MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #402 (2001) Online

© 2001 WILEY-LISS, INC.

Received 11 September 2000; revised article accepted 22 January 2001.

Molecular Basis of Recessive CongenitalMethemoglobinemia, Types I and II: Exon Skippingand Three Novel Missense Mutations in the NADH-Cytochrome b5 Reductase (Diaphorase 1) GeneWilfried Kugler*, Arnulf Pekrun, Petra Laspe, Bernhard Erdlenbruch, and Max Lakomek

Universitäts-Kinderklinik, Robert-Koch-Str. 40, D-37075 Göttingen, Germany

*Correspondence to: Dr. Wilfried Kugler, Universitäts-Kinderklinik, Robert-Koch-Str. 40, D-37075 Göttingen,Germany; Tel: (49)-551-39-6216; Fax: (49)-551-39-6231; E-mail: wkugler@med.uni-goettingen.deContract grant sponsor: Deutsche Forschungsgemeinschaft, DFG; Contract grant numbers: La 527/3-1 and Pe374/2-3.

Communicated by Mark H. Paalman

Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into an erythrocyte type (I) and ageneralized type (II). We investigated the b5R gene of three unrelated patients with types Iand II and found four novel mutations. The patient with type I was homozygous for ac.535G→→ A exchange in exon 6 (A179T). The patients with type II were found to behomozygous for a c.757G→→ A transition in exon 9 (V253M) and compound heterozygous fortwo mutations, respectively. One allele presented a c.379A→→ G transition (M127V). Thesecond allele carried a sequence difference at the invariant 3’ splice-acceptor dinucleotide ofintron 4 (IVS4-2A→→ G) resulting in skipping of exon 5. To characterize a possible effect ofthis mutation on RNA metabolism, poly(A)+ RNA was analyzed by RT-PCR and sequencing.The results show that RNA is made from the allele harboring the 3’-splice site mutation.Furthermore, western blot analysis revealed a complete absence of immunologicallydetectable b5R in skin fibroblasts of this patient. The compound heterozygosity for the splicesite and the missense mutations apparently caused hereditary methemoglobinemia type II inthis patient. © 2001 Wiley-Liss, Inc.

KEY WORDS: hereditary methemoglobinemia; RCM; NADH-cytochrome b5 reductase; diaphorase 1; DIA1; exonskipping; mutation analysis

INTRODUCTION

Recessive congenital methemoglobinemia (RCM) due to a deficiency of reduced nicotin amide adeninedinucleotide (NADH)-cytochrome b5 reductase (b5R; EC 1.6.2.2.), also known as diaphorase 1 (DIA1), is anautosomal disorder. Two forms of this enzyme are known, a membrane-bound form mainly found in the outermitochondrial membrane and the endoplasmatic reticulum of all investigated tissues and a soluble form present inerythrocytes [Bulbarelli et al., 1998]. Both isoforms are produced by a single gene (DIA1, MIM# 250800;GenBank: NM_000398) about 31 kb in length with nine exons and eight introns, and encoded on chromosome22q13 [Bull et al., 1988]. Clinically, two types of RCM have been classified [Jaffé and Hultquist, 1995]: type I, in

2 Kugler et al.

which cyanosis due to methemoglobinemia is the single clinical symptom (erythrocyte type), and type II, in whichcyanosis is associated with severe mental retardation and neurologic impairment (generalized type). The enzymedeficiency is restricted to the red cell soluble b5R in type I, whereas in type II, the enzyme defect is generalized toall tissues, involving both soluble and membrane-bound forms of b5R.

In the present study, we have analyzed the b5R gene of three patients with RCM types I and II. We identifiedfour novel mutations: three missense mutations in exons 5, 6, and 9, as well as an exchange of the consensus A atthe 3’ splice acceptor site in intron 4.

