Methionine synthase reductase deficiency (CblE): A report of two patients and a novel mutation M. Ruiz-Mercado 1, M. T. Vargas 1, I. Pérez de Soto1, C. Delgado Pecellín 2, M. Conde Sánchez 2, M. A. Bueno Delgado 3, R. Bernal Ruiz 1, J. A. Pérez-Simón1, A. Herrera Díaz-Aguado 1 1
Hematology Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/ CISC, Seville, Spain, 2Clinical Laboratory Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CISC, Seville, Spain, 3Pediatrics Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CISC, Seville, Spain Importance: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment. Clinical presentation: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them. Intervention: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677–1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency. Conclusion: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme. Keywords: Methionine synthase reductase, Cobalamine E deficiency, Hyperhomocysteinemia, Megaloblastic anemia, Hypomethioninemia
Introduction Inborn errors of intracellular metabolism of vitamin B12 include a wide spectrum of congenital disorders of autosomal recessive inheritance that greatly vary from clinical and therapeutic perspectives. The three groups are: (1) methylmalonyl-CoA reductase, which uses adenosylcobalamin as a cofactor (CblA, CblB, and CblD subtype 2), (2) methionine synthase using methylcobalamin as a cofactor (CblD subtype 1 CblE and CblG), and (3) involvement of both enzymatic pathways (CblC, CblD, CblF, and CblJ).1–3 The methionine synthase reductase enzyme is required for the reductive methylation of methionine synthase (MTR) – which uses CblG as a cofactor – responsible for remethylation of homocysteine to methionine.2,4 The subtype CblE, rarely reported in Correspondence to: A. Herrera Díaz-Aguado, Hospital Universitario Virgen del Rocío-Virgen Macarena, Avda. Manuel Siurot, s/n° 41013 Seville, Spain. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845415Y.0000000017
literature, is due to mutations in the gene that encodes for methionine synthase reductase enzyme (MTRR). To date, 19 different mutations have been identified.5 It mainly manifests as a macrocytic anemia, with or without megaloblastic features, estaturo-ponderal retardation, and neurological disorders, encompassing from cognitive disorders to neurodegenerative encephalopathy.6,7 The most striking finding in all patients is the presence of hyperhomocysteinemia with normal levels of methylmalonic acid and usually low levels of methionine. This classic triad defines the subtypes CblG and CblE whose final diagnosis must be confirmed by molecular assays8 (see Fig. 1). Early treatment with intramuscular hydroxocobalamin improves the outcome by correction of the macrocytic anemia and the almost complete disappearance of cytological dysmorphias and achieves transfusion independence. However, the impact is lower in biochemical parameters and generally
Hematology
2015
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Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
Figure 1 Algorithm for the diagnosis of disorders of intracellular cobalamin metabolism. CblC: cobalamin C, CblD: cobalamin D, CblF: cobalamin F, CblA: cobalamin A, CblB: cobalamin B, CblE: cobalamin E, CblG: cobalamin G, AdoCl: adenosil-cobalamin, and MetilCl: metil-cobalamin.
hyperhomocysteinemia persists over time.9 The established neurological disorders do not reverse although their prevention is possible with the early administration of specific treatment.10 In the current report we describe the clinical, molecular, metabolic and cytological characteristics of two new cases of MTRR deficiency. Given the finding of high levels of serum homocysteine, the study focused on congenital disorders of intracellular cobalamin metabolism. The diagnosis was confirmed by molecular assays, revealing, in one case, a new mutation unknown to date associated to more benign course.
Case presentation Case 1 The patient, an 8-year-old girl with non-consanguineous parents and non-family history of genetic disorders, presented at 3 months of age a severe anemia requiring several blood transfusions. The laboratory findings are as follows: hemoglobin (Hb) 45.8 g/l, mean corpuscular volume (MCV) 84.2 fl, mean corpuscular hemoglobin (MCH) 30.1 pg/l, and reticulocytes 71 × 10 e9/l. The leukocyte and platelet counts were normal. The erythroid morphology highlighted an important poikilocytosis with macro-ovalocytes, basophilic stippling, and 3% of erythroblasts. In addition, total bilirubin was 5.7 mg/dl with indirect bilirubin of 3.8 mg/dl and haptoglobin of 2 mg/dl.
