mosaicism

New Tech Helps At-Risk Couples Ensure Their Kids Don’t Inherit Their Illnesses

Thanks to technological advances, at-risk couples undergoing IVF can have their embryos subjected to genetic screening before being implanted in the womb.

To be born with the right genes used to be a blessing. But thanks to technological advances, it is possible today for at-risk couples undergoing in-vitro fertilisation (IVF) to have their embryos subjected to genetic screening before being implanted in the womb. This way, the couple will know the child doesn’t have any genetic or chromosomal defects.

Dr Nandita Palshetkar, president of the Federation of Obstetric and Gynaecological Societies of India, shared a case study with The Wire. Two partners – both carrying the genes for thalassemia, a life-limiting blood disorder – had tried to have a baby thrice but failed. The first two pregnancies had to be medically terminated after doctors diagnosed the foetuses with thalassemia and the third was a miscarriage.

Doctors then advised the couple to undergo IVF followed by pre-implantation genetic diagnosis (PGD). Two IVF cycles later, they had six embryos at the day-5 stage. An embryologist performed biopsies and sent the cell samples to a genetic lab to be analysed; the embryos were kept frozen. When the results arrived, they showed three embryos were ‘normal’ and three had inherited the genes for thalassemia. So doctors transferred the ‘normal’ embryos to the womb. Later, an amniocentesis confirmed that the child did not have thalassemia, and the mother delivered a healthy baby.

A diagram illustrating how IVF works: After ovulation, the egg is collected from the woman’s reproductive organs, fused with sperm and the resulting fertilised ovum is reinserted into the uterus. Image: Manu5/Wikimedia Commons, CC BY-SA 4.0

“Any additional embryos that are free of genetic problems are kept frozen for possible use later while embryos with problematic genes are destroyed,” Dr Palshetkar said.

“PGD enables identification of genetic defects in the early embryo before the embryo is transferred to the uterus in an IVF program,” Dr N. Pandiyan, the head of the department of reproductive medicine at the Chettinad Super Speciality Hospital and Research Institute, Chennai, explained.

Dr Sasikala Natarajamani, founder and scientific director of Crea Conceptions, an assisted reproduction centre in Chennai, said that, “according to the Human Fertilisation and Embryology Authority, UK, PGD has been approved for testing over 600 genetic conditions.”

Also read: After U-Turn on Rare Disease Policy, Health Ministry Assures New One in 9 Months

There are “numerous tests,” in Dr Pandiyan’s words, that enable PGD. For example, scientists developed PGT-M – pre-implantation genetic testing for monogenic defects – to prevent the birth of a baby with genetic defects “when the previous baby was affected” or when the parents have “a history of inheritable disease in the family.”

Another kind is PGT-A, where the ‘A’ stands for aneuploidy, a condition where the body’s cells don’t have the normal number of chromosomes, 46. This gives rise to chromosomal disorders like Down syndrome. In PGT-A, embryos from parents presumed to be ‘chromosomally normal’ are screened to check whether the cells in each embryo contain 46 chromosomes. Such screening is called pre-genetic screening (PGS).

“At present, PGS and PGD are the only options available to parents trying to avoid the risk of having a child affected with a genetic disease, prior to implantation,” Dr Natarajamani added.

PGD has been gaining traction of late. “Screening the embryo for possible life-threatening and debilitating genetic diseases before transfer changes reproductive options for families at risk,” Dr Palshetkar told The Wire. “In some cases, further testing is needed during pregnancy to ascertain if a genetic factor is still possible.”

But “overall, PGD has been able to diagnose genetic defects with approximately 98% accuracy,” she added.

This is the result of major advancements in the last three decades, such as being able to visualise specific chromosomes using fluorescence in situ hybridisation, to produce thousands of copies of a DNA fragment using the polymerase chain reaction, and to profile genomes rapidly using next-gen sequencing (NGS).

This isn’t to say the procedure is entirely free of issues. For example, according to a review published by the American Society for Reproductive Medicine (ASRM) in 2017, IVF embryos sometimes have a condition called mosaicism – where all cells in the embryos don’t have identical sets of chromosomes. When these abnormal cells proliferate, it could result in a miscarriage or severe birth defects.

