Mitochondrial Neurogastrointestinal Encephalopathy Due to Mutations in RRM2B

OBSERVATION Mitochondrial Neurogastrointestinal Encephalopathy Due to Mutations in RRM2B

Aziz Shaibani, MD; Oleg A. Shchelochkov, MD; Shulin Zhang, MD, PhD; Panagiotis Katsonis, PhD; Olivier Lichtarge, MD, PhD; Lee-Jun Wong, PhD; Marwan Shinawi, MD

Background: Mitochondrial neurogastrointestinal en- of our patient’s condition. Sequencing of genes associ- cephalopathy (MNGIE) is a progressive neurodegenera- ated with mitochondrial DNA depletion—POLG, PEO1, tive disorder associated with thymidine phosphorylase ANT1, SUCLG1, and SUCLA2—did not reveal deleteri- deficiency resulting in high levels of plasma thymidine ous mutations. Results of sequencing and array com- and a characteristic clinical phenotype. parative genomic hybridization of the mitochondrial DNA for point mutations and deletions in blood and muscle Objective: To investigate the molecular basis of MNGIE were negative. Sequencing of RRM2B, a gene encoding in a patient with a normal plasma thymidine level. cytosolic p53-inducible ribonucleoside reductase small subunit (RIR2B), revealed 2 pathogenic mutations, Design: Clinical, neurophysiological, and histopatho- c.329GϾA (p.R110H) and c.362GϾA (p.R121H). These logical examinations as well as molecular and genetic mutations are predicted to affect the docking interface analyses. of the RIR2B homodimer and likely result in impaired Setting: Nerve and muscle center and genetic clinic. enzyme activity.

Patient: A 42-year-old woman with clinical findings Conclusions: This study expands the clinical spectrum strongly suggestive for MNGIE. of impaired RIR2B function, challenges the notion of lo- cus homogeneity of MNGIE, and sheds light on the patho- Main Outcome Measures: Clinical description of the genesis of conditions involved in the homeostasis of the disease and its novel genetic cause. mitochondrial nucleotide pool. Our findings suggest that patients with MNGIE who have normal thymidine lev- Results: Identification of mitochondrial DNA deple- els should be tested for RRM2B mutations. tion in muscle samples (approximately 12% of the control mean content) prompted us to look for other causes Arch Neurol. 2009;66(8):1028-1032

ITOCHONDRIAL NEURO- notype associated with multiple mtDNA gastrointestinal en- deletions3 or mutations in POLG4 have cephalopathy(MNGIE) been reported. is an autosomal reces- Although mtDNA depletion is one of the sive disease character- characteristic molecular findings in MNGIE, Author Affiliations: Nerve and ized by gastrointestinal dysmotility, oph- mtDNA depletion is also associated with Muscle Center of Texas, M thalmoplegia, ptosis, cachexia, peripheral mutations in nuclear genes that affect mi- Houston (Dr Shaibani); 1 Departments of Medicine neuropathy, and leukoencephalopathy. It tochondrial nucleotide metabolism and (Dr Shaibani), Molecular and is caused by mutations in TYMP, a gene mtDNA replication. These genes exert their 2 Human Genetics encoding thymidine phosphorylase. De- action by either directly affecting the mtDNA (Drs Shchelochkov, Zhang, ficiency in thymidine phosphorylase leads replication fork (POLG, POLG2, and PEO1) Katsonis, Lichtarge, Wong, and to marked elevation of thymidine and de- or regulating the mitochondrial deoxy- Shinawi), Biochemistry and oxyuridine levels, resulting in imbalance nucleotide triphosphate pool (TYMP, TK2, Molecular Biology of the mitochondrial nucleotide pool. It has DGUOK, SUCLA2, SUCLG1, and ANT1).5 (Dr Lichtarge), and been hypothesized that alteration of de- Mutations in RRM2B (GenBank AB036532) Pharmacology (Dr Lichtarge) oxynucleotide triphosphate homeostasis were recently implicated in a novel mito- and Medical Genetics results in improper mitochondrial DNA chondrial depletion syndrome presenting Laboratories (Drs Zhang and Wong), Baylor College of (mtDNA) replication that in turn causes with early-onset seizures, hypotonia, diar- Medicine, Houston; and Texas mtDNA deletion, point mutations, and rhea, renal tubulopathy, lactic acidosis, and 2 6-9 Children’s Hospital, Houston depletion. Although TYMP is the only early lethality. (Drs Shchelochkov and gene known to be associated with typical Here we describe a 42-year-old woman Shinawi). MNGIE, a few cases of MNGIE-like phe- with clinical findings strongly suggestive

