- Email: [email protected]
Hb M-Iwate in an Indian family
Clinica Chimica Acta 446 (2015) 192–194
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Hb M-Iwate in an Indian family Ganesh Kumar V a, Prashant Sharma a, Sanjeev Chhabra a, Jasbir Kaur Hira a, Amita Trehan b, Reena Das a,⁎ a b
Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 21 April 2015 Accepted 21 April 2015 Available online 27 April 2015 Keywords: Hemoglobin M-Iwate Methemoglobin Alpha globin variant Congenital cyanosis Polymerase chain reaction restriction fragment length polymorphism Gene sequencing
a b s t r a c t Background: High performance liquid chromatography in a newborn girl with congenital cyanosis and a unilateral cleft palate revealed a variant hemoglobin with retention time of 4.8 min, similar to hemoglobin Q-India. Since hemoglobin Q-India did not explain the cyanosis, further investigations were initiated. Methods: Sequencing of α-globin genes revealed hemoglobin M-Iwate ([α87 (F8) His→Tyr]) that was confirmed on restriction enzyme analysis. Results: Hemoglobin M-Iwate is a rare methemoglobinemic variant formed due to a point mutation in the α-globin gene. Primarily reported from the Iwate prefecture of Japan, there have been occasional case reports from other regions as well. Inherited methemoglobinemia finds only rare mention in Indian literature while hemoglobin M-Iwate has not been reported from India. Conclusions: This case illustrates the step-wise logical diagnostic approaches necessary to elucidate the cause of methemoglobinemia in an otherwise healthy child with cyanosis.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction Common causes of methemoglobinemia include oxidant drugs and chemicals, especially in the setting of G6PD deficiency. Rare cases are due congenital deficiency of nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase or due to the presence of variant hemoglobins (HbMs) [1,2]. A total of 10 variant HbMs are described of which 4 are β-globin variants (Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, Hb M-Milwaukee-2), 4 are α1 or α2 globin variants (Hb M-Boston, Hb Auckland, Hb M-Iwate, Hb M-Yantai) [3] and 2 are G γ globin variants (Hb F-M-Osaka, Hb F-M-Fort Ripley) [4]. Mutations in HbM result in altered amino acid constitution of the heme-binding pocket of the hemoglobin (Hb) molecule. The majority of these mutations lead to substitution of the proximal or distal histidine residues with tyrosine [1]. Amino acid substitutions at these critical hemebinding sites result in irreversible or prolonged oxidation of iron from ferrous (Fe2 +) to ferric (Fe3 +) state. The amino acid substitution as well as the ferric iron alters the physicochemical properties of the variant hemoglobins like oxygen affinity (P50), Bohr effect, electrophoretic migration, chromatographic affinities and absorption spectra [2]. HbM is a rare event and, to the best of our knowledge, only three case reports have been described from India [5]. We recently ⁎ Corresponding author at: Department of Hematology, Level 5, Research Block A, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India. Tel.: +91 172 2755128; fax: +91 172 2744401. E-mail address: [email protected] (R. Das).
http://dx.doi.org/10.1016/j.cca.2015.04.031 0009-8981/© 2015 Elsevier B.V. All rights reserved.
