Transcript
TK2d symposium at ICNMD 2026
Supported by UCB Biopharma SRL
Yolanda Cámara, PhD, Cristina Domínguez-González, MD, PhD, and Caterina Garone, MD, PhD
All transcripts are created from interview footage and directly reflect the content of the interview at the time. The content is that of the speakers and is not adjusted by Medthority.
- Good afternoon and welcome to the symposium on advancing TK2d management from natural history to clinical evidence and real world opportunities. Those are my disclosure and the agenda. I will have the honor to have with me Dr. Yolanda Cámara from Barcelona and Dr. Cristina Domínguez-González from Madrid. I will briefly introduce you the symposium and then I'll give an overview of instructive case. So in my introduction, I will briefly introduce you to why we are here and the key learning points of this symposium. Thymidine kinase 2 deficiency is an autosomal recessive disorder in which a nuclear TK2 gene impacts the mitochondria DNA replication. It is an enzyme that phosphorylates deoxycytidine and deoxythymidine to their monophosphate, that are the building block for novel mitochondrial DNA. So if we have a loss of TK2 activity, we will have a defect in the quantity and the quality of the mitochondria DNA and the multiple oxphos deficiency. The level of the mitochondria DNA copy number and the quality of the mitochondria DNA set the age of onset for thymidine kinase 2 deficiency and also the type of motor milestones loss and progression of the disease. Overall, all patients experience respiratory dysfunction and diaphragm pulsing. The disease has a high mortality, and this is particularly true in patients with age of onset less than 12 years. 50% of them die within a few years of disease onset. But morbidity is common between early and late onset, and specifically for complication that require invasive ventilation and gastrostomy. Patients with TK2 deficiency still experience a diagnostic odyssey.
They are seen by multiple consultants before a TK2 gene analysis is suggested, and some of them undergo several panels that do not include the TK2 gene. So we have a delay in the diagnosis and this will impact the outcome after treatment. A couple of examples, this is a patient that had age of onset of 2 years, she started to have difficulty in climbing stairs and frequent falls after normal psychomotor development. She had the first muscle biopsy suggesting mitochondrial myopathy, but the first exome analysis was done 24 years after disease onset. Inbetween she lost the ability to walk, she required invasive ventilation, and the TK2 gene was confirmed only when she was 26 years old. The second patient had very early onset, in the first month of life. He lost the ability to control his head when he was three months old, and then he was seen by several child neurology consultants. And then the first hypothesis was a neurodevelopmental disorder because of hypotonia. Then a third clinical center identified an increase of CK suggest for trio exome, and the TK2d diagnosis was confirmed. But the disease was so progressive that he had CNS involvement, and he arrived to our attention at a very late stage of the disease, and so the treatment was not effective anymore. So why are we here? Because since the TK2 gene discovery in 2001, we had the opportunity to dissect the disease mechanism in the mouse model to develop experimental therapy, specifically the nucleoside treatment, and translate the treatment after a natural history study into human use thanks to compassionate use and a clinical program. And late in 2025 and early in 2026, we obtained both EMA and FDA approval. So now is the time for the real world evidence. And I am very honored to leave the floor to Dr. Yolanda Cámara that will go into the detail of the disease mechanism. Thank you Caterina, and thanks to Medthority for having me today. We are going to look closely at the biochemistry of TK2 deficiency. Here you have my disclosures.
And let me just start by reminding you that mitochondria are actually considered a central hub for the regulation of many essential biological processes. Among them, probably the most important and the best known is energy production, since you know that they are the powerhouses producing most of the ATP to meet cellular energy demand. And to do so, mitochondria have their own genome, mitochondrial DNA which is a double-stranded circular molecule of DNA of about 16 kilobases that encodes only 13 of the subunits that form the respiratory chain complexes. It's also good to remember that every mitochondria has several copies of mitochondrial DNA. So all other respiratory chain subunits, assembly factors, mitochondrial enzymes, transporters, as well as even factors that are involved in the mitochondrial DNA expression, are actually encoded in the nuclei. Need to be synthesized in the cytosol, and then imported by mitochondria where they will play their role. So it's very important in mitochondrial function that there is a good crosstalk between both genomes, the nuclear and the mitochondrial DNA. So today we are talking about a particular subset of mitochondrial diseases, because we usually say and we know that mitochondrial diseases are genetic diseases that can be due to mutations in both genomes, the nuclear and the mitochondrial DNA. But in this subset that are mitochondrial DNA depletion and deletion syndromes, or MDDS, these diseases are caused by mutations in nuclear encoded genes that synthesize or encode proteins that are involved in the regulation of mitochondrial DNA replication. So in MDDS, it's important to know that they are highly heterogeneous, both clinically and also genetically. So they are autosomic recessive or dominant traits, but these defects, mutations, in many different genes can lead to MDDS. And although... Although there is a common patho-mechanism here because there is dysregulation of mitochondrial DNA replication, mutations in different genes can actually manifest with different tissue specificity. So we can focus on and say that there are three main presentations: encephalomyopathic, hepatocerebral or myopathic syndrome.
