new variant of cytochrome b5 reductase deficiency (b5rkurashiki) in red cells, platelets,...

American Journal of Hematology 40:299-305 (1992)

New Variant of Cytochrome b5 Reductase Deficiency (b5RKurashiki ) in Red Cells, Platelets, Lymphocytes,

and Cultured Fibroblasts With Congenital Methemoglobinemia, Mental and Neurological Retardation,

and Skeletal Anomalies

Yoshihito Yawata, Lily Ding, Kiyoh Tanishima, and Akio Tomoda Division of Hematology, Department of Medicine, Kawasaki Medical School, Kurashlki (Y Y ), Department of Clinical Laboratory Sciences, School of Allied Medical Professions, Kanazawa University, Kanazawa (L D , K T ), and Department of Biochemistry,

Tokyo Medical College, Tokyo (A T ), Japan

A Japanese man with cytochrome b5 reductase (b5R) deficiency in various blood cell lineages (red cells, platelets, and lymphocytes) and in cultured fibroblasts demonstrated congenital methemoglobinemia associated with mental and neurological retardation, and various skeletal anomalies, such as spondylosis deformans and finger joint deformations, which have never been described in association with this enzyme deficiency. Cy- tochrome b5 reductase deficiency was most severe in red cells (0.34%) and less marked in platelets (13-27%), lymphocytes (1 8-31%), and fibroblasts (50%). The present case appears to be a new variant of cytochrome b5 reductase deficiency (b5R,,,,,,i,i). 'b 1992 Wiley-Liss, Inc.

Key words: cytochrome b5 reductase deficiency, methemoglobinemia, neuro-mental retardation, skeletal anomalies

INTRODUCTION

Hereditary methemoglobinemia is an autosomal reces- sively inherited disorder with deficiency of NADH-cytochrome bS reductase (b5R; E.C. 1. 6 . 2. 2.; NADH: ferricytochronie bS oxido-reductase) [ 1,2]. This disorder has previously been reported as a deficiency of inethemo- globin reductase, NADH-diaphorase, or fenihemoglobin reductase.

Cytochrome b5 reductase, a flavoprotein with FAD as a prosthetic group, catalyzes the reduction of cytochrome bS. This enzyme consists of two forms: I ) a membrane- associated form located in the cytoplasmic portion of the endoplasmic reticulum of tissue cells, and 2) a soluble form in red cells [3]. Structurally, the former form of cytochronie b5 reductase in somatic cells consists of 300 amino acid residues including 2.5 residues with hydropho- bic amino acids, which is equivalent to the membrane domain [4,5]. The membrane-associated form of this enzyme contributes to lipid metabolism: e.g., to desaturation and elongation of fatty acids. and to cholesterol biosynthesis [6-81. The latter form (the soluble form) of

0 1992 Wiley-Liss, Inc.

cytochrome b5 reductase with 275 hydrophilic amino acids is derived from the catalytic domain of the primary translational compound (the membrane-associated form) by proteolytic cleavage. The soluble form of this enzyme exists in red cells with the function of methemoglobin reduction. At the gene level, it has been elucidated that both forms (the membrane-associated form and the soluble form) of cytochrome bS reductase are produced from a single gene [9] approximately 3 1 kb long and with nine exons and eight introns [ 10, I I ] .

Clinically, three phenotypes have been reported [ I ] . Type I is a red cell type with mild cyanosis due to deficient b5R, which has only been detected in the red cells.

Received for publication October 3 . 1991; accepted January 30. 1992.

Address reprint requests to Yoshihiro Yawata, M.D., Professor of Medicine, Division of Hematology, Department of Medicine. Ka- wasahi Medical School. 577 Matsushirna, Kuritshibi 701-01, Japan.

300

Type I1 is known as a generalized type with deficiency of b5R in various tissues resulting in cyanosis and severe mental retardation with neurological manifestations [ 12- 151. Type 111 is a hematopoietic phenotype with deficiency of b5R activities in red cells, leukocytes, and platelets. without mental retardation [ 15,161.

The causal relationship between gene anomalies and clinical expressions in the b5R deficiency has recently been clarified in part. In type I (erythrocytic), a point mutation (57 GGC-GAC: arginine to glutamine) was dis- covered at codon 57 in exon 3 of the gene [ 171. In a patient with type I1 (generalized), a substitution of cy- tosine for thymine ( 127 TCT-CCT: serine to proline) was reported at codon 127 in exon 5 of the gene [ 18). In type 111 (hematopoietic), a point mutation ( 148 CTG-CCG: leucine to proline) was detected at codon 148 in exon 5 of the gene [ 171.