However, Dr Natarajamani said, “Techniques like NGS have allowed for very high-fold coverage,” allowing scientists to detect “even low levels of mosaic mutations” and ensure only the healthiest embryos are transferred. In general, according to the review, NGS can eliminate mosaic cells when they make up fewer than a fifth of all cells in the embryo but fail when the extent of mosaicism crosses 50%. In the latter case, the embryo is deemed aneuploid and discarded.

According to Dr Palshetkar, the most frequently diagnosed disorders include those of blood and muscles, and other rare conditions such as cystic fibrosis, beta thalassemia, sickle-cell disease, spinal muscular atrophy, myotonic dystrophy, haemophilia A and Duchenne muscular dystrophy.

Since the requisite testing facilities aren’t widely available in India, results can take up to three weeks to arrive. But this hasn’t put people off from opting for it, especially since the procedure seems safe. According to the Centre for Advanced Reproductive Services, Connecticut, there has thus far been no increase in the rate of congenital abnormalities or pregnancy complications due to embryo biopsies.

Nonetheless, Dr Natarajamani hopes that non-invasive techniques will replace biopsies soon, given the inescapable risk the latter procedure carries in some cases.

Also read: Widely-Available Genetic Risk Tests Aren’t Always Useful – and Could Even be Harmful

Then there are the potential ethical issues. However, Dr Pandiyan is quick to defend PGD and draws a line between PGD and PGS as well as other, more morally fraught practices.

“PGD is clearly and absolutely indicated in certain circumstances, like in couples with a previous child with abnormalities, with a family history of abnormalities, etc.,” he said. “The debate is only about routine screening of all embryos, as in PGS, before embryo transfer or when [the technique] is used to identify the sex of the embryo.”

In his telling, the doctors’ decisions aren’t arbitrary but are guided by the recommendations of bodies like the ethics committee of the ASRM. For example, in an opinion published last year, entitled ‘Disclosure of sex when incidentally revealed as part of … PGT’, this committee reached the following conclusion:

“When patients undergo [assisted reproductive therapy] and PGT for medical reasons, embryo sex could be a common secondary finding. Patients should be informed of this possibility before undergoing PGT. Patients have the right to information about an embryo’s sex, as well as the right to request not to be given this information. Clinics may have policies not to take an embryo’s sex into account in making transfer decisions. Clinics must have nondiscrimination policies regarding embryo transfer when the sex of the embryo is known. Patients should be informed about these policies.”

Indeed, all genetic labs in India are required to follow guidelines set by the Pre-Conception and Pre-Natal Diagnostic Techniques (Prohibition Of Sex Selection) Act 2003.

Dr Palshetkar, like Dr Pandiyan, has no doubts: “PGD is an amazing technology and the application of next-gen sequencing [has been] a game-changer. It will reduce the incidence of genetic diseases and complications [associated with] multiple births.” She added that many IVF centres around the world have also been able to achieve “fantastic birth rates” after transferring “only one genetically normal embryo” into the womb.

Sudha Umashanker is a Chennai-based freelance journalist, author and award-winning storyteller.

How a Battleground Between Bacteria and Viruses Is Being Used for Human Health

Viruses can’t replicate on their own, so they infect other organisms and hijack their cellular machinery to produce viral offspring. To defend themselves, bacteria evolved CRISPR/Cas.

Evolution is both fascinating and horrifying in its ruthlessness. The strong survive and proliferate, while the weak are mercilessly done away with. All around us, billions of microorganisms such as fungi, bacteria, and viruses are constantly at war in environments that have limited nutrients. In fact, understanding these war tactics enabled the discovery of penicillin, a chemical compound produced by a fungus called Penicillium chrysogenum. This compound inhibits the growth of pathogenic bacteria, such as Staphylococcus, by weakening the structure of their cell walls, eventually causing cell death by explosion. Viruses are no less vicious; since they cannot replicate on their own, they infect other organisms, hijack their cellular machinery to produce viral offspring, and kill their hosts. To defend themselves against viral infections, bacteria evolved the CRISPR/Cas system.