A common molecular feature that is distinctive of these diseases, is that they all have the particularity that mitochondrial DNA accumulates aberrations in affected tissues. Why is this? Because when there is a normal mitochondrial DNA replication, we have a normal number and size of mitochondrial DNA molecules. But whenever mutations affect these factors that are involved in the regulation of mitochondrial DNA replication, mitochondrial DNA can accumulate somatic point mutations along its sequence, or can, if for instance the replication process is not successfully completed, then we accumulate shorter molecules, so multiple deletions. Or we can even have a loss of mitochondrial DNA molecules, what we call mitochondrial DNA depletion. So as we said earlier, mutations in many different genes have been associated with MDDS. We find genes encoding proteins directly involved in the process, such as for instance, the mitochondrial polymerase or helicase. We also have genes encoding proteins involved in the regulation of mitochondrial dynamics, since we know that the organization of the mitochondrial network is important for controlling the distribution of mitochondrial DNA molecules between different mitochondria. There is also another group of genes encoding proteins related with mitochondrial DNA replication by a yet unknown patho-mechanism. And the last group of genes encoding proteins involved in deoxynucleotide triphosphate or dNTP homeostasis. That was actually the first group to be associated with MDDS. And this is the most important for us today, because within this group is where we find thymidine kinase 2. Mutations in TK2 are associated usually with a myopathy, and they are inherited as an autosomic recessive trait. And it is actually not difficult to understand or to imagine why dNTP homeostasis is that important for mitochondrial DNA replication, since all of you know that dNTPs are the building blocks for DNA synthesis. So actually, in order to keep its mitochondrial DNA, mitochondria have their own dNTP pool. So within the cell, we have two main compartments that serve as a storage of dNTPs, the cytosol and the mitochondria. But these compartments that are independent are actually interrelated and exchange components. So there are two main metabolic pathways that contribute to the synthesis of dNTPs. The de novo pathway, that builds up dNTPs started from smaller molecules, for instance, amino acids, and also the salvage pathway that recycles deoxynucleosides from the diet and also from the catabolism of nucleic acids. In proliferating cells, we need a large amount of dNTPs to keep up with nuclear genome replication so the de novo pathway is highly active. But in non-proliferative cells, the de novo pathway is markedly down regulated and there is actually a decrease in the dNTP pool by 10 fold. And the most active pathway is the salvage pathway. So in postmitotic tissues, as, for the instance, in the skeletal muscle, the salvage pathway is actually the most important pathway as I was saying. So mitochondria, not only the cell, but also mitochondria, depend heavily on this pathway. What is this pathway? So the salvage pathway consists of two sets of deoxynucleoside kinases, two within mitochondria and two in the cytosol.
The mitochondrial ones are thymidine kinase 2, TK2, and deoxycytidine kinase. You have them here. And in the cytosol, there is another set of deoxynucleoside kinases that catalyze equivalent reactions, thymidine kinase 1, and deoxycytidine kinase. So deoxynucleoside kinases catalyze the first and rate limiting phosphorylation of deoxynucleosides, here, catalyze the first and rate limiting phosphorylation of deoxynucleosides to deoxynucleoside monophosphate. Then by successive phosphorylations, we end up with three phosphates that are actually the building blocks for DNA synthesis. So deoxynucleoside kinases, this family of proteins, usually are active as homodimers and have this particular structure with five parallel beta sheets and eight to 10 alpha helices. The catalytic core usually sits in the cleft between these structures. Okay, so somewhere around here is where the substrate binds. We can also identify another region, which is the P loop, where the phosphate donor will sit. Usually it is ATP that acts as the phosphate donor. Also something that is particular to this deoxynucleoside kinases is despite having or sharing a highly conservative structure, they have substrate specificity, which actually depends on very few residues. So while TK2 phosphorylates thymidine and deoxycytidine, deoxyguanosine kinase phosphorylates the purine nucleosides, deoxyguanosine and deoxyadenosine. This is in the mitochondria. In the cytosol, deoxycytidine kinase phosphorylates the purine deoxynucleosides plus deoxycytidine. TK1 has the same substrate specificity as TK2, but in the cytosol. And they are all regulated negatively by the triphosphates. So if we look at TK2 now more closely, we can recognize the same structures that we have just seen. And we can see that it phosphorylates both deoxycytidine and thymidine, but it has a preference and performs better with thymidine, which acts as a competitive inhibitor, for deoxycytidine phosphorylation. And also, as mentioned, it is inhibited by the three phosphates. So, what we would expect in the skeletal muscle of a patient harboring mutations in TK2, what we can see is that there is a lower phosphorylation of both thymidine and deoxycytidine. And this is concomitant to a decrease in the monophosphates, diphosphates, and finally, the triphosphates. And this dTTP and dCTP insufficiency, in the case of TK2 leads to multiple deletions or a loss of mitochondrial DNA molecules; depletion. Of course, these aberrations in mitochondrial DNA affect the performance of all mitochondrial dependent actions. So you can expect a low ATP production, but also an increased ROS production, activation of stress responses, altered mitochondrial dynamics, impaired mitophagy, etc. So even if TK2 is constitutively expressed, it affects, skeletal muscle including respiratory muscles, sometimes also brain.
