1. Genetics and Genomics
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Repurposing eflornithine to treat a patient with a rare ODC1 gain-of-function variant disease

  1. Surender Rajasekaran  Is a corresponding author
  2. Caleb P Bupp
  3. Mara Leimanis-Laurens
  4. Ankit Shukla
  5. Christopher Russell
  6. Joseph Junewick
  7. Emily Gleason
  8. Elizabeth A VanSickle
  9. Yvonne Edgerly
  10. Bryan M Wittmann
  11. Jeremy W Prokop
  12. André S Bachmann  Is a corresponding author
  1. Pediatric Critical Care Medicine, Helen DeVos Children’s Hospital, United States
  2. Spectrum Health Office of Research and Education, United States
  3. Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, United States
  4. Medical Genetics, Spectrum Health and Helen DeVos Children’s Hospital, United States
  5. Department of Pharmacy, Helen DeVos Children’s Hospital, United States
  6. Department of Diagnostic Radiology, Spectrum Health and Helen DeVos Children's Hospital, United States
  7. Metabolon, United States
  8. Department of Pharmacology and Toxicology, College of Human Medicine, Michigan State University, United States
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Cite this article as: eLife 2021;10:e67097 doi: 10.7554/eLife.67097

Abstract

Background:

Polyamine levels are intricately controlled by biosynthetic, catabolic enzymes and antizymes. The complexity suggests that minute alterations in levels lead to profound abnormalities. We described the therapeutic course for a rare syndrome diagnosed by whole exome sequencing caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC), characterized by neurological deficits and alopecia.

Methods:

N-acetylputrescine levels with other metabolites were measured using ultra-performance liquid chromatography paired with mass spectrometry and Z-scores established against a reference cohort of 866 children.

Results:

From previous studies and metabolic profiles, eflornithine was identified as potentially beneficial with therapy initiated on FDA approval. Eflornithine normalized polyamine levels without disrupting other pathways. She demonstrated remarkable improvement in both neurological symptoms and cortical architecture. She gained fine motor skills with the capacity to feed herself and sit with support.

Conclusions:

This work highlights the strategy of repurposing drugs to treat a rare disease.

Funding:

No external funding was received for this work.

Introduction

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines (putrescine, spermidine, spermine), which orchestrate essential physiological and pathologic processes including embryogenesis, organogenesis, and neoplastic cell growth (Bello-Fernandez et al., 1993; Pendeville et al., 2001). We recently described a new autosomal dominant genetic disorder (Bachmann-Bupp syndrome, OMIM #619075) caused by a heterozygous de novo variant in the ODC1 gene in a 3-year-old girl with phenotypic features that included alopecia universalis and global developmental delay (Figure 1; Bupp et al., 2018). The nonsense variant caused premature abrogation of 14-aa residues in the C-terminus of the protein (ODC, p·K448X, Figure 2), leading to enhanced function. Red blood cells from the patient exhibited elevated ODC activity and putrescine levels compared to healthy controls. Four additional patients with similar mutations and phenotypic features of this syndrome have since been reported (Rodan et al., 2018) and at least four more cases have been identified.

Patient phenotypes and metabolites before and after eflornithine treatment.

Panel A shows the timeline of events for the patient with milestones marked on the top and clinical observations below. Panels B-C show hair growth and muscle tone are the most noticeable phenotype changes with treatment. Follicular cysts recurred on back, neck, and posterior scalp (bottom left images). First hair growth was eyebrows 1 month into treatment (bottom right images). Panel D shows MRI before and after eflornithine treatment. Neonatal: Axial T1 (TR 483 ms, TE 9 ms, and flip angle 63 degrees), T2 (TR 3250 ms, TE 220 ms, and flip angle 90 degrees), and T2-FLAIR (TR 8002 ms, TE 122 ms, and flip angle 90 degrees) show marked abnormal signal of cerebral white matter (*) and several subependymal cysts (arrows). Five years of age: Axial T1 (TR 809 ms, TE 16 ms, and flip angle 111 degrees), T2 (TR 4850 ms, TE 107 ms, and flip angle 142 degrees), and T2-FLAIR (TR 6002 ms, TE 91 ms, and flip angle 90 degrees) show decrease in cerebral white matter volume, but normalization of signal and resolution of subependymal cysts.

ODC1/ODC clinical variant c.1342 A > T/ p.K448X and support for eflornithine treatment.