MATERIALS AND METHODS

Patients

Case1: The male newborn of Turkish origin developed cyanosis within one hour after birth. Methemoglobinconcentration amounted to 25 % of total hemoglobin. After i.v. administration of methylene blue (0.5 mg/kg)cyanosis disappeared at once and methemoglobin concentration fell to below 2 %. After a few days a 2nd rise ofmethemoglobin to ∼30 % occurred. Ascorbic acid was given at a dosage of 20 mg/kg/day leading to a reduction ofmethemoglobin to ∼5-10 %. The neurologic development is normal up to the present age of eight months.

Case 2: The 49 year old german patient developed slight cyanosis in early infancy. During the next few years hesuffered from amaurosis and severe retardation of neuropsychologic development. Methemoglobin was firstmeasured at the age of 49 years and found to be increased to 7.5 % of total hemoglobin.

Case 3: The propositus is a four year old boy of German ancestry. Within minutes after birth dark grey cyanosisof skin and mucous membranes was noted. Methemoglobin concentration was increased to ∼15 % of totalhemoglobin. Methemoglobin reductase deficiency was diagnosed about three weeks after birth. Further course wascharacterized by an early strong psychomotor developmental delay. The boy started walking at the age of threeyears and the Denver Developmental Screening Test revealed a delay of two years at chronological age of fouryears. Nuclear magnetic resonance tomography at the age of 11 months showed slightly reduced myelinization andsome atrophy of the corpus callosum.

Biochemical Assays

Methemoglobin reductase activity and methemoglobin concentration were measured by the methods of Caweinet al. [1984] and Gerald [1958]. Other red cell enzymes including glucose-6-phosphate dehydrogenase wereestimated by routine methods [Beutler et al., 1977]. Hemoglobin electrophoresis was performed according toMarengo-Rowe [1965].

Non-Radioactive Single-Strand Conformation Polymorphism (SSCP) Analysis

Genomic DNA was prepared from whole blood of the patients and their parents using QIAamp spin columns(Qiagen). b5R primer sets were designed for the SSCP screening (Table 1) and analysis was performed on acommercially available pre-cast 15% polyacrylamide gel (CleanGel DNA-HP) using the DNA-DELECT buffer kit(ETC) at 15 °C as described (manuscript in preparation).

Table 1. Oligonucleotides and enzymes used for SSCP analysis, sequencing andconfirmation of the novel b5R (DIA1) mutations

Fragment size (bp)

Mutation Exon Primer pair Ta Enzyme PCR Normal Mutant

M127V 5 CCT GGA GCT GGG GAC TGA GG (s)GTG TAA CCA AGG GAT TCC GAC CCG (as)

62 BsaXI 200 200 30, 69,101

A179T 6 CCG GGC CTC ACC CCT TCT CT (s)GGA CGC CTC AGT GGG GGG TT (as)

62 BclI 140 140 95, 45

V253M 9 GCC ACA CGT CAG CTT ACC TGG (s)AGG TGA CTG GGT GAG CGT GAA (as)

62 BspHI 280 280 58, 222

Ta, annealing temperature; s, sense primer; as, antisense primer

Molecular Study in Patients with Methemoglobinemia 3

Conventions

The translating initiating ATG will be referred to as codon/amino acid no. 1 and the corresponding adenine asnucleotide no. 1. Reference sequence is GenBank: NM_000398.

Direct Sequencing of Amplified Genomic DNA and Mutation Analysis

SSCP analysis was followed by bidirectional BigDye terminator cycle sequencing (25 cycles at 96 °C for 30sec, 58 °C for 15 sec and 60 °C for 4 min) of the exons in which mobility shifts or additional bands were observed.The amplified DNA was sequenced on an ABI PRISM 377 DNA Sequencer by using Amplitaq DNA Polymerase(PE Biosystems). All new mutations were confirmed by restriction digestion (Table 1) with the appropriateenzyme (New England Biolabs).