And to a lesser extent, it has been proposed also other tissues may be affected. When we look at the histopathological features, it's very common to find ragged red fibers indicating accumulation of abnormal mitochondria, and negative cytochrome oxidase staining. This is also typical in other mitochondrial diseases. However, we also find some traits that are more typical of muscle dystrophies, such as fibrosis, necrotic fibers, inflammatory infiltrates, etc. The patients present, and my colleagues will explain much better, but they present within a continuous and broad clinical spectrum. But there is not a clear correlation between genotype and phenotype. And actually, you see patients that display a very low TK2 activity, but present with a relatively mild phenotype. What find is that there is quite a good correlation between mitochondrial DNA copy number and the age at onset, or even the severity of the disease. So the higher the depletion, the more severe presentation. So what can we do to rescue thymidine kinase 2 deficiency? It was proposed to administer nucleosides, the deoxynucleosides as precursors of the insufficient dNTPs. So, although we mentioned that TK2 is no longer working, the enzyme can still have some residual activity. In addition, there are the cytosolic kinases, TK1 and dCK, that can also phosphorylate the deoxynucleosides. So luckily, there were two mouse models of the disease that were developed in parallel in Karolinska Institute and also at Columbia University, and they both develop a very severe phenotype. The most severe resembling the most severe phenotype that humans with TK2 deficiency can present, that is encephalomyopathy.
They have a short lifespan of about two weeks, and show a marked mitochondrial DNA depletion in several tissues. The oral administration of these deoxynucleosides, successfully expanded the survival of these mice in a dose-dependent manner. And also, treatment rescued mitochondrial DNA depletion in skeletal muscle. But only in early aged mice. At later ages it was observed, that the treatment lost efficacy. We believe this is mainly due to the lower activity of cytosolic kinases in postmitotic tissues, and also to a lower bioavailability of the administered deoxynucleosides because of increased catabolism. I don't have time to go into detail. So what we think is that this may also be an issue in humans that may have low availability of the cytosolic alternative kinases. We have to keep in mind that most identified mutations today are missense mutations or deletions of only one amino acid. Frameshift or nonsense mutations that could be models to use are found mostly in heterozygosis. And most mutations then are compatible with the expression of a truncated or a mutant protein that may have residual activity. This needs to be tested, but it could be that the residual activity of the mutant enzyme could still contribute to the therapeutic efficacy of administered thymidine and deoxycytidine, especially in non-proliferating tissues. And I just want to finish by thanking our collaborators, especially Dr. Anna Karlsson, who sadly left us recently, and who made important contributions, very important contributions to the nucleotide metabolism field. And also to patients and their families and to all of you for your attention. And I leave the floor to Dr. González. Good afternoon everyone. Now we are going to present a description of the clinical characteristics of patients with TK2 deficiency across the whole spectrum of the disease. And also I'll try to summarize the more important results of the nucleoside therapy. These are my disclosures. As has already been discussed, TK2 deficiency primarily affects the skeletal muscle. Therefore, it manifests as a progressive muscle disease, and it is characteristic that these patients have an early bulbar and respiratory involvement. Importantly, the disease can start at any age and progress at different rates. The phenotype that was first associated with TK2 variants and is more frequently reported in literature is the infantile onset disease. These patients begin with symptoms during the first two years of life, and they are characterized by the presence of a profound mitochondrial DNA depletion in the skeletal muscle. These patients progress quite rapidly and have short survival.
