HUMAN MUTATION 23:496^505 (2004)

RESEARCH ARTICLE

Genetic Characterization of Myeloperoxidase

Deficiency in Italy
Caterina Marchetti,1,2 Pierluigi Patriarca,2 G. Pietro Solero,3 Francisco E. Baralle,1 and
Maurizio Romano1,2n
1

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2Department of Physiology and Pathology, University of Trieste,
Trieste, Italy; 3Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica e Microscopia Clinica, Verona, Italy
Communicated by Jan Kraus
Hereditary myeloperoxidase (MPO) deficiency (MPOD) is the most common neutrophil biochemical defect,
and is characterized by a lack of peroxidase activity. In order to extend the epidemiological studies on hereditary
MPOD in Italy, a population screening was carried out to detect mutations in the MPO gene. Of approximately
40,000 individuals analyzed, seven partial and eight total MPO-deficient subjects were identified. The genetic
characterization of the subjects showed the presence of three already-known mutations (c.752T4C,
c.1705C4T, and c.1566_1579del14) and six novel mutations: four missense mutations (c.995C4T,
c.1112A4G, c.1715T4G, and c.1927T>C), then a deletion of an adenine within exon 3 (c.325delA) and
a mutation within the 30 splice site of intron 11 (c.20312A4C). The novel missense mutations cause the
substitution of the residues p.A332V, p.D371G, p.L572W, and p.W643R, respectively, and the potential
structural changes are discussed. The c.325delA deletion causes a shift of the reading frame with the
occurrence of a premature stop codon within the propeptide. Then, considering the difficulty in obtaining bone
marrow samples from MPO-deficient subjects to study MPO mRNA splicing in vivo, we set up an eukaryotic
expression system to investigate how the c.20312A4C mutation alters the MPO pre-mRNA splicing. The
activation of a cryptic 30 splice site located 109nt upstream of the authentic 30 splice site was observed. The
109nt-insertion causes a shift in the reading frame that should lead to the generation of an abnormal MPO
precursor lacking the enzymatic activity. Hum Mutat 23:496505, 2004. r 2004 Wiley-Liss, Inc.
KEY WORDS:

myeloperoxidase; MPO; MPO deficiency; MPOD; missense mutations; splicing mutation; Italian

DATABASES:

MPO OMIM: 606989, 254600 (MPOD)

INTRODUCTION

Hereditary myeloperoxidase (MPO) deficiency

(MPOD; MIM# 254600) appears to be the most
common biochemical defect of neutrophils and is not
geographically restricted. An estimated prevalence of 1
out of 2,000 individuals has been reported in the United
States and of approximately 1 out of 4,000 individuals in
Italy [Cramer et al., 1982; Dri et al., 1982]. MPOD is the
result of mutations in the MPO gene (MIM# 606989).
Studies have reported an increased susceptibility to
infections in some MPOD patients, particularly those
infections caused by Candida albicans [Lehrer and Cline
1969; Cech et al., 1979]. The patients in these studies
also suffered from diabetes and, hence, the increased
susceptibility to the infections could not be attributed
only to MPOD. Other studies, however, have found an
association between MPOD and pustular candidal
dermatitis [Nguyen and Katner, 1997] or systemic
infections (candidiasis and bacteremia) in nondiabetic
MPO-deficient patients [Parry et al., 1981]. The case of
a young MPO-deficient female who developed osteomyelitis with no evidence of diabetes mellitus has also
r2004 WILEY-LISS, INC.

been reported [Weber et al., 1987]. It has not yet been

possible to establish a clear correlation between MPOD
and infection onset in all reported cases. It has been
suggested that MPOD itself does not cause an increase of
susceptibility to infections, but does synergistically work
with other pathologies in order to decrease the ability of
the host to defend against microorganisms.
The possible relationship between neutrophil MPOD
and occurrence of neoplasm was investigated in a
population-screening study [Lanza et al., 1987]. Of
The Supplementary Material referred to in this article can be found
at http://www.mrw.interscience.wiley.com/suppmat/1059 -7794/
suppmat/index.html
Received 28 July 2003; accepted revised manuscript 19 November
2003.
n
Correspondence to: Dr. Maurizio Romano, International Centre for
Genetic Engineering and Biotechnology, Padriciano 99, 34012 ^
Trieste, Italy. E-mail: mromano@icgeb.org
Grant sponsors: Commissario del Governo nella Regione Friuli-Venezia Giulia (FondoTrieste); Ministero dellIstruzione, dellUniversita'
e della Ricerca (ex 60%).
DOI 10.1002/humu.20027
Published online in Wiley InterScience (www.interscience.wiley.com).