CRISPR stands for clustered regularly interspaced short palindromic repeats, a mouthful for an elegant mechanism of self-defence. Consider the following situation: Team A and Team B are locked in a perpetual battle. Team A is always trying to invade Team B’s territory and capture all its resources. Every member of Team A carries identical ID cards and when one member of Team A invades Team B’s territory, Team B captures the ID card and stores a copy in its database. The next time another member of Team A tries the same thing, Team B compares the two ID cards. If it’s a perfect match, the invading Team A member is destroyed. This is essentially what happens between bacteria (Team B) and phages (Team A), the viruses that infect bacteria.

Figure 1: How CRISPR/Cas immunity works. Credit: Club SwiWri

Bacteria (Team B) make use of the fact that a phage (Team A) injects its genetic material during an infection. The bacteria cut the material in specific locations to create protospacers. These protospacers are then copied and modified to produce spacers, which are integrated into the bacterial genome within the CRISPR locus. Therefore, the bacteria are able to permanently maintain a record of the information carried in the spacer sequence. CRISPR refers to the short, repetitive sequences that separate each unique spacer sequence. As shown in the figure, Spacer 1, Spacer 2, and Spacer 3 can come from several different phages, therefore each bacteria has a unique sequence stored in its genetic database. Upon subsequent infection, the bacteria utilise the Cas proteins, such as Cas9, to compare the spacer sequences they acquired from the previous infection to the sequence of the invading phage. If the copies match, the bacteria proceed to destroy the invading genetic material by cutting it up completely.

There are several diverse uses of CRISPR sequences- since they are unique, the spacers can be used to identify the bacteria that carry them. For example, although the current methods of detecting Salmonella infections are extremely accurate, they are technically demanding; hence, difficult to implement in poorer countries. Additionally, these methods are non-automated, which delays detection. However, identification of ‘signature’ CRISPR sequences can be automated, thus helping detect bacterial pathogens and thereby the corresponding bacterial subtype, quickly and accurately. CRISPR sequences have also found a place in the dairy industry – for cheese production. The cheese-making process requires very high densities of bacterial cultures, thus making them extremely vulnerable to phage infections, which can kill the entire bacterial population. The introduction of CRISPR sequences into these bacteria makes them resistant to phage infections thereby eliminating production problems.

The most exciting applications of the CRISPR/Cas9 technology, however, are based on the fact that Cas9 proteins can create precise double-stranded DNA breaks. Therefore, these proteins can be guided to precisely edit host DNA sequences resulting in insertion, deletion, or other changes in the genetic sequence. Furthermore, since the technology is simple, it can be used to modify several targets simultaneously, and can be used in a wide range of eukaryotic organisms, ranging from yeast to humans. A further improvement on this system is the use of a protein called Cpf1, instead of Cas9, which enables new targeting possibilities and improves the efficiency of genetic insertions.

Figure 2: Types of DNA manipulation that can be accomplished with the CRISPR/Cas9 system. Credit: Club SciWri

The DNA of interest is cut using Cas9. The eukaryotic cell can then process the DNA via either of two distinct pathways: Pathway 1 that joins cut ends, which can either result in a loss of genetic information (deletion) or insertion of a new sequence between the cut ends or Pathway 2 where the missing DNA sequence is rebuilt by using the other copy of host DNA which contains the intact sequence as a template.

Where can we use this gene manipulating ability of the CRISPR/Cas9 system? One of the most exciting applications is to kill bacterial pathogens. Antibiotic resistance is becoming one of the most pressing health problems in the world today. One potential way to deal with these drug-resistant bacteria would be to make specific deletions in their sequence, say by targeting essential genes, using CRISPR/Cas9. To do so, a cocktail of phage that contain the CRISPR message would be introduced into the body, the phage would then target the pathogenic bacteria, therefore leading to their destruction. Of course the irony is that the CRISPR system, which bacteria use to kill phage, would now being harnessed to make phage kill bacteria. The added advantage of this system would be that the natural, non-pathogenic bacteria in the body would remain untouched, which is not the case when antibiotics are used.

