Through a case-based, evidence-driven approach, this Neuroscience Grand Rounds session highlights how clinicians can differentiate among neuromuscular conditions, localize disease processes and identify those disorders where timely treatment can significantly alter disease trajectory. Emphasis is placed on integrating current research, multidisciplinary care strategies, and evolving therapeutic options into routine clinical practice to improve long-term neurologic and functional outcomes for pediatric patients.
Learning Objectives:
Recognize the clinical presentation of treatable pediatric neuromuscular disorders.
Describe the treatments available for pediatric neuromuscular disorders.
I was kind of giving everybody a minute. OK, perfect. Um, good morning, everyone. Thanks for joining us for grand rounds. Um, I'm excited to, um, introduce today's speaker, so Doctor Justin Hoffman. He's our pediatric neuromuscular fellow. So, Doctor Hoffman did his Bachelor of Science and Health Sciences in Psychological Sciences at the University of Arizona in Tucson. And after graduation, he did research at the University of Arizona. Um, looking at novel therapeutics for asthma, headache, and chronic pain. And then he returned to Midwestern University for medical training, and he completed his residency in child neurology here at Phoenix Children's, and now he is doing our fellowship here in pediatric neuromuscular neurology, and we're excited to have him join us after fellowship later this year. All right. Good morning, everyone. Thanks so much uh for, for joining me this morning. Uh thanks, Doctor Chow for the introduction. I appreciate it. I'm looking forward to talking to you a little bit today about some treatable pediatric neuromuscular disorders. Aces here. I have nothing to disclose and our objectives today are pretty simple. Uh, we're gonna recognize some clinical presentations of, of treatable pediatric neuromuscular disorders and we'll talk a little bit about the treatments, um, and I'll talk about these things by going through a couple of cases, uh, of patients that I've either taken care of or been somewhat involved in their care to some degree, uh, during this year to help illustrate some of these things. But before we hop into the cases, just a little bit of background about pediatric neuromuscular disorders. There's over 400 of them, and they can affect uh the, the nervous system anywhere from the anterior horn cell all the way down to the muscle in and of itself and can be split up into two different types, either acquired disorders or inherited disorders, and the acquired The ones are things that we might see commonly in the hospital like myasthenia, GBS, inflammatory etiology such as dermatomyositis and things like nutritional metabolic deficiencies. Whereas the inherited disorders um include things more commonly like Duchenne muscular dystrophy, uh, spinal muscular atrophy, congenital myasthenia. And uh of these 400 different disorders, there's over 500 genes and that number is probably lower than the true number right now, and it's exploded um with the implementation of more advanced genetic testing platforms with uh high throughput next generation. Uh, sequencing techniques, the ubiquity of whole genome sequencing and RNA sequencing techniques to define some novel, uh, very rare variants, and, and the number of genes is, I mean, like you can see over 200 some odd in, in the decade between 2010 and 2019. So, really exciting time for us. Now, many of these acquired disorders have disease modifying treatments available, and I've talked about a couple of these uh in my grand rounds the last couple of years with GBS and myasthenia. Uh, but the availability of disease-modifying therapies for inherited disorders is a much smaller group. Um, and these can either be FDA or off-label uses, and I've included some of these, uh, diseases here like spinal muscular atrophy, Duchenne muscular dystrophy, uh, Pompeii disease, um, and the fact that we have these is, is exciting, but, but, you know, with Gene discovery of new genes and, and uh looking into new therapeutic targets and improving our understanding of mechanisms that underlie these uh diseases uh with things like proteomics, transcriptomis, RNA sequencing, uh are really gonna be able to enable us to develop novel uh therapies for, for new diseases and improve therapies uh for ones where, for diseases where therapies already exist. Now, when we look at, you know, treating patients with neuromuscular disorders, we can break it down into something pretty simple. We have to be able to recognize the disorder and then Uh, implement a treatment, uh, for it should one be available, but it's pretty challenging to diagnose some of these pediatric neuromuscular disorders, and that can be for a multitude of reasons. It might be that we're unfamiliar with some of the clinical presentations of these more rare disorders. It might be because there's a quite a significant overlap in the presenting symptoms, um, of many diseases. Proximal weakness, uh, can be the presentation for lots of different neuromuscular disorders, for example. There's a pretty wide heterogeneity of phenotypical clinical presentations based on a single genotype, uh, that can be with the symptoms of patient, um, displays or the, uh, variability of the severity of those symptoms or how rapidly they progress and even at sometimes, um, a different clinical presentation for the same gene based on the age of onset of, of the disease for the patient. Limited or lack of disease biomarkers is something that sort of plagues the neuromuscular field and There can be negative or inconclusive initial testing, so genetic testing is great, but many times might not reveal an etiology immediately, and so you really have to understand the, the possible phenotypes or know them to know when to pursue additional testing and how to interpret the testing results of those tests and to consider initiation or continuation of empiric therapy, uh, despite potentially negative diagnostic results. Those are the challenges to the diagnoses and there's also challenges to the development of therapeutics. Um, the limited understanding of the exact underlying disease mechanisms is something that's a problem and, and is, is getting better as technology improves, but there really is a lack of natural history studies, uh, for many of the rare disorders and specific biomarkers to help us track disease progress. Clinical trials certainly do exist. There's many ongoing out there in the field right now, uh, but they can be tedious and really difficult designs. It's hard to power trials appropriately, especially when diseases are rare, uh, to recruit enough patients, and We don't always know exactly what functional measures to take a look at. So for example, if we were to look at a 100 m walk time and force vital capacity for a particular disease, but it's grip strength that really makes a difference uh with the treatment, then we might miss out on knowing that the that the medicine is, is, is helpful for this disease. And these trials can be kind of long as well. Um, it can be really difficult to see meaningful changes from placebo groups, especially in slowly progressing disorders. However, There are many therapies that have been recently approved and many things in the pipeline. These are listed just a few, uh, a new treatment for Barth syndrome, uh, new, uh, gene therapy for patients over 2, for spinal muscular atrophy, there are upcoming FDA approval dates for cell-based therapies for Duchesne and Myostatin inhibitors for SMA and plenty of things that are up and coming. For more common neuromuscular diseases like limb girdle, uh, Charcore