MPO DEFICIENCY IN ITALY

148,000 subjects screened, 36 unrelated individuals with

neutrophil MPOD were studied. The high incidence of
malignancy in patients with complete MPOD suggested a
relationship between a defective MPO system and
neutrophil-mediated tumor cell cytotoxicity [Lanza
et al., 1987].
Genetic studies have found that multiple genotypes
might cause human MPOD. One research group
suggested a pretranslational mechanism for the defect,
since mRNA could not be detected in the granulocyte
precursors [Tobler et al., 1989]. In contrast, another
group observed normal levels of MPO mRNA in
neutrophil precursors and the presence of MPO
immunorelated peptides in mature neutrophils in other
MPO-deficient subjects. This finding suggested a
posttranslational nature for the defect [Nauseef et al.,
1983].
Hereditary MPOD has generally been considered an
autosomal recessive trait with highly variable expression.
Four genotypes responsible for hereditary MPOD have
been identified, three of which are missense mutations in
the coding region of the gene: c.1705C4T (g.7906C4T;
p.R569W) [Nauseef et al., 1994], c.752T4C
(g.1626T4C; p.M251T) [Romano et al., 1997], and
c.518A4G (g.1215A4G; p.Y173C) [DeLeo et al.,
1998]. Another mutation in the MPO gene responsible
for hereditary MPOD is a 14-base deletion in exon 9 of
the gene, c.1566_1579del14 [Romano et al., 1997].
Several studies have suggested that most MPOD
patients are compound heterozygotes (i.e., they have a
different mutation on each allele) [Nauseef et al., 1994,
1998; Romano et al., 1997; Patriarca and Romano,
2000]. Similarly to other genetic diseases, a number of
different allele combinations can lead to the MPOD
phenotype, and this may partially explain the variability
of the clinical features. Some mutations result in
posttranslational defects, while others seem to cause
pretranslational defects that are possibly due to structural
alterations in the regulatory parts of the MPO gene
[Selsted et al., 1993]. Overall, the genetic basis of this
condition is now thought to be quite heterogeneous and
complex, and consequently further studies are required
to shed light on the nature and indeed the clinical
consequences of the defect. In order to extend the
studies on the genetic basis of MPOD in Italy, we carried
out a population screening that led to the identification
of eight total and seven partial MPO-deficient subjects
from unrelated families and we explored their structure
function correlation. We now report 12 new genotypes
arising from the different combinations of three
known mutations (c.1705C4T, c.752T4C, and
c.1566_1579del14) and six new mutations causing the
phenotype of complete or partial MPOD.
MATERIALS AND METHODS
Screening for MPO-Decient Subjects
In collaboration with the Clinical Chemistry Laboratory of the
G.Rossi Hospital in Verona, Italy, we carried out a population
screening using the Technicon-H3 system (Bayer Diagnostic,
Tarrytown, NY). The Bayer Technicon H3 uses cytochemical

497

staining of leukocytes; cells are stained for peroxidase and then

analyzed using light scatter and absorbance patterns [Gulley et al.,
1990]. Reference values ranged between 10 and +10 and a mean
peroxidase index (MPXI) of o34.5 was used as a threshold to
identify MPO-deficient neutrophils.
The instrument was calibrated with a set point hematology
calibrator (Bayer Technicon, www.bayerdiag.com) and day-to-day
calibration was done with a 3*in*1 test point calibrator (Bayer
Technicon) that includes the NEUTx and NEUTy.
Mutation Detection
DNA was extracted from peripheral blood leukocytes by
standard laboratory methods. In order to analyze the MPO coding
sequence, each MPO exon and the MPO intronic flanking regions
(GenBank accession number J02694.1 for cDNA and X15377.1
for genomic DNA) were amplified by PCR (941C for 30 sec, 561C
for 1 min, 721C for 1 min; 35 cycles) using approximately 200400
ng of genomic DNA as a template. After amplification, the PCR
products were purified through a MicroSpin S-400 HR Column
(Amersham Pharmacia Biotech, Uppsala, Sweden) and were then
directly sequenced by a CEQ 2000 sequencer (Beckman-Coulter,
Fullerton, CA) according to the manufacturers instructions.
When a mutation was found within any MPO amplified sequence,
the corresponding PCR product was cloned into the Sma I site of
pUC19 plasmid in order to analyze the two alleles separately.
Sequence analysis of the plasmid DNA was performed by the CEQ
2000 sequencer both for sense and anti-sense strand. The
oligonucleotides listed in Supplementary Table S1 (available
online at http://www.mrw.interscience.wiley.com/suppmat/10597794/suppmat/index.html) were used to amplify and sequence all
the 12 MPO exons including the intronic flanking regions. The
presence of each mutation was confirmed by sequencing of
amplicons obtained in an independent experiment of PCR amplification. For genomic DNA and cDNA sequences, the A of the
ATG of the initiator Methionine codon is denoted nucleotide +1.
Characterization of the Eects of the c.2031^2A4C
Substitution on Splicing
Constructs. An in vitro system has been set up to analyze the
effects of the c.20312A4C mutation on splicing. The 2,257-bp
human MPO genomic region encompassing exon 10, exon 11,
and exon 12 was amplified by PCR (941C 30 sec, 581C 1 min,
721C 2 min 30 sec; 35 cycles) from a MPO wild-type allele and
from a c.20312A4C allele. The following oligonucleotides were
used for PCR amplification: Ex 10 Hind dir, 50 -ACGAAGCTTATGAGTGGCATTGACCCCATCCTC-30 and Ex 12 Xho rev,
50 -CGTCTCGAGTTACCTGGCCTCTAGGAGGCTTCCCT-30 .
The PCR product was blunt-end ligated into a pUC 19 Sma I cut
plasmid. Sequencing excluded the presence of mutations within
the insert. The insert including MPO exon 10-exon 12 genomic
region was Hind III/Xho I excised from pUC 19 vector and ligated
in pcDNA3 expression vector for cellular transfection (Invitrogen
Italia, Milan, Italy). The identity of all constructs was checked by
sequencing.
Transfections. Human hepatocarcinoma Hep3B cell line was
grown in Dulbeccos modified Eagles medium supplemented with
4.5 g/l glucose, 10% fetal calf serum, 50 mg/ml gentamicin, and
4 mM glutamine. The DNA used for transfections was purified
with JetStar columns (Genomed, GmbH, Wielandstrasse, Germany). Liposome-mediated transfections of 3
105 Hep3B cells
were performed using DOTAP (Alexis Biochemicals, Lausen,
Switzerland), according to the manufacturers instructions. A total
of 5mg of DNA construct was used for each transfection. After 12
hr the medium was replaced with fresh medium and 24 hr later the
cultures were terminated. The RNA was extracted using RNAwiz
solution (Ambion, Austin, TX). Each transfection experiment was
repeated three times.