Panel A shows the gene structure for ODC1 (ornithine decarboxylase 1) with active site amino acids labeled in blue and the last exon identified. In the last exon cluster, a mouse model variant and four different patient variants including our patient’s K448X variant are described (Panel B, red). The patient variant falls on the disordered C-terminus of ODC, where the two active sites are composed of amino acids from each of two ODC proteins forming a dimer (Panel C). The patient with K448X variant displays alterations of metabolic pathways (Panel D) including polyamines (triangle), urea (square), and others (circle). Metabolites measured are marked in cyan and those altered by K448X with red arrows based on direction of changes seen in the patient. Panel E shows changes in metabolite levels during treatment with eflornithine, with elevated levels of N-acetylputrescine and acisoga decreasing on therapy.

Remarkably, these patients all represent human phenotypes of a transgenic mouse described in 1996, overexpressing C-terminally deleted ODC in the dermal tissue, leading to higher ODC enzyme activity and increased putrescine biosynthesis (Soler et al., 1996). The phenotypic changes first described in a mouse model included hair loss that was reversible with ODC inhibitor α-difluoromethylornithine (DFMO; common name eflornithine) (Soler et al., 1996). Experiments with the patient’s cultured primary dermal fibroblasts showed eflornithine reduced ODC activity, resulting in putrescine levels comparable to controls without affecting cell morphology or inducing cell death (Schultz et al., 2019).

Based on previously published murine data with eflornithine for gain-of-function variants (Soler et al., 1996), multiple long-term safety studies for clinical use in African sleeping sickness (trypanosomiasis), colorectal cancer, and neuroblastoma (Alirol et al., 2013; Priotto et al., 2009; Saulnier Sholler et al., 2015; Meyskens et al., 2008), and the absense of toxicity in the patient’s primary cell culture inresponse to eflornithine (Schultz et al., 2019), we surmised eflornithine might be a novel therapy for patients with this syndrome.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Homo sapiens)ODC1NCBI GeneGene ID: 4953https://www.ncbi.nlm.nih.gov/gene/4953
Chemical compound, drugEflornithine (DFMO)Sanofi Aventis Supplied for studyhttps://pubchem.ncbi.nlm.nih.gov/compound/Eflornithine
Biological sample (Homo sapiens)Blood EDTA tubesFreshly isolated blood from patient
Software, algorithmYASARAYASARAhttp://www.yasara.org/Protein modelling
Commercial assay or kitLiquid chromatography paired massspectrometryMetabolon, Morrisville, NChttps://www.metabolon.com/

Study participants and consent 

Request a detailed protocol

Following FDA approval of our single-patient Investigational New Drug (IND) Application (144022) as a compassionate use treatment protocol, the study was further reviewed and approved by the Spectrum Health Institutional Review Board (IRB). IRB approval for sample collection with informed consent was received to conduct global metabolomics that included, among others, the polyamine metabolites spermidine and N-acetylputrescine.

The patient first presented at Spectrum Health, Helen DeVos Children’s Hospital (Grand Rapids, MI) at 11 months of age (Figure 1A). We diagnosed the ODC C-terminal deletion at age 19 months through whole exome sequencing and characterized the metabolic dyshomeostasis by 32 months (ODC protein and polyamine abnormalities). Eflornithine oral solution was prepared by diluting the lyophilized powder with purified water to a final concentration of 100 mg/mL. At age 4 years and 8 months, we started eflornithine (Sanofi Aventis) treatment with 500 mg/m2/dose bid twice daily via a gastrostomy tube along with a low polyamine diet on November 14, 2019, for 3 months, increasing to 750 mg/m2/dose twice daily, and a final increase to 1000 mg/m2/dose twice daily after 3.5 months. Dosing was based on what had been demonstrated to be safe in pediatric patients in maintenance therapy for neuroblastoma treated with eflornithine (Saulnier Sholler et al., 2015).

Blood collection and processing

Request a detailed protocol

EDTA blood tubes collected from the patient were mixed by inversion 8–10 times, centrifuged at 1000×g for 10 min at 4°C to separate plasma (minimum of 0.25 mL, free of hemolysis from red blood cells) from cellular fraction, and both fractions were immediately placed at −80°C. The plasma specimens were coded and anonymized, kept frozen, and shipped in batch to Metabolon, Morrisville, NC, for metabolomics analysis. N-acetylputrescine, the only polyamine that meets CAP/CLIA standards in this analysis, served as the primary indicator of putrescine levels. N-acetylputrescine and additional metabolites meeting CAP/CLIA standards (Figure 2E) and supplemental metabolites were measured in the EDTA plasma samples using ultra-performance liquid chromatography paired with mass spectrometry and Z-scores were calculated for each metabolite against a reference cohort of 866 pediatric patients as described previously (Squitti et al., 2019).