encountered three generations of a family who presented with familial congenital central cyanosis. They were diagnosed as HbM-Iwate that is previously undescribed from India. The rarity of the condition and the overlapping high performance liquid chromatography (HPLC) pattern with Hb Q-India are discussed in this report. 2. Case report A newborn girl of the Punjabi Chhimba community, born from a non-consanguineous marriage after an uneventful pregnancy was noticed to have unilateral cleft palate and central cyanosis. She was otherwise healthy with a normal birth weight, APGAR score with no other obvious congenital anomalies. The father reported similar longstanding cyanosis as did the paternal grandmother, indicating a likely autosomal dominant disorder. Neither had been worked up previously, and both were in good health. Hemoglobin HPLC of the child showed fetal hemoglobin (Hb F) 56.1% (normal for age), elevated Hb A2 of 4.2% and a small variant peak (3.7%) with retention time 4.8 min (Fig. 1A). Further work-up could not be done as the child was lost to follow-up. She was brought again to the hospital at the age of 3-y for surgical correction of the cleft palate and was referred to the Hematology Department since the cyanosis persisted. There was no history to suggest a cardiac/respiratory condition and drug or chemical-induced cyanosis was unlikely in view of its persistence over 3 y. On examination, in addition to that palatal cleft, the child had mild pallor and brownish to slate-gray discoloration of skin and mucous
G. Kumar V et al. / Clinica Chimica Acta 446 (2015) 192–194
A
B
193
C
Fig. 1. A: Hemoglobin HPLC of proband at 10 days shows predominantly HbF (normal for age). HbA2 is raised (4.2%). A variant Hb peak (3.7%) is seen with retention time of 4.8 min. B: Hemoglobin HPLC of proband at three years of age. This shows a variant hemoglobin peak of 13.2% with retention time of 4.8 min. The HbA2 level (3.4%) is normal at this stage. The amount of HbA0 is 75.8%. The hemoglobin HPLC of the father and paternal grandmother were essentially similar. C: Hemoglobin HPLC of a heterozygous Hb Q India for comparison. Heterozygous state for Hb Q India has similar HPLC peak, retention time and relative hemoglobin percentage and is difficult to distinguish from HbM-Iwate based on HPLC findings alone.
membranes. Other general physical and systemic examinations were normal. Specifically, no icterus, hepato-splenomegaly, edema, raised jugular venous pressure, cardiac murmurs or basal crepitations were noted. Chest roentgenogram, electrocardiogram and echocardiogram were normal. Hemogram at 10 days of age had shown Hb 156 g/l with normal RBC indices for age. Peripheral blood showed normocytic normochromic red cells. Complete blood counts repeated at 3 years showed Hb 119 g/l, red cell count 5.93 × 1012/l, mean corpuscular volume (MCV) 65.1 fl, mean corpuscular hemoglobin (MCH) 20.1 pg, mean corpuscular hemoglobin concentration (MCHC) 30.9 g/l, and red cell distribution width (RDW-CV) 36.8% with normal reticulocyte count, total and differential leukocyte counts and platelet count. A blood film showed mild red cell anisopoikilocytosis with hypochromic microcytes and a few ovalocytes. Complete blood counts of the father and paternal grandmother were within normal limits. Hemoglobin HPLC was performed again at the second presentation twice using Bio-Rad® Variant™ and Variant II™ platforms (Bio-Rad laboratories) utilizing the β-Thalassemia Short™ program. It now showed 75.8% HbA0, normal HbA2 (3.4%) and a variant Hb peak (13.2%) with retention time of 4.80 min (Fig. 1B). An unstable Hb (unlikely due to the absence of reticulocytosis) was excluded by negative heat and isopropanolol instability tests. The low percentage of the unknown HPLC peak suggested an α-globin chain variant. One possibility was that the HPLC had simply uncovered an incidental heterozygous state for HbQ-India, an asymptomatic α-chain variant relatively common in northern India, whose retention time is usually around 4.73 min. However, Hb Q-India did not explain the child's cyanosis. Hb electrophoresis at alkaline pH performed in an Interlab Genio S® automated electrophoresis instrument (Via Rina Monti) showed a faint fast-moving band just anodal and almost merging with the HbA band. Electrophoresis at acidic pH was normal. Hb HPLC and electrophoreses of the father and maternal grandmother were essentially similar to that of the child done at 3 y. Spectrophotometry on the total Hb was performed with readings from 400 to 700 nm and the result appeared normal. Since the exact nature of the variant remained unclear, genetic studies were initiated. Genomic DNA was extracted from peripheral blood leukocytes of the child using phenol-chloroform technique. HBB, HBA2, and HBA1 genes were amplified with specific primers. Automated DNA sequencing (ABI 3130 Genetic Analyzer, Applied Biosystems) was performed for the β-globin gene and both α1 and α2 globin genes for the entire coding exons 1–3 including the consensus sequences, promoter region and the 3′ poly-A tail. Electrophoregram of exon 2 of the α1 globin gene showed a heterozygous mutation CAC N TAC at codon 262 (His N Tyr). Literature search revealed this to be the responsible mutation for the methemoglobinemic variant HbM-Iwate, thus explaining the child's cyanosis. The variant