tooth, as well as myotonic dystrophy. So maybe if I get invited back sometime, that this might be the, the scope of the, of the next talk, um, but really, uh, an exciting time for us. OK. So kind of with that background in mind, I'm gonna go through a couple of cases here now and um I just wanna keep in mind that certainly some of the cases have been condensed for clarity, um, and, and sort of simplified uh for, for the context of this talk. So the first case um is a case of persistent stridor, and this is a previously healthy 12 month old male who came into the ED here at Phoenix Children's for congestion and worsening noisy breathing. His noisy breathing had been going on for several months and he was actually previously diagnosed with bronchitis and trialed on steroids with some improvement, but his symptoms persisted. Uh, the noisy breathing was present when he was sleeping in with activity and, and wasn't triggered by any sort of symptoms of illness. His prior history was relatively unremarkable, a termed child with no really prior medical history and uh no reported family history of respiratory or neurological disorders. In the ED he was stridorous on exam but well appearing and didn't have any sort of neurological deficits. It was really suspected that his stridor was anatomical in nature and he was referred to the outpatient ENT clinic. Where he showed up a couple of days later and underwent flexible laryngoscopy and, and was found to have bilateral vocal, total vocal cord paralysis and directly admitted to PCH from the clinic due to the risk of, of airway demise. He was pretty well appearing when he came into the hospital, but actually less than 24 hours later. um, underwent a code blue, uh, was very cyanotic, had oxygen saturations in the 70s, uh, became pulseless and actually required CPR for a couple of minutes before they got a, a rhythm back and uh was sent to the ICU and intubated for respiratory support. In the ICU he underwent an MRI brain and neck for evaluation uh for his vocal cord paralysis, and it was unremarkable. He improved and was extubated, uh, but did have aspiration on his swallow evaluation. His course in the ICU was complicated by a norovirus infection requiring a brief uh spell of NG tube feed support before he was able to get back to oral feeds, uh, but did have persistent vocal cord paralysis at discharge and, and was undergoing some dexamethasone treatment for it and it was thought to be due to a viral etiology. He came back about 5 days later, worsening stridor, increased work of breathing, none neurological defects, and thought to be having ongoing complications of a viral process and was readmitted to the ICU. Uh, he underwent a right vocal cord lateralization to help with his, uh, airway patency, but was still noted to continue to have stridor continuously, uh, and, and increased, uh, work of breathing with agitation. He underwent a reintubation for hypercarbic respiratory failure in the setting of an infection around the surgical site and was subsequently extubated again. And this is when neurology became involved in his care, about 3 days after he was extubated for concerns for persistent vocal cord paralysis and persistent hypotonia after he was extubated. So this was about 2 months after he initially presented. He was able to sit and crawl prior to the onset of his symptoms, uh, but now he was having more difficulty with these, uh, functions, and family told the neurology team that his muscles really seemed to be looser with his recent illness. He was having some difficulty keeping his eyes open while awake and they did not completely close while he was asleep, and family was maybe suggesting that he could have had a little bit of mild facial weakness over the past few months. He was examined by the team uh on a few occasions, uh, over the next few weeks, and this is just a summary of the exams, um, where he was noted to have significant findings, uh, including, uh, facial weakness, maybe some difficulty holding his head up. Eyelid lag, uh, inability to fully bury his sclera when he was abducting his eyes bilaterally. He was noted to be globally hypotonic but moving his extremities anti-gravity and with no neck weakness, um, and had normal reflexes. Uh, some exams, and some exams he was felt to be hyporeflexic. So he underwent a pretty significant diagnostic workup, uh, and I've just highlighted some of the, the highlights here. Uh, his CK was normal. Uh, acetylcholine receptor antibodies looking for myasthenia were also normal. Looking at his lactic acid, ammonia, azylcarnitine profile, metabolic workup, unremarkable in that regards. However, his urine organic acids were positive for hexano uh hexanoglycine and superroglycine, um, and, and overall had a pyruvic aciuria, um, with intermediates, the citric acid cycle being elevated, um, and a dicarboxylic aciduria that maybe could be due to his dietary supplementation. Um, his echocardiogram, MRI brain, um, an EMG and nerve conduction study, including studies for high and low frequency, uh, repetitive nerve stimulation to evaluate for a, a, a neuromuscular junction defect were normal. I was quite surprised, uh, when, when we did the study. Uh, but he, he, he had another swallow evaluation that showed pretty significant dysphagia. OK, so genetics was consulted. He had a whole exome sequencing obtained. He had an NG tube placed because of his dysphagia and, and went home, um, uh, for unclear reasons, but came back three days later for concern for pneumonia. And his genetic testing resulted shortly following his admission showing a normal mitochondrial DNA sequence, but his rabbit hole genome sequence showed a homozygous deletion in SLC 52A3 that is seen with riboflavin transporter deficiency. And that's what I'll talk about here in the coming slides, but first, I just wanna talk just briefly about his clinical course. He was started on high-dose riboflavin, uh, when, when the genetic results came back, his riboflavin level was normal, which can be seen in this disorder. He underwent a, a bear which showed no Hearing loss and I'll come back to that, but had persistent vocal cord paralysis and some feeding difficulties and underwent tracheostomy and G tube placement. He, he's still currently hospitalized and discharge planning is ongoing, and he remains on high dose riboflavin. Uh, escalating his dose, uh, with a goal dose of 50 mg per kilogram per day. But importantly, it's not been documented that I can see to have any sort of progression of his ocular bulbar, um, symptoms that were noted on his neurological exam or any sort of extremity weakness. OK, so let's talk just a little bit about the uh riboflavin transporter deficiency, the mechanism behind it cause I think that's really important to understanding uh why therapies work and um why patients actually have the symptoms that they, they clinically show uh to us in the hospital or in the clinic setting. So, so in riboflavin transport deficiency, there's an insufficient transport of riboflavin into cells. In mammals, humans, we, we can't make vitamin B2 uh de novo, so transport uh from exogenous sources is really important. Uh, we take this in in our diet, certain meats, fish, pork, uh, greens like spinach, dairy, high in B2 and really necessary, uh, to be able to, uh, have sufficient B2 intake to build biological co-factors, uh, that participate in a variety of, uh, energy and, uh, metabolic processes within the cell. And really when these processes are disrupted, tissues that are highly metabolic, like the nervous tissue or muscle tissue can be quite susceptible. So there's 3 riboflavin transporters uh in the family that's shown here in the bottom right. I