Blood draws for polyamine levels were obtained at initiation of therapy, 1-week post-initiation, immediately prior to each dose increase and then 7 days after each dose increase. These draws were performed in conjunction with safety screening, which included a complete blood count, liver function test, lactate dehydrogenase, complete metabolic panel, calcium, magnesium, and phosphate.

Results

The novel treatment of this ultra-rare (less than 10 known cases) genetic syndrome presented unique challenges for monitoring efficacy over time. Growth parameters and metabolite levels were monitored easily, whereas others such as cognitive and motor functioning proved challenging, making us dependent on her standardized neurological examination.

Eflornithine improves clinical findings

The patient was born with a full head of silver-blond hair similar to a previously described murine phenotype (Soler et al., 1996), which fell out in early months and she remained hairless other than a few scattered, long hairs on the scalp. One month into treatment, hair growth was noted, with eyebrows appearing first (Figure 1B–C). Two months into treatment, scalp hair began to diffusely appear in the normal pattern of hair distribution increasing to resemble normal growth for age (Figure 1). She had a history of recurring follicular cyst formation and enlargement. Multiple lesions located on the posterior scalp and back that first were small maculopapular pustules slowly increased in size to approximately 6–7 cm in diameter (Figure 1B). These lysed spontaneously, but some would enlarge until painful, requiring surgical removal. Upon initiation of eflornithine, the formation of cysts ceased immediately (Figure 1B).

Prior to therapy, she had delayed development which manifested with no standing, cruising, or sitting, and limited fine motor skills. Her BMI increased during eflornithine treatment from 25th percentile to 90th percentile primarily due to increase in weight. This quantitative change was not accompanied by any change in body habitus but rather an increase in muscle bulk. She gained muscle strength demonstrated by acquisition of her ability to hold up her head without support (Figure 1B). As the video file shows after 4 months of eflornithine therapy, she was able to sit unsupported and maintain posture with the physical therapist providing resistance, use a walker, and feed herself with a spoon with some assistance. Video 1 allows for optimal visualization of this rapid improvement of our patient with this gain-of-function mutation in the ODC1 gene. The drastic external change in hair growth, and visible improvement in coordination, attention, and interaction can be clearly seen.

Video 1
Treatment progression after 4 months of eflornithine therapy.

A neonatal brain MRI showed abnormal cerebral white matter and subependymal cysts (Figure 1D). Repeat MRI done at the end of the 9-month treatment trial with eflornithine demonstrated normalization of the cerebral white matter signal with decrease in volume with white matter loss and resolution of all previously noted cysts. Post-treatment magnetic resonance spectroscopy was also performed showing normalization of the N-acetylaspartate and choline signals relative to creatine Figure 1D.

Eflornithine normalizes metabolomic findings

N-acetylputrescine, the only polyamine metabolite measurement that is CAP/CLIA-certified, was quantified in addition to others using a global metabolomics approach (Figure 2) before and after initiating therapy with eflornithine. Metabolite levels from a reference cohort of 866 pediatric patients were converted into Z-scores, a calculation of standard deviations from the mean of the reference populationthat our patient’s values are compared to. In the global analysis of 915 metabolites of the patient before treatment, a total of 16 had values above the 97.5th percentile and 38 below the 2.5th percentile, with a noted difference in polyamine connected metabolites (Source data 1) without any marked disruption of any other metabolic pathways on treatment. The initial elevation of both N-acetylputrescine as well as the polyamine metabolite N-(3-acetamidopropyl) pyrrolidin-2-one (acisoga), which were above the 97.5th percentile, decreased at initiation of therapy and remained reduced at all time points (Figure 2), indicating that eflornithine treatment had the expected effect. Ornithine and N-acetylarginine were below the 2.5th percentile at start of therapy and normalized to the larger pediatric values over the course of therapy. Urea cycle components citrulline and arginine, along with other metabolites, remained at 1 to −1 standard deviation throughout the study period (Figure 2).

Discussion

The introduction of both whole genome and exome sequencing into clinical practice has led to rare diseases being diagnosed at rates never before seen (Turro et al., 2020; Tarailo-Graovac et al., 2016; Splinter et al., 2018). There are over 6000 rare diseases (incidence of greater than 1 in 2000 people) with over 300 million people worldwide affected (Nguengang Wakap et al., 2020). Though collectively common, each rare disease is unique making it challenging to develop specific therapies.