Preparation of poly(A)+ RNA and Reverse Transcription (RT)-PCR analysis

Poly(A)+ RNA was prepared from total fibroblast RNA with the Dynabeads mRNA DIRECT kit. Samples ofpoly(A)+ RNA were reversed transcribed using an exon 7 specific primer, 5’-CTGGTTGGCAAAGAGCAGG-3’.PCR was performed by 35 cycles (93 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec) with the primers5’-TCATCAGCCATGACACCCG-3’ (s) and 5’-CTTTTTGTCAGGTCGGATGG-3’ (as). The products wereanalyzed on a 2.5% agarose gel.

Culture of Skin Fibroblasts and Preparation of Fibroblast Whole Homogenates

Fibroblasts of patient 3 were grown from skin biopsy in DMEM, supplemented with 15% FCS. Confluent cellmonolayers were detached by brief trypsinization, collected by centrifugation, and washed two times with PBS.The cell pellets were then resuspended in lysis buffer (30 mM Tris-Cl, pH 7.5, 150 mM NaCl, 3 mM MgCl2,)containing 1% NP40 and a protease inhibitor cocktail. The lysate was incubated on ice for 15 min and centrifugedat maximum speed in a table-top centrifuge (4 °C). The supernatant was used as a source in western blotting.

SDS-PAGE and Western Blot Analysis

Fibroblast lysates were analyzed on a 12% polyacrylamide gel as described [Kugler et al., 2000] using amonoclonal antibody against human b5R, kindly provided by Drs. Lan and Zhu.

RESULTS AND DISCUSSION

Methemoglobin reductase activity was reduced to ∼20 % in patients 1 and 2 and to below 10% of normal inpatient 3. This holds true even considering the slightly reduced normal range in infants compared to adults. Theparents of the patients each showed reduced enzyme activities compatible with the heterozygous carrier state.There was no evidence for the presence of any abnormal hemoglobin as judged by electrophoresis. No other redcell enzyme deficiency was detected. Hematological data including hemoglobin concentration and reticulocytecount were normal in all probands.

The mutations responsible for the b5R deficit in our patients were identified by sequencing of the b5R exonswhich revealed differences in SSCP analysis (Fig. 1A). To exclude the possibility that the mutations identified arecommon variants in the population, identical genomic fragments were amplified from 60 unaffected subjects. Thebase substitutions detected in all patients were not found in any of these DNA samples, thus making the existenceof a polymorphism unlikely. Patient 1 is homozygous for a c.535G→A exchange in exon 6 (A179T). Thissubstitution is situated in Nβ2 within the NADH domain (residues 119-272). As a result the mutant enzyme has arelatively subtle change as Ala and Thr are both nonpolar and uncharged amino acid residues. One would expectalmost normal kcat/Km values and roughly normal catalytic activity of the mutant enzyme. This would well explainwhy the patient’s symptoms were so mild. A similar amino acid change, A179V, has already been described[Higasa et al., 1998]. The present finding of the A179T mutation, together with the reported mutations at codon204 [Wang et al., 2000; Vieira et al., 1995], make codons 179 and 204 the only ones within the b5R gene wheremore than one mutation has been identified.

Patient 2 was shown to be homozygous for a mutation in exon 9 (c.757G→A) resulting in a replacement ofvaline by methionine (V253M) which is located between Nβ4 and Nα5 within the NADH domain.

In patient 3 we detected a heterozygous c.379A→G change in exon 5 (M127V). The mutation is situated inbetween the two strands of a small antiparallel β-sheet (residues 120-124 and 128-130). This hinge region behind alarge cleft appears to determine the relative position of the FAD and NADH domain [Nishida et al., 1995]. Due to

4 Kugler et al.

the somewhat bulkier shape of valine compared to methionine the two strands of the β-sheet may be shifted fromtheir normal position. This in turn would influence the width of the cleft and the relative position of the twodomains resulting in a modified conformation of mutant b5R from this allele. An additional sequence differencewas found at the invariant 3’-splice site of intron 4 (IVS4-2A→G). His father is heterozygous for the mutation inexon 5, his mother for the splice site mutation.