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RT-PCR. In order to synthesize cDNA, 3 mg of total RNA

extracted from cells was mixed with Not I-d(T)18 primer in a final
volume of 20 ml. PCR was carried out for 35 cycles (941C 30 sec,
561C 45 sec, 721C 45 sec; 40 cycles) in 50 ml reaction volumes
using these specific oligonucleotides: Ex 10 Hind dir and Ex 12
Xho rev. The PCR products were separated on a 2% agarose gel
and stained with ethidium bromide. The amplicons were purified
from the gel and directly sequenced.
Secondary Structure Prediction
The possible impact of the missense mutations on the secondary
structure of the MPO protein was tested using the Advanced
Protein Secondary Structure Prediction Server (APSSP2, www.
imtech.res.in/raghava/apssp2/). This method uses three steps for
protein secondary structure. First, it uses the standard neural
network and multiple sequence alignment generated by PSIBLAST instead of a single sequence. In the second step, it predicts
the secondary structure of proteins using the modified examplebased learning (EBL) technique. In the third step, the secondary
structures predicted from the first two steps are combined in order
to predict the final structure. The combination of the two steps
based on reliability score.
The probability of correct prediction of the structure state of
relevant residues ranged between 0.7 and 0.9 (where the
maximum possible score was 1.0).
RESULTS
Identication of 15 Total or Partial MPOD Subjects

In order to extend the epidemiological studies on

hereditary myeloperoxidase deficiency in Italy, a popula-

tion screening using the Bayer Diagnostic Technicon-H3

system was performed in the Clinical Chemistry
Laboratory of the G.Rossi Hospital in Verona. This
system is currently in use in clinical laboratories to obtain
the leukocyte differential-count based on a continuous
flow cytometric assay of peroxidase activity (Fig. 1). After
4 months, seven partial and eight total MPOD subjects
were identified out of approximately 40,000 individuals
analyzed.
Genetic Characterization of a Group of MPOD Subjects

Using the genomic DNA of all the subjects as a

template, we amplified by PCR and directly sequenced all
12 MPO exons and their intronic flanking regions. After
the identification of mutations, the PCR products were
cloned in a vector in order to analyze the two alleles
separately and confirm their presence. Initially, we looked
for the presence of the already-known mutations and, in
order to identify possible new mutations, we then
screened all the MPO exons and their flanking intronic
regions.
The sequence of exon 9 and 10 showed that three
subjects were heterozygous and one homozygous for the
c.1566_1579del14 mutation. Moreover, one subject was
heterozygous for the c.752T4C substitution and another individual was a compound heterozygote for the
c.752T4C and c.1566_1579del14 mutations. Then, the

Leukocyte counts determined by automated ow cytometric analysis of peroxidase-stained samples of venous blood.The
cytograms were obtained in Verona Hospital Clinical Laboratory using aTechnicon-H3 analyzer (Bayer Diagnostic). Normal cytogram represents normal neutrophil peroxidase activity. Partial and total cytograms are examples of abnormal proles of neutrophils
counts in the MPOD subjects. Partial, partial MPO deciency; Total, complete MPO deciency.The position of dierent leukocytes
within the cytogram are indicated. NEUT, neutrophils; EOS, eosinophils; LYMP, lymphocytes; LUC, large unstained cells.

