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Neurotoxicity Research

, Volume 33, Issue 3, pp 681–692 | Cite as

Experimental Evidence that In Vivo Intracerebral Administration of L-2-Hydroxyglutaric Acid to Neonatal Rats Provokes Disruption of Redox Status and Histopathological Abnormalities in the Brain

  • Rafael Teixeira Ribeiro
  • Ângela Zanatta
  • Alexandre Umpierrez Amaral
  • Guilhian Leipnitz
  • Francine Hehn de Oliveira
  • Bianca Seminotti
  • Moacir Wajner
ORIGINAL ARTICLE

Abstract

Tissue accumulation of L-2-hydroxyglutaric acid (L-2-HG) is the biochemical hallmark of L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic inherited disease characterized by neurological symptoms and brain white matter abnormalities whose pathogenesis is not yet well established. L-2-HG was intracerebrally administered to rat pups at postnatal day 1 (P1) to induce a rise of L-2-HG levels in the central nervous system (CNS). Thereafter, we investigated whether L-2-HG in vivo administration could disturb redox homeostasis and induce brain histopathological alterations in the cerebral cortex and striatum of neonatal rats. L-2-HG markedly induced the generation of reactive oxygen species (increase of 2′,7′-dichloroflurescein-DCFH-oxidation), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione-GSH) and sulfhydryl content in the cerebral cortex. Alterations of the activities of various antioxidant enzymes were also observed in the cerebral cortex and striatum following L-2-HG administration. Furthermore, L-2-HG-induced lipid peroxidation and GSH decrease in the cerebral cortex were prevented by the antioxidant melatonin and by the classical antagonist of NMDA glutamate receptor MK-801, suggesting the involvement of reactive species and of overstimulation of NMDA receptor in these effects. Finally, L-2-HG provoked significant vacuolation and edema particularly in the cerebral cortex with less intense alterations in the striatum that were possibly associated with the unbalanced redox homeostasis caused by this metabolite. Taken together, it is presumed that these pathomechanisms may underlie the neurological symptoms and brain abnormalities observed in the affected patients.

Keywords

L-2-hydroxyglutaric aciduria L-2-hydroxyglutaric acid Redox homeostasis Cerebral cortex Striatum Histopathology 

Notes

Funding Information

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico #404883/2013-3, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul #2266-2551/14-2, Pró-Reitoria de Pesquisa/Universidade Federal do Rio Grande do Sul #PIBIC 27613, FIPE/HCPA, and Financiadora de Estudos e Projetos/Rede Instituto Brasileiro de Neurociência #01.06.0842-00.

Compliance with Ethical Standards

Ethical Approval

The experimental protocol was approved by the Ethics Committee for Animal Research of the UFRGS, Porto Alegre, Brazil, and followed the “National Institutes of Health Guide for the Care and Use of Laboratory Animals” (NIH Publications No. 85–23, revised 2011).