hemoglobin can arise due to this point mutation either in α1 or α2 globin genes [6]. To confirm the sequencing finding, a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) test for the Hb M-Iwate mutation was also performed as described by Horst et al. [6]. The enzyme endonuclease RsaI does not digest the wild HBA1 gene while a restriction site (GT^AC) is created by the mutation. Digestion of the mutant allele results in 2 extra bands which were detected in our case. 3. Discussion Hb M Iwate arises due to a point mutation in either α1 or α2 globin chains causing substitution of a thymidine (T) instead of cytosine (C) in codon 262 i.e. HBA1 (or HBA2): codon 262C N T and a resultant substitution of tyrosine instead of histidine in position 87 of the α-globin protein [4,6,7]. It was first characterized in the Iwate prefecture of Japan in 1960 where terms like ‘black mouth’, ‘black blood’ or ‘black child’ were used historically to denote the cyanosis in these individuals [2,8,9]. A few case reports have subsequently also been published from Europe [10,11,12], Turkey [13] and Brazil [14]. Hb M-Iwate was previously also christened as Hb M-Oldenburg, M-Kankakee and M-Sendai, all of which after molecular characterization were found to be the same variant [4,15,16]. HbMs show autosomal dominant transmission as was observed in this family. Congenital central cyanosis is a known phenomenon in affected individuals. Congenital cyanosis is the usual presentation although many individuals remain undiagnosed till late in their lifetime as there is no significant impairment of day-to-day activities [17]. Unlike the usual cyanosis due to deoxyhemoglobin, in HbMs, the altered visible spectrum of the abnormal hemoglobin is responsible for the brownish/ slate-gray skin color. Hence, the term “pseudocyanosis” has been used [2,9]. Hb M-Iwate however, has reduced oxygen affinity and therefore some degree of true cyanosis is also present [9]. Unlike methemoglobinemia due to oxidant damage or enzyme deficiency, HbMs do not cause severe symptoms. However, it is important to correctly identify them as it helps avoid misdiagnosis, overinvestigation and unnecessary treatment [1]. Even for individuals with obvious cosmetic effects, unfortunately, no definite treatment is possible for HbM. Methylene blue used as an antidote for methemoglobinemia arising due to oxidant damage is ineffective in HbM [18]. Literature on HbMs from India is limited to only a few case reports [5,19,20]. A β-globin variant, Hb M-Ratnagiri [5] was previously reported as arising from the substitution of histidine by tyrosine at the β63 position, similar to Hb-M Saskatoon. Our report of HbM-Iwate from India highlights the familial autosomal dominant inheritance and the clinical presentation of congenital cyanosis. The Hb HPLC pattern of HbM-Iwate can be confused with HbQ-India which is fairly commonly encountered
194
G. Kumar V et al. / Clinica Chimica Acta 446 (2015) 192–194
in northern India. This highlights the importance of confirming all screen-detected abnormal hemoglobins by a second independent technique. Indeed, a complete work-up is important even for minimally symptomatic Hb variants, as rendering an accurate diagnosis of HbMIwate avoided further needless work-up of our patient. And finally, the case serves as a reminder that even among genetic diseases like hemoglobinopathies that show regional specificity, exotic variants are occasionally encountered, presumably arising from de novo mutations. Acknowledgment We thank Dr. Preethy J. Mathew, Associate Professor, Department of Anaesthesia, Postgraduate Institute of Medical Education and Research, Chandigarh, India who first suspected an inherited hemoglobinopathy in this child and initiated the diagnostic work-up. References [1] Steinberg MH. Disorders of hemoglobin: genetics, pathophysiology, and clinical management. 2nd ed. Cambridge: Cambridge University Press; 2009. [2] Wintrobe MM, Greer JP. Wintrobe's clinical hematology. 12th ed. Philadelphia l London: Wolters Kluwer Health/lippincott Williams & Wilkins; 2009. [3] Sun Y, Wang P, Li Y, et al. Familial congenital cyanosis caused by HbM(Yantai)(alpha-76 GAC – N TAC, Asp – N Tyr). Genet Mol Biol 2010, Jul;33(3): 445–8. [4] University PS. HbVar: a database of human hemoglobin variants and thalassemias. [cited 2014 Aug 04]; Available from: http://globin.bx.psu.edu/cgi-bin/hbvar/ counter; 2014. [5] Kedar PS, Nadkarni AH, Phanasgoankar S, et al. Congenital methemoglobinemia caused by Hb-MRatnagiri (beta-63CAT– N TAT, His– N Tyr) in an Indian family. Am J Hematol 2005, Jun;79(2):168–70.