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Figure 1. Axial fluid-attenuated inversion recovery, T2-weighted magnetic resonance images of the brain showing symmetric, hyperintense, nonenhancing lesions in the basal ganglia (arrows) (A) and patchy signals throughout the white matter (arrows) (B).

for MNGIE but whose plasma thymidine level was nor- showed mild elevation of the levels of total protein, IgG, mal. Further analysis showed severe mtDNA depletion myelin basic protein, and lactate. Funduscopic evaluation, in muscle tissue, and sequencing analysis revealed 2 patho- echocardiography, and needle electromyography results were genic mutations in the RRM2B gene. normal. Nerve conduction studies revealed normal sural potentials, generalized motor slowing (32-37 m/s), and pro- longed distal latencies consistent with demyelinating neu- METHODS ropathy. Brain magnetic resonance imaging showed increased T2-weighted signal in the basal ganglia and patchy CLINICAL PRESENTATION T2-weighted signals throughout the periventricular and sub- cortical white matter (Figure 1). The muscle biopsy revealed A 42-year-old woman of mixed African American and Latin Ameri- mild variation of fiber size (Figure 2A), increased endo- can descent was referred to us for evaluation of ophthalmople- mysial connective tissue, rare ragged red fibers on modified gia, ptosis, gastrointestinal dysmotility, cachexia, peripheral neu- Gomori trichrome stain (Figure 2B), and numerous ragged ropathy, and brain magnetic resonance imaging changes blue fibers on succinate dehydrogenase reaction (Figure 2C). (Figure 1). She was in good health until age 30 years, when she The overall findings were consistent with mitochondrial developed recurrent and severe episodes of nausea and vomiting myopathy and mild neurogenic atrophy with reinnervation. due to gastrointestinal dysmotility. In the same year, she was hos- The activity of the respiratory chain complexes in muscle was pitalized with systemic bacterial infection and received intrave- within the normal range (data not shown). nous gentamicin sulfate, which was followed by the development of sensorineural hearing loss. Owing to her gastrointestinal problems, she lost significant weight; in fact, she weighed 30 kg ANALYSES (body mass index [calculated as weight in kilograms divided by height in meters squared], 12.5) a year before her presentation Spectrophotometric analysis of the respiratory chain com- to us. Since age 37 years, she had progressive restriction of eye plexes was performed according to previously described pro- movements, ptosis, micronystagmus, dysarthria, unsteady gait, tocols.10 The coding exons and the immediate flanking in- generalized muscle weakness, and loss of deep tendon reflexes. tronic sequences of RRM2B, POLG1, ANT1, PEO1, SUCLG1, Her sensorium was preserved. Her family history was negative and SUCLA2 were amplified by polymerase chain reaction and for similarly affected relatives. sequenced in the forward and reverse directions using auto- Her initial diagnostic evaluation revealed normal levels of mated fluorescence dideoxy-sequencing methods. The poten- plasma thymidine (100 mmol/L; reference range, Ͻ150 tial effects of mutants on the protein structure were estimated mmol/L), serum aminotransferases, creatine phosphokinase, by comparing the local environments of mutants11 among the and plasma amino acids as well as a normal acylcarnitine pro- homologs of the ribonucleoside reductase small subunit (RIR2B) file. Her plasma lactate level was mildly elevated (27.9 mg/dL protein family. Nuclear DNA and mtDNA copy numbers were [to convert to millimoles per liter, multiply by 0.111]; refer- determined by real-time quantitative polymerase chain reac- ence range, 1.8-18.0 mg/dL). Cerebrospinal fluid analysis tion according to a previously validated protocol.12