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126.  https://doi.org/10.1016/S0076-6879(84)05016-3 PubMedCrossRefGoogle Scholar
  2. Aghili M, Zahedi F, Rafiee E (2009) Hydroxyglutaric aciduria and malignant brain tumor: a case report and literature review. J Neuro-Oncol 91(2):233–236.  https://doi.org/10.1007/s11060-008-9706-2 CrossRefGoogle Scholar
  3. Aksenov MY, Markesbery WR (2001) Changes in thiol content and expression of glutathione redox system genes in the hippocampus and cerebellum in Alzheimer’s disease. Neurosci Lett 302(2-3):141–145.  https://doi.org/10.1016/S0304-3940(01)01636-6 PubMedCrossRefGoogle Scholar
  4. Anghileri E, Bertolino N, Salsano E, Antelmi L, Carpinelli P, Castellotti B, Zucca I, Gellera C, Bisogno R, Caccia C, Cuccarini V (2016) In-vivo brain H1-MR-spectroscopy identification and quantification of 2-hydroxyglutarate in L-2-Hydroxyglutaric aciduria. Brain Res 1648(Pt A):506–511.  https://doi.org/10.1016/j.brainres.2016.08.013 PubMedCrossRefGoogle Scholar
  5. Anisimov VN, Popovich IG, Zabezhinski MA, Anisimov SV, Vesnushkin GM, Vinogradova IA (2006) Melatonin as antioxidant, geroprotector and anticarcinogen. Biochim Biophys Acta 1757(5-6):573–589.  https://doi.org/10.1016/j.bbabio.2006.03.012 PubMedCrossRefGoogle Scholar
  6. Balaji P, Viswanathan V, Chellathurai A, Panigrahi D (2014) An interesting case of metabolic dystonia: L-2 hydroxyglutaric aciduria. Ann Indian Acad Neurol 17(1):97–99.  https://doi.org/10.4103/0972-2327.128565 PubMedPubMedCentralCrossRefGoogle Scholar
  7. Barbot C, Fineza I, Diogo L, Maria M, Melo J, Guimarães A, Melo Pires M, Luis Cardoso M, Vila rinho L (1997) L-2-Hydroxyglutaric aciduria: clinical, biochemical and magnetic resonance imaging in six Portuguese pediatric patients. Brain Dev 19(4):268–273.  https://doi.org/10.1016/S0387-7604(97)00574-3 PubMedCrossRefGoogle Scholar
  8. Barth PG, Hoffmann GF, Jaeken J, RJA W, Duran M, Jansen GA, Jakobs C, Lehnert W, Hanefeld F, Valk J, RBH S, Trefz FK, Hartung HP, Chamoles NA, Sfaello Z, Caruso U (1993) L-2-hydroxyglutaric acidaemia: clinical and biochemical findings in 12 patients and preliminary report on L-2-hydroxyacid dehydrogenase. J Inherit Metab Dis 16(4):753–761.  https://doi.org/10.1007/BF00711907 PubMedCrossRefGoogle Scholar
  9. Browne RW, Armstrong D (1998) Reduced glutathione and glutathione disulfide. Methods Mol Biol 108:347–352.  https://doi.org/10.1385/0-89603-472-0:347 PubMedGoogle Scholar
  10. Carlberg I, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490.  https://doi.org/10.1016/S0076-6879(85)13062-4 PubMedCrossRefGoogle Scholar
  11. Circu ML, Aw TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48(6):749–762.  https://doi.org/10.1016/j.freeradbiomed.2009.12.022 PubMedPubMedCentralCrossRefGoogle Scholar
  12. Cubells JF, Rayport S, Rajendran G, Sulzer D (1994) Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress. J Neurosci 14(4):2260–2271PubMedGoogle Scholar
  13. da Rosa MS, João Ribeiro CA, Seminotti B, Teixeira Ribeiro R, Umpierrez Amaral A, de Moura Coelho D, de Oliveira FH, Leipnitz G, Wajner M (2015) In vivo intracerebral administration of L-2-hydroxyglutaric acid provokes oxidative stress and histopathological alterations in striatum and cerebellum of adolescent rats. Free Radic Biol Med 83:201–213.  https://doi.org/10.1016/j.freeradbiomed.2015.02.008 PubMedCrossRefGoogle Scholar
