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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 252-257
Somatic Triple Mosaicism in a Carrier of X-Linked Chronic
Granulomatous Disease
By
Martin de Boer,
Egbert Bakker,
Stefaan Van Lierde, and
Dirk Roos
From the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service, and Laboratory for Experimental and Clinical
Immunology, Academic Medical Centre, University of Amsterdam,
Amsterdam, The Netherlands; the Department of Human Genetics,
University of Leiden, Leiden, The Netherlands; and the Department of
Pediatrics, University Hospital Leuven, Leuven, Belgium.
 |
ABSTRACT |
The X-linked form of chronic granulomatous disease (CGD) is caused
by mutations in the CYBB gene, which encodes the 91-kD subunit of the
flavocytochrome b558, a component of the
superoxide-generating nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase in phagocytic leukocytes. Mutations in
this gene are very heterogeneous and often unique for one family. Here
we report on a family with two patients (brothers), one with a 3-kb
deletion comprising exon 5 and the other with a 3.5-kb deletion
comprising exons 6 and 7 of the CYBB gene. Sequence analysis of
polymerase chain reaction (PCR)-amplified genomic DNA proved these
deletions to be overlapping for 35 bp. Analysis by restriction fragment
length polymorphism of genomic DNA from the mother's leukocytes showed
her to be a carrier of both deletions in addition to the normal CYBB
sequence. This triple somatic mosaicism was confirmed with
PCR-amplified genomic and complementary DNA. The presence of the
normal CYBB gene in the mother was also proven by the finding of
normal superoxide-generating neutrophils in addition to cells lacking
this ability. Triple X syndrome was excluded. These findings suggest
that the mutations are the result of an event in early embryogenesis of
the mother, possibly involving a mechanism like sister chromatid
exchange.
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INTRODUCTION |
CHRONIC granulomatous disease (CGD) is a
severe clinical syndrome characterized by recurrent, life-threatening
bacterial and fungal infections.1 The disease is caused by
failure of the nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase enzyme in phagocytic leukocytes (neutrophilic and eosinophilic
granulocytes, monocytes and macrophages) to generate superoxide, a
precursor of microbicidal oxygen metabolites.2 This enzyme
is composed of several constituents. The flavocytochrome
b558 is located in the plasma membrane of the
phagocytes and consists of two subunits, a 91-kD glycoprotein called
gp91phox (from phagocyte oxidase) and a
22-kD protein called p22phox.3-5 This
flavocytochrome is the actual enzyme that accepts electrons from NADPH
in the cytosol and transmits these to oxygen at the other side of the
membrane, ie, in the phagosome that contains the microorganisms
ingested by the phagocytes. However, under resting conditions, the
oxidase is not active. Only when the phagocytes have bound
microorganisms or soluble stimuli, such as high concentrations of
chemotaxins, the flavocytochrome is converted into an active enzyme.
This activation is brought about by translocation of two additional
oxidase components from the cytosol to the flavocytochrome, probably by
inducing a conformational change in the flavocytochrome.6
These cytosolic oxidase components are a 47-kD protein called
p47phox and a 67-kD protein called p67phox. A
third cytosolic p40phox protein probably stabilizes
p47phox and p67phox in resting
phagocytes.7 Finally, a membrane-bound and a cytosolic
guanosine triphosphate (GTP)-binding protein and several regulating
proteins are involved in fine-tuning the activation
process.8-10
CGD is caused by mutations in gp91phox,
p22phox, p47phox, or
p67phox.11 The CYBB gene encoding
gp91phox is located at Xp21 and contains 13
exons.12 The genes for the other oxidase components are
located on autosomes. Mutations in the CYBB gene, which are found in
about two thirds of all CGD patients, are very heterogeneous,
comprising deletions from one to several million base pairs, insertions
up to 2 kb, splice site mutations, missense mutations, and nonsense
mutations.11 Usually, these mutations are family-specific,
but now, after analysis of about 250 X-CGD families, a few `hot
spots' of point mutations can be recognized, most of them in CpG
dinucleotides.13 In contrast, the origin of the deletions
in the CYBB gene is not clear, but occasionally a family presents with
a defect that gives some insight into this phenomenon. We report here
on a family in which two sons suffer from X-linked CGD due to two
different deletions in the CYBB gene, while their mother carries the
same deletions in her somatic (hair root and white blood) cells in
addition to the normal CYBB sequence. This indicates that the mother is
a triple mosaic in her somatic cells, possibly due to breakage and
reunion of single strands of the sister chromatids early in her
embryonic development.