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Figure 2. Muscle biopsy suggestive of mitochondrial dysfunction. A, Decreased cytochrome-c oxidase staining (original magnification ϫ100). B, Modified Gomori trichrome staining showing a ragged red fiber (original magnification ϫ400). Mild variation in fiber size is seen. C, Succinate dehydrogenase staining showing multiple ragged blue fibers (original magnification ϫ40). These findings were suggestive of the mitochondrial dysfunction. Other findings included rare denervated fibers and several small fiber-type groups (data not shown).

quencing and deletions by array comparative genomic S112 V115 Q113 hybridization. A homoplasmic unclassified missense vari- V117 Q116 E114 ant, m.15077GϾA (E111K, CytB), was detected and pre- P118 viously reported in the Polysite database (http://www F111 .genpat.uu.se/mtDB/) with a frequency of 2702:2 (A:G). A homoplasmic variant, m.16017TϾC, in transfer RNA E119 R186 located at the first base of the stem region of the amino A120 acid acceptor arm was detected. The clinical conse- R121 S181 quences of these nucleotide changes are unknown but C122 likely represent benign variants. Sequencing of POLG, R179 F123 ANT1, PEO1, SUCLG1, and SUCLA2 did not identify deleterious changes. Sequencing of RRM2B revealed Y124 A177 c.329GϾA (p.R110H) and c.362GϾA (p.R121H) muta- R110 tions. Analysis of parental DNA confirmed that the iden- I176 E109 tified mutations were in trans configuration.

V108 mtDNA DEPLETION

L107 The mtDNA content in blood and muscle was measured in duplicates and confirmed by repeat runs. Analysis of the mtDNA content performed on the muscle biopsy N106 specimen showed 12% of the control mean content, but no depletion was detected in peripheral blood.

PREDICTED STRUCTURAL CHANGES ON RIR2B R110 environment R121 environment R110 and R121 environment To evaluate the effect of the mutations R110H and R121H, we examined the evolutionary history of each mutant in its local structural environment composed of the residues Figure 3. Consequences of the R110H and R121H mutations on the structure of the ribonucleoside reductase small subunit (RIR2B). R110H and within 0.50 nm. The analyzed homologs consisted of 256 R121H (in red) are within 0.75 nm of each other and straddle the outlined sequences coming from 165 different sources (including homodimer interface. Each has 13 structural neighbors (light green) that animals, plants, bacteria, and viruses). The metazoa se- overlap at F111, S112, Q113, and E114 (dark green). The few instances of homologs carrying the R110H substitution have at best only 6 identical quences clustered in the phylogenetic tree and consisted structural neighbors (N106, L107, E109, F111, E114, and R186), and the of 60 sequences from 32 different species. Substitution homologs with the R121H substitution have at best just 1 identical structural R121H occurs in only 2.3% of homologs, which share less neighbor (Y124). The figure was generated by using the PyMOL program than 30% of the total sequence identity and only 8% of the (DeLano Scientific LLC, Palo Alto, California) and the 2vux Protein Data Bank file (doi:10.2210/pdb2vux/pdb). sequence identity among the contacts in position 121. Sub- stitution R110H is seen in 1.2% of homologs sharing less than 70% of the total and 46% of the local sequence iden- tities. Both positions are almost invariant in RIR2B in meta- RESULTS zoa. No homologs simultaneously carried histidine in both positions. Residues R110 and R121 are located in close prox- DNA ANALYSIS imity to each other and to the known homodimer RIR2B docking interface (Figure 3). Together these data are con- Results of mtDNA analysis in peripheral blood and muscle sistent with R121H and R110H being jointly sufficiently were negative for deleterious point mutations by se- deleterious to affect the protein function.