FIGURE 1.

MPO DEFICIENCY IN ITALY

sequencing of exon 6 and 4, where the c.752T4C and

c.518A4G mutations were located, led to the identification of four subjects heterozygous for the c.752T4C
substitution.
Finally, the sequence of the remaining exons led to the
identification of four new missense mutations in exons 7,
10, and 11, a deletion of 1 bp within exon 3, and a
mutation within the 30 splice site of intron 11. In fact, we
identified two point mutations in exon 7: the C>T
transition in position 995 of cDNA (c.995C4T) that

499

causes the replacement of the alanine 332 with a valine

(p.A332V) (data reviewed but not shown) and the
A4G transition in position 1112 (c.1112A4G) that is
responsible for the substitution of the aspartic 371 with a
glycine (p.D371G) (data reviewed but not shown). The
third new missense mutation was a T4G substitution in
position 1715 of cDNA (c.1715T4G in exon 10) that
causes a replacement of the residue leucine 572 with a
tryptophan (p.L572W) (data reviewed but not shown).
We subsequently found a T4C transition in position
1927 of cDNA (c.1927T4C) of the coding sequence in
exon 11 that causes the substitution of the tryptophan
643 with an arginine (p.W643R) (data reviewed but not
shown). Two subjects with partial MPOD were heterozygous, one for the mutation c.995C4T and one for the
mutation c.1112A4G. Two patients with total MPOD
presented a compound heterozygosity, one for
the c.995C4T and the c.1715T4G mutations and
the other for the c.1927T4C and c.1566_1579del14
mutations.
A single nucleotide deletion in position 325 within
exon 3 was observed in another subject with partial
MPOD (c.325delA) (Fig. 2A), causing a shift of the
reading frame and the occurrence of a premature stop
codon at position 113 within the propeptide. Finally, a
splice-site mutation was showed by our sequencing
analysis: an A4C substitution in position 2 of exon
12 (c.20312A4C) that involves the 30 splice site of
intron 11 (Fig. 2B). The analysis indicated that two
subjects were homozygous for the c.20312A4C mutation and one presented a compound heterozygosity for
the c.20312A4C mutation and the c.1566_1579del14
deletion.
The genetic characterization of the group of MPOD
subjects including the new mutations responsible for
hereditary MPOD is summarized in Table 1.
Structural Implications of the New Missense Mutations

Sequence of novel c.325delA deletion and c.2031^

2A4C mutation. A: MPO exon 3 wild-type and mutant allele
(c.325delA; g.836delA) sequences are shown (GenBank accession number J02694.1). The c.325delA deletion is indicated by
an asterisk. B: Eect of the c.325delA deletion on exon 3 reading
frame.The occurrence of a premature stop codon at position 113
is shown. C: IVS 11 wild-type c.2031^2A and c.2031^2A4C alleles are shown. The A4C substitution in position 12554 (GenBank accession number X15377.1) is underlined. D: The
mutation involves the 3 0 splice site of MPO intron 11. Intron sequences are in lower case while exon sequences are in upper
case. Both the wild-type and the mutated nucleotides are boldfaced and indicated by an arrow.
FIGURE 2.

After genetic characterization of the MPOD subjects

and the discovery of six new mutations of the MPO gene
responsible for the hereditary MPO deficiency, we
focused our attention on the missense mutations position
within the MPO molecule. The protein fold of MPO
[Zeng and Fenna 1992; Fiedler et al., 2000] provides the
data required to localize the new missense mutations
within the MPO molecule and suggests their possible
effects on the MPO secondary structure (Fig. 3A).
The secondary structure of the MPO molecule is
largely a-helical [Zeng and Fenna 1992]. Figure 4B
summarizes the localization of the new and the known
missense mutations within MPO a-helices (H) and
locally folded regions (LR). We observed that the
missense mutations p.A332V and p.D371G are located
in a short strand of an extended chain that links the two
domains LR1 to LR2 together, and in the LR2 domain,
respectively. More specifically, we noticed that the
mutation p.A332V is located in the strand of the protein
chain that links H2 to the H3 a-helix, while the
mutation p.D371G is located in the strand of the protein

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TABLE 1. Genetic characterization of

Patient
number

the group of 15 MPO decient subjects

Name

Sex

MPO
status

Genotypea

Eects of mutations

A.P.

[c.2031^2A4C]+[c.2031^2A4C]

2
3
4
5
6
7

A.A.
Z.A.
B.M.
B.N.
C.E.
C.M.

F
F
F
M
F
F

T
P
T
P
P
T

[c.1566_1579del14]+[c.1705C4T]
[c.752T4C]+[WT]
[c.1566_1579del14]+[c.1566 _1579del14]
[c.995C4T]+[WT]
[c.325delA]+[WT]
[c.2031^2A4C]+[c.2031^2A4C]

8
9
10
11
12
13
14
15

C.G.
C.A.
D.D.
L.G.
M.A.
R.M.
S.I.
V.O.