So why is there controversy surrounding the CRISPR/Cas9 system? As discussed before, CRISPR/Cas9 can make very specific genetic modifications. This trait can be immensely useful when the technique is used to cure diseases such as cataract disorders, Duchenne muscular dystrophy and treat viral infections such as HIV and hepatitis B. However, this ability to manipulate genes may lead to potentially unethical practices, such as eugenic selection of traits in human embryos.

This genetic modification process may also have off-target effects, where changing one gene may lead to unintended changes in other genes. Although there is no convincing data that indicate such undesired effects, they still remain a concern. With these issues in mind, in 2015, the members of the national scientific communities of America, Britain and China agreed that the technology would only be used for basic research provided that the legal and ethical guidelines were followed. Further, the technology would be utilised only for somatic cells, where only a single individual would be affected, rather than for germ cells, where gene modifications would be inherited by the offspring and could therefore affect human evolution.

A more concerning problem was recently published, where researchers found that CRISPR could lead to large-scale DNA deletions and rearrangements. Granted that this unwanted side effect is not unique to CRISPR and can happen with other gene-editing tools, the finding calls for increased vigilance among researchers. Usually CRISPR is used to create small deletions to knock out the effect of a gene. However, close investigation seems to suggest that larger deletions are present in the vicinity of the target gene. Although these side effects are concerning, careful experimental design and sequencing of the target DNA including the surrounding sequences should go a long way in avoiding this danger. Nevertheless, the applications of CRISPR/Cas9 are exciting and can have far-reaching medical uses, which can make (many) life-threatening genetic conditions a thing of the past.

This article was originally published on Club SciWri and has been republished here with written permission.

All You Need to Know: The Latest Gene Editing Breakthrough

The most risky aspect of gene editing is accidentally editing the wrong gene, causing problems that can be passed down generations.

Credit: qimono/pixabay

What happened?

Reproductive biologist Shoukhrat Mitalipov led a team of scientists from Oregon Health & Science University, Portland, and the Salk Institute, California, as well as collaborators in South Korea and China, to make a pretty huge breakthrough in gene editing. They targeted a dangerous mutation in a gene which makes seemingly healthy adults prone to sudden and fatal heart attacks. Using their technique, they were able to get rid of the mutation in embryos that would’ve gone on to become a baby that would’ve grown up without the disease. So, thanks to Mitalipov & co., it would now seem that we are closer to a future where disease-causing mutations in genes can be corrected to ensure healthy babies. As if the nature of his research wasn’t newsworthy enough, there was a further flutter of excitement thanks to a mysterious leak.

What was the leak?

Mitalipov’s paper was due to be published in Nature journal on August 2 (until the date and time of publishing, studies are under strict embargo). But somehow, parts of the media caught wind of it almost a week earlier (examples here and here). There is still no clarity on how this happened. The jury is also out on what the implications of such high profile leaks in scientific research are.

What is the gene that was edited?

The gene is called MYBPC3. We all have two copies of it in each of our cells. It plays a major role in maintaining the structure and function of the heart. About 1 in 500 (0.2%) of us inherits a mutation in one of our MYBPC3 genes. This makes us susceptible to a condition called hypertrophic cardiomyopathy (HCM). It manifests in young, otherwise healthy, carriers in the form of sudden heart attacks. In fact, HCM is known as the most common form of sudden death among young athletes and is even more alarmingly common among Indians. Some studies say that more than 4% of Indians carry this mutation.

A parent who is a carrier of a mutated gene has a 50% chance of passing the defect down to his or her offspring – provided the other parent’s gene does not have the mutation.

When do you edit a gene?

An adult human body has billions of cells in it, so correcting a gene defect in adults is not an option. An ideal patient for gene editing would have just one cell so that, if the mutation in that single cell is corrected, the newly healthy cell can divide to more healthy cells and eventually develop into a healthy individual. The single cell from which the ball of development starts rolling is called a zygote. It is formed from the fusion of the egg and the sperm cells. It then multiplies to form an embryo and, over the next six weeks, a developing foetus.

How did Mitalipov do it?