[6] Horst J, Assum G, Griese EU, Eigel A, Hampl W, Kohne E. Hemoglobin M Iwate is caused by a C––T transition in codon 87 of the human alpha 1-globin gene. Hum Genet 1987, Jan;75(1):53–5. [7] Orisaka M, Sasaki T, Kato J, Harano K, Harano T. [Hb M-Iwate [alpha 87 (F8) His– N Tyr]: analysis of the genomic DNA and biosynthesis]. Rinsho Byori 1995, Mar;43(3):295–9. [8] Percy MJ, McFerran NV, Lappin TR. Disorders of oxidised haemoglobin. Blood Rev 2005, Mar;19(2):61–8. [9] Bain BJ. Haemoglobinopathy diagnosis. Malden, Mass2nd ed. . Oxford: Blackwell; 2006. [10] Pik C, Tonz O. Nature of haemoglobin M-Oldenburg. Nature 1966, Jun 11;210(5041): 1182. [11] Maggio A, Massa A, Giampaolo A, Mavilio F, Tentori L. Occurrence of Hb M Iwate (alpha 2 87 His leads to Tyr beta 2) in an Italian carrier. Hemoglobin 1981;5(2): 205–8. [12] Mayne EE, Elder GE, Lappin TR, Ferguson LA. Hb M Iwate [alpha (2)87His––Tyr beta 2]: de novo mutation in an Irish family. Hemoglobin 1986;10(2):205–8. [13] Ozsoylu S. Congenital methemoglobinemia due to hemoglobin M. Acta Haematol 1972;47(4):225–32. [14] Viana MB, Belisario AR. De novo alpha 2 hemoglobin gene (HBA2) mutation in a child with hemoglobin M Iwate and symptomatic methemoglobinemia since birth. Rev Bras Hematol Hemoter 2014, May-Jun;36(3):230–4. [15] Steffens G, Buse G. Hemoglobin M Oldenburg identified as HB alpha 2 87(F8)His replaced by Tyr beta 2. Hoppe Seylers Z Physiol Chem 1977, Jan;358(1):35–8. [16] Heller P, Weinstein HG, Yakulis VJ, Rosenthal IM. Hemoglobin M Kankakee, a new variant of hemoglobin M. Blood 1962, Sep;20:287–301. [17] Kuji A, Satoh Y, Kikuchi K, Satoh K, Joh S. The anesthetic management of a patient with hemoglobin M(Iwate). Anesth Analg 2001, Nov;93(5):1192–3 (table of contents). [18] Melarkode K, Prinzhausen H. Hemoglobin M variant and congenital methemoglobinemia: methylene blue will not be effective in the presence of hemoglobin M. Can J Anaesth 2008, Feb;55(2):129–30. [19] Bajaj RT, Malik RM, Desai M, Sukumaran PK. Hemoglobin-M disease. Indian Pediatr 1973, Jun;10(6):383–5. [20] Das KC, Bidwai PS, Mahapatra SS, Sukumaran PK. Haemoglobin-m disease in a Punjabi Hindu family. Indian J Pathol Bacteriol 1975, Jan;18(1):54–60.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Hb M-Iwate in an Indian family Ganesh Kumar V a, Prashant Sharma a, Sanjeev Chhabra a, Jasbir Kaur Hira a, Amita Trehan b, Reena Das a,⁎ a b
Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 21 April 2015 Accepted 21 April 2015 Available online 27 April 2015 Keywords: Hemoglobin M-Iwate Methemoglobin Alpha globin variant Congenital cyanosis Polymerase chain reaction restriction fragment length polymorphism Gene sequencing