hope you can see my cursor. Um, riboflavin transporter 12, and 3, Genetically coded by SLC 52A1, 2, and 3 respectively. Uh, riboflavin transporter 1, really highly expressed in the intestine and the placenta, uh, riboflavin 2 in all tissues, but particularly important in the nervous tissue, and riboflavin, uh, transporter 3 also expressed in the intestine. Now, riboflavin transporter 2 and 3 are associated with riboflavin transporter deficiency. Riboflavin one really isn't. There's a single case report in the literature and some reports of some mothers who are homozygous, uh, for the, the deficiency who had inadequate riboflavin and taken pregnancy and had infants that were transiently symptomatic for, for a couple of years before improving. OK, so what happens once these transporters bring riboflavin into the cell? The uh riboflavin is converted into these biological flavin, um, cofactors, flavin mononucleotide and flavin adenine dinucleotide FMN and FAD. That participate in a multitude of, of, of biological reactions. So, they're highlighted in a few different areas of the figures here, and I'll just point out that these um biological cofactors participate in redox reactions and are really important in, in complex one and two in the respiratory chain as part of uh carbohydrate metabolism. Um, they participate in other areas of carbohydrate breakdown. Uh, they're important in steroid metabolism, fatty acid, acid oxidation, DNA synthesis and repair. Uh, they've been, uh, associated with, um, axonal outgrowth, although that's a little bit, um, debated in the literature. So, the overall point is that the the reduced flavins lead to disruption of all of these processes and cause problems. So, here in this slide, I've, I've highlighted uh some figures from a paper that was uh pretty important in showing that like kind of the mechanism behind how this works biologically, and it was this group, Mano that publishes paper on brain looking at this model in Drosophila, um, and shown over here on the, the left are graphs. That depict the two Drosophila models that are crossbred to produce this knockdown or knockout riboflavin transport deficiency model, and they looked at FMN FAD, the biological sort of intermediate levels, and looked at respiratory chain activity. And the big takeaway here is that these uh flavin levels are diminished. Uh, the electron transport chain activity in complex one and two is significantly diminished in these uh drosophila, uh, nerve nervous tissue or brain tissue, and they highlighted that this really does seem to affect the mitochondria with these figures here on the right. So, this figure here This shows the uh control model. This shows the riboflavin transporter knockdown model in the drosophila brain tissue. Here we have very nice mitochondrial cristae, uh, nice mitochondrial structures in these control models, but you get these really odd deformed, vaculated, uh, abnormal mitochondria when you, uh, knock out, these are knock down this riboflavin transporter. Um, and so they really did show that, that, that this is sort of the, the biological mechanism behind uh riboflavin transport deficiency, and they also did, uh, show this in the human fibroblast cells, again, showing the biological flavins, FMN and FAD, the complex activity of the electron transport chain, uh, that was knocked down in all facets, um, in patients that had riboflavin transporter deficiency. All right, so let's get into some of the clinical things, uh, as far as the disorder goes. So this was first described in 1894 by Violetto and later by a couple of others, and, and termed this Brown Violetto-Venler disorder or the Zio-Londe syndrome for those that might have heard of this before, but these are have ultimately been recognized as different spectrums of the same disorder, and now they're just classified as riber flavored transport deficiency. These are autosomal recessive changes in the, uh, the two genes here that encode the riboflavin transporters 2 and 3, and it's fairly rare. It's estimated 1 in a million cases, but possibly underestimated um because of the, the rareness and sort of heterogeneity of the presentation. The bottom line is it sort of manifests as a neuromuscular disorder with variable age of onset, clinical manifestations and rate of progressions, uh, that's shown over here in this table, and I know it's kind of busy, but I just wanna show a couple of things. Um, both, uh, both people that have riboflavin transporter 2 and 3 deficiency, uh, will have bulbar weakness, um, optic atrophy, although that's more common in riboflavin transporter 2. Hearing loss is a hallmark for this disorder and, and if you see a patient with hearing loss and weakness, really put this on your differential. Uh, patients might have facial weakness, although it's a little more common in people that have type 3 mutations. Uh, ataxia or sensory abnormalities is a little bit more common in 2. Both patients, type, uh, both genetic mutations rather have feeding difficulties as well as respiratory symptoms. Patients can have Um, EMG, uh, abnormalities and MRI abnormalities that I'll detail here in the following slides. I just wanted to, to highlight here at the bottom of the figure that this is from a, a review paper that pulled different cases of a patient with riboflavin transporter 3 deficiency. Like the case that I presented, who presented with a facial palsy as well as vocal cord paralysis like our patient. Actually, patients that have type 3 transporter deficiency are a little bit more likely to have a stridor as part of their clinical presentation. So as far as neurodiagnostic findings, the EMG and nerve conduction study for the EMGers out there can show an axonal motor neuropathy with evidence of chronic deinnervation and can actually sort of mimic what you might see in a patient with motor neuron disease or a sensory motor axonal neuropathy can also be seen. As far as the MRI brain and spine, they're typically normal and cognition is typically normal in these patients. However, you can see abnormal T2 signal, um, in different areas of the brain, both the cerebellum as well as uh the cortex and deep gray, and sort of these symmetrical lesions I don't have pictured here in the, in the brain stem, uh, targeting the vestibular nuclei, as well as the central tegmental tracts. So here at the right is shown as someone with a normal or patient with a normal MRI brain, uh, but when we start to look at the, uh, spinal cord, we see some abnormalities. So, in patients, you might see this, this abnormal T2 signal that can be seen in the ventral nerve roots, which is pointed out here in the cervical spine of this patient as well as the, the dorsal aspect of the spinal column. The nerve roots you can see here are, are quite profoundly enlarged here on the sagittal section, um, and they're just re-demonstrated down here in, in part D. You can also have some uh sort of enhancement of the, the caudal, uh, nerve roots in the lumbar spine can also be seen. Pictured here below is, is a, is a patient with a, a finding that has been shown in, in multiple patients in the literature where you get this abnormal T2 signal that's this longitudinal, it's kind of faint, but longitudinal uh T2 abnormality in the cervical spine. So how about the serum laboratory studies? They're really not consistently abnormal like we saw in the, the patient that, that I presented in the case. And so things like azylcarnitine profiles, plasma flavin levels, urine organic acid levels, they're variably abnormal within patients and really can't be used to diagnose this condition, but can be used to support a diagnosis if you're clinically suspicious. Genetic testing