In the present communication, a new variant of b5R deficiency ( b5R,,,,,,i,i) with characteristic features of cyanosis, mental retardation, and neurological nianifesta- tions and with numerous skeletal anomalies is reported. This case appears to differ from the classical type I1 from the standpoint of the extent of the deficiency of bSR activities, which varies (3-50% of normal control) among several tissue cells. This b5R deficiency was also proved in cultured fibroblasts obtained from the skin of the patient, which may explain the defects in his mesenchymal tissues.

Case Report: Yawata et al.

MATERIALS AND METHODS

Blood specimens were obtained from normal volun- teers and one patient with congenital methemoglobinemia by venipuncture with disodium EDTA as an antico- agulant, as described later in detail. The blood samples were centrifuged to fractionate blood cells. Platelets were obtained from the platelet-rich plasma by centrifugation of whole blood at 120 g for 15 min. Lymphocytes were separated by a modification of the method of Boyum 1191. Red cell fractions were washed three times with 0.154 M Na/K phosphate-buffered saline solution containing glucose (250 mg/dl). These blood cells were uti- lized immediately after their separation. Fibroblasts were cultured from the biopsy specimens of the skin by the method of Arakawa et al. [20]. Pure cell lines of fibroblasts were obtained.

Enzymatic assays were performed using cell lysates prepared with Triton X- 100 (Wako Pure Chemical Indus- tries Co. Ltd., Tokyo) in a final concentration of 0.1% as a detergent. The enzymatic activities of diaphorase, ferrihemoglobin reductase, ferricyanide reductase and cytochrome bS reductase were determined by the methods of Scott and Griffith [21], Hegesh et al. [22], Omura and Takesue 1231 and Hultquist and Passon [24], respec- tively. Cytochrome bS as an electron acceptor was pre-

pared from pig liver. Assays of these enzymatic activities were performed at 30°C. Electrophoretic studies were carried out in red cell hemolysates by the isoelectric fo- cusing method on Ampholine polyacrylamide gel plates (pH 3.5-9.5, Pharmacia LKB Biotechnology, Uppsala, Sweden). The enzymatic activities on electrophoresis were visualized by the staining method of Kaplan and Beutler [25].

Hemoglobin contents were determined by the conven- tional ferricyanide method with commercial reagent kits (Wako Pure Chemical Industries Co. Ltd., Tokyo, Ja- pan). Protein concentrations in the homogenates of lymphocytes and platelets were determined by the modified method of Lowry et al. [26] with bovine serum albumin as a standard.

CASE REPORT

The patient was a 43-year-old male of Japanese origin. No consanguinity was detected in his trait. Cyanosis of a moderate degree was detected at birth without other significant symptoms. During his childhood and adoles- cence, various skeletal anomalies were noted by his mother, such as developmental retardation of the extrem- ities including finger joint deformations. He had no drug history of any kind. He has never been married. He first visited our outpatient clinic with a chief complaint of cyanosis. The patient was extraordinarily short ( I 39 cm) but his body weight was inappropriate (55.0 kg) for his height. His pulse rate was 76/niin, respiratory rate 161 min, body temperature 37.0"C, and blood pressure 138/ 98. The presence of cyanosis was easily detected facially. Physical examination revealed no anemia, no jaundice, and no lymphadenopathy. Examination of the chest and abdomen showed no abnormalities. Neurological examination revealed a tandem to clumsy gait and mental retardation (IQ 70), but normal motor and sensory functions. Hand grips were markedly decreased (Rt 12 kg, Lt 18 kg), which is normally 50 kg. Electroencephalogram was interpreted as borderline, demonstrating a basic a wave (3 to 10 Hz) with positive a-attenuation. Brain computed tomography revealed a moderate atrophy of the cerebral cortex. The patient has no brothers and sisters, and, un- fortunately, his parents refused to be examined. although neither of them appeared to have any symptoms including cyanosis when seen on several occasions for examination of their son and for history-taking.