There are also other patients that are more rare with a more severe phenotype with symptom onset during the first months of life that may have central nervous system involvement with encephalopathy and epilepsy, but are less frequent. Patients with later onset with symptoms starting at the age of between two and 12, childhood onset disease or juvenile onset disease, do have a mainly pure progressive myopathy with very few extramuscular manifestations. Patients with childhood onset disease may have hearing loss and patients with juvenile or adult onset disease may have axonal sensory polyneuropathy, but is usually subclinical. These patients have progressive myopathy. In childhood onset, patients lose the ability to walk independently, whereas patients with juvenile onset disease usually retain the ability to walk. However, the main cause of morbidity and mortality in patients of all ages is respiratory involvement. Patients with very late onset disease, patients with symptom onset after the age of 40, may have ophthalmoplegia as the main clinical manifestation, or thermoplegia with ptosis, facial weakness, and also cervical weakness. However, in our experience, these patients also die due to respiratory problems. UCB has built the largest international data set that includes 257 patients with TK2 deficiency. They gathered the information through literature review, but also from retrospective analysis of chart reviews and also from the pre-treatment period of several clinical studies. And they built this natural history data set to be able to describe the prognosis, survival, and the progression of the disease. In this data set, as you can see here, most of the patients have an early onset disease. And the median age of onset of these patients was 1.4. And they were able to describe that patients usually develop normally during the first months of life. In fact, more than 90% of the patients initially achieved independent sitting, and also more than 80% of patients are able to walk independently before disease onset. However, after disease onset, they experience motor regression that occurs quite rapidly, and more than 80% of patients lost at least one motor milestone previously achieved. And 40% of the patients lost at least four motor milestones. This includes the ability to walk, to climb stairs, to stand, to sit or to hold their head upright. This is an example of a patient with an infantile onset disease, a characteristic patient that's initially developed normally after birth, but then when the disease starts, progression occurs rapidly. And within two months, reached this very severe stage with hypotonia, generalized muscle weakness. And these patients usually die in the next few months after disease onset. The median age at death in these patients with symptom onset of less than two years is 1.8.
So there is a very high risk of mortality during the first months after the disease onset. Patients with childhood onset disease that starts later between the ages of two and 12 have a slower progression, but this is also a very severe disease, very progressive disease. Patients usually lose the ability to walk and usually die due to respiratory failure. Generally, 10 years after birth. So this is also a very severe progressive condition. They have proximal muscle weakness, that can mimic many other different muscle diseases. But characteristically these patients usually develop ptosis and facial weakness over time. If we take together the infantile onset and the childhood onset disease, the median time from birth to death is about four years of age, so quite a severe progressive disorder. Patients with juvenile or adult onset disease that are less frequently described, but probably underdiagnosed, these patients have a slower progression, although also a bad prognosis due to respiratory involvement. These patients have muscle symptoms due to myalgia, exercise intolerance, extreme fatigue, but also progressive muscle weakness. The weakness usually involves the ocular muscles. They all have ptosis. They all have facial weakness and cervical weakness, and half of them have ophthalmoplegia. They also have proximal upper limb and lower limb muscle weakness and weakness of the extensors of the fingers. As I said, these patients may also have subclinical axonal sensory neuropathy. And they may also have dysphagia, although in general this is mild. However, the more important sign in these patients is the presence of respiratory involvement, because as I said, this occurs very early in the disease course. In fact, in our experience, patients already require mechanical ventilation from the time of diagnosis. And while they still retain the ability to walk or even to run, as you can see in this example, this patient is still able to run, but already needs mechanical ventilation at night due to diaphragmatic weakness. In fact, many patients are diagnosed only after an acute respiratory failure. And without therapy, in these patients forced vital capacity declines an average of 8% per year. So quite a progressive disorder. And this progression is independent from the muscle weakness of the lower limbs. What clues are there to recognize this disease earlier? Well, these patients have high CK levels. This is different from other mitochondrial myopathies where the CK levels are usually not very high, but patients with TK2 deficiency do have high or very high CK levels from hundreds to thousands. But there are also other biomarkers in blood that reflect mitochondrial dysfunction such as GDF15, which is growth differentiation factor 15. It's a cytokine that is induced by the presence of mitochondrial dysfunction. And levels of GDF15 are normal in non-mitochondrial myopathy and are high or very high in patients with mitochondrial myopathies in general. In TK2d, we have seen that GDF15 levels correlate with the age at onset and also with the severity of the disease. As has already been shown, the morphology of the muscle biopsy, is also useful in the differential diagnosis, because in addition to the common ragged red COX-negative fibers that are present in all patients, in pediatric patients we can also find the myopathic and dystrophic changes. If we do a muscle MRI in adults, we have identified a characteristic pattern of involvement with an early alteration of the gluteus maximus that is the muscle that is more affected, and also the sartorius muscle. This pattern is quite characteristic, and in fact, the amount of fat replacement correlates with the muscle function in patients. This pattern allows you to differentiate these patients from other patients with muscle diseases and similar clinical characteristics. For example, some patients are diagnosed as facioscapulohumeral muscular dystrophy due to the severe facial weakness, or of oculopharyngeal muscular dystrophy due to the ptosis and dysphagia. And the pattern of involvement seen by MRI is very useful to differentiate these patients from TK2d.