a b s t r a c t Background: High performance liquid chromatography in a newborn girl with congenital cyanosis and a unilateral cleft palate revealed a variant hemoglobin with retention time of 4.8 min, similar to hemoglobin Q-India. Since hemoglobin Q-India did not explain the cyanosis, further investigations were initiated. Methods: Sequencing of α-globin genes revealed hemoglobin M-Iwate ([α87 (F8) His→Tyr]) that was confirmed on restriction enzyme analysis. Results: Hemoglobin M-Iwate is a rare methemoglobinemic variant formed due to a point mutation in the α-globin gene. Primarily reported from the Iwate prefecture of Japan, there have been occasional case reports from other regions as well. Inherited methemoglobinemia finds only rare mention in Indian literature while hemoglobin M-Iwate has not been reported from India. Conclusions: This case illustrates the step-wise logical diagnostic approaches necessary to elucidate the cause of methemoglobinemia in an otherwise healthy child with cyanosis.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction Common causes of methemoglobinemia include oxidant drugs and chemicals, especially in the setting of G6PD deficiency. Rare cases are due congenital deficiency of nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase or due to the presence of variant hemoglobins (HbMs) [1,2]. A total of 10 variant HbMs are described of which 4 are β-globin variants (Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, Hb M-Milwaukee-2), 4 are α1 or α2 globin variants (Hb M-Boston, Hb Auckland, Hb M-Iwate, Hb M-Yantai) [3] and 2 are G γ globin variants (Hb F-M-Osaka, Hb F-M-Fort Ripley) [4]. Mutations in HbM result in altered amino acid constitution of the heme-binding pocket of the hemoglobin (Hb) molecule. The majority of these mutations lead to substitution of the proximal or distal histidine residues with tyrosine [1]. Amino acid substitutions at these critical hemebinding sites result in irreversible or prolonged oxidation of iron from ferrous (Fe2 +) to ferric (Fe3 +) state. The amino acid substitution as well as the ferric iron alters the physicochemical properties of the variant hemoglobins like oxygen affinity (P50), Bohr effect, electrophoretic migration, chromatographic affinities and absorption spectra [2]. HbM is a rare event and, to the best of our knowledge, only three case reports have been described from India [5]. We recently ⁎ Corresponding author at: Department of Hematology, Level 5, Research Block A, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India. Tel.: +91 172 2755128; fax: +91 172 2744401. E-mail address: [email protected] (R. Das).
http://dx.doi.org/10.1016/j.cca.2015.04.031 0009-8981/© 2015 Elsevier B.V. All rights reserved.
