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RED CELLS
From the Central Laboratory of the Netherlands Blood
Transfusion Service (CLB), and Laboratory for Experimental and Clinical
Immunology, Academic Medical Center, University of Amsterdam,
Amsterdam, and Department of Biomolecular Sciences, Laboratory of
Biochemistry, Wageningen University, Wageningen, The Netherlands;
Hospital de la Santa Creu I Sant Pau, Barcelona, Spain; Departments of
Chemical Pathology and Paediatrics, Prince of Wales Hospital, Shatin,
NT, Hong Kong.
Cytochrome b5 reductase (b5R) deficiency
manifests itself in 2 distinct ways. In methemoglobinemia type I, the
patients only suffer from cyanosis, whereas in type II, the patients
suffer in addition from severe mental retardation and neurologic
impairment. Biochemical data indicate that this may be due to a
difference in mutations, causing enzyme instability in type I and
complete enzyme deficiency or enzyme inactivation in type II. We have
investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations
predicted amino acid substitutions at sites not involved in reduced
nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide
(FAD) binding, as deduced from a 3-dimensional model of human b5R. This
model was constructed from comparison with the known 3-dimensional
structure of pig b5R. The seventh mutation was a splice site mutation
leading to skipping of exon 5 in messenger RNA, present in heterozygous
form in a patient together with a missense mutation on the other
allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in
nonessential regions of the enzyme. In contrast, 2 other substitutions,
known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH
binding. Thus, these data support the idea that enzyme inactivation is
a cause of the type II disease, whereas enzyme instability may lead to
the type I form.
(Blood. 2001;97:1106-1114) Nicotinamide adenine dinucleotide reduced (NADH)
cytochrome b5 reductase (b5R; EC 1.6.2.2) is a
300-amino acid, membrane-bound enzyme localized in the endoplasmic
reticulum of all tissue cells. This enzyme transfers electrons from
NADH to cytochrome b5 via its flavin adenine
dinucleotide (FAD) prosthetic group.1 Reduced cytochrome
b5 then donates its electrons to various
acceptors involved in elongation and desaturation of fatty acids,
cholesterol biosynthesis, and cytochrome P-450-mediated drug
metabolism.2-5 In erythrocytes, a 25-amino acid shorter,
soluble form of b5R exists, which is functional in methemoglobin
reduction.6,7 Both forms of b5R are derived from a single
gene,8 but recent studies have indicated that an
erythroid-specific transcript generates the soluble form of b5R in
humans.9
The primary structure of b5R is highly conserved among mammalian
species.10 The 31-kd hydrophilic form of the human enzyme shows 92% sequence identity with the solubilized form of pig liver b5R, for which a 3-dimensional model of the crystal structure (1ndh) at
2.4 Å resolution is available.11 A preliminary account of
the molecular structure of the human enzyme at 2.5 Å has also been
published.12 The crystallographic analysis has revealed that 1ndh consists of 2 domains that are separated by a large interdomain cleft. The N-terminal domain, which harbors the FAD, is structurally related to the FAD-binding domain of
ferredoxin-NADP+ reductase (FNR).13
The C-terminal domain of 1ndh is involved in NADH binding, and its
structure is somewhat different from the corresponding FNR domain.
Despite the presence of a Rossmann fold14 and the
structural homology with several other flavoenzymes,15-25 the exact mode of NADH binding in 1ndh is unknown. Nevertheless, it has
been suggested that the large interdomain cleft permits the
nicotinamide moiety of NADH to access the re side of the
isoalloxazine ring of the flavin without a conformational change of the
C-terminal region.11 This would be different from the
situation in FNR, where a displacement of a tyrosine residue is
required for direct hydride transfer from NADPH to
FAD.21,26
Deficiency of b5R may lead to 2 different clinical phenotypes. In type
I methemoglobulinemia, cyanosis is the only clinical symptom.27,28 In type II methemoglobulinemia, cyanosis is
accompanied by severe mental retardation and neurologic
impairment.29,30 In type I, the b5R enzyme deficiency is
profound in the erythrocytes (< 10% of normal activity) and moderate
in other blood and tissue cells (20%-60% of normal
activity).31-34 In type II, the b5R deficiency is
generalized to all tissues.