  14. da Silva CG, ARF B, Schuck PF, Leipnitz G, CAJ R, CMD W, ATS W, Wajner M (2003) L-2-hydroxyglutaric acid inhibits mitochondrial creatine kinase activity from cerebellum of developing rats. Int J Dev Neurosci 21(4):217–224.  https://doi.org/10.1016/S0736-5748(03)00035-2 PubMedCrossRefGoogle Scholar
  15. Fourati H, Ellouze E, Ahmadi M, Chaari D, Kamoun F, Hsairi I, Triki C, Mnif Z (2016) MRI features in 17 patients with l2 hydroxyglutaric aciduria. Eur J Radiol Open 3:245–250.  https://doi.org/10.1016/j.ejro.2016.09.001 PubMedPubMedCentralCrossRefGoogle Scholar
  16. Grings M, Moura AP, Parmeggiani B, Pletsch JT, Cardoso GMF, August PM, Matté C, Wyse ATS, Wajner M, Leipnitz G (2017) Bezafibrate prevents mitochondrial dysfunction, antioxidant system disturbance, glial reactivity and neuronal damage induced by sulfite administration in striatum of rats: implications for a possible therapeutic strategy for sulfite oxidase deficiency. Biochim Biophys Acta 1863(9):2135–2148.  https://doi.org/10.1016/j.bbadis.2017.05.019 PubMedCrossRefGoogle Scholar
  17. Haliloglu G, Jobard F, Oguz K, Anlar B, Akalan N, Coskun T, Sass J, Fischer J, Topcu M (2008) L-2-hydroxyglutaric aciduria and brain tumors in children with mutations in the L2HGDH gene: neuroimaging findings. Neuropediatrics 39(02):119–122.  https://doi.org/10.1055/s-2008-1081217 PubMedCrossRefGoogle Scholar
  18. Halliwell BGJ (2015) Cellular responses to oxidative stress: adaptation, damage, repair, senescence and death. In: Free radicals in biology and medicine. Oxford University Press Inc, Oxford, pp 199–283.  https://doi.org/10.1093/acprof:oso/9780198717478.003.0005 CrossRefGoogle Scholar
  19. Hoffmann GF, Meier-Augenstein W, Stockler S, Surtees R, Rating D, Nyhan WL (1993) Physiology and pathophysiology of organic acids in cerebrospinal fluid. J Inherit Metab Dis 16(4):648–669.  https://doi.org/10.1007/BF00711898 PubMedCrossRefGoogle Scholar
  20. Jellouli NK et al (2014) Founder effect confirmation of c.241A>G mutation in the L2HGDH gene and characterization of oxidative stress parameters in six Tunisian families with L-2-hydroxyglutaric aciduria. J Hum Genet 59(4):216–222.  https://doi.org/10.1038/jhg.2014.4 PubMedCrossRefGoogle Scholar
  21. Jovic NJ, Kosac A, Koprivsek K (2014) L-2-Hydroxyglutaric aciduria: a case report. Srp Arh Celok Lek 142:337–341PubMedCrossRefGoogle Scholar
  22. Junqueira D, Brusque AM, Porciúncula LO, Rotta LN, CAJ R, MES F, CSD F, CMD W, ATS W, Souza DO, Wajner M (2003) Effects of L-2-hydroxyglutaric acid on various parameters of the glutamatergic system in cerebral cortex of rats. Metab Brain Dis 18(3):233–243.  https://doi.org/10.1023/A:1025559200816 PubMedCrossRefGoogle Scholar
  23. Koeller DM, Woontner M, Crnic LS, Kleinschmidt-DeMasters B, Stephens J, Hunt EL, Goodman SI (2002) Biochemical, pathologic and behavioral analysis of a mouse model of glutaric acidemia type I. Hum Mol Genet 11(4):347–357.  https://doi.org/10.1093/hmg/11.4.347 PubMedCrossRefGoogle Scholar
