Transcript
TK2d symposium at EPNS Congress 2025
Supported by UCB Biopharma SRL
Cristina Domínguez González, MD, PhD; Caterina Garone, MD, PhD; Michio Hirano, MD
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.
- Welcome to this independent medical event focused on TK2 deficiency, from early recognition to advances in disease management. I'm Christina Dominguez and I am a neurologist from Spain and I'll be hosting this meeting. These are my disclosures. I have worked with UCB in the development of the nucleoside therapy. And these are some disclaimers. The more important one is that there is no drug approved yet for the treatment of TK2 deficiency. The learning objectives for today are first of all being able to recognise symptoms and signs of TK2 deficiency to being able to diagnose our patients early. And also to describe the importance of the genetic testing for this early detection. Finally, we'll summarise the current standard of care and the results from clinical trials in the management of the disease. This is the agenda. I'm joined by excellent speakers with great knowledge on the topic and after a brief introduction on the basis of the disease by myself, then I will hand over Caterina Garone, professor from Bologna University, who is going to describe the TK2 manifestations and natural disease course of TK2 deficiency. Following her, Dr. Hirano, from Columbia University in New York is going to review the current status of TK2d management and the results of the clinical trials. And in my final presentation, I'll review some key learnings on the early diagnosis. We'll have some time at the end of the session for some questions. So let's get started as an introduction. TK2 deficiency is included in the group of mitochondrial DNA depletion and deletion syndromes, the mitochondrial DNA maintenance problems, which are a heterogeneous group of mitochondrial diseases that are caused by mutations in nuclear genes involved in the synthesis and repair of the mitochondrial DNA. Therefore, they are characterised by the presence on the affected tissues of a reduction of the total amount of mitochondrial DNA, which is called depletion, or by the presence of an accumulation of incomplete molecules of mitochondrial DNA, which is called multiple deletions or by both of them. And importantly, this quantitative or qualitative defect is the responsible for the mitochondrial dysfunction in the affected tissues. The severity of these biochemical defect is closely related with the severity of the phenotype. And classically it has been said that patients with severe depletion with severe reduction of the mitochondrial copy number in the affected tissues do have an early and severe phenotype. While patients without a significant reduction of the mitochondrial DNA copy number, but the presence of multiple deletions have a milder disease with a later onset. This is true for all maintenance syndromes and also for TK2 deficiency. And how TK2 deficiency produces this alteration of the mitochondrial DNA is because TK2 enzyme plays an important role in the synthesis of the mitochondrial deoxynucleotides. It catalyses the first phosphorylation of the pyrimidine deoxynucleosides, deoxycytidine and deoxythymidine. And its deficiency produces a disruption of the nucleotides available in the mitochondria for the synthesis of the mitochondrial DNA. And in fact in the graphic on the right you can see that patients with TK2 deficiency do have a significant reduction of the copy number of the mitochondrial DNA in the muscle. Most of the patients have this depletion, even patients with a juvenile or adult onset disease. In terms of frequency, current estimates suggest that there are approximately 1.64 patients per million worldwide, although this is probably an underestimation. In fact, there are differences between countries that are not well understood and as an example, in Spain we have identified more than 60 patients to date, mainly late onset patients. And also in Brazil they have described recently a cohort of more than 30 patients. And in both countries we have found a wide range of genetic variants and therefore a foundry effect doesn't explain fully this clustering of cases. In my opinion, an increased awareness of the disease is crucial for the patient identification. And with that, I'd like to hand it over to Professor Garone to explain the clinical manifestations natural course of the disease.