The human b5R gene DIA1 is localized at chromosome 22q13 and
contains 9 exons.8 Until now, 7 different mutations in
this gene have been described to lead to type I b5R
deficiency,33-39 and 11 different mutations to the type II
disease.31,32,34,40-44 In general, type I mutations were
found in "marginal portions" of the enzyme, giving rise to
enzymatically active, but unstable proteins. Type II mutations cause
exon skipping in messenger RNA (mRNA) and premature protein synthesis
termination, or are localized in consensus FAD- or NADH-binding sites
of b5R, leading to loss of protein expression or to expression of
enzymatically inactive proteins. Due to the long half-life of
erythrocytes and the lack of protein synthesis in these cells, type I
b5R deficiency is mainly found in the red blood cells, whereas type II
b5R deficiency is apparent in all tissue cells, with much more
detrimental effects.
We now describe 7 new mutations in the b5R gene leading to
type I methemoglobulinemia. These and previously published
mutations were tested in a 3-dimensional structural model of human
b5R for their effects on structure and function of b5R.
Patients were classified as having methemoglobinemia when
cyanosis was apparent, methemoglobin (metHb) in the blood was above 5%
and/or metHb reductase in the erythrocytes was below 0.5 IU/g Hb. The
patients were regarded to suffer from methemoglobinemia type I if
neurologic abnormalities had not become manifest at the age of 6 to 9 months. EDTA or citrated blood samples were washed 3 times with 0.9%
NaCl solution. MetHb reductase activity in the red cells was measured
with the method of Hegesh and coworkers45 in a Beckman
DU-65 spectrophotometer (Beckman Coulter, Fullerton, CA). The metHb
levels were measured by the method of Evelyn and Malloy46
with a Hewlett Packard 8451A diode-array spectrophotometer or with an
OSM3 Hemoximeter from Radiometer (Copenhagen, Denmark). A Puregene DNA
isolation kit (Gentra Systems, Minneapolis, MN) was used to isolate DNA
from leukocytes. RNA was isolated from leukocytes by the method of
Chirgwin and colleagues,47 followed by synthesis of
complementary DNA (cDNA) as described by Bolscher and
associates.48
DNA amplification and sequencing
Exon 1 of the b5R gene is very GC-rich and was therefore
amplified with the following adaptations: 0.45 µL MgCl2
25 mM, 0.6 µL 7-deaza-dNTP solution 75% (3.75 mM 7deaza-dGTP, 1.25 mM dGTP, 5 mM dATP, 5 mM dCTP, 5 mM dTTP; Boehringer Mannheim,
Mannheim, Germany) and addition of 3 µL GC-melt (5 M GC-melt, Clontech).
The PCR equipment consisted of an Air Thermo Cycler (ATC) (type: 1605, Idaho Technology, Idaho Falls, ID).
The cycle conditions were denaturation 5 seconds at 96°C, annealing
20 seconds at 65°C, and elongation 20 seconds at 72°C, for 40 cycles. Fragments 5 and 6,7 had annealing temperatures of 62°C and
68°C, respectively. Fragment 1 had an annealing/elongation time of 80 seconds at 72°C and was amplified for 60 cycles.
The oligonucleotide flag on the forward PCR primers was
complementary to the cDNA amplification and sequencing
Restriction fragment analysis Restriction fragment analysis was performed with the following enzymes.NciI (BcnI) cuts after C' in CC'GGG,
indicative for the mutation G BsiEI (Bsh1285I) cuts after G' in
CG'GCCG, indicative for the mutation C ApaI (Bsp1201) cuts after C[prime in
GGGCC'C, indicative for the mutation C BstUI (Bsh1236I) cuts after G' in
CG'CG, indicative for the mutation G Pag1 (BspH1) cuts after T' in
T'CATGA, indicative for the mutation G The assays were performed with 1 µL restriction enzyme, 1 µL restriction enzyme buffer, 4 µL demineralized water, and 5 µL of the PCR product. Digestion took place overnight at 37°C. The products were loaded on a 1% agarose gel with ethidium bromide and were run for 30 minutes at 100 mA. Structure homology modeling The globular fold of human b5R was predicted with MODELLER49 using the CHARMM forcefield.50 The 3-dimensional model of the crystal structure of pig liver b5R at 2.4 Å resolution (Brookhaven Protein Data Bank file: 1ndh; reference 11) served as the template file. The stereochemical quality of the homology model was verified by PROCHECK51 and the protein folding was assessed with PROFILE52 and PROSAII,53 which evaluate the compatibility of each residue to its environment independently. After this initial verification, incompatible regions of the model were optimized with the simulated annealing procedure (molecular dynamics) of the XPLOR package,54 thereby fixing the other parts of the protein. A simulated annealing calculation was performed for 1000 steps at 900 K, with each step taking 0.5 femtosecond. Before and after molecular dynamics the model was energy minimized with the conjugate gradient algorithm of XPLOR, the minimization converged after 2000 cycles (gradient: 0.1 kcal/mol). Again, the model was checked and verified. NADH docking was performed with the program O,55 based on the mode of NADP(H) binding in the related structure of ferredoxin-NAD(P)+ reductase.26 NADH forcefield constants for energy minimization were derived from CHARMM. Finally, the model was verified after several rounds of energy minimization.