encountered three generations of a family who presented with familial congenital central cyanosis. They were diagnosed as HbM-Iwate that is previously undescribed from India. The rarity of the condition and the overlapping high performance liquid chromatography (HPLC) pattern with Hb Q-India are discussed in this report. 2. Case report A newborn girl of the Punjabi Chhimba community, born from a non-consanguineous marriage after an uneventful pregnancy was noticed to have unilateral cleft palate and central cyanosis. She was otherwise healthy with a normal birth weight, APGAR score with no other obvious congenital anomalies. The father reported similar longstanding cyanosis as did the paternal grandmother, indicating a likely autosomal dominant disorder. Neither had been worked up previously, and both were in good health. Hemoglobin HPLC of the child showed fetal hemoglobin (Hb F) 56.1% (normal for age), elevated Hb A2 of 4.2% and a small variant peak (3.7%) with retention time 4.8 min (Fig. 1A). Further work-up could not be done as the child was lost to follow-up. She was brought again to the hospital at the age of 3-y for surgical correction of the cleft palate and was referred to the Hematology Department since the cyanosis persisted. There was no history to suggest a cardiac/respiratory condition and drug or chemical-induced cyanosis was unlikely in view of its persistence over 3 y. On examination, in addition to that palatal cleft, the child had mild pallor and brownish to slate-gray discoloration of skin and mucous
G. Kumar V et al. / Clinica Chimica Acta 446 (2015) 192–194
A
B
193
C
Fig. 1. A: Hemoglobin HPLC of proband at 10 days shows predominantly HbF (normal for age). HbA2 is raised (4.2%). A variant Hb peak (3.7%) is seen with retention time of 4.8 min. B: Hemoglobin HPLC of proband at three years of age. This shows a variant hemoglobin peak of 13.2% with retention time of 4.8 min. The HbA2 level (3.4%) is normal at this stage. The amount of HbA0 is 75.8%. The hemoglobin HPLC of the father and paternal grandmother were essentially similar. C: Hemoglobin HPLC of a heterozygous Hb Q India for comparison. Heterozygous state for Hb Q India has similar HPLC peak, retention time and relative hemoglobin percentage and is difficult to distinguish from HbM-Iwate based on HPLC findings alone.
membranes. Other general physical and systemic examinations were normal. Specifically, no icterus, hepato-splenomegaly, edema, raised jugular venous pressure, cardiac murmurs or basal crepitations were noted. Chest roentgenogram, electrocardiogram and echocardiogram were normal. Hemogram at 10 days of age had shown Hb 156 g/l with normal RBC indices for age. Peripheral blood showed normocytic normochromic red cells. Complete blood counts repeated at 3 years showed Hb 119 g/l, red cell count 5.93 × 1012/l, mean corpuscular volume (MCV) 65.1 fl, mean corpuscular hemoglobin (MCH) 20.1 pg, mean corpuscular hemoglobin concentration (MCHC) 30.9 g/l, and red cell distribution width (RDW-CV) 36.8% with normal reticulocyte count, total and differential leukocyte counts and platelet count. A blood film showed mild red cell anisopoikilocytosis with hypochromic microcytes and a few ovalocytes. Complete blood counts of the father and paternal grandmother were within normal limits. Hemoglobin HPLC was performed again at the second presentation twice using Bio-Rad® Variant™ and Variant II™ platforms (Bio-Rad laboratories) utilizing the β-Thalassemia Short™ program. It now showed 75.8% HbA0, normal HbA2 (3.4%) and a variant Hb peak (13.2%) with retention time of 4.80 min (Fig. 1B). An unstable Hb (unlikely due to the absence of reticulocytosis) was excluded by negative heat and isopropanolol instability tests. The low percentage of the unknown HPLC peak suggested an α-globin chain variant. One possibility was that the HPLC had simply uncovered an incidental heterozygous state for HbQ-India, an asymptomatic α-chain variant relatively common in northern India, whose retention time is usually around 4.73 min. However, Hb Q-India did not explain the child's cyanosis. Hb electrophoresis at alkaline pH performed in an Interlab Genio S® automated electrophoresis instrument (Via Rina Monti) showed a faint fast-moving band just anodal and almost merging with the HbA band. Electrophoresis at acidic pH was normal. Hb HPLC and electrophoreses of the father and maternal grandmother were essentially similar to that of the child done at 3 y. Spectrophotometry on the total Hb was performed with readings from 400 to 700 nm and the result appeared normal. Since the exact nature of the variant remained unclear, genetic studies were initiated. Genomic DNA was extracted from peripheral blood leukocytes of the child using phenol-chloroform technique. HBB, HBA2, and HBA1 genes were amplified with specific primers. Automated DNA sequencing (ABI 3130 Genetic Analyzer, Applied Biosystems) was performed for the β-globin gene and both α1 and α2 globin genes for the entire coding exons 1–3 including the consensus sequences, promoter region and the 3′ poly-A tail. Electrophoregram of exon 2 of the α1 globin gene showed a heterozygous mutation CAC N TAC at codon 262 (His N Tyr). Literature search revealed this to be the responsible mutation for the methemoglobinemic variant HbM-Iwate, thus explaining the child's cyanosis. The variant