The final diagnosis is genetic. This is a recessive condition. And as I said, since the clinical symptoms may be quite similar to other neuromuscular diseases, you need to make sure that the TK2 gene is included in your panels of neuromuscular diseases, not only congenital myopathies, but muscular dystrophies, and also congenital myasthenia syndromes. Moving now to the results of the therapy with oral nucleosides, the rationale for the use of oral nucleosides has already been described in detail. And soon after the description of the benefits of the nucleosides in the mouse model, several patients around the world started taking oral non-GMP nucleosides under compassionate conditions. In 2017, 16 patients from five different countries were receiving nucleosides, five of them with early onset rapid progressive disease. After initiation of the therapy, we saw that survival increased substantially. And in fact, all these patients are still alive more than 10 years after treatment initiation. But patients not only survived, but also recovered motor function. Meaningful motor functions such as, for example, three out of eight patients regained ambulation. In one out of nine patients, mechanical ventilation could be removed. And also in four out of five, the gastrostomy could also be removed. So quite remarkable improvement without relevant side effects. The only side effect described was dose-dependent diarrhea. With these results, the industry became involved to obtain the regulatory approvals. All patients transitioned from the non-GMP nucleosides to the GMP nucleosides. And as they could not do a randomized placebo controlled clinical trial due to these preliminary results and their compassionate use conditions, they decided to compare the outcomes of treated patients with the outcomes of the data set shown before; untreated patients age match historical controls. And they selected the survival and also the regain of motor milestones as the main clinical outcomes to show the benefit of the therapy. And this was done due to the age at onset of the patients that received therapy were, most of them with symptom onset of less than 12 years. The median age at onset of the patients that received therapy was 1.5 years. With this approach, they were able to demonstrate a substantial decrease in the risk of mortality by more than 90%, and not only survival, but also patients regained motor function. 75% regained at least one motor function, 30% of them two motor milestones, and 20% at least four motor milestones. And again, the safety profile was acceptable. The more frequent side effect was dose-dependent diarrhea, well controlled by reducing the dose. Although some patients experienced an increase in transaminase levels so liver function should be monitored if initiating therapy. And with these results, the treatment was approved by the regulatory agencies, but only to treat patients with symptom onset of less than 12. What about the rest of the patients? Well, outcomes selected were not appropriate to prove effectiveness of the therapy in adult patients with a slower disease, but we have experienced treating under compassionate conditions of adults with juvenile or adult onset disease. And we've seen that even in patients with a very late onset presentation, this patient started with symptoms after the age of 40, the therapy is able to promote motor improvement, Which you can see this in the six-minute walk test and in these videos. There was also an improvement in the forced vital capacity. Historically, we saw a decline in 8% per year, and in this case, forced vital capacity improved by 5% around four years of therapy. In fact, in the muscle MRI, after four years of treatment, we can see that the disease has stopped progression and the fat replacement remained completely stable. And with this result in mind, we decided to perform a clinical trial.
And currently we have enrolled eight patients in our site with late onset TK2 disease. And I can show you some preliminary results of the first year of treatment. And as you can see, the biomarker that reflects mitochondrial dysfunction that correlates with the severity of the disease, within a few months of treatment, this declines significantly, and even reaches normal values in patients with better improvements. In parallel of the decrease of the biomarkers, we can see an improvement in the motor function measured using different scales, the MRC strength score, but also North Ambulatory Assessment. And as you can see in these videos, patients recover the strength of individual muscles. For example, here, the biceps and also the extensors of the fingers. This improvement is seen in just the first weeks of therapy, and also they recover some functionality. We've seen an increase of six points in the North Star Ambulatory Assessment during the first nine months of therapy. So quite remarkable improvements. Again, side effects were the same, just dose-dependent diarrhea. So in conclusion, TK2 deficiency is a very severe disease that manifests as a continuous clinical spectrum, and the earlier disease onset is associated with a more severe prognosis. However, patients with late onset presentations also have a poor prognosis due to their respiratory involvement. Oral nucleosides is the first approved treatment to treat TK2 deficiency, but only for patients with symptom onset of less than 12. In this group of patients, the treatment has demonstrated that is able to substantially decrease the risk of mortality with an acceptable safety profile. And with that, I'd like to thank you all for your attention.