M
M
M
M
F
F
F
F

T
P
T
P
P
T
P
T

[c.1705C4T]+[c.752T4C]
[c.752T4C]+[WT]
[c.995C4T]+[c.1715T4G]
[c.1566_1579del14]+[WT]
[c.752T4C]+[WT]
[c.1566_1579del14]+[c.2031^2A4C]
[c.1112A4G]+[WT]
[c.1566_1579del14]+[c.1927T4C]

[r.2031-109_2031-2ins; r.2031-2A4C]
+[r.2031-109_2031-2ins; r.2031-2A4C]
[r.1545_1622del]+[p.R569W]
[p.M251T]+[WT]
[r.1545_1622del]+[r.1545_1622del]
[p.A332V]+[WT]
[p.A108fs5]+[WT]
[r.2031-109_2031-2ins; r.2031-2A4C]
+ [r.2031-109 _2031-2ins; r.2031-2A4C]
[p.R569W]+[p.M251T]
[p.M251T]+[WT]
[p.A332V]+[p.L572W]
[r.1545_1622del]+[WT]
[p.M251T]+[WT]
[r.1545 _1622del] +[r.2031-109_2031-2ins; r.2031-2A4C]
[p.D371G]+[WT]
[r.1545 _1622del] +[p.W643A]

MPO status:T, complete deciency; P, partial deciency; WT, wild-type allele.The genotype of each patient (fth column) and the eects of mutations
(sixth column) at RNA (plain text) or at protein level (bold text) are shown.
a
GenBank accession numberJ02694.1 for cDNA.The A of the ATG of the initiator methionine codon is denoted nucleotide +1.

FIGURE 3. Localization of the amino acids aected by the novel point mutations within the MPO dimer. A: The small and the large
polypeptides of the two halves are shown with the heme groups (Fe) and the calcium ions (Ca). Each amino acid aected by the new
point mutations within the MPO molecule is specied and indicated by arrows.The amino acid numbers refer to the propeptide sequence. B: Localization of the missense mutations within the MPO structure. Both genomic (rst column) and exonic (second column) localization of each mutation are indicated.The position of each amino acid substitution caused by each mutation is referred
both to pro-MPO (third column) and mature MPO protein (fourth column).The position of the mutations within MPO locally-folded
regions (LR) and within MPO a-helices is shown. C: APSSP2 prediction of the possible changes of the secondary structure induced by
the indicated point mutations. MPO p.A332, p.L572, and p.W643 are the wild-type protein sequences and structures while MPO
p.A332V, p.L572W, and p.W643R are the mutants. The amino acids involved by the point mutations are underlined in bold. C,
random coil; H, alpha helix; E, extended strand.

MPO DEFICIENCY IN ITALY

chain that links H3 to the H4 a-helix (Fig. 3B). In order

to evaluate the possible effects of each amino acid
substitution on MPO secondary structure, we used the
secondary structure program APSSP2 (Advanced Protein Secondary Structure Prediction Server) [Kaur and
Raghava, 2002]. This prediction shows that the presence
of a valine in position 332 may cause the change of the
normal coil fragment in an a-helix region (Fig. 3C). On
the other hand, the substitution of an aspartic acid with a
glycine seems to disturb the alpha helix that encompasses
the region with residue 371 (Fig. 3C). The p.L572W
substitution occurs just downstream of the carboxylterminus of the H11 helix located within the LR4
domain (Fig. 3B). The APSSP2 secondary structure
prediction for p.L572W suggests that the tryptophan in
position 572 might cause the change of the normal coil
fragment into an a-helix (Fig. 3C). Finally, the
tryptophan 643 affected by the p.W643R mutation is
located in the H16 helix of the LR5 domain that
encompasses the carboxyl-terminal portion of the
large polypeptide (Fig. 3B). The APSSP2 secondary

501

structure prediction for the p.W643R substitution

suggests that it might disrupt the H16 a-helix
conformation (Fig. 3C).
Eects of the c.2031^2A4C Substitution on Splicing

MPO mRNA is actively synthesized only in neutrophil

granulocyte precursors [Cowland and Borregaard, 1999]
and thus studies of the defects of pre-mRNA processing
in myeloid precursors of MPOD subjects are not simple.
Therefore, in order to evaluate the possible effects of the
c.20312A4C mutation on MPO splicing, we generated
an eukaryotic mini-gene expression system carrying the
genomic region of human MPO spanning from exon 10
to exon 12. We amplified by PCR the 2,257-bp genomic
region encompassing MPO exons 10, 11, and 12 from a
wild-type allele and from a c.20312A4C allele. The
two amplicons were then cloned into an eukaryotic
expression vector according to the original reading frame.
Two constructs, IVS11-WT and c.20312A4C, were
generated carrying the IVS11 wild-type or the c.2031
2A4C allele, respectively (Fig. 4A). IVS11-WT and