- So good afternoon. Thanks the organiser for giving me the opportunity to talk about the natural course of thymidine kinase 2 deficiency, and to Professor Dominguez for the kind introduction. Those are my disclosure and same disclaimers as Professor Dominguez. So thymidine kinase 2 deficiency is an autosomal receptor disorder in which a nuclear DNA gene encode for a mitochondrial protein thymidine kinase 2 that is responsible of the conversion of the deoxycytidine and deoxythymidine into the monophosphate. When we have a defect in thymidine kinase 2 we have a reduction of the pyrimidine phosphates that are essential for the mitochondrial DNA replication. So the fascinating part of thymidine kinase 2 is that in nuclear DNA defect impact the mitochondrial DNA in term of quantitative defect when we have reduction of the copy number and so depletion or a qualitative defect when we have multiple deletion and this lead to a defect, a biochemical defect in the mitochondrial respiratory chain activity with multiple oxphos defect. The main clinical symptoms that are responsible for the tissue specificity of thymidine kinase 2 is muscle weakness. And we have different motor outcome and motor milestone loss depending of the age of onset. So in patient that have infantile onset between zero and two here, they lose the ability to stand, to crawl, to sit or to control their head. When there is a childhood onset between two and 12 years, patient lose the ability to walk to step on stairs or to play with pairs. When stand we have a late onset after 12 years of age, patient lose the ability to run and they cannot independently take care of themselves. This clinical spectrum is associated with a molecular genetic spectrum with depletion in the most severe case and early onset and multiple deletion in the late onset. So the three clinical form that I presented here, you should keep in mind that there is no clear cut between them but you to believe them as a continuum of defect. I like to present some patient that I chance to follow in the last year with a clinical presentation TK2 before 12 year of age. So the first patient had a very early onset, a six month of age he was able to control his head and to crawl, but then it became within two months as a floppy baby. The second patient instead reached the ability to stand and to do few steps at 18 month. And then again he had a severe motor regression and became completely hypotonic. The third patient was able to walk independently at two year of age, but then she became very weak. She was not able to crawl anymore and she was very wobble while doing her step and she presented fatigue. The third instead had growth failure, scapular wings and muscle weakness. And when we ask her to stand up from a sitting position, you certainly will appreciate a positive Gowers sign. So from this clinical spectrum, you can also understand the thymidine kinase 2 deficiency is mimicking other neuromuscular disorder and so might be underdiagnosed. I won't go into the detail for the late onset patients since this is a paediatric meeting. But also when we have a late onset, we have muscle weakness with ptosis and external ophthalmoplegia. Facioplegia and dysphagia. Some sign of the disease may be very subtle in the earlier age. It's important to make the right question when we do the clinical history. Muscle MRI can be helpful and also again we have differential diagnosis with other neuromuscular disorder. In term of motor milestone loss, patient that have an early onset before two year age have a very severe progressive disorder with more than one motor milestone loss in 80% of the cases and more than four motor milestone loss in 40% of case. While the late onset have a milder progression. Another key sign and symptom for thymidine kinase 2 deficiency is the respiratory muscle weakness that is leading to different ventilatory support. In the earlier onset cases, 80% of patient require 12 hours per day of ventilatory support, while in the late onset we have 23% of patients that require a minimum of seven hour. In the infantile onset some patient may require tracheostomy. And another important key point is that in the late onset the respiratory function may be very helpful for the diagnosis. In a very small subgroup of patient between 25 and 30% of case that are reported in the literature and they have a early onset of the disease. We also have brain involvement. And I like to alight that in general the brain disease is presenting with seizure within two months after the muscle weakness and the motor regression. From the literature we don't have detail about the type of seizure and the progression of the brain disease. But I like to report a recent case that we follow in our clinic and the important message from this case is that sometimes muscle weakness is so pronounced that is difficult to understand that patient have seizure. So a 24 hour EEG recording will be important to understand the some subdural myoclonus or spasm are related to abnormal electro clinical activity. And in our patient we were able to diagnose a focal migrating epilepsy in this patient. In term of brain MRI, again, we don't have a case serious study, but in our case we were able to demonstrate that after three month after the disease onset patient had a severe involvement of the brain grey and white matter. And after three month there was an extensive progression of the disease. The spectroscopy was quite clear with a huge lactate peak in the lesion. So how we diagnostic TK2d? Definitely we must recognise sign and symptoms of the disease. We have some biomarker in which I will not go into the detail, but it's important to remember that thymidine kinase 2 to deficiency is one of the few disease in which we have an increase of CK and plasma lactic acid. And there is an important biomarker