BioQuakes

AP Biology class blog for discussing current research in Biology

Tag: gene mutation

Researchers Discover Hacking Enzymes as New Cancer Treatment

We all know that mutations occurring in the synthesis of our cells lead to cancer, whether that be via ultraviolet light radiation, the inhalation of cigarette smoke over a long period of time, or otherwise. But how do these mutations actually occur, and if modern science knows that much, why can’t scientists step in before the mutation occurs in the cell and stop the creation of a cancerous one altogether? While the answer to this is evidently easier said than done, researchers such as Szymon Barcawz, Rahul Bhomick, Malgorzata Clausen, Marisa Dinis, Masato Kanemaki, Ying Liu, Katrine Lundgaard, and Wei Wu have found a way to limit the success of cancer-yielding cell mutations. 

In this study titled, ‘Mitotic DNA Synthesis in Response to Replication Stress Requires Sequential Action of DNA Polymerases Zeta and Delta in Human Cells,’ researchers studied the replication process of cells, also known as mitosis, in human body cells (all human cells except gametes, sex cells). In order to understand the study fully, a few biological concepts should be covered first; For starters, the activation of the oncogene in relation to developing cancer. ‘Oncogene’ is simply a term for a mutated cell which turns cancerous. The activation of such creates disorder to cells going through mitosis called DNA replication stress, the name of which essentially reveals its effect: when genetic material is being synthesized under these conditions, it is extremely difficult for the mitotic cell to correctly replicate, causing faulty, under-replicated DNA regions (UDRs) to be built. Since DNA replication is completed in the S phase of interphase, which technically is before the commencement of mitosis in a cell; enough genetic material needs to be available for the cell to split in order for it to be replicated. Therefore, if UDRs are going to occur in a cell, they are created during this time. 

However, our cells have developed clever adaptations to attempt to fight this type of cellular mistake. The strategy includes performing “‘unscheduled’ DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated” genetic material (Barcawz et al.). In studying this cellular defense mechanism, these researchers have discovered how exactly cells make up for a faulty S phase (the phase which copies DNA during mitosis) utilizing DNA gap-filling mechanisms (REV1 and Pol ζ) and DNA polymerases (group of enzymes) whose sole purpose is to replicate unfinished genomes (Pol δ). The study’s main goal, however, was to reveal which of these polymerases was the most crucial in the “rescuing” of under-developed genetic material, which were not, and which were not really necessary at all. 

The researchers were most interested in studying POLDI (a subunit of  Pol δ), REV 1, and REV 3 / REV 7 (both subunits of Pol ζ).  These are all different polymerases whose main job is to “[promote] the bypass of damaged DNA sites” (Barcawz et al.). Each one works to solve a different issue within DNA replication that could lead to a mutation. For example, a TLS polymerase called Pol ζ4 is better at “bypassing bulky regions” of genetic material than the others (Barcawz et al.); this can be defined as Pol ζ4’s ‘role.’  

A crucial realization in this study was that the polymerases Pol ζ and Pol δ may actually be switching roles at some point within the rescuing process by switching their subunits, which we defined earlier as POLDI, and REV3 / REV 7. But, this still doesn’t answer the question of whether or not all the aforementioned polymerases are essential in the process of fixing mutations in the copying of genetic material during mitosis. 

The study at hand was successful at answering this question. It found that POLDI, REV1, and REV 3 are crucial to MiDAS, while REV7 is not at all. Additionally, it was discovered that POLDI and REV1 colocalize with another substance (FANCD2) in mitosis, which reveals how they both indeed play a role in the ‘rescue’ of under-replicated regions” (Barcawz et al.).

However, something unexpected about REV1 was also discovered. While it was found to be useful in mending UDRs in conjunction with POLDI and FANCD2, it actually does more harm than good: When REV1 was removed from the rescuing process in a situation where all the cell’s defense mechanisms failed at stopping the synthesis of a cancer cell, cancer cells were much less likely to survive in the human body. This suggests that it is very possible for a new and effective way to treat cancer to be the inhibition of the presence of REV1 polymerase. 

In the coming years, if the inhibition of REV1 is found to be possible and turns out to be a promising way of preventing cancer cells from surviving in the body, we could be looking at a groundbreaking advancement to modern medicine and the world of cancer treatment as we know it changing forever.

Cancer cells

Real image of cancer cell under a microscope.

A Life Saving Treatment: CRISPR Gene Editing

A proud, hard-working father is what Paddy Doherty looked up to all of his life until a sudden heart attack that took the life of his dad. What would you do if someone you love is unexpectedly gone without a goodbye?

His father had a career in construction and various home improvement projects which kept him active until his 60s until Doherty first caught glimpses of a worrying decline in his dad’s health. “I noticed him getting breathless on walks. He’d stop for a while and maybe make an excuse for stopping, saying, ‘Oh, isn’t that a lovely tree’ or whatever,” said Doherty, who lives in Ireland. Doctors chalked it up to angina, or chest pain caused by reduced blood flow to the heart, symptomatic of an underlying heart problem.

After his dad died, the true cause was discovered: a rare disease called transthyretin (ATTR) amyloidosis, characterized by a misfolded protein that builds up in the heart and interferes with normal function. As learned in AP Biology, misfolded proteins are caused by the lack of chaperonins that are present in cells to provide a secure hydrophilic environment. The misfolded proteins cannot achieve their native state and are contorted into shapes that are unfavorable to the environment it’s in. The formation of oligomers and aggregates occurs in the cell when a critical concentration of misfolded protein is reached. Aggregated proteins inside the cell often lead to the formation of an amyloid-like structure, which eventually causes different types of degenerative disorders and ultimately cell death.