FIGURE 4. Eects of the mutation c.2031^2A4C on MPO intron 11 splicing. A: Diagram of the IVS11-WTand the c.2031^2A4C constructs.White boxes are MPO exons, the lines are MPO introns. Both the wild-type sequence (CAG) and the c.2031^2A4C mutation
(CCG) are indicated. Hind III and Xho I are the restriction sites used for cloning in the pcDNA 3 expression vector. B: RT-PCR assay
performed on transfection experiments of Hep3B cell line using the IVS11-WT and the c.2031^2A4C constructs. The RT-PCR products were resolved on 2% agarose gel electrophoresis. Lane IVS11 WT: 650-bp RT-PCR fragment corresponding to exon 10 +11+12
mRNA. Lane c.2031^2A4C: 759-bp RT-PCR fragment corresponding to exon 10+11+12 mRNA plus the inclusion of 109 nt of intron
11. Control: RT-PCR negative control. Marker:1-kb molecular weight markers. C: Eect of the mutation c.2031^2A4C on MPO gene
translation.The 30 end of exon 11 and exon 12 sequences are in upper case while intron 11 is in lower case.The normal 3 0 splice site of
intron 11 is underlined (cag). In the WT sequence the normal 30 splice site is recognized, while in the c.2031^2A4C sequence the 30
splice site of intron 11 is mutated and another cryptic splice site located 109 nt upstream is selected in bold.

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c.20312A4C plasmids were used for transient transfection experiments in the Hep3B cell line. After termination of the cell cultures, RT-PCR analysis was carried out
using specific primers for the constructs and the splicing
pattern was analyzed by running the PCR products on
2% agarose gel stained with ethidium bromide (Fig. 4B).
Transfection of the IVS11-WT construct resulted in the
expression of a 650-nt mRNA corresponding to the MPO
exons 10, 11, and 12, while transfection of the c.2031
2A4C construct resulted in the expression of a 759-nt
mRNA, corresponding to the MPO exons 10, 11, and 12,
plus the inclusion of 109 nt of intron 11 (Fig. 4B). The
identity of the RT-PCR products was verified through
cloning and sequencing. This experiment showed that
the c.20312A4C mutation activates a cryptic 30 splice
site located 109 nt upstream of the normal 30 splice site
of intron 11. These 109 nt are inserted within the MPO
mRNA between exon 11 and 12 (r.2031-109_2031-2ins;
r.2031-2A4C).
This insertion causes a shift in the reading frame with
the generation of a stop codon after codon 749 that
should be translated into a MPO precursor with a
completely subverted sequence of the 72 amino acids
carboxy-terminal tail downstream of the activated cryptic
splice site (Fig. 4C).
DISCUSSION

The genetic basis of the MPO deficiency seems to be

quite complex, as most patients are compound heterozygotes, which means that they are affected with an
autosomal recessive disorder having two different mutations in homologous genes [Nauseef et al., 1998]. Since a
number of genetic mutations resulting in MPOD have
been identified in the past years, we hypothesized that
other mutant alleles might occur within the MPO gene,
concluding that epidemiological studies are necessary to
assess the possibility that known genotypes are limited to
certain geographic regions. For these reasons, we decided
to extend the epidemiological studies on hereditary
MPOD to the Veneto region in Italy by a full-MPO gene
screening for old and new mutations responsible for the
disease. In collaboration with the Laboratory of Clinical
Biochemistry of Verona G. Rossi hospital, we performed a population screening of approximately 40,000
individuals. After 4 months, 15 subjects with partial or
total MPOD were identified. Thus, the prevalence of
MPOD was 1 out of 2,500 individuals, as reported in
previous studies [Parry et al., 1981; Cramer et al., 1982].
The patients were healthy or in any case showed no
diseases correlated with MPOD. Only the genomic DNA
of the subjects was available for the study and it was used
for the genetic characterization.
The first step of the genetic analysis, consisting of the
verification of the presence of the already known
mutations in the MPO gene, showed the c.1705C4T
mutation in two subjects, the c.752T4C mutation in
four subjects, and the c.1566_1579del14 deletion in five
subjects. This result indicates that the point mutation
c.1705C4T, which was originally identified in the