for the severity of the disease, that is GDF15. Thymidine kinase 2 deficiency is also the only one mitochondrial myopathy in which we can recognise an histochemical defect, but also dystrophic changes which are very unusual for a mitochondrial disorder. We can decide in our diagnostic protocol to go first with a muscle biopsy and look also for depletion and deletion, but the confirmation comes from a genetic diagnosis. So we need to find the biallelic pathogenic variant to have a confirmation TK2d. Muscle biopsy might be important even after the genetic diagnosis to predict the severity of the disease. Before or during the management of patient. A deep phenotyping is extremely important to understand other signs and symptom associated with the disease like the cardiac involvement or the presence of peripheral neuropathy. And also we have to understand the diseases very progressive in the early on and is reducing the survivals with 50% of patients with age of less than two year that die with two year from the diagnosis. There is no worldwide distribution in term of the epidemiology equal distribution for thymidine kinase 2 deficiency and especially in my country in Italy, we have very few cases. So we try to understand why there are this difference. And so we also hypothesise that this is an undiagnosed disorder. So we decide collaboration with the UCB to run a study called TK2 FINDER and reanalyze genome and exome data in order to identify patient that have a potential mitochondrial myopathy with no diagnosis. And interesting, analysing more than a 1,000 NGS data. We already identified eight patient with single het variant in coding and non-coding region that are predict or known to be pathogenic. So I will look for the missing variant. Definitely there are some technology that are already available in our diagnostic labs especially for gene data and structural variant. But the future or the present for some of us is the third generation exome sequencing with long-read that will allow us to identify TK2 variant variability in the methylation or other variant in regulatory region. And also the multi approach with the transcriptomic proteomic and functional study may help us to increase the diagnostic rate also in thymidine kinase 2 deficiency. Another important factor in term of clinical symptoms in thymidine kinase 2 deficiency is that there is a clinical variability even with the same family. So this is a patient that I used to follow since the beginning of my career in this disease. He had an onset at two year age, he lost the ability to work at eight year of age and then thanks to the treatment that Professor Hirano will illustrate later, he recovered the ability to work but is still very weak. The sister was coming to the clinic, is the older sister, she was completely fine, but at 14 year age she start to have some milder sign of the disease. So proximal muscle weakness and the increase of the CK and transaminases. We made the genetic diagnosis as she had the same biallelic variant. We started the treatment since we were able to treat the brother and now she has a completely normal motor function. She's completely independent and able to work and do sport and every activity. So how do we explain? There is no genotype phenotype correlation or at least from the study that we had done in 2018, we didn't find any genotype phenotype correlation. So we try to understand definitely there might be some modifier gene that can be responsible of this variability. But from our TK2 FINDER study and by analysing the allele-frequency of non-pathogenic variant in non database, we also understood that there is a variability in term of allele-frequency in different population. So in our first study we claim that the mutation in the position 108 change at tryptophan to methionine was the most common one. But unfortunately looking at the current genomic database, we can see that in the European population, this mutation is not the most frequent. So there might be other factor that are influencing the variability in term of epidemiology of the disease. And with this last slide, I'd like to thank all of you for the attention and my clinical and research team and the organiser for this meeting.
- I'm gonna talk about the current status of TK2d management. And I would also like to thank the organisers for inviting me to speak here today. Okay, so these are my disclosures. I should mention that I am listed as an inventor of this therapy for which there's a patent and that is now in the hands of UCB. And we already heard that there are no approved drugs for the treatment of TK2d. However, there are some in development. We already heard about the clinical spectrum of TK2, where we can have a very early onset in the first year of life, infantile onset through adult onset and the severity of the mitochondrial DNA damage, the depletion correlates with the young patients and the deletions with the older patients. And similarly, the progression and survival are related to the age and onset Early onset is more rapidly progressive and with earlier fatality. And as we heard earlier from Professor Garone that the cognitive involvement in seizures are most frequent in the early onset patients, as many as 25 to 30%. Whereas the older patients tend to have purer myopathies. So management of these various symptoms is at this point largely symptomatic. The myopathy is treated as with any other myopathy with assessments of the strength by a neuromuscular specialist and physical and occupational therapy. It's important to initiate the therapy early because the patients often develop joint contracture and scoliosis, which can be alleviated by the early initiation of therapy. And difficulty swallowing and speaking are often present in these patients. So it's important to evaluate swallowing because that could