  24. Koeller DM, Sauer S, Wajner M, de Mello CF, Goodman SI, Woontner M, Mühlhausen C, Okun JG, Kölker S (2004) Animal models for glutaryl-CoA dehydrogenase deficiency. J Inherit Metab Dis 27(6):813–818.  https://doi.org/10.1023/B:BOLI.0000045763.52907.5e PubMedCrossRefGoogle Scholar
  25. Latini A, Scussiato K, Borba Rosa R, Leipnitz G, Llesuy S, Belló-Klein A, Dutra-Filho CS, Wajner M (2003) Induction of oxidative stress by L-2-hydroxyglutaric acid in rat brain. J Neurosci Res 74(1):103–110.  https://doi.org/10.1002/jnr.10735 PubMedCrossRefGoogle Scholar
  26. LeBel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5(2):227–231.  https://doi.org/10.1021/tx00026a012 PubMedCrossRefGoogle Scholar
  27. Leong SF, Clark JB (1984) Regional development of glutamate dehydrogenase in the rat brain. J Neurochem 43(1):106–111.  https://doi.org/10.1111/j.1471-4159.1984.tb06684.x PubMedCrossRefGoogle Scholar
  28. Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357PubMedCrossRefGoogle Scholar
  29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  30. Mannervik B, Guthenberg C (1981) Glutathione transferase (human placenta). Methods Enzymol 77:231–235.  https://doi.org/10.1016/S0076-6879(81)77030-7 PubMedCrossRefGoogle Scholar
  31. Marcel C, Mallaret M, Lagha-Boukbiza O, Kremer S, Echaniz-Laguna A, Tranchant C (2012) L-2-hydroxyglutaric aciduria diagnosed in a young adult with progressive cerebellar ataxia and facial dyskinesia. Rev Neurol 168(2):187–191.  https://doi.org/10.1016/j.neurol.2011.06.002 PubMedCrossRefGoogle Scholar
  32. Marklund SL (1985) Product of extracellular-superoxide dismutase catalysis. FEBS Lett 184(2):237–239.  https://doi.org/10.1016/0014-5793(85)80613-X PubMedCrossRefGoogle Scholar
  33. Moroni I, Bugiani M, D’Incerti L, Maccagnano C, Rimoldi M, Bissola L, Pollo B, Finocchiaro G, Uziel G (2004) L-2-hydroxyglutaric aciduria and brain malignant tumors: a predisposing condition? Neurology 62(10):1882–1884.  https://doi.org/10.1212/01.WNL.0000125335.21381.87 PubMedCrossRefGoogle Scholar
  34. Navarro-Gonzalvez JA, Garcia-Benayas C, Arenas J (1998) Semiautomated measurement of nitrate in biological fluids. Clin Chem 44:679–681PubMedGoogle Scholar
  35. Niizuma K, Endo H, Chan PH (2009) Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem 109(Suppl 1):133–138.  https://doi.org/10.1111/j.1471-4159.2009.05897.x PubMedPubMedCentralCrossRefGoogle Scholar
  36. Olivera-Bravo S, Fernandez A, Sarlabos MN, Rosillo JC, Casanova G, Jimenez M, Barbeito L (2011) Neonatal astrocyte damage is sufficient to trigger progressive striatal degeneration in a rat model of glutaric acidemia-I. PLoS One 6(6):e20831.  https://doi.org/10.1371/journal.pone.0020831 PubMedPubMedCentralCrossRefGoogle Scholar
  37. Olivera-Bravo S, Isasi E, Fernández A, Rosillo JC, Jiménez M, Casanova G, Sarlabós MN, Barbeito L (2014) White matter injury induced by perinatal exposure to glutaric acid. Neurotox Res 25(4):381–391.  https://doi.org/10.1007/s12640-013-9445-9 PubMedCrossRefGoogle Scholar
  38. Olivier P, Fontaine RH, Loron G, van Steenwinckel J, Biran V, Massonneau V, Kaindl A, Dalous J, Charriaut-Marlangue C, Aigrot MS, Pansiot J, Verney C, Gressens P, Baud O (2009) Melatonin promotes oligodendroglial maturation of injured white matter in neonatal rats. PLoS One 4(9):e7128.  https://doi.org/10.1371/journal.pone.0007128 PubMedPubMedCentralCrossRefGoogle Scholar