The process of developing treatment options for these rare diseases starts with a description of the genetic abnormality and developing an understanding of the molecular disruptions downstream from the affected protein. Once the biochemical perturbations are identified, then the quest to identify a drug that will return molecular pathways to normal begins. In genetic diseases, the correct mechanism to adopt could be challenging as many options exist such as activating or repressing pathways, enzyme blockade therapy, gene therapy regimens (Mendell et al., 2017), and potentially circumventing or correcting a genetic mutation such as treatments for muscular dystrophy (Iyer et al., 2019). Taking a genetic defect to human trials requires cell cultures, animal studies, and phased trials to determine safety and efficacy of such therapies. For multiple patients, often with unique genetic variants, to see benefit from this process could take the best part of a decade even as identification of successful drugs has been enhanced by the Orphan Drug Act (Augustine et al., 2013; Griggs et al., 2009).

We show a more rapid strategy of matching a patient with an ultra-rare newly identified syndrome to a drug with subsequent treatment being able to safely correct many phenotypic features. Once the whole exome identified the biochemical pathway, we used data from a previously described transgenic mouse model and our published cell culture study to surmise, eflornithine therapy could be of benefit to the patient. Though experimental data suggested that eflornithine could be beneficial, there is a chasm between ex vivo and in vivo studies with difficulties in extrapolating efficacy or safety from a fibroblast study alone (Schultz et al., 2019). We were fortunate that studies existed for eflornithine in a large enough population to suggest dosage and safety (Alirol et al., 2013; Priotto et al., 2009; Saulnier Sholler et al., 2015; Meyskens et al., 2008).

Once therapy was initiated, some neurological improvement in the patient was noted with better posture, weight gain, reduction, elimination of cyst formation, and significant hair growth. Six months into therapy, she had fine motor capability that she previously lacked, such as the capacity to feed herself and sit with some support. Brain imaging also showed changes that are beyond what would be explained merely by the passage of time, suggesting improvement related to eflornithine treatment.

While COVID-19 restrictions interrupted neurological assessments over the treatment period, the improvements noted throughout the relatively short treatment period of 6 months are truly remarkable, especially given the neurological deterioration in the patient prior to eflornithine therapy. This could be especially consequential if we could initiate therapy in a neonate diagnosed early before neurological damage occurs. We are now aware of other patients identified that present with similar gain-of-function ODC variants and polyamine abnormalities such as elevated N-acetylputrescine (Rodan et al., 2018). The therapy outlined here should allow for replication of the findings with a promise for significant improvement in quality of life for these patients. For such patients we recommend continued monitoring of multiple metabolites including N-acetylputrescine and acisoga to ensure that eflornithine dosing and urea/polyamine metabolite levels stay within normal ranges. The advent of global metabolomics presents a unique opportunity not only to develop a complete understanding of the dyshomeostasis prior to therapy but also a way to appreciate the drug’s impact on interconnected metabolic cycles simultaneously and perhaps a means of identifying disruptions early and predicting adverse effects. This may lead to earlier initiation of therapy in future patients, thereby perhaps avoiding some of the neurological delay that has come to characterize the disease in our patient.

Conclusion

In this study we have laid forth a promising example of going from first publication of a new syndrome to FDA-approved single-patient investigational repurposed drug treatment in 16 months, a methodology and speed rarely seen in the clinical science of rare diseases.

Data availability

Data is provided in Source data 1.

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Decision letter

  1. Goutham Narla
    Reviewing Editor; University of Michigan, United States
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The study elegantly demonstrates the use of an FDA-approved drug, Eflornithine, to treat, with considerable success, the neurological and skin manifestations of a child with a gain of function mutation of the ODC1 gene. The authors have addressed all of our concerns/comments and the manuscript in its revised form is now acceptable for publication at eLife.Decision letter after peer review:

Thank you for submitting your article "Repurposing Eflornithine to Treat a Patient with a Rare ODC1 Gain of Function Variant Disease" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

There was significant interest in the work overall as noted below from comments from both reviewers – the repurposing of FDA approved to treat existing diseases is of great interest to the scientific and medical community. There are however several issues that need to be addressed prior to the acceptance of this manuscript to eLife.

1) Where there side effects associated with administration of the drug and if so those be noted and described in detail with laboratory values/data provided when available

2) While the work is of interest a single case is presented, were additional patients treated and if so what was their outcome?