contribute to aspiration, pneumonia and malnutrition in patients. And we heard that respiratory involvement is very common in these patients and it comes on early, very much like Pompe disease. So it's very important to look for respiratory involvement and especially in the children where it's very difficult to assess if their breathing is impaired. So it's important to do sleep studies in young children or other studies that perhaps Dr. Dominguez González will tell us about. But early intervention with a non-invasive ventilation can be lifesaving in patients with this disease. The central peripheral nervous systems can be affected as Dr. Garone mentioned, the seizures can be diagnosed by EEG and seizure medications of course are very important in controlling that. It's very rare to have peripheral neuropathy and sometimes these patients can present with EMG findings very much like spinal muscular atrophy. So it's good to do electrophysiological tests to diagnose these situation. But ultimately once you diagnose the phenotype, it's important to do the genetic testing as we heard. Cardiomyopathy is very uncommon, but it's important to screen for cardiomyopathy by echocardiogram or cardiac MRI, check for arrhythmias and of course do EKGs as well. And in general medical issues, they often have manifestations beyond muscle in the nervous system. They can have a hepatopathy occasionally, so it's good to check by liver ultrasound. We often see a fatty liver. They may have gastrointestinal dysmotility. Again, you can assess them through a gastroenterologist. And a lot of the patients have osteoporosis or osteopenia in part because of the immobility of the disease. But also perhaps it may be part of the disease. And sometimes this can lead to early pathological fractures so it's important to check their bone health. These are how the symptomatic treatments that we initiate early. But we're getting into the era now where we may be able to have disease modifying therapies. We were fortunate to have a donation from a family affected with TK2 to develop a TK2 knocking mouse, which mimicked the disease in the infantile onset that form the disease. And these mice stopped gaining weight at about 10 days of life. And they only survived about two weeks. So this is a very severe disease in the mouse. And they have very reduced activities of the TK2 enzyme activity in all tissues. And as a consequence, they had a lack of DTTP in brain and in liver they had both TTP and CTP reduced and that led to the depletion of mitochondrial DNA. But the mice were different from humans. The mice developed more central nervous system problems with severe depletion in the brain and spinal cord as opposed to the patients we see who have predominantly myopathy. So while we were experimenting with the mice, we decided to try a therapy. We started with a monophosphate therapy giving deoxyuridine and deoxythymidine to bypass the defect. And we were able to, oops, initiate that end the mice and they lived longer, two to three fold longer depending on the dose of the therapy. But when we measured the metabolites in the blood, we found that when we were giving the monophosphates, they were being rapidly converted back to the nucleosides thymidine and the deoxyuridine. And so we tried to give the nucleosides. So instead of giving the products of the enzyme, we were giving the substrate. And that also worked and improving the mouse phenotype. And here you can see that with the therapy, the mice begin to grow a little better and they live two to threefold longer with the thymidine and deoxyuridine nucleoside therapy. And it did partially rescue the mitochondrial DNA depletion in the tissues, but only transiently, they lived... At 13 days, you can see that the mitochondrial DNA levels were higher, but at 29 days, the mitochondrial DNA levels dropped again. So it was a partially effective treatment in the mice. Meanwhile, while we were treating the mice, we were treating patients as well. And Dr. Dominguez González and colleagues in Spain treated a couple of patients in 2011. We treated our first patient in 2012 along with Dr. Garone and one patient in Italy. And we were reported the results of the first 16 patients who were treated with the deoxynucleosides or deoxynucleoside monophosphates. And so we found that in the early onset patients with onset at before age two, that their survival was dramatically improved. All five patients who were treated, were alive compared to historical controls. Were showed this kind of survival curve. So there's a dramatic improvement in survival of the early onset patients. And in some of the later onset patients, we saw that there were improvements in the six minute walk test, particularly when they had an impairment. When they were walking less than 300 metres at baseline, they became able to walk better. So this was all very encouraging, but this was done under compassionate use. We were fortunate that a company at that point called Modus Therapeutics decided to participate and take over the treatment of these patients in a phase two rollover study. And this here in the 102 study. The 101 study is the patients who were treated early on with the compassionate use. Some other patients treated through compassionate use in the 107. And then a company sponsored an early access programme. So over a 100 patients were treated with the nucleoside therapy. And just focusing on the patients who had onset before age 12 compared to matched historical controls. We see that the survival overall was dramatically improved. This is survival curves of the treated patients in the upper line here and the untreated patients are historical matched controls matched by age at onset. And you can see that there was a between 87 to 95% reduction in the risk of death among these patients. So this has a dramatic