2
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Folic acid levels were 7 ng/ml and vitamin B12 205 ng/l, both within the normal range. Study of hemoglobin by high-pressure liquid chromatography (HPLC), direct Coombs test, glucose 6-phosphate dehydrogenase, sucrose, and Ham tests were all normal or negative. Bone marrow aspirate revealed normal cellularity, properly represented hematopoiesis, and 17% of erythroid series with macrocytic features, chromatin remains, and basophilic stippling in orthochromatic normoblasts, multinucleated cells being outstanding. These findings supported a non-classifiable congenital dyserythropoiesis (see Fig. 2A). In the analytical follow-up, hemoglobin ranged from 103 to 136 g/l with a median of MCV 103.6 fl and red cell morphology with predominance of macro-ovalocytes. The patient was re-examined, extending the study of anemia to determine the cause of persistent megaloblastosis. Homocysteine levels were 50.7 μmol/l (normal: 0–16 μmol/l), methionine 10.6 μmol/l (15–27 μmol/l) while normal levels of methylmalonic acid in urine and serum acylcarnitines were found. These data suggested a genetic disorder involving intracellular metabolism of cobalamin probably due to methionine synthase (CblG) or methionine synthase reductase (CblE) deficiency. Genomic DNA was isolated from skin fibroblasts. PCR products were purified and sequenced by Sanger sequencing. Molecular studies identified two
Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
vascular, skeletal, or ocular symptoms and she has not required blood transfusions.
Case 2
Figure 2 (A) Bone marrow aspirate. May-Grünwald-Giemsa ×1000. Bone marrow smear showing erythroblasts with defective hemoglobinization and containing chromatin remains. (B) Bone marrow aspirate. May-Grünwald-Giemsa ×1000. Macrocytic erythroblasts with marked dysmorphia, chromatin remains, and less frequently basophilic stippling in orthochromatic erythroblasts.
mutations in the MTRR gene, one already described, c.1361C > T, whose predictable effect on the protein is the change of serine to leucine in position 454 of the protein ( p.Ser454Leu) and another mutation, not previously described in literature, c.1677-1G > A. The novel mutation leads to a frameshift and to a formation of a premature termination codon ( p.Glu560fs), confirming CblE deficiency. Treatment with hydroxicobalamin 1 mg intramuscular daily and folic acid 5 mg orally once a day was established, with partial correction of hematimetric parameters ( persistently elevated MCV, gradual disappearance of dysmorphia in peripheral blood smears, and increased levels of hemoglobin – median of 112 g/l). However, due to the persistence of hyperhomocysteinemia, betaine at 75 mg/kg/day was orally administered, subsequently achieving normal methionine levels (16 μmol/l) but maintaining elevated homocysteine levels (87 μmol/l). Currently, the patient is 8 years old and remains asymptomatic, with normal estaturo-ponderal development and school adjustment. She has not developed neurologic,
A 27-year-old female non-consanguineous parents with non-family history of genetic disorders, presented at 2 years of age a severe macrocytic anemia with hemoglobin 74 g/l, MCV 124 fl, reticulocytes 31.4 × 109/l, and normal platelets and leukocytes counts. Pronounced macro-ovalocytes and poikilocytosis with basophilic stippling in erythrocytes was observed. Indirect bilirubin was 3.2 mg/dl, lactate dehydrogenase 344 U/l, haptoglobin 7 μmol/l, vitamin B12 313 ng/l, and folic acid 6.1 ng/ml. Additionally, direct Coombs test was negative and both enzymatic and osmotic fragility tests and hemoglobinopathies by HPLC were normal. Bone marrow examination showed well-represented normocellular hematopoiesis and 41% of erythroid series with marked dysmorphia, macrocytosis, chromatin remains, and, less frequently, basophilic stippling and multinucleated cells, considering the possibility of a congenital dyserythropoiesis (see Fig. 2B). At the age of 10 years, she developed a progressive cognitive impairment mainly affecting executive functions and attention. She also developed a mixed axonal polyneuropathy predominantly restricting the ability to walk. The magnetic resonance imaging revealed cortical atrophy and hyper-intense periventricular lesions in white matter classified as non-specific ischemic leukoencephalopathy. She received red blood cells transfusions with development of secondary hemochromatosis. The patient underwent treatment with intramuscular cyanocobalamin and pyridoxine irregularly. Due to increased transfusion requirements in the last 2 years, the diagnosis assays focused on congenital disorders involving intracellular metabolism of vitamin B12. Increased homocysteine levels (117 μmol/l) and hipomethioninemia (3 μmol/ l), as well as normal excretion of methylmalonic acid in urine were found. Genetic analysis of MTRR gene identified a single homozygosis mutation, c.1361C > T ( p.Ser545Leu). DNA was isolated from venous blood using the QIAamp DNA Blood. PCR products were purified and subsequently sequenced by Sanger sequencing. Her parents displayed the same heterozygous mutation, which helped to ensure the diagnosis of methionine synthase reductase deficiency. Treatment with intramuscular hydroxocobalamin 10 mg weekly and folic acid 5 mg daily was initiated without reaching steady levels of hemoglobin out of transfusional range. Therefore, the dose of hydroxocobalamin was increased up to 20 mg weekly, and betaine 1500 mg twice a day and 15 mg folinic acid per day were added, all of which allowed