Reviewer #1:

In the present study, Rajasekaran et al. describe the clinical findings resulting from repurposing Eflornithine (ODC inhibitor), an FDA approved drug, for a metabolic rare disease caused by pathogenic gain-of-function variants in ODC1. Based on their previous case report (PMID: 30239107), the authors observed that the clinical findings of their patient (alopecia, brain and scalp cystic lesions, and a variety of metabolites) improved after Eflornithine treatment. The study provides intriguing new insights into the treatment of this ultra-rare condition, and highlights the success of repurposing drugs for metabolic disease, but is limited by the single patient treated and lack of information about side effects.

1. Eflornithine is an inhibitor of ODC. Were there any side effects observed in the patient during the treatment process, especially during the period of dosage adjustment?

2. Myelosuppression is common in patients taking Eflornithine. Were complete blood cell counts measured before, during, and after the treatment? These should be included in the report.

3. Did the protein level of ODC change before and after treatment?

4. In the Discussion section, the authors mention the possibility of replication of findings in the current study in other patients with ODC1 gain-of-function. There is another report of 4 individuals with ODC1 gain-of-function variants (PMID: 30475435) which highlights the variable expressivity present among patients with this disorder. Replicating their findings in some of these other patients seems necessary before drawing conclusions about the efficacy of Eflornithine for effectively treating the clinical features of ODC1 gain-of-function variants. This is especially important since the authors were unable to perform neurological assessments for their study.

Reviewer #3:

This is an excellent paper which describes a rational and effective treatment for a very rare inherited condition.

The authors explain clearly the reasons for their using the drug selected based on the available literature with a transgenic mouse model and show very encouraging preliminary results that it will be effective.

There are a few corrections/additions which should be made to the manuscript.

1. The sentence starting on line 58 is ambiguous. Are there for additional patients with similar mutations that have been reported and four more cases or is the total number of cases actually known at present.

2. Some details of the low polyamine diet that was co-administered with the drug should be given and some discussion of whether this is likely to be a critical issue in the success of the treatment are needed. Since, as described in the paper, polyamine levels are intricately controlled by regulation of synthesis, degradation and uptake, it seems unlikely that uptake dietary polyamines would be a major issue in this condition which involves high levels that would be expected to suppress uptake.

3. Figure 2C would be very confusing to anyone not familiar with the ODC structure. It needs a longer explanation in either the text or the figure legend explaining that the protein is made up of two identical subunits which form to active sites at their interface. Showing the two subunits in different colors would be helpful. it is also confusing to show the two ends in different formulations without clearly explaining why this is done. The text would better if it referred to the two ODC monomers than two ODC proteins

A reference to the structure is needed eg for human (Almrud, J. J., Oliveira, M. A., Kern, A. D., Grishin, N. V., Phillips, M. A., and Hackert, M. L. (2000) Crystal structure of human ornithine decarboxylase at 2.1 Å resolution: structural insights to antizyme binding. J. Mol. Biol. 295, 7-16 ) or mouse (Kern, A. D., Oliveira, M. A., Coffino, P., and Hackert, M. L. (1999) Structure of mammalian ornithine decarboxylase at 1.6 Å resolution: stereochemical implications of PLP-dependent amino acid decarboxylases. Structure 7, 567-581).

4. A reference to the original work of the very gifted chemists and pharmacologists that lead to the conceptualization and production of DFMO would be appropriate. For example, Metcalf, B. W., Bey, P., Danzin, C., Jung, M. J., Casara, P., and Vevert, J. P. (1978) Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E. C. 4. 1. 1. 17) by substrate and product analogues. J. Am. Chem. Soc. 100, 2551-2553.

https://doi.org/10.7554/eLife.67097.sa1

Author response

Essential revisions:

There was significant interest in the work overall as noted below from comments from both reviewers – the repurposing of FDA approved to treat existing diseases is of great interest to the scientific and medical community. There are however several issues that need to be addressed prior to the acceptance of this manuscript to eLife.

1) Were there side effects associated with administration of the drug and if so those be noted and described in detail with laboratory values/data provided when available

Additional information about the safety screening procedure for this treatment is now included. No abnormalities were noted from any of the laboratory values monitored, and there were no clinically apparent side effects observed throughout this treatment. An updated paragraph detailing this information is now included both in methods section and the patient’s experience in the result section of the manuscript.

Methods section “These draws were performed in conjunction with safety screening that included at baseline, weekly after each dose increase and then twice monthly, which included a complete blood count, liver function test, lactate dehydrogenase, complete metabolic panel, calcium, magnesium, and phosphate. Audiograms were obtained pretreatment and then monthly. Electrocardiograms were done pretreatment, 1 month after first dose, and every 6 months, all based upon guidance from previous pediatric treatment trials with DFMO10

In the Results section “All pre-treatment testing results were within normal limits, and through the duration of treatment, no abnormalities were noted in any of the described safety monitoring. This includes no gastrointestinal adverse effects or myelosuppression, previously noted in patients treated with DFMO.”