effect on survival as was presented in this meeting. But in addition, we also see that there were improvements in the functions of these patients, the ability to hold their head up, the motor milestones, hold their heads, sit, stand, walk, run, climb stairs. Here we see the motors milestones that were lost, prior to the initiation of therapy and even after the therapy lost some motor milestones here in the purplish bars. But after the initiation of therapy, not only did these patients stabilise, they regained and over 70% of the patients regained one or more motor milestones. And untreated patients very, very rarely regained motor milestones. In fact one patient lost the ability to run for one day and then regained it. So it was counted as a regain of a milestone. But in fact, really I don't think that's significant. So we see that there's a dramatic change in the functions of these patients with these nucleoside therapy. This therapy is currently in trial now and applications to regulatory agencies, including the FDA and EMA have been submitted, so. But there are no approved treatments yet. Here's to illustrate one of the expanded access patients we treated early on. This is a child that came to us from Korea. He had onset at age 10 months. He lost ability to sit, stand and walk within six months and he was very floppy. And after one month of therapy you can see that he's beginning to move his arms a little better and his CHOP-INTEND score, which is a motor functional scale improved. And after three months of treatment, you can see he's moving much more vigorously and he's able to transfer the toy from one hand to the other and he's almost able to roll over. And here you can see he's not able to pull himself up to sit, but once he's put in that position he's able to sit and use his arms to play with the toy. So he's regaining motor functions very rapidly as we often see with these early onset cases. And after six months of therapy, he's able to walk with a little bit of assistance. And with a little propping he can, oops, there he goes, take a couple of steps to his mom, okay? And after 10 months of therapy, you can see he's smiling a bit, he's much happier and he's able to walk around the room nonchalantly with his arms behind his back. So that's dramatic improvements. So that's therapy as I mentioned, under development in clinical trial and we hope to hear from regulatory agencies soon. So stay tuned for that. But in the meantime we are also exploring gene therapy as another alternative therapy. And in our lab led by Carlos Lopez Gomez, started treating newborn mice with the gene therapy using an AAV9 vector to deliver human TK2 to the mice. And we were able to improve the survival of the mice, here I've shown two different doses in the blue bars the low dose 4.2x10 to the 10th viral genomes and with a higher dose in order of a magnitude higher, we see the survival increases from about 45 days to about almost 90 days here. And that red line represents the nucleoside therapy, which is very similar to the efficacy to the low dose gene therapy. So that's very encouraging. Obviously it would be great to be able to give a one shot gene therapy. We found that if we gave a second dose of booster dose of the gene therapy, we could improve the survival even further from mean survival of 88 or 90 days to 120 days. And what was even more striking is that when we gave a combination of the gene therapy and pharmacological therapy, so we're giving the gene therapy, restoring the enzyme, and we're giving more of the substrate, the nucleosides, we were able to increase the lifespan of these mice to 180 days. Compared to the untreated mice, which only lived about 14 days. So it's a dramatic improvement and one mouse lived to 450 days. So we were very excited about the possibility of using gene therapy either alone or in combination with the pharmacological therapy. But that's something that's still in development pre-clinically. So I think that's the end of my slides and I think that many people who contributed to this work. Internationally, it's been a great collaboration as you see with with Drs. Garone and Dominguez González and many others around the world. As well as the many people who've worked in my lab on the gene therapy. So with that, I thank you for your attention and I'll turn it over to Christina.
- Thank you Dr. Hirano. Now in this last presentation, I'd like to highlight some key learnings for timely identification and diagnosis, which is in my view a critical step toward better outcomes. So clinical manifestations have already been described previously, but I just wanted to revisit some of the key warning signs that should always raise suspicion of TK2 deficiency. I'll also talk about some of the biomarkers that are useful in the differential diagnosis of our patients with muscle problems. And finally I'll talk briefly about the potential role of the muscle biopsy in this context. So regarding the clinical manifestations as has already been said, the infantile onset disease is the more frequent presentation of TK2 deficiency. And most patients develop normally during the first months of life and then after the disease onset they experience motor regression with rapid progressive disease. And this rapid progression of the motor problems should raise always suspicion of TK2 deficiency. Since it's quite rare in other genetic muscle diseases at this age of onset. Patients with childhood or juvenile onset disease could be more challenging to diagnose since the progression is more slower and they could mimic many other different genetic myopathies In these cases, the signs that should raise suspicion of TK2 deficiency are first of all the presence of ptosis of facial weakness and of facioplegia. Although some of these signs only appear over the course of the disease and are