Hematology
2015
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Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
the hemoglobin levels to reach over 101 g/l. Moreover, partial improvements were detected in levels of metabolic disorders, with methionine levels in the range of 13 μmol/l with persistence of high levels of homocysteine (91 μmol/l in her last follow-up). The neurological disorders did not recover with treatment.
Discussion The etiology of macrocytic anemias in childhood is extensive. Once vitamin B12 and folate deficiencies and non-classifiable congenital dyserythropoietic anemias are discarded,11 inherited disorders of intracellular metabolism of vitamin B12 and folic acid should be considered with the corresponding metabolic studies, mainly including the determination of amino acids in blood and organic acids in urine, as well as the supplementary determination of acylcarnitines by tandem mass spectrometry. From a clinical point of view, there is a wide spectrum of manifestations. In this regard, the difference in the clinical course of both patients presented in the current report is noteworthy. The first one was completely asymptomatic and the second developed a severe neurological disorder. Noteworthy, the homozygous mutation c.1361C > T has been associated with mild neurological disorders,12 which is in contrast to the severity of the case provided. A persistent hyperhomocysteinemia, high MCV and/or therapeutic refractory even in the absence of neurological symptoms, may indicate a mild form of CBlE. CBlE-affected individuals require treatment with high doses of parenteral hydroxocobalamin,13 which acts through the MTR, inducing remethylation of homocysteine to methionine, regardless of the decreased MTRR activity. The normalization of methionine levels is possible while normal homocysteine levels are more difficult to achieve.14 More complex is to obtain an improvement in neurological signs and symptoms once established.15 In case 2, the absence of neurological symptoms in early life and the initial misdiagnosis of congenital dyserythropoietic anemia led to a delay in the initiation of specific therapy. These aspects could contribute to the neurological impairment and therefore, despite the introduction of treatment, the neurologic damage has persisted. Nevertheless, in case 1, early therapeutic initiation has been able to prevent the onset of these symptoms. The persistence of hyperhomocysteinemia may justify the addition of betaine treatment, which would force its remethylation to methionine.16 Betaine is typically given as betaine anhydrous form. This substance acts as methyl group donor and substrate for the enzyme betaine-homocysteine methyltransferase that catalyzes remethylation of homocysteine to methionine by a cobalamin-independent pathway, offering an
4
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additional therapeutic alternative. However, the normalization of homocysteine levels is not always achieved.14 At birth, performing the expanded screening of inborn errors of metabolism by tandem mass spectrometry could detect a decrease in methionine, but its sensitivity is low and it is always necessary to carry out other confirmatory tests. In the case of MTRR deficiency, hyperhomocysteinemia associated to hypomethioninemia and normal levels of methylmalonic acid in urine are characteristic of this CBlE subtype. The contribution of new cases of this rare disease will allow more accurate and early diagnosis, and the administration of treatment that could limit the development of irreversible organ damage.
Disclaimer statements Contributors Ruiz-Mercado collected data and wrote the manuscript. Herrera Díaz-Aguado, Vargas, and Pérez-Simón contributed to the writing and critical reading of the manuscript. Bernal made photographs and outlined the basic ideas of the paper. Pérez de Soto, Bueno Delgado, and Delgado Pecellín participated in the management, treatment, and diagnosis of patients. Conde Sánchez conducted the biochemical analysis. Funding None. Conflicts of interest The authors declare that they have no conflict of interest. Ethics approval Ethical approval was not required. The article does not contain any studies with human or animal subjects performed by the any of the authors. All authors concur with the submission. This material has not been previously published and is not under consideration for publication elsewhere. Permission for the publication of personal information form and informed consents have been completed by both patients.