really is mean and is the definitive diagnosis. Um, although not all patients will test positive, I can think of at least one case I've heard of here at PCH where a patient never tested positive and certainly benefited from treatment for this disorder. So, uh, this, this, these genes, uh, they can be found on whole exome and genome sequences, obviously, but also on a multitude of panels, uh, for things like hearing loss, motor neuron disease, and inborn errors in metabolism. Things that might mimic this disorder would be things like Guillain-Barre, uh, congenital myasthenic syndrome that can cause ocular bulbar weakness, SMA that can cause a, a progressive, um, you know, myopathy or progressive extremity weakness. Um, ALS certainly think about that in older patients and certain inborn errors in metabolism. All right, so the treatment, high dose riboflavin, uh, in the literature, uh, that really wide range, 6 to 80 mg per kilogram per day, um, in, in patients treated for this. And the real big take-home message is that the patients that have been treated, there are no deaths that are reported in the literature, but over half of the untreated patients reported in the literature or in, in, in many studies, more or less die, uh, cause this is a progressive disorder. There were no randomized clinical trials for riboflavin. Uh, it's a rare rare disorder and, and treatment was found to be effective, so that kind of leads away from, you know, these trials are difficult to, to organize and treatment's effective, but its use has been supported in many case reports series, as well as uh open label studies. Here's an example of a dose regimen of sort of a gradual increase up to a goal dose. Um, and the treatment is typically well-tolerated with very minimal side effects, GI side effects, discolored urine, uh, but overall, very well tolerated. It is really important to treat all suspected or confirmed cases very early and to continue treatment until an alternative diagnosis is found if initial testing is negative. These certain delays in treatment might lead to irreparable loss of motor and sensory neurons that patients can't get back, and patients might not respond right away. So don't give up if patients don't respond right away as it's been well shown that there can be a latency in response to treatment. And not all patients will improve with treatment. Some patients might just stabilize and that is an improvement over what is sort of the natural history of a progressive nature of this disorder. It is a little bit difficult to find um really sort of comprehensive, well put together retrospective reviews, but, but one in the literature here, the Fennay et al. group, they, they had 94 patients that they found and they were pretty well split between type 2 and type 3, and they noted that amongst all the studies that they collected to make up this retrospective review, that there were problems because only short-term outcomes. were evaluated and there really wasn't a lack of, of uniform um monitoring and assessing changes across different domains, and I've listed some of those here, motor strength, ambulation, ataxia, hearing loss, bulbar palsy, and you might notice that the end values for patients that were assessed are quite different and that leads to the sort of heterogeneity and how different groups um assess these patients. But what I want to point out is that across these domains, patients in general, either improved and the majority of patients improved in things like limb motor strength, ambulation, uh, bulbar palsy, and respiratory dysfunction, and, uh, a small percentage of patients didn't improve but stabilized. Now, hearing loss is probably the thing that was least sort of improved overall when looking at the studies together and, and patients tended to have a, a sort of residual hearing loss despite treatment. Really remarkably with the treatment, the, the 4 patients that were reported to have feeding tubes, 3 were able to have them removed and of the 10 patients that had traches, 8 of those were able to be removed, and I would argue that those are substantial improvements. However, these treatments, I don't wanna oversell this, that many patients still have significant deficits and, you know, a lot of the patients they looked at still required a mobility device or wheelchair for ambulation and as, as I mentioned before, several patients continue to have profound hearing loss. But the bottom line is that the treatment makes a difference and is life-saving. OK, a 2 for this time. We'll talk about 2, case 2A and 2B. Case 2A is the case of rapidly progressive weakness, and 2B limb girdle weakness and abnormal gait. So I'll present them side by side here and we'll, we'll kinda compare the two presentations. So Case 2A was a 3 year, 6 month old who presented with progressive weakness, extremity pain, and an elevated creatine kinase into the thousands. He was born at term and walked on time and began to have weakness at 2.5 years of age, but by the time he showed up in clinic, he was having more rapidly progressive weakness. He had difficulty rising from the floor, getting upstairs, and actually getting his head off the bed. No fevers or rashes, no cardiac pulmonary renal dysfunction as part of his history. He had normal vision and hearing. No family history other than that parents were second cousins. Case 2B presented 5 years after 2A to the neuromuscular clinic, and she was an unrelated 23 month old female who showed up with abnormal gait and weakness. She was also born at term and walked on time and was noted to have a normal gait until she was about 20 months of age. And now she's having problems, um, getting up from a se to a standing position and having to, to walk her hands up her legs to do so. Family also noted these sort of unprovoked episodes where she seemed to have problems with strength and coordination since she was 20 months old. However, these episodes haven't significantly progressed um over the past 3 months. The episodes don't have a clear provoking event or episodes or sorry, I guess her weakness in general doesn't have Any sort of clear inciting event. She also did not have any extremity pain, fevers, vision or hearing problems, and she showed up to the clinic with a normal plasma CK that was checked by her pediatrician. So as far as the TA's exam, he did not have any sort of ophthalmoplegia, nystagmus or facial weakness, but did have very significant neck flexor weakness, uh, but was able to lift both hands above his head. His distal strength appeared to be somewhat compromised, although difficult to assess in the younger child without any sort of obvious sensory deficit. His DTR's reflexes were difficult to obtain, and his gait was quite abnormal, uh, walking with a, uh, waddling gait with a lumbar lordosis. He was not able to get up off the floor with the use, uh, without the use rather of a Gower's maneuver. Patient 2B also had no uh facial weakness, eye symptoms. She did not have any neck flex or weakness, but her reflexes were reduced bilaterally. She also was noted to have normal sensation just like uh 2A, and she also had an abnormal gait with lumbar hyperloroidosis with a little bit of a steppage quality. She too had a Gower's maneuver when she rose from the floor. So I have some videos of the exam of, of what these patients looked like when they, they came to the clinic. I'd like to play those for you now and point out some features. So this is 2A walking down the hall. Sort of a waddling gait, mildly circumducting his hips. A little bit of steppage quality to it, but does walk with a heel toe strike. A little bit of wobbity, excuse me, waddling character to his gait. All