiofacial syndrome (22q11)

Wolf Hirschhorn syndrome (4p-)

Answers:-
1. D
An EDTA tube cannot be used. The correct blood tube to use for karyotype analysis is lithium-heparin. Samples for array CGH are collected in an EDTA tube however.

2. B
For karyotype preparation chromosomes are arrested in metaphase.

3. D
One copy of chromosome 22 looks shorter, and one copy of chromosome 9 appears longer. This translocation gives rise to an oncogenic fusion gene bcr-abl, causing chronic myeloid leukaemia (CML).

4. D
The short arm of the left-hand chromosome 4 is shorter in the top image than in the bottom image, due to a 4p deletion. This is associated with Wolf Hirschhorn, a developmental disorder characterised by a distinctive facial appearance, a (usually) severe intellectual disability and sometimes seizures.

Source:-

Simple question and answers: Fluorescence in situ hybridisation (FISH)

1.Which of the following statements about FISH probes is false?

Alpha satellite probes bind to centromeric repetitive DNA sequences

Beta satellite probes identify sequences present in only one copy number per chromosome

Unique sequence probes detect a specific chromosome region or gene

Fluorescent in situ hybridisation (FISH)

Fluorescent in situ hybridisation, or FISH is a technique that uses molecular DNA probes to detect complementary sequences along a chromosome. Unlike whole genome analyses, such as karyotyping or array CGH, FISH uses DNA-specific probes and will provide information both about the copy number of a chromosome region-- so that's whether a region's deleted or duplicated-- but also about the locational position of that region.

Let's spend a bit of time thinking about how FISH works. A FISH probe is designed which is complementary to the strand of DNA which you want to identify. The probe is labelled or tagged with a coloured fluorochrome. The chromosomes of the target cells are arrested in metaphase.

The cells in the chromosomes are fixed onto the surface of a glass slide. The slide is treated so that the genomic DNA is denatured into single strands. The slide is then washed with the single strand probe DNA. And the probe anneals, or hybridises, to the single-stranded target DNA, which is still in its natural position on the chromosome. That's a really key feature of FISH. So it is looking at the target DNA in situ. And because the probe is being tagged with a fluorochrome, which is a molecule which fluoresces when it is excited by a particular wavelength of light, it can be visualised using a fluorescent microscope.

Indeed, several different probes can be hybridised, observed, and then compared simultaneously by using different colours and combinations of these fluorochromes for each probe.

There are four major groups of FISH probes, which anneal to different types of DNA. Alpha satellite probes are complementary to the repetitive DNA sequences found at the centromere. Although there is considerable overlap between the DNA content at the centromere, there's also usually enough differences between chromosomes that individual chromosomes can be distinguished. However, that's not true for chromosomes 13 and 21. And it's also very difficult to distinguish chromosome 14 from chromosome 22 with an alpha satellite probe.

And as is often the case, it's actually usually really important to distinguish chromosomes 13 and 21, for instance, if a trisomy is suspected prenatally. And so in this situation, it's important to use other more specific probes that are specific to that chromosome region. Beta satellite probes are designed to anneal to the non-transcribed sequences of the acrocentric chromosomes. And these are chromosomes 13, 14, 15, 21, and 22. Beta satellite probes might be used to interrogate, for instance, extra material on the short arm of an acrocentric chromosome. Unique sequence probes are designed to anneal to specific chromosome regions. And these probes identify sequences usually present in only one copy number per chromosome.

Unique sequence probes are therefore locus specific and can detect a specific chromosome region or gene. And then finally, whole chromosome paints consist of a number of probes, which cover the whole of the chromosome other than the centromere region and contain a mixture of overlapping probes.

And this is a picture of a whole chromosome paint. And you'll see that it will paint the entire length of a chromosome, and can be used to identify the origin of unidentified material or rearranged chromosomes or marker chromosomes.

So let's think a bit about the applications of FISH. Historically, FISH was used in a wide variety of applications, many of which are listed here on this slide. However, the technique is time-consuming, costly, and laborious.

In many areas, it's now been superseded by newer, faster techniques. For instance, prenatal aneuploidy screening is now commonly done using QF-PCR. And array CGH has superseded FISH as the go-to test for microdeletion and telomere screening. Nevertheless, FISH still has a role in confirming array CGH findings. And it can be used to clarify the nature of a balanced or unbalanced translocation, and has found an increasing variety of applications in hemato-oncology.

Let's have a quick look at how FISH was used for aneuploidy screening. So I think this nicely shows the visual nature of FISH. The image here shows a slide with trisomy 21. You'll see that two chromosome-specific probes are used. The green probe anneals to unique regions of chromosome 13, whereas the red probe is specific to chromosome 21. And on this slide, you'll see that there are three red signals, whereas there are only two green signals. So the three red signals are indicative of there being trisomy 21, or Down syndrome. Common microdeletion syndromes with consistent break points usually arise due to non-homologous recombination. And there are various commercially produced probes for these different microdeletion syndromes.

So here we have an image showing DiGeorge, or velocardiofacial syndrome, caused by a deletion at 22q11. The green probe is a satellite probe identifying chromosome 22. And the red probe is specific for the commonly deleted region.

The normal image on the left shows two red signals for the 22q11 region. And the image on the right shows a single red signal for the 22q11 probe, indicating the presence of a deletion. And this one on the right can be seen in both the interphase and the metaphase nucleus.

But one of the main uses for FISH is in the confirmation of array CGH finding. And this shows a 1q21 deletion, which has been picked up on array CGH. And that's shown on the left-hand side of this slide, where you have the ideogram, and then next to it a graph with a peak representing the 1q21 deletion. So to replicate the finding, a suitable FISH probe has been chosen. And the FISH image here on the right shows a single red signal representing the 1q21 region and therefore confirming the result.

FISH can also be used in the identification of marker chromosomes. The karyotype here shows an extra piece of chromosome material, labelled here as mar, standing for marker. And the suspicion looking at the karyotype was that the marker came from chromosome 2. So FISH was used to confirm this.

The image on the left shows whole chromosome painting revealing that the marker chromosome is derived from chromosome 2, and that's painted red. And the right-hand image shows a green alpha satellite probe for chromosome 2, again confirming its origin.

FISH is now widely used in hemato-oncology. And that's to aid diagnosis, estimate prognosis, and guide appropriate drug therapy. And the image shows the use of FISH in the diagnosis of chronic myeloid leukaemia, or CML. Probes to BCR and ABL regions are used. And this shows the classic translocation between chromosomes 9 and 22. And this is known as the Philadelphia chromosome, which is pathognomic of CML.

The arrows point to the translocated chromosome, where the red and green signals can be seen to bind side by side. So having thought a little bit about how FISH works and then the clinical applications of FISH.

limitations of FISH

Let's think, If the “standard” FISH probe is between 100 and 200 kb, what do you think the limitations of FISH might be? One of the key limitations of FISH is that it might not detect deletions that are smaller in size than the FISH probe used. This is because the probe can “bridge” the deletion.
What other disadvantages, not related to size are there of using FISH?
As well as the limitations relating to size, there are also other disadvantages associated with using FISH:
• The technique is time-consuming and relatively expensive.
• It requires a fresh blood sample (in a lithium heparin tube); the analysis can’t be undertaken on stored DNA.
• The technique is not useful to detect small tandem duplications where the signals will be overlapping and not therefore distinguishable from each other.
What information might FISH give you that is not available from other techniques to look for dosage abnormalities such as array CGH and MLPA?
FISH will give you positional as well as dosage information. MLPA and array CGH will only tell you about the dosage of a given chromosome locus.