References 1 Michael Whitehead V. Acquired and inherited disorders of cobalamin and folate in children. Br J Haematol. 2006;134(2): 125–36. 2 Watkins D, Rosenblatt DS. Inborn errors of cobalamin absorption and metabolism. Am J Med Genet C Semin Med Genet. 2011;157C(1):33–44. Doi:10.1002/ajmg.c.30288. Epub 2011 Feb 10. 3 Watkins D, Rosenblatt DS. Lessons in biology from patients with inborn errors of vitamin B12 metabolism. Biochimie 2013;95(5):1019–22. Doi:10.1016/j.biochi.2013.01.013. Epub 2013 Feb 10. 4 Gherasim C, Lofgren M, Banerjee R. Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin. J Biol Chem. 2013;288(19):13186–93. Doi:10.1074/jbc.R113.458810. Epub 2013 Mar 28. 5 Zavadakova P, Fowler B, Suormala T, Novotna Z, Mueller P, Hennermann JB, et al. CblE type of homocystinuria due to methionine synthase reductase deficiency: functional correction by minigene expression. Hum Mutat. 2005;25(3):239–47.
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6 Skovby F. Disorders of sulfur amino acids. In: Blau N, Duran M, Blackovies ME, Gibson KB, (eds.) Physician’s guide to the laboratory diagnosis metabolic diseases. Berlin: Springer-Verlag; 2003. p. 243–60. 7 Palanca D, García-Cazorla A, Ortiz J, Jou C, Cusí V, Suñol M, et al. CblE- type homocystinuria presenting with features of haemolytic-uremic syndrome in the newborn period. JIMD Rep. 2013;8:57–62. Doi:10.1007/8904_2012_161. Epub 2012 Jul 21. 8 Watkins D, Rosenblatt DS. Genetic heterogeneity among patients with methylcobalamin deficiency. Definition of two complementation groups, cblE and cblG. J Clin Invest. 1988; 81(6):1690–4. 9 Müller P, Horneff G, Hennermann JB. A rare inborn error of intracellular processing of cobalamin presenting with microcephalus and megaloblastic anemia: a report of 3 children. Klin Padiatr. 2007;219(6):361–7. 10 Harding C, Arnold G, Barness L, Wolff J, Rosenblatt D. Functional methionine synthase deficiency due to cblG disorder: a report of two patients and a review. Am J Med Genet. 1997; 71(4):384–90.
Methionine synthase reductase deficiency (CblE)
11 Renella R, Wood WG. The congenital dyserythropoietic anemias. Hematol Oncol Clin North Am. 2009;23(2):283–306. Doi:10.1016/j.hoc.2009.01.010. 12 Vilaseca MA, Vilarinho L, Zavadakova P, Vela E, Cleto E, Pineda M, et al. CblE type of homocytinuria: mild clinical phenotype in two patients homozygous for a novel mutation in the MTRR gene. J Inherit Metab Dis. 2003;26(4):361–9. 13 Watkins D, Rosenblatt DS. Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity. Am J Med Genet. 1989;34(3):427–34. 14 Schiff M, Benoist JF, Tilea B, Royer N, Giraudier S, Ogier de Baulny H. Isolated remethylation disorders: do our treatments benefit patients? J Inherit Metab Dis. 2011;34(1):137–45. Doi: 10.1007/s10545-010-9120-8. Epub 2010 May 21. 15 Huemer M, Bürer C, Ješina P, Kožich V, Landolt MA, Suormala T, et al. Clinical onset and course, response to treatment and outcome in 24 patients with the cblE or cblG remethylation defect complemented by genetic and in vitro enzyme study data. J Inherit Metab Dis. 2014. [Epub ahead of print]. 16 García MC, Baldellou A. Homocystinurias, a great stranger? Keys for the primary care diagnosis. Acta PediatrEsp. 2009;67(11):535–41.