United States [Nauseef et al., 1996], is not limited to

the United States, but it is also present in Europe. On
the other hand, the two mutations c.752T4C and
c.1566_1579del14 that were identified for the first time
in Italy in a family from Padua (Italy) [Romano et al.,
1997] are also present in the Verona area. Therefore,
these findings suggest that mutations c.752T4C and
c.1566_1579del14 are not private mutations. Moreover, these mutations seem to have a relatively high
allelic frequency, since c.752T4C was found in 4 out of
30 alleles and c.1566_1579del14 was found in 6 out of 30
alleles.
This is the first time that the c.1705C4T mutation
has been reported in Italian subjects. Conversely, the
c.752T4C and c.1566_1579del14 mutations were not
found in the United States. However, it should be noted
that the population screening performed in the United
States was set up only for the detection of the
c.1705C4T mutation and not for the simultaneous
verification of the presence of other mutations [Nauseef
et al., 1998]. Thus, it cannot be excluded that either
c.752T4C or c.1566_1579del14 might be present also
in the United States.
The second step of the study was aimed at screening
for novel mutations in the MPO gene and allowed
the identification of six new mutations: a single
nucleotide deletion (c.325delA), four missense
mutations (c.995C4T, c.1112A4G, c.1715T4G, and
c.1927T4C), and the mutation c.20312A4C within
the 30 splice site of the last MPO intron.
In particular, c.995C4T and c.20312A4C were
present in different subjects. In fact, two subjects were
heterozygous for the c.995C>T substitution. On the
other hand, two patients were homozygous and another
one was heterozygous for the c.20312A4C mutation.
The study revealed the presence of seven heterozygous, three homozygous, and five compound heterozygous subjects for the mutations within the MPO gene.
The particular combination of old and new mutations
correlates with the level of MPO enzymatic activity
exhibited by peripheral blood neutrophils. In fact, the
flow cytochemistry analysis of neutrophils from the 15
subjects indicated that seven individuals had partial
MPOD and eight had total MPOD.
Considering the literature data and the results of our
work, it is possible to conclude that the genetic basis of
hereditary MPOD seems to be extremely heterogeneous.
Until now, it has not been possible to distinguish a
predominant mutation among the 11 mutant alleles
characterized by a variable frequency. However, a further
extension of the epidemiological studies might be helpful
for the creation of a more comprehensive catalog of
MPO gene mutations.
New Deletion/Missense Mutations and
Structure-Function Relationships

In vivo studies of previously identified mutations have

highlighted critical features of normal biogenesis of MPO
[Nauseef et al., 1996, 2000; DeLeo et al., 1998]. The

MPO DEFICIENCY IN ITALY

identification of additional genotypes of MPOD and the

recent solution of MPOs crystal structure [Zeng and
Fenna, 1992; Fiedler et al., 2000] provides powerful
information for the extension of the knowledge regarding
the structure-function relationships for MPO. The
mutation c.325delA is located within exon 3 and consists
in a single nucleotide deletion that causes a shift of the
reading frame of that exon, leading to a premature stop
codon within the propeptide. The aberrant MPO mRNA
originated in this way might undergo selective nonsensemediated decay of RNA, as described for other models
[Cheng and Maquat, 1993; Belgrader et al., 1994;
Maquat, 1995; Aoufouchi et al., 1996], thus causing
MPOD.
The four newly-discovered missense mutations provided more information about the fragments and amino
acids of the protein that are essential for its enzymatic
activity. Since the overall protein fold of MPO has been
described [Zeng and Fenna, 1992; Fiedler et al., 2000],
we located the new missense mutations within the MPO
molecule (Fig. 3A). The a-helical core of the MPO
molecule, including the heme-binding pocket, is the
most important part of the molecule with regard to its
enzymatic activity. We observed that at least one of the
missense mutations (p.L572W) was located in the close
proximity of the heme-core region.
In order to assess the effect of the mutations on MPOs
secondary structure, we also performed secondary
structure predictions using APSSP2 (Advanced Protein
Secondary Structure Prediction Server) [Kaur and
Raghava, 2002]. The two missense mutations p.A332V
and p.D371G are located within the amino-terminal
portion of the large peptide that covers the upper region
of the heme-core region [Zeng and Fenna, 1992; Fiedler
et al., 2000].
The properties of the amino acids suggest that even if
the mutation p.A332V is conservative, as both alanine
and valine have a hydrophobic lateral chain, the steric
hindrance of the two amino acids is different. In fact, the
predictions of the possible impact of the amino acid
substitutions on the secondary structure suggest that the
presence of a valine in position 332 might cause the
change of the normal coil fragment in a a-helix region,
where residue 332 is located. Moreover, the p.A332V
substitution might interfere with calcium-binding ability,
as the residue alanine 332 is adjacent to threonine 334,
for which the hydroxyl oxygen atoms are possible calcium
ion ligands within the LR2 loop [Zeng and Fenna, 1992].
On the other hand, the mutation p.D371G is a
nonconservative substitution, since it replaces a hydrophilic and acid residue with a hydrophobic one. Thus,
this mutation may affect the polarity of the involved
protein region, as also suggested by the computer
prediction.
Subsequently, we observed that leucine 572 is one of
of the
the 24 residues containing atoms within 4.5 A
heme atom [Zeng and Fenna, 1992]. All members of the
mammalian peroxidase family share a high degree of
homology within the heme pocket, and the leucine 572
residue is totally conserved in all four mammalian