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 CLINICAL OUTCOME The clinical presentation in our patient closely resembled MNGIE but there were subtle differences.15 The patient received vitamin C (500 mg 3 times a day), The nerve conduction velocities were decreased but vitamin E (400 units twice a day), coenzyme Q10 (200 not to the degree typically seen in patients with mg twice a day), and levocarnitine (1 g 3 times a day). MNGIE, and brain magnetic resonance imaging find- Within 6 months from starting the treatment, she showed ings were patchy as compared with confluent white normalization of the plasma lactate level and improve- matter abnormalities seen in most patients with ment in weight, muscle strength, eye movements, and MNGIE. The normal plasma thymidine level in our daily life activities. Her hearing and deep tendon re- patient prompted us to look for other molecular expla- flexes did not improve. nations of her disease. When mitochondrial depletion was demonstrated in her muscle, we extended the sequencing to genes associated with mtDNA depletion COMMENT syndromes. Identification of RRM2B mutations in a patient with classic features of MNGIE expands the The RRM2B gene encodes protein RIR2B (p53R2), which molecular repertoire of this genetic disorder. The role is transcriptionally regulated by the tumor suppressor of RIR2B in de novo deoxynucleotide triphosphate syn- TP53 and plays a key role in the regulation of stress re- 13 thesis suggests that there could be multiple mechanisms sponse to various cell-damaging stimuli. Mutations in resulting in MNGIE phenotype and that depletion of RRM2B have been reported to cause mtDNA depletion 6 14 the intramitochondrial nucleotide pool is the common in human subjects and a murine model, demonstrat- biochemical abnormality underlying the neurological ing its essential role in mtDNA replication and repair. abnormalities in this condition.

EXPANDING THE CLINICAL SPECTRUM CONCLUSIONS OF RRM2B-RELATED DISORDERS We conclude that mutations in RRM2B may result in a To our knowledge, a total of 14 patients from 10 nonre- phenotype clinically similar to MNGIE. Therefore, RRM2B lated families and carrying a total of 16 unique muta- sequencing should be considered in patients with MNGIE tions in the RRM2B gene have been described to date.6-9 with normal thymidine levels. Development of symp- The spectrum of clinical findings includes muscle hypo- toms in midadulthood expands the spectrum of age at tonia, seizures, neonatal-onset vomiting and diarrhea, lac- presentation in RRM2B-related disorders. Aminoglyco- tic acidosis, renal tubulopathy, and early childhood mor- sides should be cautiously used in patients with severe tality. The oldest described patient, aged 36 months, had gastrointestinal dysmotility as this can be the first clini- a more benign clinical course that correlated with the cal presentation of MNGIE. higher mtDNA content, 11% of the control mean content.7 Skeletal muscle biopsy in the studied patients dem- Accepted for Publication: March 24, 2009. onstrated severe mtDNA depletion, ranging between 1% Correspondence: Marwan Shinawi, MD, Department of and 11% of the control mean content. The activities of Molecular and Human Genetics, Baylor College of Medi- respiratory chain enzymes containing mtDNA-encoded cine, One Baylor Plaza, Room T619, Houston, TX 77030 subunits were markedly reduced in most but not all of ([email protected]). the patients with RRM2B mutations. Author Contributions: Drs Shaibani and Shchelochkov Our patient is the oldest described patient with RRM2B contributed equally to this work. Study concept and de- mutations with the highest mtDNA content (12% of the sign: Shchelochkov, Wong, and Shinawi. Acquisition of control mean content). She initially presented with gas- data: Shaibani, Shchelochkov, Zhang, Wong, and trointestinal dysmotility at age 30 years. We hypoth- Shinawi. Analysis and interpretation of data: Shaibani, esize that the severe vomiting and diarrhea observed in Shchelochkov, Zhang, Katsonis, Lichtarge, Wong, and infants with RRM2B mutations reflect a fundamental gas- Shinawi. Drafting of the manuscript: Shchelochkov, trointestinal dysfunction and mirror the gastrointesti- Katsonis, Lichtarge, and Shinawi. Critical revision of the nal dysmotility seen in older patients with MNGIE. Al- manuscript for important intellectual content: Shaibani, though our patient developed sensorineural hearing loss Shchelochkov, Zhang, Wong, and Shinawi. Statistical after gentamicin treatment, results of her whole mito- analysis: Katsonis and Lichtarge. Obtained funding: chondrial genome sequencing were negative for Wong. Administrative, technical, and material support: m.1555AϾG, m.961delTϩ(C)n, and m.7443AϾG mu- Shchelochkov, Zhang, Wong, and Shinawi. Study super- tations associated with mitochondrial nonsyndromic hear- vision: Shaibani, Wong, and Shinawi. ing loss and deafness. This raises the possibility that pa- Financial Disclosure: None reported. tients with RIR2B dysfunction could be susceptible to Funding/Support: Dr Lichtarge is supported by grants aminoglycoside-induced deafness. R01-GM066099 and R01-GM079656 from the National Institutes of Health. Dr Katsonis has a fellowship from EXPANDING THE MOLECULAR the Pharmacoinformatics Training Program of Keck Cen- MECHANISMS OF MNGIE ter of the Gulf Coast Consortia (grants 5 R90 DKO71505-03 and 5 R90 DKO71505-04 from the Na- Nishino et al2 established the causal link between the tional Institutes of Health); the program is supported by dysfunction of thymidine phosphorylase and MNGIE. a National Institutes of Health–funded training grant.