Family studies In family V, both parents showed half-normal metHb reductase activity, correlating with the G757A (exon 9) mutation in one of their alleles (Figure 1A, Table 2). This mutation leads to the replacement of Val to Met at position 252. The 2 affected children were homozygotes for the G757A mutation and showed almost no metHb reductase activity. Their brother lacked the mutation and was unaffected.
Family A had the same mutation as found in family V. Both parents as well as one child were heterozygotes. These 3 individuals showed decreased enzyme activity. The 2 patients in the family were homozygotes for the mutation, showed very low metHb reductase activity and had about 10% metHb in their red cells (Figure 1A, Table 2). Mr T, whose relatives were not available for investigation, was a homozygote for the G757A mutation and had almost no metHb reductase activity (Figure 1A, Table 2). As far as we know, Mr T and the families V and A are not related to each other. In all 3 of these families, the mutation was confirmed by restriction analysis and was excluded as a polymorphism in 50 control DNA samples (100 alleles). In family W, a C to T mutation in exon 5 was found by sequencing
(Figure 1B). This C434T mutation predicts the substitution of a proline
to leucine at position 144. Also this mutation was confirmed by
restriction analysis. Unfortunately, we could not investigate the DNA
of the presumably homozygous patient II.3 (Figure
2), because he died between the time of
enzyme analysis and the time of DNA analysis. In his family members,
the presence of the heterozygous C434T mutation correlated with a metHb
reductase activity of half the control value (Table 2). The family
members who lacked the mutation showed a normal metHb reductase
activity, indicating that the C434T mutation was the cause of the
enzyme deficiency.
Mutation C434T was excluded as a polymorphism in 50 control DNA samples (100 alleles). Only in one control sample, the exon 5 PCR product was not totally digested by ApaI. When this sample was further investigated by sequencing, it became clear that the enzyme was not capable of digesting one allele because a polymorphic C432T was present. The normal sequence is 429GGGCCC434; the C434T mutation leads to 429GGGCCT434 and the polymorphism to 429GGGTCC434. The metHb reductase activity of this variant sample was normal. In family Am from Spain, we found (Figure 1C) the parents to be heterozygotes, each for a different mutation, viz. G149A (predicting substitution of arginine for glutamine at position 49 in exon 2) and C194T (predicting substitution of proline for leucine at position 64 in exon 3). Two of their children proved to be compound heterozygotes for these mutations. Only in child 1, a girl, this situation was manifested by 6% metHb, low metHb reductase activity, and slight cyanosis in her lips (Table 2). Child 2, her brother, had 2% metHb and half-normal metHb reductase activity, without clinical signs. The third child, a girl, proved to be a heterozygote for the G149A mutation and had low normal metHb reductase activity. The fourth child was enzymatically and genetically unaffected. These mutations were confirmed by restriction analysis and excluded as polymorphisms by analysis of 50 control DNA samples. Family L from Hong Kong had an almost 2-month-old baby with severe cyanosis. This patient had 9% metHb in her erythrocytes and very low metHb reductase activity (Table 2). The parents had half-normal metHb reductase activity. In exon 8 we found a T716G mutation heterozygous in the father and in the child (Figure 1D). This mutation predicts the substitution of a leucine to an arginine at position 238. In 50 control DNA samples, the T716G mutation was excluded as a polymorphism. In the child and in the mother, another mutation was found at the 3' end of intron 4, where the normal sequence cagGTTTAC was replaced on one allele by caaGTTTAC (Figure 1D). This acceptor splice site mutation predicts skipping of exon 5 during mRNA processing. Indeed, in the mother's cDNA the sequence of exon 4 was followed by a mixture of exon 5 and exon 6 sequences (not shown), indicating that she was heterozygous for a mutation that caused skipping of exon 5. From the child, no RNA or cDNA was available for these investigations. Both mutations in this family were confirmed by restriction analysis. The child showed no neurologic abnormalities at the age of 1 year. In family O, of North African origin, a newborn girl presented with severe cyanosis. This patient had 31% metHb in her erythrocytes and very low metHb reductase activity (Table 2). In her DNA we found an apparently homozygous G535A mutation in exon 6 of the DIA1 gene (Figure 1B), predicting an A178T substitution. Both parents proved to be heterozygous for this mutation; their metHb percentage was low and their metHb reductase activity was below the normal range (Figure 1B, Table 2). The G535A mutation was confirmed by restriction analysis and excluded as a polymorphism in 50 control DNA samples. The child showed no signs of neurologic abnormalities at the age of 9 months. Structural properties Figure 3 shows the sequence alignment of human b5R and the solubilized form of pig liver b5R. From this alignment it is obvious that both structures are highly homologous. The amino acid differences between both enzymes are spread throughout the structure and are mainly localized in flexible regions near the protein surface. None of the variable residues are directly involved in FAD or NADH binding.