503

peroxidases [Zeng and Fenna, 1992]. Therefore, the

substitution of leucine, which is an aliphatic residue with
a long side chain, with tryptophan, which is a huge
aromatic residue, might alter the structure of the two
largely hydrophobic helices where residue 572 is placed.
The APSSP2 secondary structure prediction strengthens
this hypothesis. In fact, it suggests that the mutation
might cause the modification of the normal coil fragment
that includes leucine 572 into a a-helix structure. Since
this change involves the immediate environment of the
heme, it is likely that the enzymatic activity might be
severely altered.
Finally, we observed that the substitution of the
aromatic and hydrophobic tryptophan 643 residue with a
highly basic arginine residue is likely to cause an
alteration of the protein. In addition, APSSP2 secondary
structure prediction supports such an hypothesis, indicating that the p.W643R substitution might disrupt
the H16 a-helix conformation. The loss of the H16 ahelix could affect the MPO-heme binding that is
essential for the activation of the enzyme.
Characterization of Eects of the c.2031^2A4C
Mutation on Splicing

Several studies have found that mutations causing

alterations of pre-mRNA splicing are the growing basis of
genetic diseases [Caceres and Kornblihtt, 2002]. The
genetic characterization of the MPOD subjects has
evidenced that five c.20312A>C alleles were found
out of the 30 alleles screened, so it is one of the most
frequent mutations found among the identified MPOD
subjects. If it was apparent that the A>C substitution
interests the 30 splice site of the last MPO intron,
nevertheless the results of this intronic mutation were
not predictable on the basis of genomic DNA sequence
analysis only. For this reason, and considering the
apparent difficulty in obtaining bone marrow samples
from MPOD subjects to study MPO mRNA splicing
in vivo, we decided to set up a eukaryotic expression
system to analyze the effects of this sequence variation
on splicing. Interestingly, the approach used for the MPO
gene might be useful to explore the effect of the
mutations possibly related to splicing of other genes
when the availability of patients RNA is limited or null.
The transfection of the construct carrying the c.2031
2A4C substitution resulted in the expression of a
mRNA species carrying a 109-nt insertion between exon
11 and exon 12. This insertion was the result of the
activation of a new 30 splice site placed 109 nt upstream
of the wild-type 30 splice site of intron 11. Therefore, the
inclusion of 109 nt in the MPO mRNA causes a shift in
the reading frame with the generation of a premature
stop codon (r.2031-109_2031-2ins; r.20312A4C).
Studies on other genes carrying mutations within the
acceptor splice site of the last intron have resulted in
different abnormalities of the splicing pattern.
The G4C substitution in the splice acceptor site (AG
to AC) of the 15th and last intron of argininosuccinate
synthetase was shown to be responsible for the deficiency

504

MARCHETTI ET AL.

of this enzyme in citrullinemia patients. The mutation

leads to the production of three different aberrantly
spliced RNA and all resulted in reading frameshifting at
the C-terminal end of argininosuccinate synthetase [Su
et al., 1983; Su and Lin, 1990]. Similarly, the studies of
the defects in beta-globin genes, present in several forms
of beta-thalassemia, has provided examples of splicing
mutants of the last introns acceptor site. Mutations in
beta-thalassemia that are known to affect the 30 splice
site include a single-base mutation in the splice acceptor
(AG to GG) at the second and last intron of the betaglobin gene, resulting in activation of a cryptic acceptor
site within the second intron [Orkin et al., 1983;
Treisman et al., 1983; Antonarakis et al., 1984]. The
effects of the c.20312A4C mutation also overlaps with
that found in argininosuccinate synthetase and the betaglobin genes as it results in the activation of a cryptic site
upstream from the correct splice site with the consequent
partial-intron retention.
Previous studies on nonsense-mediated decay have
shown that other mRNA carrying non-sense or frameshift mutations do not undergo selective degradation if
the premature stop codon is placed within the last exon
[Cheng and Maquat, 1993; Belgrader et al., 1994;
Maquat, 1995; Aoufouchi et al., 1996]. It is therefore
unlikely that the alteration of the reading frame of the
12th and last MPO exon would lead to the rapid
degradation of the mRNA carrying the 109-nt inclusion.
Instead, the aberrant mRNA should be translated into a
MPO precursor with a completely subverted sequence of
the 72 amino acids carboxy-terminal tail downstream of
the activated cryptic splice site.
Likewise to the outcome of reading frame-shifting at
the C-terminal end of argininosuccinate synthetase and
beta-globin genes, the altered MPO precursor might be
degraded through the proteasome pathway or, alternatively, might be processed generating an inactive mature
MPO protein, thus causing MPOD.
ACKNOWLEDGMENTS

We thank Prof. Mario Zatti for his support in the

organization of the population screening.
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