RESULTS Clinical Data

The patient demonstrated mild erythrocytosis (RBC: 5.70 X IO"/L, Hb 16.7 g/dl, Hct 52.4%) with normal red cell indices (MCV 96.7 fl , MCH 30.8 pg, MCHC 3 1.9%), with mild reticulocytosis ( I .8%). Platelet and

Case Report: New Cytochrome b5 Reductase Deficiency 301

Fig. 1. Skeletal anomalies at the hip joints (antero-posterior) in a patient with congenital methemoglobinemia. A marked deformation was noted at the hip joints.

leukocyte counts were 186 X 109/L and 4,200 X IOh/L with normal differentials (lymphocytes 30%)). Leukocyte alkaline phosphatase demonstrated normal activity. No bleeding tendency was noted. Bone marrow examina- tions revealed normal results. Chromosomal analysis demonstrated no abnormalities (46 XU).

Hemoglobin studies showed normal composition of globins, such as HbA 96.59%, HbF 0.43, and HbAz 2.98 with a normal synthetic rate (a@ ratio 1.01). Hemoglo- bin denaturation tests were negative. The content of methemoglobin was markedly increased (15.4%). HbM was not detected.

Blood gas analysis and respiratory tests demonstrated normal results. Serum chemistry revealed also normal results in serum proteins and lipids.

Skeletal anomalies were noted at the hip joints (Fig. I ) , the hands, the elbows, the knees, and other locations. X-ray studies demonstrated the presence of spondylosis deformans and developmental retardation of the extremi- ties with marked deformation of various joints.

Electrophoretic Pattern of the Hemolysates

The hemolysates of normal controls and of the patient were subjected to electrophoresis. The presence of methemoglobin was obvious in the patient’s hemolysate, as shown in Figure 2.

Enzymatic Activities of Methemoglobin Reductase in Various Blood Cells

As shown in Table I , the enzymatic activities of ferrihemoglobin reductase and of diaphorase in red cells were markedly reduced to approximately 3% of the normal

control. It should be noted that the enzymatic activities in red cells did not appear to be completely deficient.

In platelets, the enzymatic activities were also signifi- cantly decreased to about 15-30% of the normal control, but again they were not completely deficient. The activity of ferricyanide reductase was also reduced to around one- third of the normal control.

In lymphocytes, the enzymatic activities of ferrihemoglobin reductase, diaphorase, and ferricyanide reductase were clearly diminished to one-third to one-fifth of the normal control.

Electrophoretic Patterns of Enzymatic Activities of NADH-Diaphorase, NADH-Cytochrome b5 Reductase, and NADH-Ferricyanide Reductase

The hemolysates were subjected to electrophoresis for NADH-diaphorase. The enzymatic activity was markedly reduced in the patient’s hemolysate. as shown in Figure 3. The hemolysates were also subjected to electrophoresis for NADH-cytochrome b5 reductase (Fig. 4) and for NADH-ferricyanide reductase (Fig. 5).The activities of these enzymes were also obviously diminished.

Enzymatic Activities of NADH-Methemoglobin Reductase in Cultured Fibroblasts

A skin biopsy was performed on the patient and normal controls, and fibroblasts were cultured at 37°C for 4 weeks. The enzymatic activities of NADH-methemoglobin reductase were determined in these cultured fibroblasts. Results are shown in Table I. An approximately 50% decline in the enzymatic activities of cytochrome b5 reductase and of diaphorase was observed in the patient,

302 Case Report: Yawata et at.

Fig. 2. Electrophoretic pattern of the hemolysate in a patient with congenital methemoglobinemia. Electrophoresis was performed by the method of Scott and Griffith [21]. Methe- moglobin is clearly shown at the middle specimen of the patient. Other minor subcompo- nents of hemoglobin are shown.

TABLE 1. NADH-Methemoglobin Reductase Activities in Red Cells, Platelets, Lymphocytes, and Cultured Fibroblasts From a Patient With Hereditary Methemoglobinemia*

Red cellsa Plateletsb Lymphocytesb Fibroblastsb

Ferricytochrome b5-reductase Patient 0.13 9.5 12.5 36.9 Controls 3.4 35.2 44.6 71.9

Patient 0.04 7.9 2.2 28.0 Controls I .2 47.7 11.5 56. I

Patient 235 86 Controls 527 243

Patient 11.9 4.7 Controls 49.7 23.0

Patient 145 245 857 Controls 144 3 90 667

Diaphorase

Ferricyanide reductase

Cytochrome b5-reductase

Lactic dehydrogenase

*Values are average in two to five determinations. anrnol/min/mg Hb. bmmol/min/mg protein.

compared to the normal results of lactate dehydrogenase activity in these fibroblasts.

eralized skeletal anomalies. The phenotypic manifestations have never been reported in association with cytochrome b5 reductase deficiency. Although information

DISCUSSION on the familial background of this patient are limited, no consanguinity was detected and no other such patients -

This patient with congenital methemoglobinemia demonstrated multi-tissue manifestations, such as cyanosis, mental retardation with neurological symptoms, and gen-

appeared to exist in his family as far as we studied. Thus, this patient’s condition appeared to be due to a de novo mutation. The enzymatic activities of cytochrome b5 re-


Fig. 3. Enzymatic activities of NADH-diaphorase in the hemolysate of a patient with congenital methemoglobinemia. Enzyme assay was carried out by the method of Scott and Griffith (211. The enzymatic activities on electrophoresis were visualized by the method of Kaplan and Beutler [25].