A 3-dimensional model of the structure of human b5R was
constructed, based on the crystal structure of the solubilized form of
1ndh. A ribbon diagram of the modeled structure of human b5R is
presented in Figure 4. From this
structural model and the sequence alignment, the location of the amino
acid substitutions in b5R of the methemoglobinemia patients can be
deduced (see "Discussion").
The NADH cofactor was modeled in an extended conformation, highly
similar to the mode of binding of NADP(H) in the related ferredoxin
NADP+ reductase.26 The NADH molecule is
situated in the interdomain cleft at the re side of the FAD
isoalloxazine ring (Figure 4). The nicotinamide ring was modeled in the
anti conformation, consistent with the proposed
stereochemistry of A-hydrogen transfer in the FNR
family.26 The pyrophosphate moiety is located near the 175 to 190 sequence of the single
In 7 families with methemoglobinemia type I, characterized by
cyanosis as the only clinical symptom, we found 6 different missense
mutations in DIA1, the gene encoding human cytochrome b5 reductase. These mutations predict amino acid
substitutions at various sites in the protein (Table
3). In addition, we found one splice site
mutation, which caused skipping of exon 5. This out-of-frame exon
skipping in itself leads to enzyme inactivation and type II
methemoglobinemia,42 but in our patient, this mutation was
present in heterozygous form, in combination with a L238R substitution. We presume, therefore, that this last amino acid substitution does not have a strong deleterious effect on the b5R
activity, because the patient did not suffer from type II symptoms
(developmental and neurologic aberrations). Likewise, in family Am, we
found 2 compound heterozygous individuals for R49Q and P64L
substitutions. Because these patients had very mild cyanotic symptoms,
we presume again that these mutations, too, should be classified as
type I mutations. The other 3 amino acid substitutions found by us were
expressed in homozygous form in type I patients.
In the literature, 7 additional amino acid substitutions have been reported in patients with type I methemoglobinemia33-39 (Table 3). Some of these mutant enzymes have been expressed in bacterial systems, purified and studied for kinetic properties, heat stability, and trypsin sensitivity.33,34,38,39 In general, the recombinant type I mutant enzymes were found to have retained about 60% to 70% of the catalytic activity expressed by the recombinant wild-type enzyme, but to be more heat labile and trypsin sensitive. Similar experiments have been carried out with mutant enzymes from patients with type II methemoglobinemia.32,33,43,59 These enzymes were found to express very low catalytic activity and to be heat labile as well. According to these principles, the P95H mutation studied by Manabe and coworkers,43 although found in a type II patient, should be classified as a type I mutation, because the recombinant enzyme showed about 60% of the wild-type activity. The patient was a compound heterozygote for this mutation in combination with a Y42stop mutation. Apparently, this combination did not leave enough enzyme activity in the tissues to prevent psychomotor and neurologic deficiencies. Arbitrarily, we have listed this mutation in Table 3 as a type I mutation. Localization and implication of amino acid substitutions in b5R Inspection of the 3-dimensional model of b5R (Figure 4) revealed that the amino acid substitutions in b5R of the methemoglobinemia type I patients described in this paper are spread throughout the structure and not directly involved in FAD or NADH binding. The R49Q replacement identified in family Am from Spain concerns a nonconservative mutation located near the protein surface in the first -strand of the FAD
domain. In 1ndh, the N of the corresponding K21 forms a strong
hydrogen bond with the carbonyl oxygen of the conserved
E103.11 From this observation and the low levels of b5R in
family Am we conclude that a positively charged residue at position 49 in human b5R is important for the structural integrity of the
FAD-binding domain by forming optimal intradomain contacts.