- So in the last presentation we will cover some experiences from the real-world with some instructive cases. These are my disclosures again. So the availability of treatment gives us an additional reason for diagnosing these patients. And we have stressed the concept that TK2d patients have a clinical heterogeneity. And even when we look at the pediatric cases of patients with disease onset of less than 12 years, you can see that we can go from severe encephalomyopathy to patients that might mimic other neuromuscular disease like spinal muscle atrophy type three. What are the red flags for TK2d? So it's definitely a more a muscle specific disorder, but we should not forget that other organs and systems might be involved. When we look at the muscles, patient may experience muscle weakness, muscle regression and dysphagia, and also the respiratory muscles might be involved in this post-differential diagnosis with other neuromuscular disease. In very early onset, CNS involvement might be present, and might mimic neurodevelopmental or encephalomyopathy, epileptic encephalomyopathy. And in late onset patients, more than 80% might also have asymptomatic sensory neuropathy. In term of lab tests, all the patients have an increase of CK. Together we test biomarkers for mitochondrial disorders that might be present, like increased lactic acid and plasma alanine, or an increase of transaminase. Definitely we need to recognize signs and symptoms of TK2d, but we should also consider TK2d in patients that have isolated hyperCKemia with or without rhabdomyolysis. And biomarkers like GDF15 are important for diagnosis, but also to follow the progression of the disease. The diagnosis is only confirmed when we have biallelic variants in the TK2 gene, and we might go straight to gene testing in order to earlier diagnose patients. And muscle biopsy might be useful for confirming variants of uncertain significance, or looking at the molecular genetic defect for prognosis. This is an example of disease trajectory. This was my patient that had normal motor development in the first two years of life, and then he started to have difficulty when climbing stairs, frequent falls, facial diplegia and dysphagia. The lab tests showed an increase of CK and transaminase, and the muscle biopsy was clearly suggestive of a mitochondrial myopathy, but there was also dystrophic changes on histology. We were able to do the diagnosis when he was four here, and between four and eight years of age he progressed with loss of ambulation and restrictive respiratory insufficiency. We were able at that time to treat the patient with non-GMP compassionate use nucleoside, and he reacquired motor milestones. The patient was improving, but as we made the diagnosis at four and he has had a long disease course, even though he has improved in muscle weakness, he still has signs of myopathy. His sister was coming to the clinic. She was perfectly fine up to 14 years of age, but then she started to have proximal muscle weakness and a reduction of respiratory function. She was diagnosed when she was 16 with thymidine kinase 2 deficiency. She has had a shorter disease course, and received treatment, and she's now completely normal in terms of motor function. So there is an intrafamilial variability, and also the earlier we treat the patient, the better the outcome. It's important to perform deep phenotyping in TK2d patients, because there are signs and symptoms that are additional, and depend of the age of onset. Specifically, in patients with the age of onset less than two years, we can have hypertrophic cardiomyopathy and also 30% of them might experience CNS involvement. This is another patient I had chance to follow recently.
He had the first brother that died with a known diagnosis. He had a normal development in the first three-four months of life, but then he lost the ability to roll over and he lost the ability to control his head. When he came to our attention, he was already very severe. He had a gastrostomy for nutrition and invasive ventilation. The patient also had subtle seizures that were chronic movement of the shoulders, the mouth, and the extremities of the feet. And indeed, when we have CNS involvement in very early onset cases, seizures are the first sign of this involvement. And they start in patients just few months or a few weeks after motor regression. In term of EEG patterns, there is no specific EEG pattern in early onset cases. What we are trying to understand is if there is an MRI pattern and it seems that the first lesion appears in the insular temporary region of the brain. The disease is heterogeneous also in terms of prevalence, and in Italy it is also ultra rare. And this is the reason why we started the study called TK2 Finder with two main aims: To analyze the allele frequency across different populations around the world, Also to reanalyze next-generation sequencing data to identify patients with TK2d and also to start reverse phenotyping and perform a functional study to validate variants of uncertain significance. So it had a retrospective phase of a re-analysis data, and also a prospective phase in which we confirm the pathogenicity of the variant. In term of allele frequency, it was very interesting to note that there are some population specific genetic factors that might influence the prevalence of the disease. And if you look, the first two populations are the Finnish, and then a mixed American population that have a high risk of developing TK2 deficiency. In term of TK2 Finder, we were able to diagnose three patients. One patient was thanks to collaboration with a colleague who reanalyzed and later identified a 26-year-old patient, but also we had chance to diagnose very early onset cases that presented with a variant, a compounded variant. In one case there was a de novo variant, and the second case was a variant that was predicted to be benign, but our study confirmed that they were pathogenic for the TK2d. So when we identify variants of uncertain significance, or if you identify TK2 gene variants that do not correlate with the most common TK2d signs and symptoms, it's very important to do reverse and deep phenotyping. And to restart the diagnostic algorithm, analyze the biomarkers, reanalyze the muscle biopsy, look for the molecular genetic defect, because those are very important to validate the variant. We also have the opportunity to run a functional study, both in muscle and fibroblasts, in which we can check TK2 activity, but also can analyze OXPHOS deficiency in which we have multiple complex activities that are reduced and are generally spare complex II that is uniquely encoded by nuclear DNA. We can analyze ATP production, the respiratory cellular activity with seahorse metabolic analyses, and altogether, these data allow us to move from a variant of uncertain significance to a pathogenic variant.