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Hematology Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/ CISC, Seville, Spain, 2Clinical Laboratory Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CISC, Seville, Spain, 3Pediatrics Department, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CISC, Seville, Spain Importance: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment. Clinical presentation: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them. Intervention: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677–1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency. Conclusion: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme. Keywords: Methionine synthase reductase, Cobalamine E deficiency, Hyperhomocysteinemia, Megaloblastic anemia, Hypomethioninemia
Introduction Inborn errors of intracellular metabolism of vitamin B12 include a wide spectrum of congenital disorders of autosomal recessive inheritance that greatly vary from clinical and therapeutic perspectives. The three groups are: (1) methylmalonyl-CoA reductase, which uses adenosylcobalamin as a cofactor (CblA, CblB, and CblD subtype 2), (2) methionine synthase using methylcobalamin as a cofactor (CblD subtype 1 CblE and CblG), and (3) involvement of both enzymatic pathways (CblC, CblD, CblF, and CblJ).1–3 The methionine synthase reductase enzyme is required for the reductive methylation of methionine synthase (MTR) – which uses CblG as a cofactor – responsible for remethylation of homocysteine to methionine.2,4 The subtype CblE, rarely reported in Correspondence to: A. Herrera Díaz-Aguado, Hospital Universitario Virgen del Rocío-Virgen Macarena, Avda. Manuel Siurot, s/n° 41013 Seville, Spain. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845415Y.0000000017
literature, is due to mutations in the gene that encodes for methionine synthase reductase enzyme (MTRR). To date, 19 different mutations have been identified.5 It mainly manifests as a macrocytic anemia, with or without megaloblastic features, estaturo-ponderal retardation, and neurological disorders, encompassing from cognitive disorders to neurodegenerative encephalopathy.6,7 The most striking finding in all patients is the presence of hyperhomocysteinemia with normal levels of methylmalonic acid and usually low levels of methionine. This classic triad defines the subtypes CblG and CblE whose final diagnosis must be confirmed by molecular assays8 (see Fig. 1). Early treatment with intramuscular hydroxocobalamin improves the outcome by correction of the macrocytic anemia and the almost complete disappearance of cytological dysmorphias and achieves transfusion independence. However, the impact is lower in biochemical parameters and generally
Hematology
2015
1
Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
Figure 1 Algorithm for the diagnosis of disorders of intracellular cobalamin metabolism. CblC: cobalamin C, CblD: cobalamin D, CblF: cobalamin F, CblA: cobalamin A, CblB: cobalamin B, CblE: cobalamin E, CblG: cobalamin G, AdoCl: adenosil-cobalamin, and MetilCl: metil-cobalamin.
hyperhomocysteinemia persists over time.9 The established neurological disorders do not reverse although their prevention is possible with the early administration of specific treatment.10 In the current report we describe the clinical, molecular, metabolic and cytological characteristics of two new cases of MTRR deficiency. Given the finding of high levels of serum homocysteine, the study focused on congenital disorders of intracellular cobalamin metabolism. The diagnosis was confirmed by molecular assays, revealing, in one case, a new mutation unknown to date associated to more benign course.
Case presentation Case 1 The patient, an 8-year-old girl with non-consanguineous parents and non-family history of genetic disorders, presented at 3 months of age a severe anemia requiring several blood transfusions. The laboratory findings are as follows: hemoglobin (Hb) 45.8 g/l, mean corpuscular volume (MCV) 84.2 fl, mean corpuscular hemoglobin (MCH) 30.1 pg/l, and reticulocytes 71 × 10 e9/l. The leukocyte and platelet counts were normal. The erythroid morphology highlighted an important poikilocytosis with macro-ovalocytes, basophilic stippling, and 3% of erythroblasts. In addition, total bilirubin was 5.7 mg/dl with indirect bilirubin of 3.8 mg/dl and haptoglobin of 2 mg/dl.