 

Structure of Wild Type Human Transthyretin in Complex with Tafamidis, PDB 6E6Z

“Patients left untreated with this type of amyloidosis develop heart failure, low blood pressure, horrible bowel disturbance, and eventually become incontinent of urine and feces,” said Julian Gillmore, nephrologist and head of the National Amyloidosis Centre at University College London. “It’s a truly awful, gradually progressive disease that is ultimately fatal.”

In February last year, Doherty began to experience the same early breathing symptoms his father had had. As an avid hiker who had trekked the Himalayas, he was surprised to find himself getting winded on local hill walks. Testing confirmed that Doherty had a hereditary form of ATTR amyloidosis.

But there was one bit of good news: If Doherty had been diagnosed even a year earlier, no treatment options would have been available to him — an all-too-common situation for over 30 million U.S. patients with rare diseases. But Gillmore, Doherty’s doctor, offered him the chance to participate in an early-stage clinical trial using CRISPR, a groundbreaking genome editing therapy with the potential to cure his ATTR amyloidosis in a single dose.

CRISPR logo

“I had no side effects and left the facility after two days,” Doherty said. “The walk that I felt breathless on, which is a steep kind of mountain walk through a forest, I’m doing that every Sunday now.” CRISPR-Cas9 allows researchers to alter the DNA of living things at will. It works like genetic scissors that can insert, repair or edit individual genes to rewrite the code of life. The system itself consists of two molecules — a protein known as Cas9 that works like scissors and a guide RNA that takes Cas9 to the right place in the genome — that can be inserted into cells or the bloodstream.

In the case of the clinical trial on patients with ATTR amyloidosis, Gillmore and his colleagues aimed to edit the malfunctioning gene itself and demonstrate for the first time that direct infusion of CRISPR molecules into the bloodstream is safe effective.

The hereditary form of ATTR amyloidosis affects roughly 50,000 people worldwide with a large cluster of patients like Doherty with roots in Donegal County, Ireland. Because circulating transthyretin is made almost entirely in the liver — and everything that enters the bloodstream is carried to the liver to metabolize — the researchers realized they could simply inject patients with the CRISPR-based therapy.

The therapy, called NTLA-2001, appeared to knock out the mutated gene as intended. Only six patients were tested in total, but the three who received the higher of two doses — including Doherty — saw their transthyretin levels drop by an average of 87 percent after 28 days. The results remain preliminary, and several more patients will need to be tested before the trial is complete.

Doherty said he hopes his family members and fellow Donegal residents will be able to benefit from CRISPR as much as he has. Fortunately, testing shows his two daughters did not inherit ATTR amyloidosis. And along with his father, Paddy’s uncle and cousin both died of the disease.

“When the trial is over, I hope that CRISPR is available and affordable for all amyloidosis patients,” Doherty said. “If a pharmaceutical company can mass-produce something like that and sell it at a good price, it would be a godsend.”

Is the Difference in Size of a German Shepherd and a TeaCup Poodle Due to a Gene Mutation?

Out of all the mammals on the planet, dogs differ in size the most. The biggest dog breeds are around 40 times bigger than the smallest breeds. A recent study has shown that this occurs because of a gene mutation that lies near a gene called IGF1. This gene was originally flagged 15 years ago as playing a major role in the variations of dog sizes. Ancient dogs that were domesticated from wolves in the past 30,000 years differ very little in size, however, in the past 200 years the largest difference in breed size has been recorded as people began to breed the more modern dog breeds during this time. 

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The IGF1 gene was studied comparing to body size of dogs and wild canids. There was one variant that stood out to researchers; this gene mutation was found in a stretch of DNA that works to encode a molecule called a long non-coding RNA. Long non-coding RNAs are a type of mammalian genome that lack protein coding capabilities. Specifically, the long non-coding RNA that was found to affect the size of dog breeds is involved with the levels of the IGF1 protein in the dogs bloodstream. As we learned in AP Biology, mutations in genes occur during the DNA replication phase of mitosis. Mitosis is the division of one mother cell into two daughter cells. DNA replication happens during the S phase of interphase. During this phase, the single stranded chromosome will duplicate and turn into two identical sister chromatids. The mutation will occur when copying the DNA, which would cause the sister chromatids to not be identical. 

This study identified that there are two alleles of this variant. Dogs carrying two copies of the small-bodied allele were most likely to weigh 15 kilograms or less, meanwhile, dogs carrying two copies of the large-bodied allele were most likely to weigh more than 25 kilograms. Dogs that carry one copy of each allele tend to be of an intermediate size. Additionally, dogs containing the larger-bodied allele contain  higher levels of the IGF1 proteins in their bloodstream compared to dogs who carry the smaller-bodied allele. Researchers also recorded a similar relationship in wild canids.

Prior to this study, researchers believed that certain dog breeds were smaller-bodied because of relatively new genetic changes. However, scientists now believe that the smaller-bodied allele is evolutionary and is actually much older than the bigger-bodied allele. They believe this to be true because the smaller-bodied allele was found in coyotes, foxes, jackals, and other smaller canids; this leads us to believe that this allele was present in one common predecessor. More studies must be done to truly determine how these variants impact the levels of  IGF1 proteins in a mammals bloodstream. The IGF1 gene only accounts for about 15% of size variation in dogs, so there is still much more research do be done. This study is just the beginning to really figuring out how we came to have dogs as large as German Shepherds and as small as TeaCup Poodles. Which allele do you think your dog has?

 

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