We also stress the concept that there is no genotype-phenotype correlation, but all the data that we have so far are retrospective data. So with the international community we are moving to another study called TK2d GEM, in which we will try to analyze all the variants, and try to clusterize those variants in order to predict prognosis and response to treatment. In the study we will also look for the lost population, meaning the population that have only one TK2 variant, they might have TK2d deficiency, but they are not diagnosed because the second variant might be a deep intronic variant or a large rearrangement. So looking at the last population and analyzing them with multiomics analysis might increase the diagnostic rate of TK2 deficiency. In terms of timeline, the TK2 GEM study will be run under the GENOMIT registry. So if you have patients, please join the mitochondrial global registry, in which we will have a subclinical TK2d registry. And this will be extremely important for having real world evidence data, not only for the genotype phenotype study, but also to understand more about the efficacy of the treatment in the long-term. So I thank you for the attention and we will move to the Q&A. Okay, so we already have some questions. So the first question is, what's the most common misdiagnosis of TK2d patients? Cristina, would you like to answer? - Yes, well, if we classify patients according to the age of onset, I would say that patients with infantile onset disease usually the first thing they think about is spinal muscular atrophy . But in patients with childhood onset disease, I've seen patients that have been diagnosed as Duchenne muscular dystrophy, especially patients without a muscle biopsy of course. And then in late onset presentations in adults, there are patients that are diagnosed as facioscapulohumeral muscular dystrophy due to the very severe facial weakness that some patients have. Although, in these patients, facial weakness is always symmetric, which is different to FSH.. And also they have respiratory problems and ptosis. So the phenotype is different, but I've seen several patients diagnosed as facioscapulohumeral muscular dystrophy. And also some of them, those with ophthalmoplegia and dysphagia, some are diagnosed with seronegative myasthenia or even oculopharyngeal muscular dystrophy. So I guess the main message is, if you have a patient with that clinical diagnosis who do not yet have a genetic diagnosis, please look into TK2d, because they might, you know, have the biallelic variant that we might able to treat them. So the second question is, "A patient with a TK2d phenotype, one mutation pathogenic and on the other allele a variant of uncertain significance, is it a TK2d patient?" So all the variants must be confirmed by a functional study. So definitely, I do believe the patient will have TK2d, but the variant must be confirmed. So muscle biopsy and fibroblasts are extremely important, a functional study is extremely important, because that will allow us also to identify other patients with the same variant.