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PATIENT HISTORIES |
Patient 1 is the oldest of two brothers, born in 1979 and presenting at
the age of 2 years with a disseminated Salmonella infection.
CGD was diagnosed with a stimulated nitroblue tetrazolium (NBT) dye
test showing 0% formazan-positive granulocytes. This result was
confirmed by subsequent NBT tests and by chemiluminescence tests.
The boy was otherwise well until the age of 5 when he had a cellulitis
of the left foot with associated lymphangitis and inguinal
lymphadenitis. At 11 years of age, he developed a first submandibular
lymphadenitis that needed to be drained. The patient was subsequently
given cotrimoxazole as prophylaxis.
When he was 13, tetracycline was given for severe acne. Two years
later, the boy developed a left upper lobe pneumonia, which was
succesfully treated with antibiotics. A few months thereafter, he had a
second episode of submandibular lymphadenitis. The lymph node was
excised, and the culture was positive for Aspergillus
fumigatus. Treatment with amphotericin B followed, and the patient
recovered. Itraconazole was given as prophylaxis.
Because of uncontrolled severe acne, isotretinoin was given at the age
of 16. This drug was discontinued after a few months because of
presumed drug-induced fever and other side effects. That same year, the
patient developed rectal bleeding due to ulcerative lesions in the left
colon. Pathologic examination showed granulomatous inflammation, but no
specific pathogen was recovered from cultures. The patient was treated
with mesalazin and steroids. This treatment was successful for the
colitis. However, after 6 weeks, a nodular pneumonia appeared.
Broncho-alveolar lavage showed no pathogens, but Aspergillus
was felt to be the most likely cause. The patient was successfully
treated for 6 weeks with amphotericin B, while tapering off the
steroids.
As of this writing, the patient is 17 years old and in good general
health. He takes cotrimoxazole and itraconazole as prophylaxis for
bacterial and fungal infections.
Patient 2 is the youngest brother, born in 1981. CGD was diagnosed in
the first weeks of life by means of an NBT dye test. The diagnosis was
confirmed by the absence of chemiluminescence after neutrophil
stimulation in vitro.
At the age of 2 months, the patient developed a submandibular
lymphadenitis, which was successfully treated with parenterally
administered antibiotics. At 6 years of age, he had a pneumonitis with
associated hilar lymphadenopathy, from which the boy recovered after a
10-day course of intravenously administered antibiotics. Three years
later he was hospitalized for a period of 3 months because of a liver
abscess, caused by Staphylococcus aureus. The course was
protracted and complicated with ulcerative duodenitis and subsequent
duodenal substenosis, drug-induced renal salt wasting with hyponatremia
and convulsions, and drug-induced leukopenia. The boy finally recovered
after surgical drainage of the abscess, prolonged courses of multiple
antibiotics, and the administration of  -interferon. While on
cotrimoxazole, rifampin, and fluconazole prophylaxis, he was readmitted
14 months later with a pneumonitis and pericarditis caused by
Aspergillus. Despite treatment with amphotericin B, the patient
deteriorated and developed a constrictive pericarditis, for which he
underwent pericardectomy 2 months after admission. Cardiac output did
not improve, and the patient died of circulatory failure at the age of
10 years and 9 months. The autopsy showed a large thrombus in the
jugular veins and in the left atrium. The epicardium was filled with
Aspergillus hyphi. Both parents had an NBT dye test. It was
normal in the father, but gave intermediate results in the mother who
showed only about 40% of NBT-positive cells after neutrophil
stimulation. Both the father and the mother are in good health. In the
mother's family, no one has a clinical history of CGD.