absent at the beginning of the disease. Facioplegia in fact is much more frequent in patients with an adult-onset disease. It has already been mentioned that their early respiratory involvement is also a critical key, not only for the diagnosis but also because it's the main cause of morbidity and mortality of TK2 deficiency in all ages. But this early involvement of the respiratory muscles should always raise suspicion of TK2 deficiency. In general, patients with a progressive muscle disease only require mechanical ventilation after losing the ability to walk. It's different in TK2 deficiency where many patients, especially those with a childhood or a juvenile onset, may need the mechanical ventilation before losing the ability to walk. So this early involvement of the diaphragmatic muscle is very important also for the differential diagnosis. This is an example of a patient with a childhood onset disease to show you how they progresses generally. After birth, he developed normally during the first years of life. At the age of four he started noticing progressive proximal muscle weakness. He lost the ability to walk at the age of 10, from the age of eight he already required nocturnal mechanical ventilation. And in his 20s he required 24 hour mechanical ventilation. In these pictures you can see that he developed the progressive ptosis and the facial weakness over the evolution of the disease. But as you can see this progression, this pure myopathy could resemble many other different genetic myopathies. So what biomarkers could we use to differentiate these patients from a patient with a mitochondrial myopathy? So first of all, the blood biomarkers. Patients do have with TK2 deficiency, high CK levels. That could range from normal which is rare to very high levels around thousands. Sometimes they have CK levels in the same range of muscle dystrophy. So this is not useful to differentiate TK2 deficiency from other genetic myopathies, but it's useful to differentiate a mitochondrial myopathy from TK2 deficiency. Since patients with mitochondrial myopathies generally don't have such a high levels of CK. Lactate levels are a classical sign of mitochondrial dysfunction. So could be very useful if they are altered. Although this is not very sensitive, the majority of the patients with TK2 deficiency have normal lactate levels and only very few had a slight increase of lactate levels. The more sensitive biomarker to detect the mitochondrial origin of a progressive muscle disease is the GDF15, which is growth differentiation factor 15. It's a cytokine that is induced in the muscle after mitochondrial dysfunction. And it has been described first in patients with TK2 deficiency. In our cohort of cases, almost 90% of the patients with a mitochondrial myopathy in general have elevated levels of GDF15. And they are normal in all non mitochondrial myopathies. So it's very useful to suspect the mitochondrial origin of a progressive muscle syndrome. In fact, in patients with TK2 deficiency, levels of GDF15 cover late with the severity of the disease and with the age of onset of the disease. In patients with an early onset and rapid progressive disease, they have an increase of 60 fold, while patients with a late onset milder phenotypes do have an increase of six fold of the GDF15. In fact, in our cohort of patients with juvenile or adult onset disease, GDF15 levels correlate with the motor performance and with the respiratory function. In our patients, levels of GDF15 inversely correlate with the North Star Ambulatory Assessment and also with the 100 metre run test. So it's a very sensitive biomarker to also predict the prognosis of the disease. Again, it correlates with the force vital capacity. So it's useful to predict the prognosis of the disease. In fact, when we introduced the treatment with oral nucleosides, the levels of GDF15 significantly decline over time, even reaching normal values after one year of treatment, especially in those cases with more effective response to the treatment. So GDF15 is useful to suspect a mitochondrial origin of a myopathic process also to predict the prognosis, to monitor the disease progression and also to evaluate the response to the treatment. Another useful tool in the differential diagnosis of a patient with a progressive myopathy is the muscle MRI. We have studied the muscle MRI only in patients with childhood onset and juvenile or adult onset disease. And we have found that they do have a specific pattern of involvement with an early alteration of the sartorius muscle and the gluteus maximus muscle. And this pattern of involvement is useful in the differential diagnosis with other myopathies with similar clinical characteristics. Although we have only studied this pattern in patients with childhood or juvenile lung disease. It is also useful to monitor the disease progression since we can't quantify the amount of fat in the muscle to quantify the amount of muscle degeneration over time. Regarding the muscle biopsy, in general it is well known that the muscle biopsy in a patient with mitochondrial disease in general is not very sensitive in paediatric cases to detect signs of mitochondrial dysfunction. However, in TK2 deficiency, this is different since the muscle is the main tissue affected of the disease. So in all patients with TK2 deficiency, the muscle biopsy is abnormal and we can see the signs of mitochondrial dysfunction in all of them. Ragged red fibres, cox-negative fibres. In this example, on the top you can see a patient with an infantile onset disease and at the bottom a patient with childhood onset disease. So you have a patient with progressive muscle weakness where the genetic test is negative or is delayed or is inconclusive. A muscle biopsy allows you to detect the mitochondrial origin of this disease and it's help you to