right, so this is an example of him getting off the floor, and I think it really highlights some of the Axial truncal weakness and neck weakness that he had. To be able to sit up. I'll play it one more time. But his neck neck flexions are really big. Like that's not like you. Let's compare that, um, or let's see what, what some of Tubby's um exam findings were when she was getting up off the floor and climbing stairs. So, I'll show you patient 2B. On your hands and knees here. And pushes herself up to a standing position with a modified or or Gower modified Gower's maneuver. Oh, she does. She was tested to climb stairs and had to utilize her hand on her thigh to be able to push herself up to be able to get up that step. So what is this? Is this some sort of muscular dystrophy that both patients have some, some type of spinal muscular atrophy that uh the patients have a myopathy, might be more rapidly progressive in one perspective. We'll find out. All right, so, the clinical, uh, sort of testing and workup, uh, 2A had elevated CK level, uh, 1700 and on, just checked multiple times and always elevated, varying between 800 and 2000. His aldelase was also elevated. His metabolic profile was normal. He had a normal lumbar puncture. MRI brain was unremarkable as well as his echocardiogram. His EMG and nerve conduction study showed evidence of an active and diffuse myopathy. Gene testing, MOPA analysis, uh, didn't show any sort of deletions or duplications for Duchesne, and his SMA testing was normal. He did undergo a biopsy showing very marked atrophy of both fiber types, ragged red fibers, mitochondrial structural abnormalities, um, and when it was evaluated for, uh, stained for respiratory chain function, cyclooxygenase or marker of, um, Respiratory chain function, uh, metabolism was significantly reduced. His genetic testing for certain mitochondrial defects, uh, things like NARP, uh, Kern Sayers, me loss was normal. His chromosomal microray showed multiple areas of homozygosity which might lend to the consanguinity in the family. Patient 2B, she had a, uh, you know, a little bit elevated CK levels, certainly not as high as 2A, and at times they were actually normal. Her metabolic workup, lumbar puncture, MRI, brain, and echocardiogram were also unremarkable. Her EMG also showed uh a myopathic process and biopsy showed, uh, both morphological and immunohistochemical features suggestive of a mitochondromyopathy with enlarged mitochondria seen on electron microscopy. His comprehensive myopathy and neuropathy panels and SMA testing was negative. So what's going on? Well, uh, genetic testing was performed on fibroblasts for patients A showing a homozygous deletion in a TK2 gene that is related to TK2 related mitochondrial depletion syndrome, and a whole genome sequence for patient 2B was also found to have a homozygous deletion in in uh TK2, this uh PT108M. I'll come back to that here shortly. Finding, um, so both patients have a TK2 related mitochondrial depletion syndrome but variable presentation, and that's what I'd like to talk about in the coming slides. All right, so again, uh, before I do that, I do wanna highlight the clinical course for these patients. Um, the case 2A at 3 years and 10 months old was non-ambulatory and unable to roll over or sit independently, just over 4 years, started to show a decline in his pulmonary function uh studies at 85 and 90% of predicted values for FEV1 and FVC. At 4 years old, uh, dysphagia and uh was noted, and at, uh, just short of 5 years old, very significant neck flexor weakness, and now his respiratory function was less than 50% of normal. And sadly, this patient passed away due to respiratory insufficiency about a month later. Now, remember, case 2B showed up about, in the clinic about 5 years after uh patient 2A presented. So she continued to have um what overall was a relatively stable course. Physical therapy was helpful for improving her motor functions, um, and she was noted to have Things like waddling gait, running awkwardly, uh, when she was seen at 5 and 6 years old, but at 8 and nearly 9 years old, she was presenting relative with just relatively stable limb girdle weakness without any concern for regression, normal pulmonary function testing. She underwent a process to request uh treatment for TK2 related mitochondrial disorder through clinical trials and IND studies, but did not qualify until she was nearly 9 years old and has subsequently started treatment and is tolerating that well when she was recently seen in the clinic at 9 years and 2 months of age with continued stable motor function and, and family saying she's, she's getting upstairs a little bit better. All right, so I know no matter what kind of background training we, we did medical school, PA school, nursing school, uh, one thing we might remember is the mitochondria is the powerhouse of the cell, and, um, that might be where our understanding is right now, and I want to go just a little bit deeper into that to appreciate the, the mechanism. Mechanism behind this. So, TK2 uh or thymidine kinase deficiency 2, I'll refer to as TK2D is a mitochondrial depletion or deletion syndrome, and it has to do with mitochondrial, um, DNA which is produced in the mitochondria, and the mitochondria have their own pool of DNA machinery and uh molecules to build their DNA that is independent of the nuclear DNA. However, many of the proteins, enzymes, and transporters that are responsible for the mitochondrial DNA synthesis are included by nuclear DNA, um, and that's shown in the table that's here on the right. So, these are the nuclear, different nuclear genes that are involved with different aspects of mitochondrial DNA synthesis, so things like replication, you might be familiar with pool, pool G. Um, things that are with the, uh, deoxy nuclear triphosphate metabolism, things like TK2D and many with an unknown, um, pat pathogenetic mechanism, um, but certainly have found to be causative of disease. So, it's these mutations in the nuclear DNA genes that really lead to problems with mitochondrial DNA. Uh, production and causes depletion syndromes, and when these depletion syndromes occur, these patients have difficulty with producing ATP, uh, energy via oxidative phosphorylation and other aspects of cellular metabolism. All right, busy figure on the right, but we'll walk through it. So, the mutations that are involved with TK2D um are, have to deal with the uh mitochondrial DNA production, and that's what's shown here on the right. So, the mitochondrial DNA production importantly requires a really balanced pool of uh deoxynucleotidede phosphates to be integrated into this mitochondrial DNA. The mitochondrial DNA precursors, so these um deoxynucleotide triphosphates can be made either de novo or through salvage pathways. So, uh, de novo pathways are shown. Here in the cytosol, and the salvage pathways are also sown in the cytosol as well as within the mitochondria in and of themselves. This de novo pathway is very much tied to the cell cycle and very active in dividing cells, and so actually really depletes the pool of uh triphosphate precursors to be able to continue to make and repair. mitochondrial DNA in quiescence, and it's in quiescence where the sal, the salvage pathways really take over, and these salvage pathways, I'll just point out the one associated with TK2D here, really depend on sequential phosphorylation of these precursor molecules to form the building blocks, these deoxyucleotide triphosphates that make up mitochondrial DNA. So here again are shown cytostolic paths, uh, the salvage pathways with thymidine kinase type 1 and deoxycytidine kinase, um, deoxyleukocytidine kinase here, um, as well