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 Additional Contributions: Amina Jinna, MD, Shweta controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA Dhar, MD, Richard Lewis, MD, Kerri Lamance, RN, and depletion. Nat Genet. 2007;39(6):776-780. 7. Bornstein B, Area E, Flanigan KM, et al. Mitochondrial DNA depletion syndrome due the Baylor College of Medicine Mitochondrial Labora- to mutations in the RRM2B gene. Neuromuscul Disord. 2008;18(6):453-459. tory contributed to the caring management of our pa- 8. Spinazzola A, Invernizzi F, Carrara F, et al. Clinical and molecular features of mito- tient. We thank the patient’s family for their kind coop- chondrial DNA depletion syndromes. J Inherit Metab Dis. 2009;32(2):143-158. eration. 9. Kollberg G, Darin N, Benan K, et al. A novel homozygous RRM2B missense mutation in association with severe mtDNA depletion. Neuromuscul Disord. 2009; 19(2):147-150. REFERENCES 10. Wong LJ, Brunetti-Pierri N, Zhang Q, et al. Mutations in the MPV17 gene are responsible for rapidly progressive liver failure in infancy. Hepatology. 2007; 1. Nishino I, Spinazzola A, Papadimitriou A, et al. Mitochondrial neurogastrointes- 46(4):1218-1227. tinal encephalomyopathy: an autosomal recessive disorder due to thymidine phos- 11. Ward RM, Erdin S, Tran TA, Kristensen DM, Lisewski AM, Lichtarge O. De- phorylase mutations. Ann Neurol. 2000;47(6):792-800. orphaning the structural proteome through reciprocal comparison of evolution- 2. Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylase gene mutations in arily important structural features. PLoS ONE. 2008;3(5):e2136. doi:10.1371 MNGIE, a human mitochondrial disorder. Science. 1999;283(5402):689-692. /journal.pone.0002136. 3. Lehnhardt F-G, Horvath R, Ullrich R, et al. Altered cerebral glucose metabolism 12. Bai R-K, Wong L-JC. Simultaneous detection and quantification of mitochon- in a family with clinical features resembling mitochondrial neurogastrointestinal drial DNA deletion(s), depletion, and over-replication in patients with mitochon- encephalomyopathy syndrome in association with multiple mitochondrial DNA drial disease. J Mol Diagn. 2005;7(5):613-622. deletions. Arch Neurol. 2008;65(3):407-411. 13. Tanaka H, Arakawa H, Yamaguchi T, et al. A ribonucleotide reductase gene in- 4. Van Goethem G, Schwartz M, Löfgren A, Dermaut B, Van Broeckhoven C, Viss- volved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature. 2000; ing J. Novel POLG mutations in progressive external ophthalmoplegia mimick- 404(6773):42-49. ing mitochondrial neurogastrointestinal encephalomyopathy. Eur J Hum Genet. 14. Kimura T, Takeda S, Sagiya Y, Gotoh M, Nakamura Y, Arakawa H. Impaired func- 2003;11(7):547-549. tion of p53R2 in Rrm2b-null mice causes severe renal failure through attenua- 5. Copeland WC. Inherited mitochondrial diseases of DNA replication. Annu Rev tion of dNTP pools. Nat Genet. 2003;34(4):440-445. Med. 2008;59(1):131-146. 15. Nishino I, Spinazzola A, Hirano M. MNGIE: from nuclear DNA to mitochondrial 6. Bourdon A, Minai L, Serre V, et al. Mutation of RRM2B, encoding p53- DNA. Neuromuscul Disord. 2001;11(1):7-10.

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