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MATERIALS AND METHODS |
This study was conducted after appropriate informed consent had been
obtained from all human subjects involved.
Granulocyte function assays.
Neutrophils were purified from venous blood as described
earlier.14 NADPH oxidase activity of intact cells was
measured by oxygen consumption, lucigenin-amplified chemiluminescence,
or NBT dye reduction14 after stimulation of the cells with
phorbol-myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine
(fMLP) or serum-treated zymosan (STZ) particles.
Western blot analysis.
Neutrophil membrane and cytosolic fractions were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
electroblotted onto nitrocellulose paper and probed with mouse
monoclonal antibodies against gp91phox or
p22phox15 or with specific rabbit antisera against
p47phox or p67phox.16 Binding of
the antibodies was detected with alkaline phosphatase-conjugated
antimouse-Ig and antirabbit-Ig, respectively.
Preparation of RNA and DNA and analysis of DNA.
RNA was isolated from blood mononuclear leukocytes as described
previously.17 First-strand cDNA was synthesized from RNA by
reverse transcription. Part of the gp91phox-coding region
(CYBB exon 4-10) was amplified from cDNA by PCR with primers specified
in Table 1, under conditions described
previously.17 The PCR products were separated on Nusieve
2:1 agarose gel and stained with 0.05% (wt/vol) ethidium bromide.
Genomic DNA was isolated from total blood leukocytes.18
Southern blot analysis of genomic DNA was performed by digestion with
the restriction enzyme EcoRI and probing with a cDNA CYBB
fragment that recognizes the entire coding sequence (code pSV-CGD,
kindly provided by Dr M. Dinauer, Indiana University School of
Medicine, Indianapolis). Fragments of the CYBB gene were amplified from
genomic DNA by PCR with the primers specified in Table 1, under
conditions defined previously.17 For nucleotide sequencing,
the direct sequencing method was used.19
X-chromosome marker analysis was performed as previously
described.20,21 Polymorphisms in the ornithine
transcarbamylase (OTC) gene, located at the proximal side of the CYBB
gene, and in the Duchenne muscular dystrophy (DMD) gene, located
distally of the CYBB gene, were used to show the inheritance of the
CYBB mutations in the investigated family.
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RESULTS |
Identification of the CGD subtype.
The patients were identified as CGD patients by complete lack of NBT
reduction by their neutrophils with PMA as the stimulus. Moreover,
lucigenin-amplified chemiluminescence of the patients' neutrophils
stimulated with either PMA or PMA followed by STZ was totally absent.
On Western blots of the patients' neutrophil membranes after SDS-PAGE,
gp91phox was not detectable, and p22phox was
only faintly visible. In the patients' neutrophil cytosols,
p47phox and p67phox were normally present.
These results suggest that the patients suffer either from the X-linked
form of CGD (gp91phox deficiency) or from the autosomal
p22phox-deficient form of CGD. These forms of CGD cannot
easily be discriminated from each other, because the
gp91phox and the p22phox subunits of
flavocytochrome b558 need each other for stable
expression in the cell. Both forms of CGD therefore lead to lack of
expression of both subunits.15 We studied also the
neutrophils of the patients' mother. These cells showed a partial lack
of NADPH oxidase activity in the oxygen consumption assay: with PMA 2.3
nmol of O2 were consumed per 106 cells per
minute (control 8.8) and with fMLP 3.1 (control 8.4). In the NBT slide
test, 42% of her PMA-activated neutrophils reduced the NBT dye to
formazan (control 98%). This proves that the mother has a mixture of
normal and oxidase-deficient neutrophils in her circulation, and
therefore is a carrier of the X-linked form of CGD. Thus, her sons were
expected to be hemizygote patients with this disease.