2
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2015
Folic acid levels were 7 ng/ml and vitamin B12 205 ng/l, both within the normal range. Study of hemoglobin by high-pressure liquid chromatography (HPLC), direct Coombs test, glucose 6-phosphate dehydrogenase, sucrose, and Ham tests were all normal or negative. Bone marrow aspirate revealed normal cellularity, properly represented hematopoiesis, and 17% of erythroid series with macrocytic features, chromatin remains, and basophilic stippling in orthochromatic normoblasts, multinucleated cells being outstanding. These findings supported a non-classifiable congenital dyserythropoiesis (see Fig. 2A). In the analytical follow-up, hemoglobin ranged from 103 to 136 g/l with a median of MCV 103.6 fl and red cell morphology with predominance of macro-ovalocytes. The patient was re-examined, extending the study of anemia to determine the cause of persistent megaloblastosis. Homocysteine levels were 50.7 μmol/l (normal: 0–16 μmol/l), methionine 10.6 μmol/l (15–27 μmol/l) while normal levels of methylmalonic acid in urine and serum acylcarnitines were found. These data suggested a genetic disorder involving intracellular metabolism of cobalamin probably due to methionine synthase (CblG) or methionine synthase reductase (CblE) deficiency. Genomic DNA was isolated from skin fibroblasts. PCR products were purified and sequenced by Sanger sequencing. Molecular studies identified two
Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
vascular, skeletal, or ocular symptoms and she has not required blood transfusions.
Case 2
Figure 2 (A) Bone marrow aspirate. May-Grünwald-Giemsa ×1000. Bone marrow smear showing erythroblasts with defective hemoglobinization and containing chromatin remains. (B) Bone marrow aspirate. May-Grünwald-Giemsa ×1000. Macrocytic erythroblasts with marked dysmorphia, chromatin remains, and less frequently basophilic stippling in orthochromatic erythroblasts.
mutations in the MTRR gene, one already described, c.1361C > T, whose predictable effect on the protein is the change of serine to leucine in position 454 of the protein ( p.Ser454Leu) and another mutation, not previously described in literature, c.1677-1G > A. The novel mutation leads to a frameshift and to a formation of a premature termination codon ( p.Glu560fs), confirming CblE deficiency. Treatment with hydroxicobalamin 1 mg intramuscular daily and folic acid 5 mg orally once a day was established, with partial correction of hematimetric parameters ( persistently elevated MCV, gradual disappearance of dysmorphia in peripheral blood smears, and increased levels of hemoglobin – median of 112 g/l). However, due to the persistence of hyperhomocysteinemia, betaine at 75 mg/kg/day was orally administered, subsequently achieving normal methionine levels (16 μmol/l) but maintaining elevated homocysteine levels (87 μmol/l). Currently, the patient is 8 years old and remains asymptomatic, with normal estaturo-ponderal development and school adjustment. She has not developed neurologic,
A 27-year-old female non-consanguineous parents with non-family history of genetic disorders, presented at 2 years of age a severe macrocytic anemia with hemoglobin 74 g/l, MCV 124 fl, reticulocytes 31.4 × 109/l, and normal platelets and leukocytes counts. Pronounced macro-ovalocytes and poikilocytosis with basophilic stippling in erythrocytes was observed. Indirect bilirubin was 3.2 mg/dl, lactate dehydrogenase 344 U/l, haptoglobin 7 μmol/l, vitamin B12 313 ng/l, and folic acid 6.1 ng/ml. Additionally, direct Coombs test was negative and both enzymatic and osmotic fragility tests and hemoglobinopathies by HPLC were normal. Bone marrow examination showed well-represented normocellular hematopoiesis and 41% of erythroid series with marked dysmorphia, macrocytosis, chromatin remains, and, less frequently, basophilic stippling and multinucleated cells, considering the possibility of a congenital dyserythropoiesis (see Fig. 2B). At the age of 10 years, she developed a progressive cognitive impairment mainly affecting executive functions and attention. She also developed a mixed axonal polyneuropathy predominantly restricting the ability to walk. The magnetic resonance imaging revealed cortical atrophy and hyper-intense periventricular lesions in white matter classified as non-specific ischemic leukoencephalopathy. She received red blood cells transfusions with development of secondary hemochromatosis. The patient underwent treatment with intramuscular cyanocobalamin and pyridoxine irregularly. Due to increased transfusion requirements in the last 2 years, the diagnosis assays focused on congenital disorders involving intracellular metabolism of vitamin B12. Increased homocysteine levels (117 μmol/l) and hipomethioninemia (3 μmol/ l), as well as normal excretion of methylmalonic acid in urine were found. Genetic analysis of MTRR gene identified a single homozygosis mutation, c.1361C > T ( p.Ser545Leu). DNA was isolated from venous blood using the QIAamp DNA Blood. PCR products were purified and subsequently sequenced by Sanger sequencing. Her parents displayed the same heterozygous mutation, which helped to ensure the diagnosis of methionine synthase reductase deficiency. Treatment with intramuscular hydroxocobalamin 10 mg weekly and folic acid 5 mg daily was initiated without reaching steady levels of hemoglobin out of transfusional range. Therefore, the dose of hydroxocobalamin was increased up to 20 mg weekly, and betaine 1500 mg twice a day and 15 mg folinic acid per day were added, all of which allowed
Hematology
2015
3
Ruiz-Mercado et al.