  39. Patay Z, Mills JC, Lobel U, Lambert A, Sablauer A, Ellison DW (2012) Cerebral neoplasms in L-2 hydroxyglutaric aciduria: 3 new cases and meta-analysis of literature data. AJNR Am J Neuroradiol 33:940–943.  https://doi.org/10.3174/ajnr.A2869 PubMedCrossRefGoogle Scholar
  40. Reiter RJ, Tan DX, Rosales-Corral S, Manchester LC (2013) The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Min Rev Med Chem 13(3):373–384Google Scholar
  41. Requejo R, Chouchani ET, Hurd TR, Menger KE, Hampton MB, Murphy MP (2010) Measuring mitochondrial protein thiol redox state. Methods Enzymol 474:123–147.  https://doi.org/10.1016/S0076-6879(10)74008-8 PubMedCrossRefGoogle Scholar
  42. Reznick AZ, Packer L (1994) Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 233:357–363PubMedCrossRefGoogle Scholar
  43. Rodrigues DGB, de Moura Coelho D, Sitta Â, CED J, Hauschild T, Manfredini V, Bakkali A, Struys EA, Jakobs C, Wajner M, Vargas CR (2017) Experimental evidence of oxidative stress in patients with l-2-hydroxyglutaric aciduria and that l-carnitine attenuates in vitro DNA damage caused by d-2-hydroxyglutaric and l-2-hydroxyglutaric acids. Toxicol in Vitro 42:47–53.  https://doi.org/10.1016/j.tiv.2017.04.006 PubMedCrossRefGoogle Scholar
  44. Rzem R, Van Schaftingen E, Veiga-da-Cunha M (2006) The gene mutated in l-2-hydroxyglutaric aciduria encodes l-2-hydroxyglutarate dehydrogenase. Biochimie 88(1):113–116.  https://doi.org/10.1016/j.biochi.2005.06.005 PubMedCrossRefGoogle Scholar
  45. Sauer SW, Okun JG, Fricker G, Mahringer A, Müller I, Crnic LR, Mühlhausen C, Hoffmann GF, Hörster F, Goodman SI, Harding CO, Koeller DM, Kölker S (2006) Intracerebral accumulation of glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency. J Neurochem 97(3):899–910.  https://doi.org/10.1111/j.1471-4159.2006.03813.x PubMedCrossRefGoogle Scholar
  46. Sauer SW, Opp S, Mahringer A, Kamiński MM, Thiel C, Okun JG, Fricker G, Morath MA, Kölker S (2010) Glutaric aciduria type I and methylmalonic aciduria: simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood-brain barrier and the choroid plexus. Biochim Biophys Acta 1802(6):552–560.  https://doi.org/10.1016/j.bbadis.2010.03.003 PubMedCrossRefGoogle Scholar
  47. Saxena G, Bharti S, Kamat PK, Sharma S, Nath C (2010) Melatonin alleviates memory deficits and neuronal degeneration induced by intracerebroventricular administration of streptozotocin in rats. Pharmacol Biochem Behav 94(3):397–403.  https://doi.org/10.1016/j.pbb.2009.09.022 PubMedCrossRefGoogle Scholar
  48. Seminotti B, Amaral AU, Ribeiro RT, MDN R, Colín-González AL, Leipnitz G, Santamaría A, Wajner M (2016) Oxidative stress, disrupted energy metabolism, and altered signaling pathways in glutaryl-CoA dehydrogenase knockout mice: potential implications of quinolinic acid toxicity in the neuropathology of glutaric acidemia type I. Mol Neurobiol 53(9):6459–6475.  https://doi.org/10.1007/s12035-015-9548-9 PubMedCrossRefGoogle Scholar