as the intracellular pathways, TK2, um, being one of those pathways. All right, so there's TK2. That's what we're gonna focus on here. All right, so, TK2D. Um, initially described in a small cohort of patients in 2001, and that's what's shown in the table here at the right, for patients, uh, that were found to have this motor regression disorder early on in life with elevated creatine kinase that had that had profound effects with two of the patients, uh, passing away and two of the patients requiring mechanical ventilation very early on. As more studies have come out, and this is, uh, more case been reported in the literature, um, we've seen that it manifests with a much more of a, uh, kind of variable symptoms. Uh, so those things can include proximal muscle weakness, hypotonia, facial weakness, um, ophthalmoplegia, seizures and encephalopathy can be important. Um, cardiomyopathy is rare but present, and there can be other sort of extra muscular manifestations in the bone and in the kidney. Again, it's very rare, uh, estimated at 1.64 per million, so if we break that down, that's probably 600 or so cases, more or less in the United States, but it's likely underestimated, uh, because of its, uh, sort of heterogeneity and presentation. All right, so here are figures from a paper that describe, um, you know, just kind of what this looks like. So even though there's sort of a variable age of onset, more patients tend to present earlier on in life, but there are late onset cases in adulthood in patients even in their 50s and beyond. The most common symptoms shown here in this review of about 82 included patients include muscle weakness, elevated CK, hypotonia, ragged red fibers, and COX deficient fibers on muscle biopsy, which we saw in our patients, as well as myogenic EMGs. Uh, these patients had respiratory and feeding difficulties, um, as well. All right, so this is a very busy figure, but what I want you to take away from this is that we can delineate TK2D into three general phenotypes, early onset, childhood onset, and late-onset disease. Uh, with this delineation between early and childhood onset at 1, although some places in the literature will argue it's 2 years old and late-onset disease being older than 12. We will go through this in the, in the coming slides here. But one of the things to take away is that the early onset TK2D, these are patients that are more likely to have mitochondrial depletions, and the patients that have more late onset disease are more likely to have mitochondrial deletions, so not depletions, uh, but deletions. And as we've aggregated more cases, uh, people have started to be able to look at the different geno phenotype correlations. And again, I know this is a very busy figure, but I wanna draw your attention here, uh, where you'll Note that there are certain um genes that are listed in different colors that might have more than one phenotype with like an infantile and childhood onset for the same genotype or an infantile and adult onset or a very heterogeneous uh phenotype. And remember, our patient had this mutation, this uh 3anine uh uh methionine um mutation here that's associated with a very heterogeneous phenotype from very early to very late onset. Um, so I think that this is pretty fascinating to see the phenotypes that come from these different genotypes. OK, let's talk about early onset. So, these are patients present with severe motor weakness, failure to thrive, feeding difficulties, um, and patients are pretty likely to have some sort of extraskeletal manifestations, and about a third of the patients have CNS involvement, uh, whether that be seizures, some sort of encephalopathy, um, The seizures can range from things like migrating focal seizures to epileptic encephalopathies, and the TK2 gene is actually on the behind the seizure panel from invita, um, for, for those that order that panel, um, and it's something to be aware of. These patients, uh, looking at these, uh, Kaplan-Myer plots here, looking at survival probability, uh, for mortality here in the early onset in blue versus the late onset in red, uh, for, uh, mortality, and then for, um, Mortality or requiring ventilator support here on the bottom. What I'd like you to appreciate is that the early onset TK2D much more likely to have early mortality, much more likely to have an event of mortality or require ventilation at a significant earlier time point than the childhood or late onset patients. All right, so this childhood onset, so both of our patients, uh, in that I presented in the cases presenting at 1 through 12, disease can really vary to place and the degree of mitochondrial DNA depletion. So you can have patients that present very severe with more depletion or maybe um more of a, a deletion phenotype and a little bit less severe course. This was kind of contrast between the two patients that I showed you. The late onset disease, so those that are over 12, the, the very variable presentation, but the bottom line is that the common features are ocular, facial, and cervical weakness. So patients that might present with ophthalmoplegia, patients that might, uh, present with bulbar weakness, patients that might, um, have sort of a limb girdle phenotype. But really, one of the things to highlight is that these patients likely have diaphragmatic involvement out of proportion to their other degree of weakness and it's something that can clue you into this diagnosis. So again, I have, there's a Kaplan-Miyer plot here, and this is the time of TK2D symptom onset to first ventilatory support in years and late onset patients, and you'll see that about 50%, 50% chance that, uh, patients will need ventilation at about 20 to 23 years after symptom onset. These authors also highlighted that the age of mechanical ventilation onset really correlated nicely with DNA copy mitochondrial DNA copy number. I recognize that there's a small n value here, as well as the age of mechanical ventilation onset correlated nicely with the age of disease onset with those with earlier onset of disease requiring earlier ventilation support. All right, so, from a muscle histopathology perspective, I just want to point out the ragged red fibers that can be seen in these patients. So here is just an H&E stain of muscle. You'll note the fiber size variability for those that look at muscle biopsies frequently. And in the air, these uh stars rather are sort of these uh ragged red or jaggedy fibers that do not look nice like uh typical muscle fibers. You can also see, which is uh what's shown here, sort of a type 1 fiber predominance. So, these are stained for uh ATP um uh at low pH highlighting uh type 1 fiber predominance and the type 2 fibers are the ones here that don't stain. You might also see this pattern in spinal muscular atrophy. Importantly, uh, patients, uh, have, uh, the cytochrome oxidase staining. Uh, this is, uh, a normal patient here and it is absent or, or near absent, significantly reduced in patients with mitochondrial depletion disorders. Laboratory findings, uh, CK is usually elevated in sort of the earlier onset, uh, patients, but the levels can range from normal to a little bit later onset, um, uh, sorry, normal to mildly, more mildly elevated in later onset patients, and there are, uh, certain biomarkers, uh, this GDF 15, which is a marker of, uh, mitochondrial dysfunction or mitochondrial, um, efficacy or function. Um, might serve as a good biomarker for this. This is something that can be ordered from, uh, Mayo Clinic Laboratories, typically very, very high in the early in childhood onset and lower, um, uh, but still elevated in the adult onset. And I just wanted to point out here quickly, um, that there