2) While the work is of interest a single case is presented, were additional patients treated and if so what was their outcome?

No, as with most rare diseases, we have a unique situation but there are new patients being identified with potential for benefit from DFMO treatment. These patients are in other locations receiving healthcare from other Institutions. The process of initiating DFMO needs careful planning and FDA involvement. We hope to continue to understand this condition with the potential for expanded treatment, one day. The publication of this report will go a long way to shed light on this rare disorder and highlight potential for therapy. To the best of our knowledge, there are currently no other patients with this disorder that have been treated with DFMO.

Reviewer #1:

In the present study, Rajasekaran et al. describe the clinical findings resulting from repurposing Eflornithine (ODC inhibitor), an FDA approved drug, for a metabolic rare disease caused by pathogenic gain-of-function variants in ODC1. Based on their previous case report (PMID: 30239107), the authors observed that the clinical findings of their patient (alopecia, brain and scalp cystic lesions, and a variety of metabolites) improved after Eflornithine treatment. The study provides intriguing new insights into the treatment of this ultra-rare condition, and highlights the success of repurposing drugs for metabolic disease, but is limited by the single patient treated and lack of information about side effects.

1. Eflornithine is an inhibitor of ODC. Were there any side effects observed in the patient during the treatment process, especially during the period of dosage adjustment?

We thank reviewer 1 for identifying this shortcoming as did the prior reviewer. Please see our response to as we did not identify any adverse effects to the patient during this treatment. Statements to that are now in both the methods and Results section.

2. Myelosuppression is common in patients taking Eflornithine. Were complete blood cell counts measured before, during, and after the treatment? These should be included in the report.

Complete blood counts were part of the safety monitoring during this treatment, and we did have access to other historical CBC results for this patient. There was no observed difference in counts during treatment, and a specific comment has been added to the manuscript noting this as it is an important matter to comment on.

3. Did the protein level of ODC change before and after treatment?

This is an excellent question and this is a limitation. We had previously measured the ODC levels of this patient in red blood cells and primary dermal skin fibroblasts before treatment and its response to DFMO. The previous publications showed both ODC proteins and enzymatic activity were significantly elevated compared to healthy controls and the levels dropped after DFMO. This is consistent with the hypothesis that the C-terminally deleted ODC leads to accumulated and active ODC protein in the patient (due to failure of the proteasome to clear the protein), resembling a gain-of-function (GOF) mutation. These observations were published prior to this study (Bupp et al., AMJG, 2018; Schultz et al., Biochem J, 2019). The use of western blot to measure protein levels has remained the traditional pathway to detect levels of intermediates before designing therapies for rare diseases. In this case we used the information from the previous study supported with global metabolomics done at different timepoints during therapy to determine that Putrescine was indeed elevated and dropped on therapy. After discussions with the FDA we pursued therapy through the more rapid compassionate use mechanism. Our hope is that this manuscript will highlight this approach as a model for using repurposed drug therapy rapidly with rapid advances in genetic diagnostics and need for rare disease treatments.

A statement is now added to the discussion as a limitation that says: “A limitation of this study is that we did not specifically measure enzymatic levels. In rare diseases, the enzyme activity and the resultant metabolic perturbation are traditionally studied in bench settings by detecting biochemical changes in the patient’s biological samples. This study used global metabolomics to measure polyamine levels serially as the patient underwent therapy.”.

4. In the Discussion section, the authors mention the possibility of replication of findings in the current study in other patients with ODC1 gain-of-function. There is another report of 4 individuals with ODC1 gain-of-function variants (PMID: 30475435) which highlights the variable expressivity present among patients with this disorder. Replicating their findings in some of these other patients seems necessary before drawing conclusions about the efficacy of Eflornithine for effectively treating the clinical features of ODC1 gain-of-function variants. This is especially important since the authors were unable to perform neurological assessments for their study.

Indeed, the phenotype may be variable, and the exact metabolic derangements need careful examination before therapy is initiated. We knew from the cell studies that she exhibited gain of function dyshomeostasis. In addition we utilized global metabolomics to map out the derangements at multiple timepoints. The presence of CAP/CLIA mandates allows the results to be reported in the Electronic Medical Record and allows treating physician to indeed initiate treatments. Our hope is that this initial manuscript highlights an approach that is more clinically practical in laying the groundwork for additional study and serves as a model for other ‘n of 1’ treatments.