The most risky aspect of gene editing is accidentally editing the wrong gene, causing problems that can be passed down generations. This forces us to confront many ethical issues (see below), and this is also why gene editing studies on human embryos is either banned or tightly regulated around the world. So first, Mitalipov had to receive the adequate permissions and scientific and ethical reviews in the US before his team was allowed to work with human embryos. The scientists used sperm samples from an HCM patient. Among the two copies of the gene this individual’s cells possessed, the MYBPC3 gene was mutated in one while the other was healthy. The eggs were obtained from a healthy female donor (both copies of whose gene were healthy), and they were fertilised with the sperm cells in the lab.

A child born from such a union would have a 50% chance of inheriting the HCM mutation from the father. At this juncture, the scientists used a five-year-old gene-editing technology called CRISPR/Cas9. CRISPR/Cas9 works like a customised pair of scissors: they programmed it such that it cut the gene at the start of the mutated part (in this case a tiny missing section in the MYBPC3 gene). The cell notices this cut and proceeds to repair it using the other copy of the gene as a template. And since the other copy doesn’t have the mutation, the cell repairs the cut into a fully healthy one. Thus, the process began with the presence of a mutation and ended without it.

Previous experiments with CRISPR-Cas9 in human zygotes have not been very successful. Sometimes the embryos of edited zygotes were found to consist of a mosaic of cells, some with the mutation and some without. To avoid this, Mitalipov started one step earlier. He injected CRISPR/Cas9 into the egg along with the sperm itself, rather than after fertilisation. In this way, he was able to produce embryos whose every cell was edited. Ergo, no mosaicism.

Ever since its discovery in 2012 by Jennifer Doudna, of the University of California, Berkeley, and Emmanuelle Charpentier, from the Helmholtz Centre for Infection Research, Germany, this technique has captured the imaginations of people around the world because of the ease, precision and efficiency with which it allows scientists to edit genes. It was even a favourite to win the 2015 Chemistry Nobel Prize, though it finally didn’t.

What was the breakthrough?

All 58 of Mitalipov’s mutation-carrying embryos were cut at the right spot. Forty-two of them were successfully edited. That means the chance of healthy offspring rose from 50% without this procedure to 72.4% with. The fact that the team was able to accomplish this without mosaic embryos (with both defective and corrected cells) is highly encouraging. If their methodology – injecting the sperm and the CRISPR/Cas9 molecule simultaneously into the egg – is tested and shown to consistently prevent mosaicism, it would mean one less giant hurdle for the future of gene editing. Also significant is the fact that they did not seem to have induced any unwanted mutations in non-target areas.

What should we watch out for?

Just because they detected no off-target mutations need not mean there weren’t any. In his blog, American biologist Paul Knoepfler expressed optimism at the quality of this study but also noted: “I do not believe they can be quite so confident about ‘no off-target activity’, when as best as I can tell they did not look thoroughly in enough embryos and cells and in an unbiased manner at the whole genome to really be sure about this.” Discussions about this are sure to follow in the coming weeks.

Mitalipov leaves no speculation about his aspirations for this research. He is known to have a strong opinion, that basic research such as this must go on to become treatments available to people who need it. His other major contribution was developing mitochondrial replacement therapy – a way to prevent babies from inheriting a life-threatening disease from the mother by using DNA from a female donor. These babies would now have DNA from three adults rather than two, earning them the tag ‘three-parent babies’. Despite the controversies that accompanied this, at least two ‘three-parent’ babies have been born in Mexico and, reportedly, in Ukraine.

Much less successful attempts to edit human embryos (until now, all from China) have been followed by deep debates about the need for such technologies. The fear of them heralding an era of designer babies, where parents can pick and choose traits considered more desirable, opens up an ocean of ethical implications. Moreover, mistakes in editing can introduce more problems that may persist in future generations. Given that the easy and efficient CRISPR/Cas9 technology is in the picture and is presumably here to stay, it is good that progress is being made to make it a safer method.

In-vitro fertilisation techniques today already make genetic screening methods for embryos available so that doctors can ensure only healthy ones are transferred into the uterus. So we may not always have to resort to gene editing. Nevertheless, the future of gene editing as a therapeutic technique will depend not just on breakthroughs like these but also on the success of studies that attempt to reproduce these results – and on all stakeholders mindfully considering the implications.