Fig. 4. Enzymatic activities of NADH-cytochrome b5 reductase in the hemolysate of a patient with congenital methemoglobinemia. Enzyme assay was performed by the method of Hultquist and Passon [24]. The enzymatic activities on electrophoresis were visualized by the method of Kaplan and Beutler [25].

ductase were deficient in various somatic cells: e.g. , in red cells, lymphocytes, platelets, and fibroblasts. The presence of cyanosis appeared to be directly related to the deficiency of the enzyme. The role of this cytochrome b5 reductase deficiency in lymphocytes and platelets, how-

ever, has not yet been determined because the patient has never had any history of recurrent infections, immune deficiency, or a bleeding tendency. The defective activity of cytochrome b5 reductase in the cultured fibroblasts may explain the presence of the numerous skeletal abnor-

304 Case Report: Yawata et al.

Fig. 5. Enzymatic activities of NADH-ferricyanide reductase in the hemolysate of a patient with congenital methemoglobinemia. Enzyme assay was carried out by the method of Omura and Takesue [23].

malities as a manifestation of mesenchymal dysfunc- tions, although it cannot be excluded that these two abnormalities may be unrelated and coincidental.

The enzymatic activities of cytochrome b5 reductase are not completely absent in this patient. The patient’s cytochrome b5 reductase rather retained some part of the enzymatic activity. different from previously published classical cases of type I1 [12-151 or type I11 [15,16] hereditary methemoglobinemia, in which the enzymatic activities are usually totally absent.

The extent of the deficiency in enzymatic activities varied widely among various tissue cells, being 97% in red cells, around 70% in lymphocytes and platelets, and about 50% in the cultured fibroblasts. These characteristic features of clinical and biochemical expression may indicate a new variant of congenital methemoglobinemia.

Based on the results of gene analyses, type I1 (serine to proline substitution at codon 127 in exon 5 of the enzyme gene) [ 181 appears to be different from type I11 (leucine to proline at codon 148 in exon 5 ) [17]. Between these two types, defects were located at the same exon, exon 5 , only 21 amino acids apart. However, the difference in clinical expression of mental retardation, such as its presence in type I1 and its absence in type 111, has still not been satisfactorily explained by these gene analysis results. In our patient, at the present time, no abnormality has been detected on exon 5 (Type I1 and 111) or on exon 3 (Type I), contrary to our expectations (Fukuniaki, 1991, personal communication). The preliminary results, however, indicate the presence of the abnormally large

mRNA without defects in the codons where alterations have been observed in other examples of cytochrome b5 reductase deficiency.

The varied extent of the deficiency in the enzymatic activities of cytochrome b5 reductase in several types of tissue cells as yet cannot be explained from the standpoint of the expression of clinical manifestations. The marked reduction in the enzymatic activity in red cells (97%) may explain the presence of cyanosis. On the other hand, the partial deficiency in cytochrome b5 reductase in the cultured fibroblasts may not satisfactorily explain the marked skeletal anomalies. The significant reduction (30-50%) in the activity in the lymphocytes and platelets does not appear to express any defect in biological functions as far as we have been able to determine. In sum- mary, the present case should provide several insights for elucidation of the relationship between the role of cytochrome b5 reductase and clinical manifestations in various tissues, and also regarding gene control on the expression of this enzyme.

ACKNOWLEDGMENTS

The present study was supported in part by a research grant for Idiopathic Disorders of Hematopoietic Organs from the Ministry of Health and Welfare of the Japanese Government, by the Grant-in-Aid for Scientific Research (No. 62570555, 01570696, 02771848 and 03671201) from the Ministry of Education, Science and Culture of the Japanese Government, by research grants from Ka-


wasaki Medical School (No. 1-30-5, 2-301 and 3-305), and by the Japan-France Joint Research Project of JSPS and CNRS.

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new variant of cytochrome b5 reductase deficiency (b5rkurashiki) in red cells, platelets,...

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