One of the main limitations in rare disease research is the recruitment of patients from all over the world even if several have been identified, the challenge becomes how to get patients’ local hospital systems interested in novel trials particularly when these are often non-academic settings and how to balance the costs to families if recruitment is out of network or requires extensive travel. Thus, our hope is that this paper could open the possibility of navigating these challenges. In addition global metabolomics can be used to characterize the dyshomeostasis in each of these patients in the original healthcare institution.

A statement has now been added to the discussion that says “There are challenges in treating patients with rare diseases as the exact phenotype may be quite variable and the exact metabolic profile needs to be first established. Once therapy is indicated then the healthcare institution needs to be willing to navigate the regulatory hurdles to make the specific agent available for a single patient.”

Reviewer #3:

This is an excellent paper which describes a rational and effective treatment for a very rare inherited condition.

The authors explain clearly the reasons for their using the drug selected based on the available literature with a transgenic mouse model and show very encouraging preliminary results that it will be effective.

There are a few corrections/additions which should be made to the manuscript.

1. The sentence starting on line 58 is ambiguous. Are there for additional patients with similar mutations that have been reported and four more cases or is the total number of cases actually known at present.

We agree that this sentence is poorly worded and confusing to the reader. There are four additional patients with similar mutations that were reported by Rodan et al. in 2018 as well as four more cases that have been identified but not yet reported. We will edit the sentence in line 58 to clarify this statement.

It now reads “Four additional patients with similar mutations and phenotypic features of this syndrome have since been reported4 with at least four more cases identified in other centers, but not yet reported”.

2. Some details of the low polyamine diet that was co-administered with the drug should be given and some discussion of whether this is likely to be a critical issue in the success of the treatment are needed. Since, as described in the paper, polyamine levels are intricately controlled by regulation of synthesis, degradation and uptake, it seems unlikely that uptake dietary polyamines would be a major issue in this condition which involves high levels that would be expected to suppress uptake.

The consideration of polyamines in diet, its impact with this syndrome, and the potential role during treatment is an excellent point. Considering this suggestion, we reviewed the diet of this patient further with her family. We provided them a list of foods that are high in Polyamines to avoid. The patient reported in this manuscript has significant developmental delay and receives most of her nutrition through formula (Pediasure) given through gastrostomy tube. Her diet remained unchanged during the study.

We added a sentence in the methods and in the discussion on our recommendation to the family to avoid dietary items that have a high Polyamine content. “The family was provided a list of high polyamine containing food to avoid and therapy with DFMO initiated”.

3. Figure 2C would be very confusing to anyone not familiar with the ODC structure. It needs a longer explanation in either the text or the figure legend explaining that the protein is made up of two identical subunits which form to active sites at their interface. Showing the two subunits in different colors would be helpful. it is also confusing to show the two ends in different formulations without clearly explaining why this is done. The text would better if it referred to the two ODC monomers than two ODC proteins

Thank You reviewer 3, This is now done. We have changed the text to refer to monomer units, changed one of the monomers to green, and added the following sentence to clarify the structure model interpretation, “The protein in figure 2C shows one of the monomers as a surface plot in gray and the second monomer in secondary structure in green, with the region of the patient removed colored in red.” We wish to point out to the reviewers that neither of the structures mentioned contain the C-terminal amino acids, and thus we did not use either of these structures alone. To get this model we utilized a nonbiased multi model homology strategy that filled in the end regions with loop samples. We have added into the methods the following sentence: “Protein model of ODC dimer was generated using a merge of PDB files 1D7K, 2OO0, and 2ON3 with samplings of other PDB files to fill in the C-terminal region, followed by relaxation of bonds with the YASARA2 force field in a water shell using YASARA tools (www.yasara.org/).”

A reference to the structure is needed eg for human (Almrud, J. J., Oliveira, M. A., Kern, A. D., Grishin, N. V., Phillips, M. A., and Hackert, M. L. (2000) Crystal structure of human ornithine decarboxylase at 2.1 Å resolution: structural insights to antizyme binding. J. Mol. Biol. 295, 7-16 ) or mouse (Kern, A. D., Oliveira, M. A., Coffino, P., and Hackert, M. L. (1999) Structure of mammalian ornithine decarboxylase at 1.6 Å resolution: stereochemical implications of PLP-dependent amino acid decarboxylases. Structure 7, 567-581).

Please see above, Thanks.