direct the genetic test. Also, when the disease progresses in more advanced disease, we can see some structural changes in the muscle biopsy, which is quite unusual In other mitochondrial myopathies, we can see the presence of myopathic or even dystrophic changes in addition to the mitochondrial dysfunction. And this is so uncommon in other mitochondrial myopathies that in some of our cases in Spain we have diagnosed a patient after the pathologist suspected TK2 deficiency based on the morphological analysis of the muscle biopsy. In addition, you have done a muscle biopsy, you can't also use it to quantify the amount of mitochondrial DNA or to look for the presence of multiple deletions. And this is useful to confirm the pathogenicity of a variant of unknown significance. So helps you to confirm the diagnosis when the genetic test is inconclusive. And in addition, you can also quantify the amount of mitochondrial DNA and with that predict the prognosis of the disease. Since we have seen that this copy number analysis is the main prognostic factor of TK2 deficiency. We have found that the copy number correlate strongly and significantly with the age of onset of the disease, but also with the age at onset of the mechanical ventilation and with the overall severity of the disease. So some of the key points of this presentation is that there are some clinical clues that allow us to early recognise our patients. And in addition, we have some biomarkers that are very useful into differential diagnosis. Not only to suspect the mitochondrial origin of a muscle problem, but also to predict the severity and to monitor the disease progression and the response to the treatment, which is GDF15. The MRI could also be useful in the differential diagnosis, although only it has been tested in patients with juvenile or childhood onset disease. But it's also a useful tool to monitor the disease progression. The muscle biopsy still plays a role when the genetic test is negative or is inconclusive or when it is delayed because allows you to narrow the differentiate diagnosis and direct the genetic test and also to have some tissue to quantify the mtDNA copy number and predict the prognosis. And finally, as you can see in many different cases, the main differential diagnosis of a patient with a TK2 deficiency is other genetic myopathies. So you need to be sure that in your genetic panels for muscle diseases, the TK2 gene is included to be able to diagnose these patients early. Take home messages from this symposium. First of all, TK2 deficiency is a very rare disease, although is probably underdiagnosed. It's a very severe disease with high mortality rates, especially in those cases with an age onset of less than 12 years. The earlier the onset, the worst the prognosis. However, we have some treatments now that is able to completely modify the natural course of the disease, is able to halt the progression and to prolong survival and improve the motor outcomes. And so it's very important to be able to early recognise our patients and to diagnose our patients from the beginning. And to do so, remember that it's important to use the genetic test and include the TK2 gene in your panels for muscle disorders. With that, I think that we can go through the questions. So we can start with some of your questions. First, a question for Professor Garone, what are the most frequent misdiagnosis for infantile onset TK2 deficiency?
- So the majority of patient, especially the early onset one, might have a diagnosis of dystrophy neuropathy because they have some dystrophic changes, although they of course the gene defect or muscle dystrophy has not been found. Or for spinal muscular atrophy type 3, which is very common. Well the juvenile onset is more for facioscapulohumeral disorder we know genetic diagnosis again.
- Question again for Professor Garone. What evidence supports the statement that a multi-omic approach can increase the diagnostic rate for TK2 deficiency versus next generation sequencing alone?
- Well, TK2 is an ultra rare disease and we know that thanks to the NGS second generation we were able to increase the diagnostic rate up to 60%. But there are still 40% of cases that does not receive diagnosis. So approach with the NGS transcriptome and metabolomic analysis may help us to identify more complex defect in the gene that might involve regulatory region or might involve the additional of other genes a like in a digenic disorder.
- Now a question for Professor Hirano. Which treatment do you recommend to treat epilepsy in TK2 deficiency?
- Treat epilepsy. Okay. Yeah, so I think we use our standard treatments, levetiracetam is often the treatment of choice initially and usually it's manageable with standard epilepsy drugs. Do you have any comments about that?
- Yeah, it really depend about the severity of the encephalopathy. In our case, unfortunately the epilepsy was untreatable, so, you know, the combination between EEG and brain MRI can help us to guide also the type of treatment that we need to use and if we need to treat the patient.
- Someone also asks is there any risk of liver failure with valproic acid?
- Yeah, well that's a well known risk for Polymer gamma disease, but I have not seen it. But I think most of us try to avoid valproic acid in these patients and certainly there are many alternatives that we can use besides valproic acid. So we do try to avoid it.
- Yes, we don't have any specific evidence for thymidine kinase 2 deficiencies. So we apply what we do for other mitochondrial epilepsy. So I agree with the Hirano we tend to avoid valproic acid.
- They asking which is the minimum age to treat?
- Zero or prenatal. So the earlier you treat the best outcome. So we are really aim to have a newborn screening soon and the patient before they have any sign or symptom of the disease.