The P64L substitution, also found in family Am, is localized in a
surface loop connecting the first 2 The P144L substitution present in b5R of some members of family W is
situated at the C-terminal end of the final The A178T substitution in family O is localized at the C-terminal end
of strand The L238R substitution in family L from Hong Kong is localized at the
C-terminal end of strand 10 of the NADH binding domain, somewhat remote
from the protein surface and relatively far away from the domain
interface. In 1ndh, L238 is conservatively replaced by a less bulky
hydrophobic residue (V210). In the structure of the pig liver enzyme,
V210 is relatively close (4 Å) to W217, which is conserved in the
human enzyme (W245). Introduction of the polar, bulky arginine side
chain at position 238 in human b5R might lead to an electrostatic
interaction with E212 (b5R numbering), which is located in the surface
loop, connecting strand The V252M substitution found in family V, family A, and Mr T is
localized near the protein surface at the end of the large surface loop
connecting strand Previously found amino acid substitutions As noted above, 8 other type I disease mutations have been found in the b5R gene (Table 3). These mutations are also spread throughout the protein structure and are mostly localized in surface loops. Based on our 3-dimensional model, new insights about some of these mutations are discussed below.The L72P replacement identified in a Chinese patient37 is
localized in a loop connecting strands Based on sequence alignments, it was predicted that V105 is part of a
Based on secondary structure analysis, it was predicted that the E212K
replacement identified in an African American patient disrupts an
In conclusion, we have shown that the novel point mutations in the b5R gene of patients with methemoglobinemia result in amino acid replacements that are distributed throughout the protein structure and not in positions directly involved in FAD and NADH binding. This is in agreement with the mild cyanotic symptoms of these patients and supports the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form. Moreover, from our 3-dimensional model of human b5R, which was constructed on the basis of the crystal structure of the pig liver enzyme,11 the effects of some other point mutations could be reinterpreted in a rational way. This knowledge, together with additional information on the interaction between b5R and its physiologic electron acceptor cytochrome b561-63 sheds more light on the molecular aspects of methemoglobin reductase deficiency.
Submitted July 26, 2000; accepted October 19, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dirk Roos, Central Laboratory Netherlands Blood Transfusion Service, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands; e-mail: d_roos{at}clb.nl.
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© 2001 by The American Society of Hematology.
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  C. Ewenczyk, A. Leroux, A. Roubergue, V. Laugel, A. Afenjar, J. M. Saudubray, P. Beauvais, T. B. de Villemeur, M. Vidailhet, and E. Roze Recessive hereditary methaemoglobinaemia, type II: delineation of the clinical spectrum Brain, March 1, 2008; 131(3): 760 - 761. [Abstract] [Full Text] [PDF]
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 M. J. Percy, M. J. S. Gillespie, G. Savage, A. E. Hughes, M. F. McMullin, and T. R. J. Lappin Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH-cytochrome b5 reductase Blood, November 15, 2002; 100(10): 3447 - 3449. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
21m13 forward fluorescent primer. Direct
sequencing of the amplified products was performed with BigDye Primer
Cycle Sequencing Ready Reaction kit 
A in exon 2 (R49Q;
single-letter amino acid codes); the PCR product with the
normal sequence will be digested.
unit of the dinucleotide-binding fold, whereas the 2-OH ribose of NADH interacts in the model with the
negatively charged D214. NADH-dependent proteins with a typical dinucleotide fold