are groups that have looked at this and the GDF 15 levels correlates with different uh motor function or functional outcomes like forced vital capacity, showing that people that have, um, I will say is lower GDF 15 fractions, so less mitochondrial evidence of mitochondrial injury, um, have better motor scores, they have better performance on, um, like timed run tests, um, and their force vital capacity is better, um, in patients that have lower GDF 15, suggesting that those patients might have more optimal mitochondrial function. Interestingly, when patients were treated for TK2D, some, some groups looked at this over time and they noted that the GDF 15 levels dropped precipitously in some patients and slowly declined in other patients when treated for TK2D. Muscle fat fraction might also serve as a, as an imaging uh biomarker uh for these patients as well. So from a neuroimaging standpoint, usually normal in most patients, but in the early onset forms, you can frequently see abnormalities, um, in the white matter, the basal ganglia, um, and, and significant cerebral atrophy. So this is from an 8 month old patient and the same patient 2 months. Later, you can see that there's some atrophy, some T2 signaling abnormalities, um, here in, in sort of the insular area, uh, again, showing cortical atrophy here. Um, and later on, we can see just two months later, progression where more temporal lobe involvement, uh, for, for this patient and then certainly some basal ganglia involvement here, uh, with the increased signal on T2 sequencing. So more likely to be seen in the younger patients. All right, so who should we consider TK2DN in the infants with the failure to thrive, feeding difficulties, hypotonia, a more rapidly progressive weakness, seizures, encephalopathy, and CNS abnormalities, in children that have progressive limb girdle and ocular bulbar weakness, dysphagia, uh, the adolescents that have progressive ophthalmoplegia, bulbar weakness, uh, particularly in those that have respiratory weakness out of proportion, uh, to weakness in other areas. I'll have to go some, a little bit briefly through here, but uh Kygevi, it was FDA approved for uh treatment of TK2D in November of 2025, and it's essentially nucleoside replacement therapy, so deoxycitidine and thymidine. Uh, are the drug and it is given to patients in high concentrations to take advantage of residual TK2 activity to help make these, um, uh, phosphorylated, uh, nucleotide, or excuse me, tri triphosphate nucleotide precursors rather, that can be incorporated into mitochondrial DNA. Um, laboratory evaluation is pretty simple. Baseline liver transaminases and bilirubin. Patients are started out and then dose escalated over the period of a couple of weeks, and it's typically well tolerated with diarrhea really being the biggest clinical side effect and in very rare cases, elevated liver function tests. Kayvi did not follow a typical pattern or phases of drug development. Um, so typically, you'll see these trials that go through phase zero, safety and, and dose escalating studies, uh, safety and efficacy studies in, in phase two trials, and then phase 3 trials where they really compare it to placebo, um, and look at side effects in depth, but that was not the case for Kay. It was, it was determined from looking at treated patients in a couple of retrospective studies in one phase two clinical trial as well as the expanded access program and comparing that to matched untreated patients uh to look for differences, and, and patient 2A was actually included in this group. All right, the bottom line is that mortality outcomes, um, greatly different. So this uh was looking at uh overall treated patients of any age and patients that had symptom onset at any age and symptom patients that had symptoms onset at less than 12 years of age. None of the treated patients died in either group, but over 50, close to 60% of untreated patients died when it was monitored for in the study. Patients that take this medicine, they have the opportunity to regain motor milestones, and that's what's shown in these figures here, um, that patients, um, before treatment lost motor milestones, so shown in the gray bars, patients, um, many patients lost at least one with several patients losing up to 4 motor milestones. But after treatment, many patients, about 70% or so, regained motor milestones with smaller percentages gaining 3 or 4 motor milestones that included things like head control, climbing stairs, ability to stand unsupported or sit unsupported, or ability to walk and run. This is also a complicated, uh, sort of table here, but it's sort of the essence of time. The things that I want to point out is that there were patients in the studies that had feeding tubes present at treatment onset, but about half of those patients, so 6 patients total, half of those patients were able to have feeding tubes removed after initiating treatment, and patients, um, Uh, had ventilatory requirements decreased. So, um, of the patients, 14, I'll show the, the data here for those less than 12 years old, um, 6, had any sort of decrease with 5 of those 14 patients having a significant decrease in ventilatory support of greater than 4 hours. So what about the most severe groups? So, that's the infantile onset TK2D. What if we separate those out? This is a small clinic, very small study uh out of Turkey that looked at 4 patients with patients that were clinically or that were genetically confirmed at a diagnosis of less than 1 year old, looking at creatine kinase levels, development of milestones, ventilatory support, and feeding status. We'll see that patients all had elevated CK levels that declined with treatment, as well as lactate levels that declined with treatment. Patients were able to develop their motor milestones on treatment, so this lists 0 through 8 for the, the total of up to 8 milestones that they monitored in the study. For the ventilatory support, there were 2 patients at baseline requiring 24 hour support who subsequently came off of ventilation. Two of those patients also, excuse me, 3 of those patients rather were also able to transition from NG tube to oral feeding. Just very briefly, I know there's a couple or probably more than a couple of people who treat adult patients or, or what, what about those patients that are older than 12 years old? Again, this medication is only FDA approved for patients less than 12 right now. This is shown in the graphs over here on the right-hand side. So what we're looking at are some motor function tests. This is the Hammersmith. This is the 6-minute walk test. These are validated motor functions in patients. This is the forced vital capacity looking at respiratory function. And of the 6 patients in the study, what I just want to point out are the trends. Patients with late onset TK2D, so over 12, can still have an improvement in motor function. Uh, is measured here by a couple of different, uh, motor function outcomes, the walk time test and the hammersmith, and it can help with improvement enforced vital capacity or respiratory function as well. Couple of patients not included in here, the most severe patients, uh, these are patients that were trach vented and bedridden. Um, did not show any improvement in respiratory function. I think might lead credence to earlier treatment is important. Once you lose a, a, a degree of critical muscle mass, treatment probably isn't very helpful. All right, so what's the bottom line for all of these things, there's many challenges to diagnosing and, and, and developing therapeutics for pediatric neuromuscular disorders. It's really important to be able to recognize the phenotypes of treatable disorders, to