Identification of the mutations.
Southern blot analysis of genomic DNA was performed after treatment of
the DNA with EcoRI and hybridization with a CYBB cDNA probe.
The results (Fig 1, lanes 3 and 4) show
that the two brothers have different deletions in the CYBB gene. In the
eldest boy, the band at 7 kb has disappeared and a new band at about
3.5 kb is visible. In the youngest boy, the bands at 7 kb and at 4.0 kb
have disappeared and a new band at about 8 kb is visible. Most
surprisingly, the mother of these two patients (Fig 1, lane 2) was
found to carry both mutations in her DNA, in addition to the normal
sequence (Fig 1, lane 1).
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| Fig 1.
Southern blot RFLP assay of genomic DNA after digestion
with EcoRI. (A) Shows the EcoRI restriction map of the
CYBB gene. Hybridization was performed with a 32P-labeled
pSV-CGD cDNA probe, containing the entire coding region of CYBB. The
length of the expected restriction fragments is indicated. (B) Shows
the results with the DNA from a healthy control donor (lane C), from
the mother of the patients (lane M), from the eldest patient (lane 1),
and from the youngest patient (lane 2). The size of the indicated
fragments was determined relative to HindIII digested
phage-lambda DNA as size markers. Arrows at right point at differences
between lane C and the other lanes.
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The existence of two different deletions was also proven by analysis of
PCR-amplified cDNA. Fragments comprising CYBB exon 4 through 10 were
amplified, separated in agarose and visualized in ethidium bromide.
Figure 2 shows that the deletions in the
patients are different in size and shows again that the mother carries
both deletions and the normal composition. From the maternal
grandmother's cDNA a fragment of normal size was obtained. Sequencing
of these PCR products showed that the eldest boy lacked exons 6 and 7
and the youngest boy lacked exon 5 (not shown).
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| Fig 2.
Size analysis of PCR products from cDNA. Primers were
chosen on exon 4 and exon 10 (Table 1). The PCR products were separated
in agarose and visualized with ethidium bromide. Lane 1 contains
material from a healthy control donor, lane 2 from the youngest
patient, lane 3 from the eldest patient, lane 4 from the mother of the
patients, and lane 5 from the maternal grandmother of the patients.
Lane M contains size markers of 100 bp.
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To characterize the deletions in detail, we amplified genomic DNA by
PCR in overlapping fragments (for primers, see Table 1) and determined
the entire nucleotide sequence of intron 4 through intron 7 of the CYBB
gene in the two brothers. In the eldest boy, we found a deletion
starting in intron 5 at about 0.2 kb 5 of the intron-5/exon-6
boundary and ending in intron 7 at about 0.9 kb 3 of the
exon-7/intron-7 boundary (Fig 3). Thus,
this deletion removes about 3.5 kb of the CYBB gene, including exons 6
and 7. In the youngest boy, we found a deletion starting in intron 4 at
about 1.2 kb 5 of the intron-4/exon-5 boundary and ending in
intron 5 at about 1.7 kb 3 of the exon-5/intron-5 boundary. As
shown in Fig 3, this deletion removes about 3 kb of the CYBB gene,
including exon 5. Figure 3 also shows that both deletions are adjacent
to each other, overlapping for 35 bp.
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| Fig 3.
Localization of deletions in the CYBB gene of the two
patients. The shaded area indicates the deletions, comprising a 3.5-kb
nucleotide stretch from intron 5 to intron 7 in patient 1 (the eldest
brother) and a 3.0-kb nucleotide stretch from intron 4 to intron 5 in
patient 2 (the youngest brother). The deletions overlap for 35 bp.