hemoglobin can arise due to this point mutation either in α1 or α2 globin genes [6]. To confirm the sequencing finding, a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) test for the Hb M-Iwate mutation was also performed as described by Horst et al. [6]. The enzyme endonuclease RsaI does not digest the wild HBA1 gene while a restriction site (GT^AC) is created by the mutation. Digestion of the mutant allele results in 2 extra bands which were detected in our case. 3. Discussion Hb M Iwate arises due to a point mutation in either α1 or α2 globin chains causing substitution of a thymidine (T) instead of cytosine (C) in codon 262 i.e. HBA1 (or HBA2): codon 262C N T and a resultant substitution of tyrosine instead of histidine in position 87 of the α-globin protein [4,6,7]. It was first characterized in the Iwate prefecture of Japan in 1960 where terms like ‘black mouth’, ‘black blood’ or ‘black child’ were used historically to denote the cyanosis in these individuals [2,8,9]. A few case reports have subsequently also been published from Europe [10,11,12], Turkey [13] and Brazil [14]. Hb M-Iwate was previously also christened as Hb M-Oldenburg, M-Kankakee and M-Sendai, all of which after molecular characterization were found to be the same variant [4,15,16]. HbMs show autosomal dominant transmission as was observed in this family. Congenital central cyanosis is a known phenomenon in affected individuals. Congenital cyanosis is the usual presentation although many individuals remain undiagnosed till late in their lifetime as there is no significant impairment of day-to-day activities [17]. Unlike the usual cyanosis due to deoxyhemoglobin, in HbMs, the altered visible spectrum of the abnormal hemoglobin is responsible for the brownish/ slate-gray skin color. Hence, the term “pseudocyanosis” has been used [2,9]. Hb M-Iwate however, has reduced oxygen affinity and therefore some degree of true cyanosis is also present [9]. Unlike methemoglobinemia due to oxidant damage or enzyme deficiency, HbMs do not cause severe symptoms. However, it is important to correctly identify them as it helps avoid misdiagnosis, overinvestigation and unnecessary treatment [1]. Even for individuals with obvious cosmetic effects, unfortunately, no definite treatment is possible for HbM. Methylene blue used as an antidote for methemoglobinemia arising due to oxidant damage is ineffective in HbM [18]. Literature on HbMs from India is limited to only a few case reports [5,19,20]. A β-globin variant, Hb M-Ratnagiri [5] was previously reported as arising from the substitution of histidine by tyrosine at the β63 position, similar to Hb-M Saskatoon. Our report of HbM-Iwate from India highlights the familial autosomal dominant inheritance and the clinical presentation of congenital cyanosis. The Hb HPLC pattern of HbM-Iwate can be confused with HbQ-India which is fairly commonly encountered
194
G. Kumar V et al. / Clinica Chimica Acta 446 (2015) 192–194
in northern India. This highlights the importance of confirming all screen-detected abnormal hemoglobins by a second independent technique. Indeed, a complete work-up is important even for minimally symptomatic Hb variants, as rendering an accurate diagnosis of HbMIwate avoided further needless work-up of our patient. And finally, the case serves as a reminder that even among genetic diseases like hemoglobinopathies that show regional specificity, exotic variants are occasionally encountered, presumably arising from de novo mutations. Acknowledgment We thank Dr. Preethy J. Mathew, Associate Professor, Department of Anaesthesia, Postgraduate Institute of Medical Education and Research, Chandigarh, India who first suspected an inherited hemoglobinopathy in this child and initiated the diagnostic work-up. References [1] Steinberg MH. Disorders of hemoglobin: genetics, pathophysiology, and clinical management. 2nd ed. Cambridge: Cambridge University Press; 2009. [2] Wintrobe MM, Greer JP. Wintrobe's clinical hematology. 12th ed. Philadelphia l London: Wolters Kluwer Health/lippincott Williams & Wilkins; 2009. [3] Sun Y, Wang P, Li Y, et al. Familial congenital cyanosis caused by HbM(Yantai)(alpha-76 GAC – N TAC, Asp – N Tyr). Genet Mol Biol 2010, Jul;33(3): 445–8. [4] University PS. HbVar: a database of human hemoglobin variants and thalassemias. [cited 2014 Aug 04]; Available from: http://globin.bx.psu.edu/cgi-bin/hbvar/ counter; 2014. [5] Kedar PS, Nadkarni AH, Phanasgoankar S, et al. Congenital methemoglobinemia caused by Hb-MRatnagiri (beta-63CAT– N TAT, His– N Tyr) in an Indian family. Am J Hematol 2005, Jun;79(2):168–70.