To further understand the underlying molecular defect, RT-PCR was performed on total skin fibroblast RNA ofpatient 3 and a healthy control. Sequencing the region from exon 3 to exon 7 revealed the exon 5 mutation in ahomozygous state. This suggests that the A→G exchange found is probably involved in a loss of exon 5 on oneallele. To analyze exactly the mode of exon 5 skipping, poly(A)+ RNA was reverse transcribed. Analysis of thePCR products revealed the expected fragment (338 bp) in the control sample (Fig. 1B). In contrast, a complexpattern of different bands was observed in the proband’s sample: one with the normal size (338 bp), and two otherswith faster migrations. Sequencing of these cDNA fragments unambiguously identified the 338 bp band as derivedfrom the allele with the missense mutation in exon 5. The 208 bp fragment was found to be completely devoid ofexon 5 and to have exon 4 precisely abutted to exon 6 (not shown). Consequently, soluble and membrane-boundb5R proteins are devoid of the NADH domain and, thus, cannot bind NADH. This analysis further demonstratesthat RNA is made from the allele harboring the 3’-splice site mutation.

The predicted translation product of this mutated mRNA is a truncated hybrid polypeptide which would not beexpected to be functional and is highly prone to intracellular proteolysis. This is concordant with the loss ofenzyme demonstrated in our western blot analysis using a monoclonal antibody against human erythrocyte b5R[Lan et al., 1996]. Fig. 1C shows that no b5R antigen was detected in the fibroblasts of patient 3. In contrast, thesample from a healthy control yielded a prominent band which can be attributed to the membrane-bound form ofb5R. However, it cannot be formally excluded that mutant b5R protein was present, but not immunologicallyrecognizable. Patient 3 is reminiscent of patient BEN [Vieira et al., 1995]. In this patient with severe RCM type II,exon 5 skipping with the precise junction of exon 4 to exon 6 was found. The only mutation detected was ahomozygous G→C transversion at position +8 downstream from the 5’ splice site in intron 5.

Figure 1 A: Silver-stained SSCP gel of a PCR-amplified DNA fragment containing exon 5 of the b5R(DIA1) gene from patient 3 (P), his parents (father, F, mother, M) and four healthy controls on a 15%polyacrylamide gel. B: PCR-amplified cDNA spanning exon 3 through exon 6 from patient 3 (P) and ahealthy control (Co) were fractionated on a 2.5% agarose gel. Fragments corresponding to the wildtype(338 bp) allele and the allele with the exon 5 skipping (208 bp) are marked. The weak fragment below338 bp was shown to be a heteroduplex with the exon 6 sequence underlying exon 5. M, 50 bp ladder.C: Western blot analysis of skin fibroblast extract from patient 3 (P) and a healthy control (Co) withanti-human erythrocyte b5R. The positions of the erythrocyte b5R and the molecular weight markerbands are indicated.

Molecular Study in Patients with Methemoglobinemia 5

To date, the highly heterogeneous molecular spectrum in the b5R gene comprises 24 different mutations inunrelated patients of different ethnic origin with RCM: 13 missense mutations, 5 nonsense mutations, 2 deletionmutations, and 4 splicing mutations [Aalfs et al., 2000; Wang et al., 2000]. Interestingly, no mutations in theupstream regulatory regions have been reported. With the exception of the mutations S128P [Kobayashi et al.,1990] and V253M [this report], RCM type I seems to be associated with amino acid substitutions only, whereasRCM type II seems to be associated with nonsense mutations or deletions. These molecular differences, which leadto variations in different enzymatic characteristics, may help to explain the clinical differences between RCMtypes I and II.

From a clinical point of view, an important practical application that the molecular knowledge can offer is ahighly reliable prenatal diagnosis, e.g. by chorion villus sampling. With a recurrence risk of 25%, prenataldiagnosis in methemoglobinemia type II may be important to parents, especially after the birth of a previous childwith RCM type II.

ACKNOWLEDGMENTS

We would like to thank Drs. Lan and Zhu (Dongfang Hospital, Fuzhou City, China) for kindly providing themonoclonal b5R antibody.

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