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To verify in yet another way that the leukocyte DNA of the mother
contained three different compositions of the CYBB gene, we devised an
allele-specific PCR to differentiate between these three forms. Primers
were chosen around the two deletions, thus leading to product formation
only when the complementary sequence was present in the genomic DNA and
the distance between the primers was not more than about 3 kb. Figure
4 confirms once more that the mother's DNA
contains both the normal CYBB sequence and the two different deleted
compositions. The maternal grandmother's DNA again showed only the
normal CYBB composition. The same test was also applied to DNA from the
mother's hair roots. Again, the triple somatic mosaicism was found
(not shown).
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| Fig 4.
Size analysis of PCR products from genomic DNA. Primer 1
(sense) was chosen on intron 4, primer 2 (antisense) on intron 5,
primer 3 (sense) on intron 5 and primer 4 (antisense) on intron 7 (for
positions, see Fig 3; for compositions, see Table 1). Primers 2 and 3
hybridize with DNA sequences within the deletions of patient 1 and
patient 2, respectively (Fig 3). Therefore, PCR (a), with primers 3 and
4, only leads to product formation with DNA from patient 1 (not with
DNA from a healthy control donor because these primers then hybridize
too far apart to lead to product formation under the PCR conditions
used). PCR (b), with primers 2 and 3, only leads to product formation
with DNA from a healthy control donor. PCR (c), with primers 1 and 2,
only leads to product formation with DNA from patient 2. The figure
shows the results of PCR (a), (b), and (c) obtained with DNA from a
healthy control donor, patient 1 (the eldest brother), patient 2 (the
youngest brother), the mother of the patients (3), and the maternal
grandmother of the patients (4). The four lanes marked M contain size
markers of 100 bp.
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Chromosomal localization of the deletions.
Karyotyping of the mother's lymphocytes in mitosis showed no
abnormalities, ie, no X-chromosome trisomy. X-chromosome marker
analysis in the family indicated that the two deletions of the brothers
originated in the same chromosome. The CYBB-containing part of the
brothers' X chromosome proved to be identical to one of their
mother's X-chromosome regions, but was not found in their maternal
grandmother (Fig 5). Hence, the mutations
took place in the CYBB allele that the mother received from her father.
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| Fig 5.
Pedigree of the CGD family. Haplotype analysis was
performed with X-chromosome-specific markers flanking the CYBB gene,
ie, two CA repeats in the DMD gene and one Msp I polymorphism
in the OTC gene.
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DISCUSSION |
X-linked CGD is caused by a large variety of mutations in the CYBB
gene.11 Usually, only one or two patients are found in
small families, due to the severity of the disease. As in other
X-linked diseases, patients are sometimes recognized without apparent
carriership in the mother or female relatives. This is usually
considered to result from a unique meiotic event, but a mitotic origin
in early embryogenesis cannot be excluded. Indeed, in some cases of
Duchenne muscular dystrophy,22 ornithine transcarbamylase
deficiency,23 and hemophilia A24 clear
indications for a mitotic origin have been obtained by recurrence of
new mutations in a sibship or by somatic mosaicism in the mother or in
the patient. If a mutation event occurs during mitosis before the
germ-line segregates from other cell lines, the germ-line and somatic
tissues can both be expected to be involved.
One family with hemophilia A deficiency has been reported25
in which one male patient had a partial factor VIII gene deletion, one
sister of the patient was a carrier of this deletion, and one other
sister was a carrier of a partial factor VIII gene duplication. Other
sisters and brothers of the patient were normal. Thus, the mother had a
germ-line mosaicism for two abnormal and the normal factor VIII gene
configuration. In the mother's leukocyte DNA, indications for the same
three configuration were found by restriction fragment length
polymorphism (RFLP) analysis. Therefore, the situation in
this family resembles the situation in the family with CGD studied by
us.