[6] Horst J, Assum G, Griese EU, Eigel A, Hampl W, Kohne E. Hemoglobin M Iwate is caused by a C––T transition in codon 87 of the human alpha 1-globin gene. Hum Genet 1987, Jan;75(1):53–5. [7] Orisaka M, Sasaki T, Kato J, Harano K, Harano T. [Hb M-Iwate [alpha 87 (F8) His– N Tyr]: analysis of the genomic DNA and biosynthesis]. Rinsho Byori 1995, Mar;43(3):295–9. [8] Percy MJ, McFerran NV, Lappin TR. Disorders of oxidised haemoglobin. Blood Rev 2005, Mar;19(2):61–8. [9] Bain BJ. Haemoglobinopathy diagnosis. Malden, Mass2nd ed. . Oxford: Blackwell; 2006. [10] Pik C, Tonz O. Nature of haemoglobin M-Oldenburg. Nature 1966, Jun 11;210(5041): 1182. [11] Maggio A, Massa A, Giampaolo A, Mavilio F, Tentori L. Occurrence of Hb M Iwate (alpha 2 87 His leads to Tyr beta 2) in an Italian carrier. Hemoglobin 1981;5(2): 205–8. [12] Mayne EE, Elder GE, Lappin TR, Ferguson LA. Hb M Iwate [alpha (2)87His––Tyr beta 2]: de novo mutation in an Irish family. Hemoglobin 1986;10(2):205–8. [13] Ozsoylu S. Congenital methemoglobinemia due to hemoglobin M. Acta Haematol 1972;47(4):225–32. [14] Viana MB, Belisario AR. De novo alpha 2 hemoglobin gene (HBA2) mutation in a child with hemoglobin M Iwate and symptomatic methemoglobinemia since birth. Rev Bras Hematol Hemoter 2014, May-Jun;36(3):230–4. [15] Steffens G, Buse G. Hemoglobin M Oldenburg identified as HB alpha 2 87(F8)His replaced by Tyr beta 2. Hoppe Seylers Z Physiol Chem 1977, Jan;358(1):35–8. [16] Heller P, Weinstein HG, Yakulis VJ, Rosenthal IM. Hemoglobin M Kankakee, a new variant of hemoglobin M. Blood 1962, Sep;20:287–301. [17] Kuji A, Satoh Y, Kikuchi K, Satoh K, Joh S. The anesthetic management of a patient with hemoglobin M(Iwate). Anesth Analg 2001, Nov;93(5):1192–3 (table of contents). [18] Melarkode K, Prinzhausen H. Hemoglobin M variant and congenital methemoglobinemia: methylene blue will not be effective in the presence of hemoglobin M. Can J Anaesth 2008, Feb;55(2):129–30. [19] Bajaj RT, Malik RM, Desai M, Sukumaran PK. Hemoglobin-M disease. Indian Pediatr 1973, Jun;10(6):383–5. [20] Das KC, Bidwai PS, Mahapatra SS, Sukumaran PK. Haemoglobin-m disease in a Punjabi Hindu family. Indian J Pathol Bacteriol 1975, Jan;18(1):54–60.