- Yeah, I think the earliest age of treatment that I'm aware of is four months. But in theory we could treat earlier if we could diagnose earlier. However, I think if there's structural brain damage, if there's atrophy of the brain, I think it's less likely to work frankly because the neuronal loss is unrecoverable.
- And Dr. Hirano, how long do you predict gene therapy in clinic? - It's hard to predict. I would hope that it can be done in a matter of years. I think the preclinical studies have almost been completed with the mouse model. So if there's interest in industry development of this, it could move forward in a matter of years. A couple of years. - We haven't mentioned. So people ask about the side effects of the therapy.
- So the most common side effect is diarrhoea in 80% of cases that actually is manageable. Patient do not need admission to the hospital or the hydration, but it's of course it's something that may impact a bit their life. But we do not have any other more severe side effect. We just had two cases that require discontinuous on treatment because of a severe increase of liver transaminase.
- Comment?
- Well these two cases with liver problems were two very late onset disease patients, two adult patients and we're using the non chemical, the grade, so we yet don't know if this treatment by UCB causes liver problems or not. At the onset of symptom, the infantile form. Do the children present with classical peripheral hypotonia with hyporeflexia or can there be already mixed hypotonia with hyperreflexia?
- The hyperreflexia is not always present so they might they still, you know, sometimes the presence of the reflex, but the hypotonia is very severe in the infantile onset.
- But theoretically with CNS involvement, of course there can be hyperreflexia and I think it may be partly due to the disease, but I think because of the early respiratory involvement, I think sometimes I've seen one case where I had a child who had a respiratory arrest before they recognised the restrictive lung disease. And I think, well afterwards that the child had cognitive issues, I suspect that it was an anoxic brain injury. So that's something that yet another reason to diagnose some respiratory problems early.
- Another questions, could nucleoside therapy be efficient in other mitochondrial DNA depletion syndromes?
- So we do have some in vitro evidence that nucleoside therapy is efficient in other mitochondrial depletion syndrome, especially affecting the same pathway for the purine and pyrimidine nucleoside. Unfortunately while pyrimidine nucleoside are stable, the purine nucleoside are very fast metabolised even in the intestinal tract. So the treatment is not yet translatable to human because of the limited bioavailability.
- They are asking about GDF15, how high exactly GDF15 is indicated deficiency? On our lab, we don't have normal values. Well it depends on the method you're using and GDF15 levels increase with age. But in general, in patients with TK2 deficiency with an infantile onset disease, they have more than 20,000 levels of GDF15 and patients with paediatric onset, childhood onset, or late onset disease, they tend to have between 2,000 and 3,000 levels of CDF15. Normal, it depends on the age, but less than 900 is the normal value. They are also asking about the CK levels, which is very unusual comparing to other mito diseases, it is already this much elevated at the onset or does it take time before the dystrophy pattern develop? Well, in my experience it's already very high at the beginning of the disease around thousands from the very early.
- May add something about the CK? That sometimes we have also the transaminase increase and that might not be related to a liver dysfunction, but an indirect sign of myolysis as well. So it's important to look at both of them. CK and transaminase level.
- As well as GGT. Yeah.
- I think the last question is what is the temporal relationship between GDF15 reduction and clinical improvement? Might it be used as an early indicator of treatment efficacy? Well, in my experience, GDF15 levels start declining after the first three months of treatment although we haven't analyses it before yet, but it's very fast, the decline in GDF15 before you can see the clinical improvements even. Our last one for Dr. Hirano. Regardless the level and inclusion criteria, don't you believe in any response for older patients know the ambulate?
- Yes, so, well you actually have more data on that than I do, but yes, we think that adults can benefit from this treatment as well. And it's not as dramatic as in the young children, but certainly we can see not only well improvements, but stabilisation is also a victory in this disease. And I can tell you anecdotally, we have one patient who had onset at age three years old, and we started treating him when he was 29 years old and the only movement he had was in his fingers, he could... But with that he was able to drive his motorised wheelchair and write two novels. So we wanted to preserve that and his ability to communicate with the outside world. We started the therapy and he's been on for three years and he's maintained that ability.
- In your patients, how long did it take for truncal weakness to resolve? - Yeah, it really depends on the age and onset and how quickly you initiate the therapy relative to the onset of the disease. So as illustrated with the video I showed, I mean that child had severe neck weakness and truncal weakness and he regained that within a matter of three to six months. So it can happen within months, but in many cases it can take longer if there's a delay in initiating treatment.
- And with that, we have finished. Thank you all for your attention.
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