And, another question, "Do you have suggestions on a re-analysis approach of our database to identify TK2d patients?" If you don't mind, I'll answer this question. TK2d FINDER was based on this approach, we reanalyzed genome and exome data. We were also looking for intronic variants and trying to run functional studies. It is indeed important to reanalyze these data and to not miss a diagnosis. Okay, so question for Yolanda I guess. "Do you foresee that this treatment may work in other genetic mitomyopathies causing problems with dNTP homeostasis like TYMP or RRM2B?" Yes, in principle, since they share a common patho-mechanism, you would think that a similar strategy would work for these other diseases with dNTP homeostasis as the problem. Although in some cases we have the challenge that while pyrimidine deoxynucleosides are quite stable, I would say, for purine deoxynucleosides we have this challenge that they are extremely low in terms of bioavailability. And so we are actually thinking of strategies to help with that problem. Yeah, I don't know if you want to add anything about that. Yes, no I totally agree. It's definitely a treatment that may be extended to other diagnoses, but we unfortunately need to work more on the catabolism of other compounds, especially purine. And it's also, it's very important to mention that the combination of the nucleosides is disease specific, because there is experience outside of our field that are trying to use deoxycytidine deoxythymidine in different diagnoses. And this has no rationale basis, and it's very important to run preclinical studies because unbalancing the nucleotide pool can cause additional disorders. So the combination is disease specific, and we are working in that direction. Question for Cristina. "Does it make sense to suspect TK2d in myasthenia gravis double negative patients?" Yeah, of course. I think that patients with ptosis, ophthalmoparesis that do not respond to any therapy, you should always think about mitochondrial myopathy or a congenital myasthenia in my experience. But patients with TK2 deficiency in general have high CK levels. Almost all of them, so it's a very useful clue. But also GDF15 we found it to be very, very useful to suspect a mitochondrial origin of any neuromuscular problem. Also in patients with ophthalmoparesis and ptosis, GDF15 is useful to differentiate myasthenia from a mitochondrial myopathy. If you don't mind Cristina, we'll continue with you with another question. "Are the adult patients on treatment maintaining improvement over time? And what's the impact on the quality of life of patient and caregiver?" Well, I can't talk about the patients that first started treatment more than 10 years ago, 15 years ago. In some patients that were treated very early after disease onset, they completely recover all motor functions previously lost and have developed completely normally. The benefit is sustained over more than 10 years without side effects. It's true that if you started the therapy when the patient already has a very severe muscle problem and muscle atrophy and fat replacement, you can stabilize the disease, but it's not as common to recover all the previous motor milestones. So the earlier you treat, the more effective the therapy.
But yes, in my experience, even in patients with late onset presentations, we see that the treatment is durable over time. And we usually use GDF15 to monitor disease progression. And we've seen that GDF15 completely normalizes in patients that improve with the therapy. And when there are some fluctuations in the response to the treatment, you can see these fluctuations also in GDF15. So it's very, very useful to monitor disease progression and also response to the therapy. And again, for quality of life, we are talking about patients that could remove mechanical ventilation. It's been seen, for example, it has been published very recently, experience from Turkey with four patients with early onset rapid progressive disease, two of them with invasive mechanical ventilation. But since they started the therapy just in the second year after the disease onset, the invasive ventilation could be completely removed. So this, we are talking about a very spectacular improvement. So the quality of life of course also is very, very much improved. Thank you. And last question, maybe a bit complicated, but we will try to answer. "During embryogenesis we do have probably the highest cell turnover in our lifetime, and during this period, de novo metabolism is dominant. Do you think when it is switched to the salvage pathway, if there is a pharmacological way to switch back to the de novo pathway?" Shall we try to answer? Yeah, yeah, I mean, I think that... I mean, you would expect that the de novo pathway is really down regulated when the cell leaves the proliferative state. So you would expect that it's also for a reason. So that is dangerous to keep a high dNTP pool in a cell that is not supporting a genomic DNA division at that time. So, but it's not that the de novo pathway is completely gone when the cell is not proliferating. It is downregulated. You can think of it perhaps as a similar strategy that we are now using the salvage pathway, but feeding the remaining de novo pathway. But I am not aware and I'm also not sure of the safety of a treatment that just completely reactivates the de novo pathway. I think that would be a bit too much. Yeah, maybe it's something to be explored with the miRNA and antisense oligonucleotides that can perhaps partially reactivate. Yes, "How do we approach treatment of patient with a strong suspicion of TK2d but an inconclusive genetic finding?" So the approach to patients that do not have a confirmed genetic diagnosis of TK2d is a similar approach for other mitochondrial myopathies. So, supportive treatment and vitamin supplementation, but I would strongly encourage looking for a diagnosis using RNAseq and other multiomics technologies. [Participant] I'm kind of worried about, you know, late onset patients that, I don't know what the situation is in Europe with coverage, but would you say that these patients, with very subtle symptoms like late fatigue or laziness in patients that in school they were very, you know, lazy doing sport or something, be allowed to be treated under the drug, or how is the situation in these cases because, you know... If you diagnose a patient as an adult but the patient experienced symptoms before the age of 12, which is quite common as you said, you'll be allowed to treat these patients.
There are only a few patients with a very late onset disease without symptoms before the age of 12. These are the less frequent phenotypes. So most patients diagnosed as adults probably had symptoms before the age of 12. [Participant] So it's important to encourage physicians to ask for that. Yes. In patients that were... very lazy doing sports or have very, very, very subtle symptoms to allow you to, you know, get the patient in the- Fatigue is a very common symptom, the fatigue or the presence of myalgia after very small effort. This is very common, and should be asked about in all patients. Agreed, thank you for the question. Okay, thank you everybody for all the questions and for participating in the discussion. We will kindly ask you to complete the evaluation to receive your CME credit, and thanks for joining us. If you have any questions in the near future for your patients, we are here to answer your questions. Thank you.
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