4. A reference to the original work of the very gifted chemists and pharmacologists that lead to the conceptualization and production of DFMO would be appropriate. For example, Metcalf, B. W., Bey, P., Danzin, C., Jung, M. J., Casara, P., and Vevert, J. P. (1978) Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E. C. 4. 1. 1. 17) by substrate and product analogues. J. Am. Chem. Soc. 100, 2551-2553.

The reviewer makes an excellent point that the addition of a reference to recognize the research of those who worked to make treatment with DFMO possible in the first place would add great value to the manuscript. This is now ref 6 and a sentence recognizing this work and a new citation have been added. The sentence in the introduction reads as “Eflornithine is the result of work done on developing inhibitors that lead to irreversible enzymatic inactivation of ODC from nearly forty years ago.6

https://doi.org/10.7554/eLife.67097.sa2

Article and author information

Author details

  1. Surender Rajasekaran

    1. Pediatric Critical Care Medicine, Helen DeVos Children’s Hospital, Grand Rapids, United States
    2. Spectrum Health Office of Research and Education, Grand Rapids, United States
    3. Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States
    Contribution
    Conceptualization, Investigation, Project administration
    Contributed equally with
    Caleb P Bupp
    For correspondence
    surender.rajasekaran@helendevoschildrens.org
    Competing interests
    Inventor on a pending US patent (US-2020215010-A1), methods for treating or preventing developmental disorders associated with mutations in the OCD1 gene.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4430-006X
  2. Caleb P Bupp

    1. Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States
    2. Medical Genetics, Spectrum Health and Helen DeVos Children’s Hospital, Grand Rapids, United States
    Contribution
    Conceptualization, Investigation
    Contributed equally with
    Surender Rajasekaran
    Competing interests
    Inventor on a pending US patent (US-2020215010-A1), methods for treating or preventing developmental disorders associated with mutations in the OCD1 gene.
  3. Mara Leimanis-Laurens

    1. Pediatric Critical Care Medicine, Helen DeVos Children’s Hospital, Grand Rapids, United States
    2. Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Ankit Shukla

    Department of Pharmacy, Helen DeVos Children’s Hospital, Grand Rapids, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  5. Christopher Russell

    Spectrum Health Office of Research and Education, Grand Rapids, United States
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
  6. Joseph Junewick

    Department of Diagnostic Radiology, Spectrum Health and Helen DeVos Children's Hospital, Grand Rapids, United States
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
  7. Emily Gleason

    Spectrum Health Office of Research and Education, Grand Rapids, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared
  8. Elizabeth A VanSickle

    Medical Genetics, Spectrum Health and Helen DeVos Children’s Hospital, Grand Rapids, United States
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5504-9248
  9. Yvonne Edgerly

    Spectrum Health Office of Research and Education, Grand Rapids, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  10. Bryan M Wittmann

    Metabolon, Morrisville, United States
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  11. Jeremy W Prokop

    1. Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States
    2. Department of Pharmacology and Toxicology, College of Human Medicine, Michigan State University, East Lansing, United States
    Contribution
    Data curation, Investigation, Visualization
    Competing interests
    No competing interests declared
  12. André S Bachmann

    Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States
    Contribution
    Conceptualization, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    bachma26@msu.edu
    Competing interests
    Inventor on a pending US patent (US-2020215010-A1), methods for treating or preventing developmental disorders associated with mutations in the OCD1 gene.

Funding

No external funding was received for this work.

Acknowledgements

The authors wish to thank the patient and family for their participation. We would like to dedicate this article to our patient who is a first in so many ways, and to her incredible family. We acknowledge the support we received from the research team at Spectrum Health. We are most grateful to Sanofi-Aventis for providing eflornithine for this study. We also thank Dr B Keith English, MD, Charles Schwartz, PhD, and Brittany Essenmacher for the critical review and editing of this manuscript, David Tack, PhD, for the design of some of the figures, and Olivia Verburg for her help with processing samples and logging samples.

Ethics

Human subjects: FDA approval (IND# 144022) was first acquired before therapy was initiated. The Spectrum Health IRB approved the study (IRB# 2019-161) and informed consent was acquired before blood sample collection. Consent was obtained for use of identifying patient images and videos within this manuscript. After study, authorization for publication was obtained from the parents of the child, now been placed in the medical records.

Senior Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. Goutham Narla, University of Michigan, United States

Publication history

  1. Received: January 31, 2021
  2. Accepted: June 16, 2021
  3. Version of Record published: July 20, 2021 (version 1)

Copyright

© 2021, Rajasekaran et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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