  49. Steenweg ME, Salomons GS, Yapici Z, Uziel G, Scalais E, Zafeiriou DI, Ruiz-Falco ML, Mejaški-Bošnjak V, Augoustides-Savvopoulou P, Wajner M, Walter J, Verhoeven-Duif NM, Struys EA, Jakobs C, van der Knaap MS (2009) L-2-Hydroxyglutaric aciduria: pattern of MR imaging abnormalities in 56 patients. Radiology 251(3):856–865.  https://doi.org/10.1148/radiol.2513080647 PubMedCrossRefGoogle Scholar
  50. Steenweg ME, Jakobs C, Errami A, van Dooren SJM, Adeva Bartolomé MT, Aerssens P, Augoustides-Savvapoulou P, Baric I, Baumann M, Bonafé L, Chabrol B, JTR C, Clayton P, Coker M, Cooper S, Falik-Zaccai T, Gorman M, Hahn A, Hasanoglu A, King MD, de Klerk HBC, Korman SH, Lee C, Meldgaard Lund A, Mejaški-Bošnjak V, Pascual-Castroviejo I, Raadhyaksha A, Rootwelt T, Roubertie A, Ruiz-Falco ML, Scalais E, Schimmel U, Seijo-Martinez M, Suri M, Sykut-Cegielska J, Trefz FK, Uziel G, Valayannopoulos V, Vianey-Saban C, Vlaho S, Vodopiutz J, Wajner M, Walter J, Walter-Derbort C, Yapici Z, Zafeiriou DI, Spreeuwenberg MD, Celli J, den Dunnen JT, van der Knaap MS, Salomons GS (2010) An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study. Hum Mutat 31(4):380–390.  https://doi.org/10.1002/humu.21197 PubMedCrossRefGoogle Scholar
  51. Topcu M et al (2004) L-2-Hydroxyglutaric aciduria: identification of a mutant gene C14orf160, localized on chromosome 14q22.1. Hum Mol Genet 13(22):2803–2811.  https://doi.org/10.1093/hmg/ddh300 PubMedCrossRefGoogle Scholar
  52. Topcu M et al (2005) L-2-hydroxyglutaric aciduria: a report of 29 patients. Turk J Pediatr 47(1):1–7PubMedGoogle Scholar
  53. Wajner M, Goodman SI (2011) Disruption of mitochondrial homeostasis in organic acidurias: insights from human and animal studies. J Bioenerg Biomembr 43(1):31–38.  https://doi.org/10.1007/s10863-011-9324-0 PubMedCrossRefGoogle Scholar
  54. Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:325–333.  https://doi.org/10.1016/S0076-6879(81)77046-0 PubMedCrossRefGoogle Scholar
  55. Wilt SG, Dugger NV, Hitt ND, Hoffman PM (2000) Evidence for oxidative damage in a murine leukemia virus-induced neurodegeneration. J Neurosci Res 62(3):440–450.  https://doi.org/10.1002/1097-4547(20001101)62:3<440::AID-JNR14>3.0.CO;2-M PubMedCrossRefGoogle Scholar
  56. Yagi K (1998) Simple procedure for specific assay of lipid hydroperoxides in serum or plasma. Methods Mol Biol 108:107–110.  https://doi.org/10.1385/0-89603-472-0:107 PubMedGoogle Scholar
  57. Yilmaz K (2009) Riboflavin treatment in a case with l-2-hydroxyglutaric aciduria. Eur J Paediatr Neurol 13(1):57–60.  https://doi.org/10.1016/j.ejpn.2008.01.003 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Rafael Teixeira Ribeiro
    • 3
  • Ângela Zanatta
    • 1
  • Alexandre Umpierrez Amaral
    • 1
    • 2
  • Guilhian Leipnitz
    • 1
    • 3
  • Francine Hehn de Oliveira
    • 5
  • Bianca Seminotti
    • 1
  • Moacir Wajner
    • 1
    • 3
    • 4
  1. 1.Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Departamento de Ciências BiológicasUniversidade Regional Integrada do Alto Uruguai e das MissõesErechimBrazil
  3. 3.Departamento de Bioquímica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  4. 4.Serviço de Genética MédicaHospital de Clínicas de Porto AlegrePorto AlegreBrazil
  5. 5.Serviço de PatologiaHospital de Clinicas de Porto AlegrePorto AlegreBrazil

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