be able to lead to timely diagnosis and and initiation of treatments when available. We're learning more and more about different genes that are important and therapeutic targets that are going to lead to more available drugs and treatments for different disorders, and we may need to use alternative processes for developing drugs, um, and improving them like uh was shown with TK2D to be able to bring some more treatments from these rare diseases to market. Riboflavin transporter deficiency is a progressive neurodegenerative sensory and motor neuronopathy, OK? It can present in an infant or child with progressive facial or extremity weakness, optic atrophy, dysphagia stridor, respiratory dysfunction, and is often associated with hearing loss. Consider this in adolescents or adults with an ALS-like presentation and hearing loss. High-dose oral riboflavin. Effective treatment makes a difference. TK2D is a mitochondrial depletion disorder, OK? Variable onset of, of, of disease, variable phenotype, but consider that in infants with the feeding difficulties, hypotonia, uh, it's rapidly progressive weakness, seizures or encephalopathy. Children with progressive limb girdle weakness or ocular bulbar weakness, OK? Or in the adolescent or adult that has neck flexor weakness, ophthalmoplegia, or weakness, um, that's out of proportion diaphragmatic weakness rather that's out of proportion, um, to weakness elsewhere. It can be treated with nucleoside replacement therapy, um, and, and it really makes a difference. So just like the oral riboflavin for, um, riboflavin transport deficiency. Um, nucleoside replacement therapy can help patients stabilize or regain motor functions, improve dysphagia, respiratory weakness, reduce the risk of death, and it makes a big difference or can make a big difference for patients and their families. All right, so I, I know I'm running kinda on the borderline of going over. I really just wanna say thanks again for watching or for listening in this morning. Um, That's all I got. These are the sources that uh helped to build this presentation, and I know everyone has to get on with their day, but I'm happy to take a question or two or you're welcome to, to email me later if um if you need, need to. Thank you. Hi Justin, good job. This is uh Shaun Car with Genetics, um. A follow-up question to some of the things that you presented, um, You mentioned with the riboflavin transporter deficiencies, um, That sometimes we don't identify uh genetic variants in these patients. Given that and given similar scenarios with other diagnoses, um, what are your thoughts about our reliance on molecular analysis to make a diagnosis right now and the consideration for empiric therapies for some of these conditions when the presentation is concerning. Uh, excellent question. Um, I think that. You know, looking at this from the perspective of some of the empiric therapies like riboflavin, um, for, for some of these disorders where you can supplement something that's sort of lower risk, well-tolerated, um, is probably, I would advocate for starting some of those things early on if it's in the differential or if you're highly suspicious, and you can always discontinue those later. I can think of at least one case here that I've seen at the Phoenix Children. I, I may not have seen as many as you, Doctor Cora, but the patient ultimately never achieved a molecular diagnosis, and there are limits to the, the, uh, knowledge of diagnostic testing. I mean, patients might need to, to have different tissues sampled. Um, to undergo things like RNA sequencing that I know you famili are familiar with, but others might not be familiar with on the, the, the call here. Um, and, and ultimately, you know, as they might have a gene that hasn't, or, or a specific mutation that hasn't been sequenced or, or known to, to, to cause disease yet. So, um, There are limits to genetic testing right now. I think being cognizant of that and treating empirically early when you're highly suspicious is a prudent thing to do. And I think it's a little bit OK to, to, to lean on those empiric therapies, um, and be cautious in digging your heels in and relying solely on the genetic testing. I agree. I think we've become a little bit too over-reliant on molecular molecular diagnosis, um, because of the rapid availability of, uh, some methods and I, I think we should consider going back to a period of time where we do start empiric therapies if we're concerned. Um, but I also wanted to take a closer look at the biochemical studies that you presented for that riboflavin patient. What was your interpretation of those? My interpretation of the, the biochemical studies, so that patient had normal ammonia azylcarnitine profile. Um, they had an abnormal, um, uh, Excuse me, I, I'm just, I'm blanking on it here. They had an abnormal, um, uh, urine organic acid study and to, to be fair with you, um, I'm not, you know, intimately familiar with the interpretation of all of those. I do know that some of those intermediates that they were positive for can be seen or, or, or they, they, they might be commonly seen with the riboflavin transport deficiency. It goes a little bit beyond my knowledge and expertise. Um, as well as, um, the, the amino acid profile, um, sort of the interpretation of, of that particular study might go just a little bit beyond my expertise. I think one of the take-home points that I, I wanted to make was that that patient's, um, Urine organic acids were abnormal, and I apologize if I didn't highlight or come back to that may have been evident in that, in that patient that that was more reflective of their underlying disease process rather than nutritional supplementation, rather than um it being due to nutritional intake. Um, but, uh, but I, I'm afraid I, I can't speak to some of the specifics and the detailed interpretation. I would request help from, from someone like yourself. Uh, you got it pretty accurate. Um, those metabolites are pretty consistent with a riboflavin metabolism disorder or, uh, the one other metabolic disorder that would be in the differential would be GA2. Um, but those dicarboxylic acids were elevated, which, as you said, can sometimes be due to dietary supplementation. Um, of MCT, but then you can always go back and see if there was actually dietary supplementation of MCT. Um, the ethylmalonic acid was also elevated. Um, depending on the degree of elevation, that should always raise concern that there's something more going on there. And while it's not a specific marker for riboflavin disorders, um, it is a marker where you should be thinking about mitochondrial disorders, GA2, and riboflavin, and a couple others. Oh, thank you for um the, the, the teaching point there, um, and again, I, I know this kind of Part, you know, goes back to your, your previous question of even if those tests are normal, um, the data shows that those tests are, those biochemical tests are not ubiquitously abnormal in patients, that even if those tests are normal and you're still highly suspicious, really consider treating. Let me check the chat here. Um, I don't think I see any other questions in the chat. Um, for, for those that are, that are interested, I'm very happy to, to extend the conversation, you know, in person around campus or, or via email. So, so thanks again, everybody, and, uh, again, thanks, uh, Thanks, Doctor Core for uh the, the thoughtful discussion regarding uh riboflavin transport uh deficiency and uh some of the uh pearls regarding uh suspicion of those dicarboxylic intermediates and ethylmalonic acid. Have a great day, everyone. Thank you, Doctor Hoffman.
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