Methionine synthase reductase deficiency (CblE)
the hemoglobin levels to reach over 101 g/l. Moreover, partial improvements were detected in levels of metabolic disorders, with methionine levels in the range of 13 μmol/l with persistence of high levels of homocysteine (91 μmol/l in her last follow-up). The neurological disorders did not recover with treatment.
Discussion The etiology of macrocytic anemias in childhood is extensive. Once vitamin B12 and folate deficiencies and non-classifiable congenital dyserythropoietic anemias are discarded,11 inherited disorders of intracellular metabolism of vitamin B12 and folic acid should be considered with the corresponding metabolic studies, mainly including the determination of amino acids in blood and organic acids in urine, as well as the supplementary determination of acylcarnitines by tandem mass spectrometry. From a clinical point of view, there is a wide spectrum of manifestations. In this regard, the difference in the clinical course of both patients presented in the current report is noteworthy. The first one was completely asymptomatic and the second developed a severe neurological disorder. Noteworthy, the homozygous mutation c.1361C > T has been associated with mild neurological disorders,12 which is in contrast to the severity of the case provided. A persistent hyperhomocysteinemia, high MCV and/or therapeutic refractory even in the absence of neurological symptoms, may indicate a mild form of CBlE. CBlE-affected individuals require treatment with high doses of parenteral hydroxocobalamin,13 which acts through the MTR, inducing remethylation of homocysteine to methionine, regardless of the decreased MTRR activity. The normalization of methionine levels is possible while normal homocysteine levels are more difficult to achieve.14 More complex is to obtain an improvement in neurological signs and symptoms once established.15 In case 2, the absence of neurological symptoms in early life and the initial misdiagnosis of congenital dyserythropoietic anemia led to a delay in the initiation of specific therapy. These aspects could contribute to the neurological impairment and therefore, despite the introduction of treatment, the neurologic damage has persisted. Nevertheless, in case 1, early therapeutic initiation has been able to prevent the onset of these symptoms. The persistence of hyperhomocysteinemia may justify the addition of betaine treatment, which would force its remethylation to methionine.16 Betaine is typically given as betaine anhydrous form. This substance acts as methyl group donor and substrate for the enzyme betaine-homocysteine methyltransferase that catalyzes remethylation of homocysteine to methionine by a cobalamin-independent pathway, offering an
4
Hematology
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additional therapeutic alternative. However, the normalization of homocysteine levels is not always achieved.14 At birth, performing the expanded screening of inborn errors of metabolism by tandem mass spectrometry could detect a decrease in methionine, but its sensitivity is low and it is always necessary to carry out other confirmatory tests. In the case of MTRR deficiency, hyperhomocysteinemia associated to hypomethioninemia and normal levels of methylmalonic acid in urine are characteristic of this CBlE subtype. The contribution of new cases of this rare disease will allow more accurate and early diagnosis, and the administration of treatment that could limit the development of irreversible organ damage.
Disclaimer statements Contributors Ruiz-Mercado collected data and wrote the manuscript. Herrera Díaz-Aguado, Vargas, and Pérez-Simón contributed to the writing and critical reading of the manuscript. Bernal made photographs and outlined the basic ideas of the paper. Pérez de Soto, Bueno Delgado, and Delgado Pecellín participated in the management, treatment, and diagnosis of patients. Conde Sánchez conducted the biochemical analysis. Funding None. Conflicts of interest The authors declare that they have no conflict of interest. Ethics approval Ethical approval was not required. The article does not contain any studies with human or animal subjects performed by the any of the authors. All authors concur with the submission. This material has not been previously published and is not under consideration for publication elsewhere. Permission for the publication of personal information form and informed consents have been completed by both patients.
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