With three different methods, we have shown that the mother of the two
male patients with CGD, each with a different deletion in the CYBB
gene, carries both deletions, as well as the normal sequence in the DNA
of her somatic cells (leukocytes and hair roots). Because she had no
other children, it cannot be established whether the triple mosaicism
is also present in her germ-line cells. In the DNA of her own mother,
only the normal CYBB sequence was found. This concurs with the lack of
the affected allele in her mother. Because the deletions are
overlapping, they probably originate from one simultaneous event.
Hence, inheritance of one of the mutations from her father or mother is
unlikely. Inheritance of both mutations from one parent (ie, from her
father) was ruled out by her normal XX karyotype. Most likely,
therefore, the mutations originated from a single mitotic event in the
early embryogenesis of the patients' mother. The nature of this event
is not known, but may involve a mechanism like sister chromatid
exchange.
Breaking and reunion of chromatids usually occur without causing any
damage. However, when a genetic defect causes genome instability with a
high incidence of sister chromatid exchange, loss of DNA may take
place.26 If, under such conditions, during late replication
of the methylated "inactive" X chromosome a single strand break
occurs in each of the newly formed chromatids at a relatively short
distance (eg, at 35 bp distance, as in our patients), these ends may
hybridize and form a stable double helix. Before repair can take place,
separation of the two chromosome halves to daughter cells may occur.
Opposite parts of DNA may be torn away and become lost during the next
round of replication.
At present it is unknown how the mutated X chromosomes are divided over
the mother's somatic cells, ie, whether they are together in one cell
(with other cells containing two normal X chromosomes) or whether they
are in separate cells (each accompanied by a normal X chromosome). In
the former case, one would expect the two mutated X chromosomes to be
present in the mother's leukocytes in equal number, whereas in the
latter case, one would expect the normal X chromosomes to make up 50%
of all X chromosomes. An indication for the existence of the second
possibility was obtained from a density scan of Fig 1B. The densities
of the 2.7/2.8-kb bands were normalized to correct for the different
amounts of DNA applied to the four lanes in the gel and the presence of
two X chromosomes in the mother's cells, but only one X chromosome in
the cells from her sons and from the (male) control donor. We then
found that the (normal) 7-kb band was present for 53% in the mother as
compared with the control, the (abnormal) 3.5-kb band for 11% in the
mother as compared with the eldest boy, and the (abnormal) 8-kb band
for 38% in the mother as compared with the youngest son. This shows
that the mother's normal CYBB allele is present in about 50% of her
X chromosomes, and that the two mutated forms of the CYBB
gene are not present in about equal amounts in her genome. These
findings indicate that each of her leukocytes carries one X
chromosome with a normal CYBB gene and another X
chromosome with either of the two mutated forms of this gene. The
3-kb deletion is present in about 75% of her leukocytes, the 3.5-kb
deletion in about 25% of her leukocytes.
Occurrence of germ-line and/or somatic mosaicism may be more
common than previously thought. Until now, we have characterized about
110 mutations in the CYBB gene. In 10 of the families involved, two or
more patients were identified. In two of these last 10 families, we
found different mutations in the patients within one family. One of
these families is reported in this article. In the other family, we
found two male patients with different deletions in the CYBB gene, ie,
one that removes the first 10 exons and intronic sequences and another
that encompasses the entire CYBB gene. Again, marker analysis indicated
that the two deletions occurred in the same allele. The mother of these
patients was found to carry a somatic mosaicism for the normal sequence
and for the first-mentioned partial CYBB deletion, but the presence of
the second, total CYBB deletion in her DNA must be studied by other
methods than PCR and Southern blotting (De Boer, Bakker, Nemet and
Roos, unpublished). Thus, the possibility of complicated mosaicisms
must be kept in mind when performing genetic studies for, eg, prenatal
diagnosis.
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FOOTNOTES |
Submitted April 28, 1997;
accepted August 25, 1997.
Supported by Grant No. 28-2167 from The Netherlands Foundation for
Preventive Medicine (Praeventiefonds), The Hague, The
Netherlands.
Address reprint requests to Dirk Roos, PhD, CLB,
Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract