Download genetic studies of the human complement c4 region in mhc class iii

FINNISH RED CROSS BLOOD TRANSFUSION SERVICE AND
DEPARTMENT OF BIOSCIENCES, DIVISION OF BIOCHEMISTRY,
UNIVERSITY OF HELSINKI, FINLAND
GENETIC STUDIES OF THE HUMAN
COMPLEMENT C4 REGION IN MHC CLASS III
Taina Jaatinen
ACADEMIC DISSERTATION
To be publicly discussed, with permission of the Faculty of Science,
University of Helsinki, in the Nevanlinna Auditorium
of the Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, Helsinki,
on June 14th, at 9 am.
Helsinki 2002
ACADEMIC DISSERTATIONS FROM THE FINNISH RED CROSS
BLOOD TRANSFUSION SERVICE NUMBER 46
SUPERVISOR
Docent Marja-Liisa Lokki, PhD
Department of Tissue Typing
Finnish Red Cross Blood Transfusion Service
Helsinki, Finland
REVIEWERS
Professor C. Yung Yu, PhD
Department of Pediatrics, Molecular Virology, Immunology and Medical Genetics
The Ohio State University
Columbus, USA
Doctor Sakari Jokiranta, MD, PhD
Department of Bacteriology and Immunology
Haartman Institute, University of Helsinki
Helsinki, Finland
OPPONENT
Docent Seppo Meri, MD, PhD
Department of Bacteriology and Immunology
Haartman Institute, University of Helsinki
Helsinki, Finland
Illustrations by the author
Book design by the author and Miikka Haimila
ISBN 952-5457-01-X (print)
ISBN 952-5457-02-8 (pdf)
ISSN 1236-0341
http://ethesis.helsinki.fi
Tummavuoren Kirjapaino Oy, Vantaa, Finland, 2002
Imagination
is
more
important
than
knowledge.
~Albert Einstein
CONTENTS
CONTENTS
PUBLICATIONS ................................................................... 6
ABBREVIATIONS ................................................................. 7
ABSTRACT ........................................................................... 8
REVIEW OF THE LITERATURE .......................................... 10
Immune system .............................................................. 10
Complement system ...................................................... 11
Classical pathway ................................................................ 14
Lectin pathway ................................................................... 15
Alternative pathway ............................................................ 16
Complement regulation ..................................................... 16
Complement and genetic deficiencies ................................ 17
Complement and infection ................................................. 19
Major histocompatibility complex ................................. 21
MHC class I and II ............................................................... 22
MHC class III ...................................................................... 23
Complement genes in MHC class III ............................... 24
C4 .................................................................................... 25
C4 isotypes and allotypes ................................................... 26
Rodgers and Chido antigens ............................................... 28
RCCX module .................................................................... 30
Genetic rearrangements in the C4 gene region ................. 31
Gene conversion .............................................................. 31
Crossover ........................................................................ 33
Conversion and crossover in C4 genes .............................. 33
Duplication, insertion and deletion ................................. 33
AIMS OF THE STUDY ........................................................ 35
MATERIALS AND METHODS............................................ 36
Ethical considerations ..................................................... 36
Study subjects ................................................................. 36
Methods .......................................................................... 37
RESULTS AND DISCUSSION............................................ 38
Family 1 (studies I & II)................................................... 38
Family 2 (study III) .......................................................... 41
Family 3 (study IV) .......................................................... 44
Genetic basis of C4 null alleles ....................................... 48
SUMMARY AND CONCLUSIONS .................................... 50
ACKNOWLEDGMENTS ..................................................... 52
REFERENCES ...................................................................... 54
6
PUBLICATIONS
PUBLICATIONS
This thesis is based on the following original publications, which have
been reproduced with the permission of the copyright holders.
ARTICLES
I
Jaatinen T, Ruuskanen O, Truedsson L and Lokki ML. Homozygous
deletion of the CYP21A-TNXA-RP2-C4B gene region conferring
C4B deficiency associated with recurrent respiratory infections.
Human Immunology 1999;60:707-714. American Society for
Histocompatibility and Immunogenetics, 1999.
II
Jaatinen T, Chung E, Ruuskanen O and Lokki ML. An unequal
crossover event in RCCX modules of the human MHC resulting in
the formation of a TNXB/TNXA hybrid and deletion of the CYP21A.
Human Immunology, in press, 2002. American Society for
Histocompatibility and Immunogenetics, 2002.
III
Jaatinen T, Lahti M, Ruuskanen O, Kinos R, Truedsson L, Lahesmaa R
and Lokki ML. C4B deficiency due to gene deletions and gene
conversions associated with severe and chronic infections. Submitted,
2002.
IV
Jaatinen T, Eholuoto M, Laitinen T and Lokki ML. Characterization
of a de novo conversion in human complement C4 gene producing a
C4B5-like protein. Journal of Immunology, in press, 2002. The
American Association of Immunologists, 2002.
ELECTRONIC PUBLICATIONS
Jaatinen T. Sequence variation of C4. Human Genome Variation Database,
2001, IND/SNP001026494-IND/SNP001026518. http://hgvbase.cgb.ki.se
In addition, some unpublished data are presented.
ABBREVIATIONS
ABBREVIATIONS
Ch
Chido antigen
CYP21
Steroid 21-hydroxylase

DHPLC/WAVE
Denaturing high performance liquid
chromatography, WAVE nucleic acid fragment
analysis system is a registered trademark of
Transgenomic Inc, Omaha, NE, USA
DNA
Deoxyribonucleic acid
Fc
Crystallizable fragment of immunoglobulins
HERV
Human endogenous retrovirus
HLA
Human leukocyte antigen
Ig
Immunoglobulin
kb
Kilobase
kDa
Kilodalton
MASP
Mannan binding lectin associated serine protease
MBL
Mannan binding lectin
MHC
Major histocompatibility complex
mRNA
Messenger ribonucleic acid
PCR
Polymerase chain reaction
PFGE
Pulsed field gel electrophoresis
RCCX
Genetic module formed by RP, C4, CYP21 and
TNX genes
RFLP
Restriction fragment length polymorphism
Rg
Rodgers antigen
SSCP
Single-stranded conformation polymorphism
7
8
ABSTRACT
ABSTRACT
The human body is constantly confronted with foreign invaders that
need to be recognized and removed, an action provided by the immune
system. The immune system is an organization of molecules, cells and
tissues with a specific function to protect from infectious disease. Defense
against microbes is mediated by the immediate innate responses and the
later responses of acquired immunity. The complement system is an
essential effector mechanism of innate immunity that also augments
humoral immunity. First line defense is provided by this antimicrobial
system consisting of a number of proteins that interact with one another
in a highly regulated manner. A cascade of reactions is formed through
complement activation, leading to the elimination of microbes and the
clearance of immune complexes.
The repertoire of various complement components is vital for the
function of the system. Lack of activation or regulation can result in
deficiency. Genetic deficiencies of complement are due to point
mutations, deletions and conversions, leading to impaired or repressed
protein synthesis. Complement component C4 plays a central role in
classical and lectin pathways of complement. There are two isotypic
forms of C4, C4A and C4B, that differ in their chemical and serological
properties. A phenotypic C4 null allele, i.e. lack of C4 protein, is quite
frequent in the general population in Finland. Homozygous C4 deficiencies
are rather rare and associate with impairment of immune complex
clearance and poor defense against microbes with exposed sugar residues.
The aim of the present studies was to characterize the genetic basis of
C4 null alleles in three families. In two families, total absence of C4B
protein was detected. The C4B deficiency was due to either a
homozygous gene deletion or a combination of gene deletion and gene
conversion. In the third family, an extraordinary C4 protein was detected.
The novel protein results from the exchange of genetic information
between maternal C4A and C4B genes through a de novo conversion.
Thus, the protein possessed characteristics of both C4A and C4B. In an
enlarged study group of 32 individuals, gene deletion was the major cause
ABSTRACT
of C4 null alleles. Gene conversion and point mutation were seen as the
basis of C4 deficiency as well.
The C4 genes possess structural, allelic and isotypic variation. Another
aim of these studies was to examine the extensive polymorphism of the
C4 genes and the C4 gene region within the major histocompatibility
complex class III. In study III, to unravel the cause of nonfunctionality of
the converted C4 gene, it was screened for mutations. No prominent
mutations were found that would conclusively explain the loss of gene
function. However, 25 novel nucleotide alterations were revealed bringing
substantiation for the vast polymorphism of the C4 genes. The
identification of structural variation in the C4 gene region confirms the
alterable nature of the C4 gene region as a whole.
C4 deficiency is often seen in association with infectious diseases. These
studies suggest that a complete lack of C4B protein is a predisposing
factor for infections. The infectious consequences are probably a result
of both the defect itself and certain secondary effects on immune
responses. Study III also revealed a heterozygous state of mannan binding
lectin, which is involved in the activation of lectin pathway of complement.
A defect in mannan binding lectin, particularly in combination with
complete C4B deficiency, may result in inadequate activation of
complement and increase susceptibility to infections.
In conclusion, these studies show that deletions, conversions and point
mutations occur frequently in the C4 gene region and lead to qualitative
and quantitative variation. Genetic rearrangements in the C4 gene region
are complex and result in deficiency states predisposing to infectious
diseases.
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REVIEW OF THE LITERATURE
REVIEW OF THE LITERATURE
Immune system
The immune system is essential for survival1,2. The tissues, cells and
molecules of this defensive network provide protection against infectious
agents and altered autologous cells. When a microbe enters the body, it
is detected by the immune system and a range of antimicrobial cells and
factors are mobilized to eliminate the microbe. Furthermore, immune
surveillance is under strict control to avoid the unnecessary destruction
of viable tissues. At the same time it participates in the clearance of
injured autologous cells and tissue debris.
The human immune system can be functionally divided into innate and
acquired immunity. Innate immunity is available at all times and it is able
to act immediately upon encounter with a microbe3. It consists of lectin
and alternative pathway complement proteins, phagocytic cells, natural
killer cells, cytokines, C-reactive protein and collectins providing fast
yet relatively nonspecific protection. Nonetheless, the actions of innate
and acquired immunity overlap and they share certain activators and
recognition molecules. Acquired immunity acts through T and B
lymphocytes. Therefore, it is very specific yet evolves slowly. T helper
lymphocytes (CD4) recognize antigens and accompanying major
histocompatibility complex (MHC) molecules with their cell surface
receptors, and activate effector T lymphocytes (CD8) and B lymphocytes
(CD19). The effector functions of activated T lymphocytes include
secretion of cytokines, induction of inflammation and target cell lysis by
helper T lymphocytes and cytotoxic T lymphocytes. Components of
the classical complement pathway, mononuclear phagocytes and natural
killer cells also participate in antigen elimination. B lymphocytes are able
to differentiate into a vast variety of plasma cells producing antibodies
against antigens. In addition, the contact with a foreign antigen is
registered by the immune system and leads to the formation of memory
B lymphocytes. This enables a more effective acquired immune response
if the same microbe is encountered again.
REVIEW OF THE LITERATURE
Complement system
The complement system aims to provide defense against foreign invaders
by marking microbes for destruction and promoting their elimination.
The complement system also augments the humoral immune responses.
Complement is a strong antimicrobial system consisting of a large number
of various plasma and cell surface proteins that become activated by
microbes resulting in a cascade of reactions. It was identified as a heatlabile substance of serum and named after its ability to complement the
action of antibodies4,5.
Complement activation leads to opsonization of microbes, chemotaxis
of phagocytic cells, direct lysis of microbes and infected cells, and
clearance of immune complexes6. Complement is effective in eliminating
gram-negative bacteria, some viruses and virus infected or apoptotic
cells7. Complement plays an essential role in inflammation through the
release of inflammatory mediators, which cause the contraction of
smooth muscle cells and the increase of vascular permeability.
Complement also participates in the generation of acquired immunity by
lowering the threshold for activation of B lymphocytes. Antibody
production and memory cell development are enhanced by the complex
of microbial antigen and C3d, which simultaneously employs membrane
immunoglobulin and complement receptor type 2 to initiate the signal
for B lymphocyte activation. C3 has been shown to be crucial for thymusdependent antibody assembly8. Further, studies on mice deficient in C3
or C4 show impaired B lymphocyte priming, diminished production of
antibodies and marked weakening in allogeneic lymphocyte response9,10.
Complement increases the antigenicity of an antigen through complement
receptors on antigen presenting cells. Under physiological conditions,
complement promotes the clearance of immune complexes11. However,
failure in this function results in the accumulation of immune complexes
and leads to constant complement activation and chronic inflammation.
In autoimmune conditions, dendritic cells internalize the body’s own
structures and present autoantigens to T lymphocytes, which in turn
activate autoreactive B lymphocytes. Mice deficient in the complement
receptors CD21/CD35 or the complement protein C4 have increased
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REVIEW OF THE LITERATURE
levels of anti-nuclear autoantibodies suggesting an important role for
complement in hindering the development of autoimmunity12.
Complement contains proteins with distinct functions, including receptors
and molecules that activate or inhibit the system. Alternative pathway of
complement constantly has a low level of activity and readily reacts to a
foreign invader. Thus, the system needs to be strictly regulated in order
to keep it from destroying viable autologous tissues 13. The severe
symptoms caused by complement deficiencies provide evidence for the
importance of complement system in host defense. The main components
of complement and their functions are presented in Table 1.
Table 1. The main complement components.
Component
C1
C1q
C1r
C1s
C4
C4a
C4b
C2
C2a
C2b
Function
Initiates the classical pathway by binding to the Fc of IgG/IgM
Binds to C1q and cleaves C1s
Cleaves C4 and C2
Acts as an anaphylatoxin
Binds covalently to microbial surface, binds C2, part of classical pathway C3/C5 convertase
Part of classical pathway C3/C5 convertase
Function not known
C3
C3a
C3b
Acts as an anaphylatoxin and chemotaxin
Binds covalently to microbial surface, binds factor B, part of C3/C5 convertase
MBL
MASP-1
MASP-2
MASP-3
Initiates the lectin pathway, activates MASP-1
Activates MASP-2
Cleaves C4 and C2
Is found together with MASP-2
Factor B
Ba
Bb
Factor D
Function not known
Part of alternative pathway C3/C5 convertase
Cleaves C3b-bound factor B
C5
C5a
C5b
C6
C7
C8
C9
Binds to C3b and becomes cleaved into C5a and C5b by C5 convertase
Acts as an anaphylatoxin and chemotaxin
Remains bound to C3b and forms a binding site to C6 and C7
Binds to C5b and forms a complex with C7
Binds to C5b6 and inserts into membrane
Binds to C5b-7 and inserts into membrane
Binds to C5b-8 and polymerizes to form membrane attack complexes
REVIEW OF THE LITERATURE
The early events of complement activation are brought about by
proteolytic steps leading to the formation of the central enzymatic
activity of complement, the C3 convertase, that binds covalently to the
target. The early events may follow either classical pathway, lectin
pathway or alternative pathway. An overview of the complement
pathways is shown in Figure 1. All three pathways converge to initiate
the late events, in which the terminal complement components cause
damage to target cells through cytolytic membrane attack complexes.
Classical pathway participates in antibody-mediated immune recognition,
whereas lectin pathway and alternative pathway provide fast recognition
for innate immunity. Complement serves as a crossing point for innate
and acquired immunity as it enhances antibody responses and
immunological memory6.
Classical pathway
Lectin pathway
Alternative pathway
Ag-Ab
Man/GlcNAc
Foreign particles
C1q+C1r+C1s
MBL+MASP-1+MASP-2
C3b/C3(H2O)+B
D
C4
C2
C4b2a
C3bB
C3
C4b2a3b
C3bBb
C3b
C3b2Bb
Opsonization and
immune clearance
C5
C4a/C3a/C5a
Inflammatory reactions
C5b+C6+C7+C8+C9
MAC
Lysis
Figure 1. Complement pathways. Enzymatic activity is indicated with a dashed line. Ag-Ab,
antigen-antibody complex; Man, mannose; GlcNAc, N-acetyl glucosamine; MAC,
membrane attack complex. Dashed lines indicate enzymatic activities.
13
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REVIEW OF THE LITERATURE
Classical pathway
Classical pathway is triggered mainly by antigen-antibody complexes. It
can also be activated by soluble immune complexes found in plasma,
C-reactive protein and apoptotic cells14,15. Specific identification of the
target is provided by antibodies, for which reason the classical pathway
is relatively slow. In a secondary encounter with the microbe, when
specific antibodies are present, the classical pathway activation is
immediate. Human IgG1, IgG3 and IgM antibodies are the most effective
in binding C1q leading to the classical pathway activation16.
The C1q interacts with the Fc part of the surface-bound antibody leading
to a conformational change in C1q and C1r. The succeeding autocleavage
of C1r results in the cleavage of C1s. Activated C1s then uses C4 and
C2 as substrates. Proteolytic cleavage of C4 results in the release of a
soluble anaphylatoxin, C4a, and the formation of C4b, which goes
through a conformational change revealing an internal thioester17. The
reactive acyl group of the thioester becomes exposed and C4b binds
covalently to the target surface with an amide or ester bond18. The
localization of the complement activity is achieved by the covalent binding
of C4b to the microbe and not to the host. Both isotypes of C4, C4A
and C4B, participate in these reactions but they have different chemical
properties affecting their binding. Attached C4b is capable of binding
C2, which becomes cleaved by C1s to produce C2a and C2b. The
complex of C4b and C2a generates the classical pathway C3 convertase.
The active serine protease part, C2a, cleaves C3 to C3a and C3b. As a
large number of C3b molecules are produced due to the effectiveness
of the C3 convertase, they become bound to the target surface. This
leads to the activation of alternative pathway through the complex of
C3b and Bb, and provides components directly for the alternative
pathway C5 convertase.
In the classical pathway activation, the main functions of C3b are to act
as an opsonin and initiate the terminal complement reactions. C3b binds
to the C4b2a complex forming the C5 convertase, C4b2a3b, which
cleaves C3b-bound C5 into C5a and C5b. C5a acts as an inflammatory
mediator, whereas C5b remains bound to C3b and serves as a binding
site for C6 and C7. The following reactions require no enzymatic activity,
REVIEW OF THE LITERATURE
but are dependent on conformational changes induced by binding. The β
chain of C8 recognizes the membrane-bound complex consisting of C5b,
C6 and C7, enabling the α chain of C8 to bind to the target surface.
Multiple C9 molecules are then polymerized around the complex to
form a fully active membrane attack complex. The terminal complement
reactions cause permeabilization of the target cell membrane leading to
the disturbance of homeostasis, a change in ion gradient, the passage of
small molecules and lysing enzymes.
There are studies demonstrating that components of different
complement pathways interact with each other. Interestingly, a bypass
pathway utilizing antibodies, C1 and components of the alternative
pathway can be activated in the complete absence of C4 or C219,20.
Weak interaction between C4b and factor B has been shown using purified
human complement components, and C4b is able to support the cleavage
of factor B by factor D, suggesting cross-reactivity between classical
and alternative pathways21. A hybrid convertase, C4bBb, is capable of
cleaving C3 in vitro indicating the presence of a C2 bypass pathway22.
Lectin pathway
Mannan binding lectin (MBL) is an acute phase protein23, which recognizes
mannose or N-acetyl glucosamine residues on microbial surfaces. Low
levels of MBL are found in normal serum at all times. The most recently
found pathway of complement, lectin pathway, becomes activated when
surface-bound MBL binds to MBL-associated serine proteases, MASP-1
and MASP-224-26. Recently, a new member of the MBL complex, MASP-3,
was found27. It is generated through alternative splicing and is found
together with MASP-2 on large oligomers. These serine proteases are
activated through a conformational change resulting from the calciumdependent binding of MBL. The complex formed by MBL and MASPs
cleaves C4 and C2 to generate the classical pathway C3 convertase,
C4b2a. Activated MASPs may also cleave C3 directly without the
generation of a C3 convertase28. The chromosomal location and structure
of MASPs imply that lectin pathway evolved before the development of
more specific immune recognition involving antibodies25.
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REVIEW OF THE LITERATURE
Alternative pathway
The alternative pathway of complement has continuously low activity in
plasma and is readily available to act on a microbe in the absence of
antibodies similarly to the lectin pathway. It is considered phylogenetically
earliest of the complement pathways and it is triggered directly on
microbial surfaces. The thiol ester bond of C3 is spontaneously hydrolyzed
causing a conformational change that allows the binding of C3(H2O) to
factor B, which in turn becomes cleaved by factor D. Together the
activation fragment Bb and C3(H2O) form the initial C3 convertase. The
C3b molecules cleaved by the C3 convertase bind to nearby target
surfaces. If such a surface is not available, C3b is hydrolyzed and becomes
turned into iC3b by factor I and the cofactor activity of factor H, factor
H-like protein 1, complement receptor type 1 or membrane cofactor
protein, preventing from unnecessary alternative pathway activation13.
Deposition of C3b on microbial surface enhances the binding of more
factor B leading to the formation of the alternative pathway C3 convertase,
C3bBb29. The C3bBb complex, stabilized by properdin, cleaves more
C3 molecules to generate C3b, establishing a positive feedback loop,
which can be further enhanced by the C3b produced by the classical
pathway. Alternative pathway also amplifies the classical pathway through
the formation of alternative C3 convertases. Some of the C3bBb
complexes bind more C3b to form C5 convertases, C3b 2 Bb 30 ,
responsible for the terminal complement reactions identical to other
pathways. However, it has been demonstrated that the alternative
pathway C5 convertase can operate without the second C3b molecule31.
The released C3a subunit acts as an anaphylatoxin producing local
inflammatory reactions.
Complement regulation
Complement is such an efficient and constantly active mechanism that it
would become used up if it was not well controlled. Also, the native
tissues of the host need to be protected from unnecessary attacking and
destruction. Excessive complement activation is inhibited by soluble
REVIEW OF THE LITERATURE
plasma proteins such as C1 inhibitor, C4b binding protein, factors H and
I, factor H-like protein 1, vitronectin and clusterin13,32. The C4b binding
protein acts as a cofactor for factor I in the inactivation of C4b. The C4b
binding protein binds to C4b without making a distinction between the
two isotypic forms of C4.
Several membrane bound regulators control the system as well.
Membrane cofactor protein (CD46) endorses the cleavage of C3b and
C4b bound to the cell membrane 33. Complement receptor type 1
(CD35) acts as a cofactor for factor I, and its receptor function promotes
the clearance of immune complexes34. Decay accelerating factor (CD55)
and protectin (CD59) are attached to the membrane via a glycosylphosphatidyl-inositol-anchor. They enhance the decay of the C3/C5
convertase and prevent the formation of membrane attack complex,
respectively34,35. On the contrary, properdin increases the efficiency of
the complement system by stabilizing the alternative pathway C3
convertase 36,37. Pathogenic organisms have also developed various
mechanisms to control complement activation on their surfaces.
Complement and genetic deficiencies
The repertoire of different complement proteins is essential for the
function of the cascade. A defect in the system can be caused by
insufficiency in activation or regulation. Genetic deficiencies of many of
the complement proteins have been described and they result in abnormal
protein synthesis or a complete lack of protein production. A majority
of these deficiencies are inherited as autosomal recessive. Deficiencies
of MBL and C1 inhibitor are inherited as autosomal dominant, and the
deficit of properdin is X-linked. MBL deficiency is predominantly caused
by point mutations leading to amino acid substitutions that hamper the
assembly of functional MBL oligomers 38. Heterozygous variants are
associated with reduced levels of serum MBL, whereas homozygotes
lack the protein almost completely39. The most common consequences
of complement deficiencies are increased susceptibility to infections,
immune complex diseases and rheumatological disorders.
17
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REVIEW OF THE LITERATURE
Disease associations of common defects in classical pathway components
are summarized in Table 2. Autoimmune disorders like systemic lupus
erythematosus, Henoch Schönlein purpura, and vasculitis are the results
of classical pathway complement deficiencies40,41. Increased susceptibility
to pyogenic infections is due to unsuccessful opsonization and may be
caused by deficiencies of C2, C3 and C4. Defects of the terminal
complement components predispose predominantly to infections by
Neisseria species.
Table 2. Summary of clinical manifestations associated with deficiencies of the classical
pathway components, topic recently reviewed by O’Neil KM42 and Frank MM43.
Component
Disease association
C1q
C1r
C1s
Autoimmune syndromes, glomerulonephritis, pyogenic infections
SLE, pyogenic infections, glomerulonephritis
SLE, pyogenic infections
C2
Autoimmune syndromes, glomerulonephritis, pyogenic infections
C3
Pyogenic infections, autoimmune syndromes, glomerulonephritis
C4A
C4B
Autoimmune syndromes, scleroderma
Infections, autoimmune syndromes
C2 deficiency is among the most common defects in the complement
system with the approximate frequency for one C2 null gene being 1%.
Two types of genetic defects have been described for C2. In type I the
C2 protein is not translated due to a gene deletion, and in type II the
defect is caused by an amino acid change and lies on the level of
secretion44-47. The majority of C2 deficient individuals have connective
tissue disorders or pneumococcal infections. Dermatomyositis and
glomerulonephritis have also been reported in association with C2
deficiency48,49.
The activation fragment of C4 forms a part of the classical pathway C3/C5
convertase, being a central component of classical and lectin pathways.
The two isotypic forms of the protein, C4A and C4B, differ in their
reactivity. This difference explains the variable symptoms caused by
deficiencies of the different C4 isotypes. Complete deficiency of C4 is
extremely rare. In the North American Caucasian population among the
REVIEW OF THE LITERATURE
150 subjects studied, homozygous deficiency of C4A was found in one
individual and a total absence of C4B was seen in two individuals50.
However, the frequency for C4AQ0 or C4BQ0 phenotype was 13% or
18%, respectively. In the Finnish population the phenotype frequencies
for C4 null alleles are higher still; 18% for C4AQ0 and 29% C4BQ051.
Thus the heterozygous state of C4 deficiency is quite frequent among
the general population in Finland. C4Q0 phenotypes are due to gene
deletion, conversion or point mutation. A substantial number of the C4
gene deletions also include the flanking steroid 21-hydroxylase (CYP21)
gene. The absence of CYP21B causes congenital adrenal hyperplasia52.
Deletions of C4A or C4B genes have been reported in association with
systemic lupus erythematosus, multiple sclerosis, vitiligo, idiopathic
membranous nephropathy, Henoch-Schönlein nephritis and EhlersDanlos syndrome 53-58. Also, complete deficiency of C4B has been
suggested to increase susceptibility to bacterial infections, especially in
children59,60.
Complement and infection
Microbes such as viruses and bacteria can activate all three pathways of
complement. Opsonization, cell activation through chemotaxis and lysis
are the major effects of complement against foreign invaders.
Complement marks virus-infected cells to be destroyed by other parts
of the immune system, or it directly enhances virus neutralization by
antibody-dependent mechanisms. Enveloped viruses can be lysed through
membrane attack complexes61,62. However, virus neutralization induced
by complement is not completely dependent on virolysis, as structural
alterations of the virion or the early complement components alone are
sufficient for neutralization62-64. Moreover, complement inhibits infectivity
in the absence of neutralizing antibody65. C1q is able to hinder the
infectivity of the human T cell leukemia virus type I by direct binding to
the extramembrane glycoprotein of the virion66. Complement is effective
in neutralization of nonenveloped viruses through C3-dependent crosslinking of viral particles67. In addition to the interactions with free viral
particles complement promotes lysis of virus-infected cells, which is
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REVIEW OF THE LITERATURE
very effective before the expression of viral proteins is initiated68. Studies
on C3 or C4 deficient mice show that complement is needed to stimulate
the memory B lymphocyte responses to herpes simplex virus69.
Also, bacteria become opsonized by complement and coating with C3
is essential for opsonophagocytosis. Certain gram-negative bacteria are
particularly vulnerable to the lytic action of complement70. Individuals
deficient in terminal complement components manifest mostly Neisseria
infections. However, there are differences in the infecting organism, time
and frequency of the infectious episode depending on the particular
section of complement influenced by the insufficiency41. Individuals with
a deficiency of early complement components typically experience
infections in early childhood and Streptococcus pneumoniae, Haemophilus
influenzae or Neisseria species account for most of the infectious episodes.
Especially the preference of C4B to form ester bonds with hydroxyl
groups exposed on sugars makes it fundamental in destroying bacteria
carrying capsular polysaccharides. Certain gram-negative bacteria require
a specific antibody for sensitization and complement deposition.
Therefore, they may be more harmful pathogens in early childhood
when a variety of specific antibodies have not been acquired41.
The evolvement of escape mechanisms to manipulate the complement
system indicates the importance of complement in host defense.
Microorganisms can escape complement by either evading appropriate
recognition or constraining the attack and destruction 71 . Some
microorganisms utilize complement regulators of the host to avoid
destruction, and some produce proteins with similar properties to the
host proteins to mimic the regulatory system. Another way to subvert
complement attack is to shed structures that activate complement and
cause effective consumption of complement components. Some
mircoorganisms have the ability to produce proteins that bind to C4b
or enhance the cleavage of C3b and C4b, thus hindering the action of
C3/C5 convertases 72. Some viruses utilize complement regulatory
molecules to repress the activation of the alternative pathway lending
more weight to the efficacy of the classical pathway and its antibodydependent activation73. To adhere to and infect cells, microorgamisms
have the ability to use complement receptors.
REVIEW OF THE LITERATURE
Major histocompatibility complex
The human MHC is an extended region containing highly polymorphic
genes, which encode proteins with essential functions in immune responses
against foreign antigens. The exceptionally well characterized MHC is
located on the short arm of chromosome six and it comprises a region
of almost 4000 kb74. The MHC has been extensively studied, yet still a
part of its genes, proteins and their functions are unidentified. According
to the Sanger Centre, 229 genes, pseudogenes or gene fragments have
been identified in the MHC (http://www.sanger.ac.uk, human MHC gene
list, updated May 31st, 2000). A substantial number of the proteins
encoded by the genes in the MHC region have a role in immune response
and inflammation, and some may confer susceptibility for cancer. Selected
loci of MHC are presented in Figure 274.
RP2
TNXA
CYP21A
RP1
C4A
CYP21B
C4B
RCCX I
DOM3Z
RCCX II
TNXA
A
PPT2
CYP21B
SKI2W
CYP21A C4B
RD
RP1 C4A
RP2
6p21.3
TNXB
C B
CREB-RP
TNXB
C2 FB C4A C4B
NOTCH4
PBX2
RAGE
DR DQ
Tel
DP
Cen
Class I
Class III
Class II
~4000 kb
Figure 2. Selected loci of the human MHC on 6p21.3. The complement genes C2, factor B,
C4A and C4B are located in class III. Together with the neighboring genes RP, CYP21 and
TNX they form a genetic unit called RCCX. The genes in RCCX modules are presented
with grey boxes, and the pseudogenes CYP21A, TNXA and RP2 are indicated with lighter
grey. The orientations of the genes in class III are indicated with arrows.
21
22
REVIEW OF THE LITERATURE
Some of the MHC genes have hundreds of alleles (http://www.ashihla.org, IMGT/HLA sequence database, release 1.13, 11/02/2002). The
high number of different HLA-DRB1 (304) and HLA-B (472) alleles offer
great variability of the residues that determine the specificity of binding
to peptides and the recognition of antigens. Many MHC haplotypes, a
set of MHC alleles, have been shown to associate with a variety of disease
conditions. Certain MHC haplotypes predispose to autoimmune diseases,
such as type I diabetes, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, and celiac disease, even though other genetic factors
are also involved75. Many other MHC-linked diseases are connected to
immunological processes as well. However, MHC alleles can also be
protective and guard against certain disease.
MHC class I and II
MHC class I and II molecules present foreign antigens to T lymphocytes76.
The classical tissue antigens are called human leukocyte antigens (HLA),
and they direct T lymphocyte selection in thymus to eliminate the cells
with too much affinity to self antigens. The HLA molecule is capable of
binding one peptide at a time and the complex persists long enough to
be recognized by a T lymphocyte.
HLA-A, HLA-B and HLA-C are grouped as class I molecules (Figure 2)
and they are expressed on all nucleated cells. Class I molecules form
heterodimers together with β2-microglobulin. They present small
peptides to CD8 T lymphocytes, which induce apoptosis and lysis of
infected cells. The amino acids determining the specificity of the class I
molecule are located in the α1 and α2 domains exposed extracellularly.
Several non-classical HLA genes with restricted expression also reside
in the class I region.
CD4 T lymphocytes recognize peptides bound to the class II molecules
and trigger the activation of B lymphocytes, T lymphocytes and
macrophages, and provoke chemotaxis and apoptosis. Class II genes
reside in the centromeric end of MHC, in the HLA-D region containing
the loci for HLA-DR, HLA-DQ and HLA-DP (Figure 2). They are
REVIEW OF THE LITERATURE
expressed mainly on antigen presenting cells. Class II molecules are
heterodimers with non-covalently attached α and β chains, that form
the peptide-binding groove with their amino terminal ends. Class II
molecules present peptides of at least 13 amino acids in size that come
from intravesicular or extracellular sources. The peptide binding depends
on the compatible amino acids on the MHC molecule and the peptide to
be presented.
MHC class III
The class III region encompasses over 60 genes with an average distance
of 10 kb (Figure 2)77. The region is very densely packed and some genes
are partially overlapping or reside within another gene. In addition,
alternative splicing of some gene transcripts brings multiplicity to the
proteins produced by class III genes. Immune-related functions are
characteristic to molecules belonging to class III as genes for some
complement proteins, cytokines and heat shock proteins lie in the region.
Many of the class III protein products have no recognized role in
immunological reactions, for instance CYP21 catalyzes steroid hormone
biosynthesis in adrenal cortex. However, the connections are complex
and the protein products of some unexpected genes may turn out to
participate in the modulation of immune responses.
In the class III region, reside many recently identified genes or expressed
sequences that are homologous to known genes, such as the protooncogene NOTCH4 encoding the human counterpart of the mouse
mammary tumor gene int-3, and the homeobox gene PBX2 (G17)
homologous to PBX1 involved in the t(1;19) translocations in acute
precursor-B-lymphocyte leukemias 78,79 . Further to the telomeric
direction lies the gene RAGE for the receptor of advanced
glycosylation end products of proteins, which is a member of the
immunoglubulin superfamily 80 , and the gene PPT2 (G14) for an
enzyme with S-thioesterase activity and partial homology to the cytokine
receptor superfamily81. In the family of genes for transcriptional regulation
is the novel Creb-rp gene (G13) encoding a general transcription factor
for glucose-regulated proteins with a leucine zipper motif common to
23
24
REVIEW OF THE LITERATURE
proteins involved in DNA binding82,83. Flanking the Creb-rp is the TNXB
gene encoding an extracellular matrix protein tenascin-X84,85. The CYP21B
gene and its related pseudogene CYP21A are 98% homologous in the
exonic regions86. CYP21B encodes for the steroid 21-hydroxylase
involved in mineralocorticoid and glucocorticoid biosynthesis in the adrenal
cortex.
Four novel genes in the region have ubiquitous gene expression and
structural features suggesting that these are probably housekeeping
genes. RP1 (G11) may be a nucleoprotein acting in transcriptional
regulation 87,88 . Additionally, two alternative transcripts of RP1
have been described, and their protein products are able to bind
adenosine 5'-triphosphate and phosphorylate serine/threonine residues
indicating a role in signal transduction. In accordance with its expression
in testis and ovary, DOM3Z may relate to growth and reproduction89.
The sequence of the gene is conserved and it has 52% sequence similarity
to a yeast homolog interacting with a 5' to 3' exoribonuclease, thus
potential to disintegrate nuclear and cytoplasmic RNA90. SKI2W encodes
a protein with a helicase domain and two leucine zipper motifs, and the
protein is able to associate with ribosomes. Hence, it is probably involved
in RNA splicing, translation and turnover. It also has similarity to a yeast
antiviral protein, suggesting a role in antiviral activities91,92. The protein
encoded by the RD gene is part of the negative elongation factor involved
in the regulation of gene transcription93,94.
Complement genes in MHC class III
The centromeric part of the class III region in the human MHC on
chromosome 6p contains the genes for the complement proteins C2,
factor B, C4A and C4B (Figure 2). These complement loci exhibit linkage
disequilibrium, i.e. certain haplotypes occur more frequently than would
be expected, based on their individual allele frequencies.
The genes encoding C2 and factor B lie in a very close proximity and
they have similar exon-intron structure. The genes share 42% identity
and 63% similarity resulting from an ancestral gene duplication 95,96.
REVIEW OF THE LITERATURE
At least six mRNA size variants lacking one or two exons have been
found in human C2. These variants are derived from differential splicing
and according to the deduced amino acid sequences they all encode
truncated C2 proteins97. Recently, a novel alternatively spliced transcript
of human factor B with intron 12 retention generating a premature stop
codon was identified98,99. Apart from its complement function, factor B
can act as a cell activator and a growth factor for the clonal expansion of
stimulated B cells.
C4A and C4B are usually tandem loci residing approximately 10 kb apart
in the genome. C4 genes can be either long (20.6 kb) or short (14.2 kb)100.
The size difference is due to an human endogenous retrovirus HERV-K(C4)
in intron 9101-103. HERV-K(C4) contains all characteristics of retroviruses
such as a primer binding site for tRNA, two long terminal repeats, and
the gag, pol and env genes responsible for the generation of virion
components102. HERV-K(C4) lies in an opposite transcriptional orientation
to the C4 genes. HERV-K(C4) antisense RNA resulting from the
transcription of the long C4 gene is found in cells expressing C4. There
are less other retroviral elements in these cells suggesting that the
retroviral insertion may provide protection against exogenous retroviral
infections104. HERV-K(C4) also contains several point mutations and
minideletions that probably render it nonfunctional. Both C4A and C4B
genes consist of 41 exons and produce a 5.4 kb transcript100,101. The
high sequence homology between the genes indicates that only a few
polymorphisms contribute to the isotype and allotype specificity. The
manifestation of duplicate C4 genes gives biological advantage in the
interaction with a wide range of antigenic structures.
C4
The activated form of C4 is a structural part of classical pathway and
lectin pathway C3/C5 convertase. It is among the most polymorphic
molecules found in plasma, excluding immunoglobulins. Mature C4 consists
of β, α and γ chains linked by disulphide bonds (Figure 3). There is an
internal thioester in the α chain, which becomes exposed upon proteolytic
cleavage of C4. C4d is formed as the C4b activation fragment becomes
25
26
REVIEW OF THE LITERATURE
cleaved by complement regulator factor I in the presence of a cofactor.
C4d is the most polymorphic domain of C4. C4 is synthesized mainly in
the liver, but it is also produced by macrophages. The C4 glycoprotein
is 202 kDa in size, synthesized from a 5.5 kb mRNA. In plasma, the C4
proteins differ in size due to incomplete processing of the single-chain
precursor protein. This does not reduce the hemolytic activity of C4,
but rather adds to the degree of polymorphism.
β chain
C
S-S
Thioester site
N
S-S
C
C4d
α chain
N
C
S-S
γ chain
N
Figure 3. Schematic structure of C4. S-S represents the interchain disulphide bonds, a
triangle identifies the internal thiester and a dashed line marks the C4d region.
C4 isotypes and allotypes
There are two isotypes of C4, C4A and C4B. They have a 99% homology
on sequence level, but the chemical and serological properties are
divergent105,106. C4A has a preference for amino groups as the acceptor
nucleophile for the reactive thioester group, whereas C4B reacts
preferentially with hydroxyl groups. Also the mechanism through which
the transacylation occurs is different18,107. C4A binding occurs directly
between the amino nucleophile and the thioester, while C4B employs a
two-step mechanism. First, an acyl-imidazole intermediate is formed
through the binding of the His1106 to the thioester. Second, the thiol
acts as a base to catalyze the attack of a hydroxyl nucleophile to the
very reactive intermediate18.
C4A is elemental in immune complex clearance. Specifically the C4d
fragment, bound covalently to immune complex antibodies or antigens,
is caught by erythrocytes and phagocytes through CR1 complement
receptors. These erythrocyte-bound immune complexes are removed
REVIEW OF THE LITERATURE
from circulation to be processed in the liver and spleen108. C4B has
more hemolytic activity than C4A due to the highly glycosylated surface
proteins with a high number of hydroxyl groups on erythrocytes. The
amino acid His1106 in C4B is essential to the preference for hydroxyl
groups105. This binding preference also accounts for the function of C4B
that leads to the destruction of microbes. The isotype specificity is
determined by four amino acids in the α chain. C4A carries
Pro 1101 Cys 1102 Pro 1103Val 1104 Leu 1105 Asp 1106 , whereas the sequence
Leu 1101 Ser 1102Pro 1103Val 1104Ile 1105His 1106 is specific for C4B 105,109,110 .
Studies by mutagenesis show that the 1106 site is the most crucial to
the functional activity of C4 proteins111.
In addition to the isotypic variation, there are roughly 40 different allotypic
variants for C4A and C4B 112 . They differ from each other by
electrophoretic mobility or hemolytic properties. The most frequent
alleles in the Finnish population are C4A3 (87%) and C4B1 (58%)51.
Altogether, six alleles for C4A (A0, A2, A3, A4, A5 and A6) and five for
C4B (B0, B1, B2, B3, B5) are represented in Finns. The most common
C4 combination is C4A3-C4B1, but C4A3-C4BQ0, C4A2-C4BQ0, and
C4AQ0-C4B1 also have frequencies over 10%113. Many of the allotypes
can be further divided into subgroups based on their serological reactivity
or DNA sequence114,115. Taken together, 27 polymorphic amino acid
residues have been reported, most of them residing in the C4d region
of the α chain100,101,116-119. The initial polymorphic residues have been
established by protein analysis from pooled serum120,121. The first DNA
sequencing studies were performed in the early 1980s, providing more
detailed data on different C4 allotypes109,110,122. Figure 4 illustrates the
extensive polymorphism of the C4 genes.
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REVIEW OF THE LITERATURE
C4d
F63(C/T)
V399A
7C>T*
32delT*
61delA*
72delC*
83G>T*
R458W*
P459L*
49-50insC*
62delT*
A476(C/T)*
Y1478D
C616S
47C>G*
Y328S
44delC*
84C>A*
P707L
D708N
T1286G
V1287G
I1298F
F811delC
V806(C/T)
25G>C
F522delC
L1669(G/A)*
DYE1401del
13(A/G)*
25-26insC*
48C>T*
68delT*
86delT*
89delT*
944(T/G)*
S1213insTC*
P1226(G/A)
S1267A
R1281V
V853A*
G863(G/T)
A888T
L1018(G/T)
S1223(G/A)*
47-48insA*
73delT*
14cgctcc/ggctc∆
205(T/C)
D1054G
G1076(C/A)
S1090I
Q1091A
P1101L
C1102S
L1105I
D1106H
18-19insC*
44-45insC*
54-55insC*
N1157S
T1182S
A1186(G/C)
V1188A
L1191R
F1159(T/C)*
Figure 4. Nucleotide and amino acid variation detected in C4 genes. The purpose of the
figure is to illustrate the multiplicity of C4 polymorphism, to emphasize the extensive
variability of the C4d region encompassing exons 23-30 and present novel nucleotide
alterations. Vertical lines designate the exonic alterations depicted in the literature.
Additionally two known variations in intron 28 are presented based on their frequent
occurrence in the literature, and they are underlined. Dashed vertical lines indicate the
variation described in this thesis (III), using human C4A3 sequences M59816 (exons 1-9)
and M59815 (exons 10-41) as the reference. Intronic sites are indicated with italics, and
the isotype specific residues with bold. An asterisk is used to mark alterations submitted
to the Human Gene Mutation Database (http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html)
or to the Human Genome Variation Database (http://hgvbase.cgb.ki.se).
Rodgers and Chido antigens
Further variation comes from Rodgers/Chido (Rg/Ch) antigens detected
on erythrocytes123,124. They are not true blood group antigens, but rather
properties provided by epitopes of C4d fragments attached to the
erythrocyte surface. Rg/Ch antigens form sequential or conformational
epitopes on C4114,125. The sequences determining the antigenicity lie
within exons 25, 26 and 28 of the C4 gene.
REVIEW OF THE LITERATURE
In principle, Rg determinants are associated with C4A and Ch
determinants with C4B, but reversed antigenicity has been observed.
Rg/Ch antigens are not clinically significant in normal blood transfusion,
yet alloantibodies against transfused erythrocytes may be produced in
individuals deficient for C4A or C4B. Some clinically significant blood
group antibodies may pass unnoticed and lead to complications if
anti-Rg/Ch antibodies interfere with the serological analysis. Also,
increased complement activation may result in the accumulation of Rg/Ch
antigens on erythrocytes126, which in the absence of control proteins
leads to unnecessary lysis, as seen in paroxysmal nocturnal hemoglobinuria.
The antigenic determinants Rg1 and Rg3 form the third Rg epitope, Rg2,
which is a conformational structure. Consecutive amino acids account
for Ch1, Ch4, Ch5 and Ch6 determinants, whereas Ch2 and Ch3 are
conformational. In addition, WH epitope is a result of the combination
of Rg1 and Ch6 127,128. Gene conversions may bring about novel
combinations and add to the variability. A model for the Rg/Ch epitopes
is presented in Figure 5.
C4
Exon 25
Rg
Ch
D
G
1101
1102
P
C
L
S
1105
1106
L
D
I
H
3
N
S
1
V
L
A
R
1054
5
Exon 26
2
4
Exon 28
1157
6
2
1188
1191
3
1
Figure 5. Schematic representation of the Rodgers and Chido determinants. The amino
acid positions are shown on the left, and the amino acids forming the conformational
epitopes are marked by dashed lines.
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REVIEW OF THE LITERATURE
RCCX module
Genetic rearrangements are common in the C4 gene region and usually
comprise the neighboring genes RP, CYP21 and TNX in addition to the
C4 gene. These four genes form a genetic unit called RCCX 84,88,129.
Ancient duplication of the RCCX has generated a bimodular structure
with duplicated C4 and CYP21 genes. RP and TNX are only partially
duplicated forming a chimeric hybrid. The duplicated CYP21A gene
carries a deletion and other mutations that render it nonfunctional86,130.
The functional genes are RP1, C4A, C4B, CYP21B and TNXB, whereas
CYP21A, TNXA and RP2 are pseudogenes. Figure 2 shows a schematic
representation of the human C4 gene region.
The RCCX may be monomodular, bimodular or trimodular. Multiple
RCCX modules originate from a duplication between TNX and RP88.
Most haplotypes in the North American Caucasian population are
bimodular (69%) with two C4 genes50. The rest of the haplotypes occur
as monomodular (17%) or trimodular (14%). Also, a haplotype carrying
four RCCX modules has been found131. Modular polymorphism is
thought to arise from the unequal crossover between RCCX modules
on sister chromosomes132,133. In a bimodular haplotype, module I usually
contains a long C4 gene, whereas the C4 gene in module II can be either
long or short50. Bimodular structures with two short C4 genes, without
the retroviral insertion, have been reported in two white individuals and
in South-Brazilian tribes only134,135.
In a normal diploid genome, the number of C4 genes varies from two to
six due to multimodularity, and indeed only 52% of North American
Caucasians carry four C4 genes50. Individuals with three or five C4 genes
comprise 25% or 17% of the population, respectively. Six C4 genes
have the frequency of 3% and two C4 genes account for 2% of the
population. Thus, there is great variation in C4 gene dosage in addition
to the size variation and nucleotide polymorphism. The length variability
in RCCX and high homology of the gene sequences causes unequal
homologous crossover at meiosis and the following exchange of genetic
information between chromosomes.
REVIEW OF THE LITERATURE
Genetic rearrangements in the C4 gene region
The C4 region is prone to genetic rearrangements due to the high degree
of variability and heterozygosity. Recombination involves the exchange
of genetic information between or within chromosomes and further adds
to the diversity and polymorphism of the gene region in question.
Recombination is not a random event, but frequently occurs between
segments combined due to linkage disequilibrium136. In humans, higher
recombination frequencies are seen in females than in males137,138. It can
have beneficial consequences such as the formation of novel alleles and
haplotypes or even functional hybrid genes. Hybrid genes result from
nonhomologous pairing as has been shown for TNX in juvenile rheumatoid
arthritis 139 , congenital adrenal hyperplasia 140 and Ehlers-Danlos
syndrome 58,141, and for CYP21 and C4 genes in congenital adrenal
hyperplasia142,143.
Sequences promoting recombination are usually found in the vicinity of
recombination hot spots and they are thought to render DNA available
for the recombination machinery144,145. Several recombination signal
sequences such as Chi sequences, long terminal repeat elements and
different tandem repeats have been found in the class II region 138 .
However, recombination may also occur in regions without specific signs
for a hot spot. Recombination does not take place between C4 genes or
its neighboring genes only. It can also happen between exons within the
genes, and the recombination sites are found at different positions146.
Also, retroviral sequences are known to affect recombination136,147. In
addition to the HERV-K(C4) in C4, retroviral elements have been found
in C2, RP and CYP21 genes88,148. Also, a number of point mutations add
to the variability in C4 genes. Recombination between misaligned
chromosomes may lead to conversion, deletion or duplication.
Gene conversion
The expression ‘gene conversion’ is generally used to describe a situation,
in which the initial step resembles the beginning of a crossover event,
but the reciprocal product is missing. In gene conversion, one strand of
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REVIEW OF THE LITERATURE
heteroduplex DNA is altered by a repair system, which removes
mismatching bases to make the sequence complementary to the other
strand. It involves the nonreciprocal transfer of information between
two chromatids. The mechanism of conversion is not known in detail,
but the double strand break repair model, simplified in Figure 6,
introduces the idea149,150. In that model, recombination is initiated by a
double strand break. The broken recipient is further digested by
exonucleases to produce a gap so that one of the 3' ends of the recipient
may attack a homologous region in the donor duplex and replace the
other donor strand. The displaced donor strand forms a D loop and
aligns with the broken recipient offering itself as a template to the
synthesis machinery. Thus, the gap in the recipient becomes replaced
with the donor sequence and two chiasmata are generated. This cross
chained structure can be resolved into a patch recombinant or a crossover
recombinant depending on how the intersections are cut. If the
recombining sequences are not identical and heteroduplexes are formed,
the repair system recognizes the mispaired bases and restores
complementarity. The hybrid DNA is usually converted to match the
intact donor sequence151.
Donor
Recipient
Double strand break is formed and
3´ end of recipient attacks donor duplex
Synthesis from 3´ end displaces the other
donor strand and D loop is formed
Recipient´s gap is filled using donor as template
and chiasmata are cut to yield recombinant
Figure 6. A model for the double strand break repair mechanism, also called the gap
repair model, introducing the idea of gene conversion.
REVIEW OF THE LITERATURE
Crossover
A crossover event begins with a double strand break to generate a site
for the genetic exchange to occur. As homologous DNA strands become
nicked the broken strands are able to cross over. Crossover point slides
by branch migration and second nicks are made in the intact strands.
The newly formed free ends cross over again and the breaks are sealed
resulting in the reciprocal exchange of genetic information.
Conversion and crossover in C4 genes
Gene conversion frequency of as high as 1/40.000 has been detected in
the MHC, and conversion acts as a mechanism of the generation of new
MHC alleles152-154. Gene conversion accounts for interallelic recombination
and is frequently seen between C4A and C4B genes as well. It does not
change the number of genes, only the information within them.
Conversion can act in a homogenizing mode and lead to transfer of a
sequence motif from one C4 isotype or allele to another rendering them
more alike155. Diversification creates novel allelic forms. Rare C4 allotypes
such as C4A1, C4A13, C4B5 and C4B12 are believed to arise from
ancient recombination events between C4A and C4B genes156-158. In
addition to interallelic recombination, the exchange of sequence motifs
may occur between different loci and it can take place at mitosis or
meiosis 154,159. Conversions are believed to arise during mitosis and
meiosis, whereas crossovers leading to duplications and deletions occur
at meiosis only.
Duplication, insertion and deletion
Primigenial gene duplication has played a major role in the generation of
the current organization and polymorphism of the RCCX129. The presence
of pseudogenes in the region indicate that the generation of functional
genes is not always a result of plain duplication. Further, these
nonfunctional genes tend to accumulate mutations as seen in connection
with CYP21 genes. The pseudogenes may provide sequence motifs that
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REVIEW OF THE LITERATURE
are used to generate novel allelic forms in their functional counterparts.
Even now, recombination between RCCX on different chromosomes
may result in duplication and produce additional variation in the length of
the C4 gene region. Depending on the location of the recombination
site, different RCCX arrangements and haplotypes are originated.
Additions or removals of nucleotides may render a gene nonfunctional.
A two base pair insertion in exon 29 of the C4 gene results in
nonexpression due to a premature stop codon in exon 30. This mutation
has been seen in both C4A and C4B genes155,160. Small deletions in exon
13 and 20 lead to early termination of translation and have been identified
in C4B and C4A genes, respectively119,161. Unequal crossover may also
lead to a large deletion. Such deletions in the C4 gene region usually
comprehend at least C4 and CYP21 genes, yet the exact boundaries for
the deletions are difficult to evaluate due to the limited group of genes
included in the studies. Various gross deletions are known to involve
either of the C4 genes and associate with many disease conditions.
AIMS OF THE STUDY
AIMS OF THE STUDY
The specific goals of this study were:
1.
To characterize the genetic alterations accounting for the C4 null
alleles in the research subjects.
2.
To study the polymorphism of the C4 genes and the C4 gene region.
3.
To evaluate the role of C4 deficiency in infections.
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36
MATERIALS AND METHODS
MATERIALS AND METHODS
Ethical considerations
The studies have been approved by the ethical committees of the
hospitals responsible for the patients’ care. Informed consent was
obtained from the patients and their family members by the attending
physicians.
Study subjects
Taken together, three families (14 individuals) and 13 different MHC
haplotypes were analyzed.
Family 1 (studies I & II). Four members of an Iraqi family were studied.
The second child of the family was admitted to Turku University Central
Hospital for immunologic evaluation at the age of two due to recurrent
respiratory infections. In addition, the proband had suffered from urinary
tract infection, several acute otitis media episodes, several pneumonia,
and asthma.
Family 2 (study III). Five members of a Finnish family were studied. The
proband had suffered from recurrent meningitis, chronic fistulas and
abscesses, and had been a patient at Turku University Central Hospital
since the age of three months.
Family 3 (study IV). The family was initially identified in Helsinki University
Central Hospital by a study on Finnish couples with a history of recurrent
spontaneous abortions in early pregnancy. The second child of the family
was found to produce an extraordinary C4 protein arousing interest in
further studies. The proband, his parents and two siblings were studied.
In addition, unpublished data on 20 Finnish individuals carrying C4 null
alleles are presented. These individuals have been revealed by C4
allotyping at the Department of Tissue Typing, Finnish Red Cross Blood
Transfusion Service.
MATERIALS AND METHODS
Methods
Methods used in the following studies (I-IV) are described in detail in the
original publications and are listed in Table 3.
Table 3. Laboratory methods used in studies I-IV45,119,155,160-180. PFGE, pulsed field gel
electrophoresis; PCR, polymerase chain reaction; RFLP, restriction fragment length
polymorphism; SSCP, single-stranded conformation polymorphism; DHPLC/WAVE,
denaturing high performance liquid chromatography.
METHOD
REFERENCE
USED IN
HLA-A, -B, -C typing
HLA-DRB1 typing
Allotyping of C4 and factor B
Factor B typing
DNA preparation
Southern blotting
Analysis of C2 gene
Amplification of CYP21
PFGE
Analysis of MBL gene
C4 isotype specific PCR
Amplification of C4d region
Cloning
Sequencing
C4 isotype specific RFLP
Detection of exon 13 mutation
Detection of exon 20 mutation
Detection of exon 29 mutation
SSCP
DHPLC/WAVE
Hemolytic analysis of C4B
Amos et al . 1969
Bidwell et al. 1990 / LIPA Kit Manual
Marcus et al. 1986, Sim et al. 1986
Jahn et al. 1994
Miller et al . 1988
Marcadet et al . 1989
Wang et al . 1998
Levo et al . 1997, Wedell et al. 1993
Yu et al. 2002
Madsen et al . 1994 and 1995
Barba et al. 1994
Lokki et al . 1999
TA/XL-TOPO Kit Manual
BigDye/Dye Terminator Cycle Sequencing Kit Manual
Yu et al. 1987
Rupert et al . 2000
Nordin Fredrikson et al. 1998
Barba et al. 1993, Sullivan et al . 1999
Witt et al . 2000
Taylor et al. 2000
Awdeh et al. 1980, O´Neill et al. 1980
I, II, III, IV
I, II, III, IV
I, II, III, IV
I, II, III, IV
I, II, III, IV
I, II, III, IV
I, III
I
II
III
III, IV
III, IV
III, IV
I, III, IV
III
III
III
III, IV
III
III
IV
37
38
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
Family 1 (studies I & II)
The complement profile evaluation of the proband revealed a constantly
low C4 level, 0.08-0.09 g/l (reference interval 0.12-0.34 g/l). No other
complement deficits were observed. Allotyping showed a total absence
of C4B protein. To characterize the genetic basis of the C4B null
alleles, Southern blot analysis was performed. Studies of the C4 gene
region revealed a homozygous deletion encompassing the genes for
CYP21A-TNXA-RP2-C4B, thus explaining the lack of C4B protein
(Figure 7). The other deleted genes are pseudogenes. The paternal and
maternal haplotypes carrying the gross deletion are [A3, B8, Cw7,
DRB1*0301; C4A3BQ0, BF*FB] and [A2, B41, Cw2, DRB1*0301;
C4A3BQ0, BF*S], respectively.
a
A3, B8, Cw7, DRB1*0301; C4A3BQ0, BF*FB
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
c
A2, B41, Cw2, DRB1*0301; C4A3BQ0, BF*S
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
b A9(24), B35, Cw4, DRB1*0301; C4A3B1, BF*FB
RP1--C4A--CYP21B--TNXB/TNXA--RP2--C4B/S--CYP21B--TNXB
d A19(29), B5(51), Cw2, DRB1*1001; C4A3B1, BF*FB
RP1--C4A--CYP21A--TNXA--RP2--C4B/S--CYP21B--TNXB
a
A3, B8, Cw7, DRB1*0301; C4A3BQ0, BF*FB
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
a
A3, B8, Cw7, DRB1*0301; C4A3BQ0, BF*FB
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
c
A2, B41, Cw2, DRB1*0301; C4A3BQ0, BF*S
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
c
A2, B41, Cw2, DRB1*0301; C4A3BQ0, BF*S
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
Figure 7. Pedigree of family I showing the structure of the C4 gene region and MHC
haplotypes. Dashed boxes indicate the deleted genes and the hybrid gene is marked with
a rectangle.
In the father, CYP21 specific PCR verified the presence of CYP21B genes
only. The total absence of CYP21A genes indicated the possibility of a
rare CYP21A deletion. However, there is very little evidence to support
a single gene deletion in the C4 gene region181. Additional southern blot
analysis suggested that the paternal haplotype [A9(24), B35, Cw4,
DRB1*0301; C4A3B1, BF*FB], not inherited by either of the children,
RESULTS AND DISCUSSION
rather had two CYP21B genes instead. The complete absence of
CYP21A is probably of minor significance as it is a pseudogene. Also, it is
not known how the high copy number of CYP21B affects steroid
biosynthesis or if it predisposes to any disorder.
It is believed that recombination occurs preferentially between segments
of several genes or frozen genetic blocks developed in ancestral
haplotypes during evolution136. Homologous recombination can happen
also between repetitive DNA sequences such as the HERV retroelements
and lead to unequal crossover resulting in duplication or deletion. HERVs
make up roughly 8% of the human genome182. It is not purely by chance
that such retroelements are present in genes with an important role in
immune defense. HERVs participate in the process of diversification which
is crucial for the effectiveness and adaptability of the immune system.
Further studies by PFGE revealed a TNXB/TNXA hybrid gene in the
father resulting from an unequal crossover between the monomodular
and bimodular RCCX haplotypes. A similar recombinant has been
reported in a patient with juvenile rheumatoid arthritis in the [A24, B8,
DR3] haplotype139. The origin of the recombinant haplotype could not
be studied, yet it has probably occurred at ancestral meiosis. Evaluation
of the outcome of the TNXB/TNXA hybrid is problematical. The
expression of such a hybrid gene may affect the expression of neighboring
genes. Overall, the regulation of gene expression may go beyond the
boundaries of an individual gene.
Deletions encompassing CYP21A and C4B genes have been described
previously, but with a different HLA allele combination to this study183.
The association of such deletion haplotypes with susceptibility to infections
has not been studied earlier. Prolonged and recurrent respiratory
infections and several pneumonia episodes were recorded during the
proband’s early childhood. A number of viruses such as respiratory
syncytial virus, adenovirus, rhinovirus, coxsackie B2 virus account for
these infections. C4B is known to be effective in destroying specifically
enveloped viruses, for instance respiratory syncytial viruses. The capside
proteins of picornaviruses such as rhinoviruses and coxsackie B viruses
form projections, the significance of which as targets for C4B is not
known. In addition, Mycoplasma pneumoniae infection was recorded.
39
40
RESULTS AND DISCUSSION
It should be noted that spasticity often causes aspiration allowing bacterial
inflow into the lungs, thus some of the pneumonia episodes may have
been associated with aspiration. The proband has also suffered from
urinary tract infection caused by Citrobacter diversus, associated with
the observed vesicoureteral reflux. Immunological evaluation revealed
no other abnormalities apart from the C4B deficiency.
Patients with inherited deficiencies of complement components are not
often described in connection with viral infections and more research is
needed to evaluate the relationship between them. Many of the previous
studies focus on the level of serum complement profile. Therefore, it is
difficult to distinguish between consumption, activation, synthesis rate
or lack of protein production.
Studies on C3 and C4 deficient mice show decreased antibody
concentrations and suboptimal immune responses9. A bone marrow
transplant is able to restore local complement synthesis and normalize
humoral immune responses184. Bone marrow cells have also been shown
to reconstitute normal serum protein levels in C1q deficient mice185. In
the future, transplanted stem cells could be used to treat patients with
severe C3 or C4 deficiencies. The studies demonstrating the rescue of
impaired humoral response by bone marrow transplantation suggested
a defect on B lymphocyte level, as T lymphocytes were fully primed9. In
contrast, the proband’s serum immunoglobulin levels and antibody
responses were normal indicating intact B lymphocyte function. T
lymphocytes responded normally in stimulation tests but the response
to pokeweed mitogen, a T lymphocyte dependent activator of B
lymphocytes, was subnormal in two independent analyses. These results
suggest a failure in T and B lymphocyte collaboration. Also, even though
the immunoglobulin levels of the proband are adequate, the repertoire
of different types of immunoglobulins may be restricted. Nevertheless,
the laboratory tests are not sensitive enough to detect minor functional
disorders.
There was a history of spontaneous abortions in the family. The first
four pregnancies lead to miscarriage during early pregnancy. Both parents
of the proband carried C4B null alleles, which occur with a slightly
increased frequency in Finnish primary abortion couples compared to
RESULTS AND DISCUSSION
healthy controls, although the difference is not statistically significant51.
The mother also had type I diabetes, an autoimmune disease, which has
been shown to associate with C4 null alleles186. The proband’s MHC
identical sister has the same deletion haplotypes. The influence of gender
and hormonal differences on disease susceptibility is not known.
In conclusion, the susceptibility to viral infections in this proband with
complete C4B deficiency may be a result of the defect itself as well as its
secondary effects on immune responses. The proband does have intact
C4A genes with the ability to produce proteins, although hemolytically
less active, to compensate the loss of C4B.
Family 2 (study III)
The proband’s total hemolytic complement was undetectable in
independent measurements on separate samples, and later the C4
concentration varied between 0.11-0.16 g/l being constantly on the lower
level of normal values. The concentrations of complement components
from C1 to C9 were normal. A total absence of C4B was detected by
allotyping. Southern blot analysis revealed a deletion of four genes
encompassing the CYP21A-TNXA-RP2-C4B loci on the maternal
chromosome (Figure 8). The paternal chromosome carried two long
C4 genes, one of which produced a C4A3 protein and the other appeared
nonfunctional in allotyping. In addition, isotype specific PCR amplification
and restriction analysis confirmed the presence of C4A genes only,
corresponding to the total absence of C4B on protein level.
The C4d region of the long C4 genes on the paternal chromosome
were cloned and sequenced to define their isotypes and allotypes. The
C4 genes at both loci were of type C4A3a. Thus the gene at the second
loci had been converted from C4B to a C4A3a-like gene. The converted
C4 gene was further analyzed to explicate the reason for the possible
nonfunctionality of the gene. A single amino acid change alone may alter
the level of complement activation, considering the importance of the
His1106 site for the hemolytic activity of C4B111. No previously known
mutations were detected, and consequently new mutations were screened
for by SSCP, DHPLC/WAVE and sequencing. Altogether, 25 novel
41
42
RESULTS AND DISCUSSION
nucleotide alterations were found, 22 of them in intronic regions.
However, none of the mutations resulted in changes that would
conclusively explain the loss of gene function. The level of expressed
C4A allotypes detected by allotyping suggests merely a minimal protein
expression by the converted C4 gene, if it is expressed at all.
Some of the intronic nucleotide alterations found in the converted gene
reside nearby the consensus and branch point sites involved in splicing
reactions. Such alterations may hamper the splicing of an intron or create
alternative splicing signals leading to abnormal protein production.
However, there is great flexibility in the putative splice site motifs in
higher eukaryotes and that complicates the evaluation of the importance
of intronic sequence variation. In the proband, the alterations found in
the intronic sequence of the converted C4 gene may cause abnormalities
in transcription or translation. Changes in the promoter region or
instability of mRNA may also confer to the loss of gene function187.
Moreover, point mutations within sequences enhancing splicing can
disturb the function of a gene. Exon skipping has been shown to result
from a small deletion in intron 3 of the growth hormone gene GH-1 and
from the disruption of an exonic splicing enhancer in the breast cancer
susceptibility gene BRCA1188,189. Different alleles vary in their ability to
lead to protein production, so the conversion may have rendered the
proband’s C4 gene inefficient.
In addition to the complete deficiency of C4B, a structurally heterozygous
MBL genotype was found. MBL deficiency is predominantly caused by
point mutations within exon 1, which result in amino acid changes and
affect the functionality of MBL oligomers. The proband had a variant
MBL allele B with an amino acid substitution at codon 54 (Asp>Gly),
which is associated with medium levels of serum MBL173,190. Also, a
polymorphism in the promoter region of the MBL gene was found, which
correlates with medium MBL concentrations. The low MBL levels have
been shown to lower the level of lectin pathway activation and increase
susceptibility to acute respiratory tract infections in children191.
The role of C4B deficiency in bacterial infections has been studied
previously, but the issue is controversial59,60,192-195. The proband of the
present studies had suffered from recurrent bacterial type culture negative
RESULTS AND DISCUSSION
a
A1, B5(51), Cw1, DRB1*1301; C4A3aBQ0, BF*FA
RP1--C4A--CYP21A--TNXA--RP2--C4A/L--CYP21B--TNXB
b A11, B35, Cw4, DRB1*1401/7; C4A4B2, BF*S
RP1--C4A--CYP21A--TNXA--RP2--C4B/S--CYP21B--TNXB
MBL: HYA, LYB
a
c
A11, B35, Cw4, DRB1*0101; C4A3aBQ0, BF*S
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
d A3, B35, Cw4, DRB1*0101; C4A3a,2BQ0, BF*FB
RP1--C4A--CYP21A--TNXA--RP2--C4B/L--CYP21B--TNXB
MBL: HYA, LYB
A1, B5(51), Cw1, DRB1*1301; C4A3aBQ0, BF*FA
RP1--C4A--CYP21A--TNXA--RP2--C4A/L--CYP21B--TNXB
b A11, B35, Cw4, DRB1*1401/7; C4A4B2, BF*S
RP1--C4A--CYP21A--TNXA--RP2--C4B/S--CYP21B--TNXB
d A3, B35, Cw4, DRB1*0101; C4A3a,2BQ0, BF*FB
RP1--C4A--CYP21A--TNXA--RP2--C4B/L--CYP21B--TNXB
d A3, B35, Cw4, DRB1*0101; C4A3a,2BQ0, BF*FB
RP1--C4A--CYP21A--TNXA--RP2--C4B/L--CYP21B--TNXB
MBL: LYB, LYB
MBL: HYA, HYA
a
A1, B5(51), Cw1, DRB1*1301; C4A3aBQ0, BF*FA
RP1--C4A--CYP21A--TNXA--RP2--C4A/L--CYP21B--TNXB
c
A11, B35, Cw4, DRB1*0101; C4A3aBQ0, BF*S
RP1--C4A--CYP21A-TNXA-RP2-C4B --CYP21B--TNXB
MBL: HYA, LYB
Figure 8. Pedigree of family II showing the structure of the C4 gene region and MHC
haplotypes. Dashed boxes indicate the deleted genes, the converted gene is marked with
a rectangle. MBL genotypes are presented undermost for each individual. B specifies the
variant MBL allele carrying the amino acid substitution at codon 54, the normal allele is A.
Polymorphic sites in the promoter region of the MBL gene are designated with HY and LY
associating with high or medium levels of MBL serum concentrations, respectively.
meningitis in early childhood. Sacral fistulas and pararectal abscesses were
recorded as well. In addition to antibiotic treatments and operations,
fresh frozen plasma infusions were given to the proband. The plasma
therapy initiated a prompt clinical response and was used again in later
years to treat recurred abscesses. Plasma infusion may be used in
replacement therapy to serve as a source of missing proteins. Studies
on MBL therapy show that plasma infusion is safe and does not lead to
the development of anti-MBL antibodies196. Previous studies suggest that
replacement therapy could be beneficial to leukemia patients with low
MBL concentrations, especially in connection with chemotherapy197. Then
again, plasma therapy is quite nonspecific, as it contains more than 100
proteins with important physiological functions. The influence of such a
variety of plasma proteins, including immunoglobulins and complement
43
44
RESULTS AND DISCUSSION
components, on immune responses is difficult to assess. In the future,
purified or genetically engineered complement components may prove
to be better and safer than plasma198. Eventually, the replaced protein
will be used up if not continuously administered. In the case of the
proband, successful plasma therapy may have induced substitutive
mechanisms to accommodate immune responses as it has had longterm effects without a continuous supply of C4B or MBL.
To conclude, the proband has a genetic basis for the complete deficiency
of C4B and the heterozygous state of MBL. Either defect alone could
lead to the reduced activation of complement through classical or lectin
pathway. The combined deficit of C4B and MBL is associated with
increased susceptibility to infections of bacterial origin. No predisposition
to viral infections was recorded.
Family 3 (study IV)
The family was initially part of a study on recurrent spontaneous abortions
in early pregnancy. Later on, the second child of the family showed
unexpected results in C4 allotyping. In convergency with the segregation
of MHC haplotypes, the proband should have inherited C4A3a,B2 from
the father and C4A3a,B1b from the mother (Figure 9). In addition to
the expected allotypes an extraordinary C4 protein was detected. It
was hemolytically active and electrophoretically similar to C4B5. This
unexpected C4 protein could not be found in the HLA identical sister or
any other family members. The C4A gene in the paternal haplotype,
inherited by the brother only, carried a two base pair insertion in exon
29 that renders the gene nonfunctional.
A hallmark of gene conversion is that even though the diversity is
increased there is no change in the gene number. Southern blot analysis
indicated no structural rearrangements in the C4 gene region. Three
different C4B genes were found by cloning and sequencing. Most of the
clones were of type C4B2 and C4B1b. One type of C4B clones carried
C4A3a specific sites in exons 28 and 29 indicating an exchange of genetic
information between C4A3a and C4B genes resulting in a de novo gene
conversion. To further characterize the proband’s C4 genes, the most
RESULTS AND DISCUSSION
a
A10(26), B40, Cw3, DR8; C4A3aB2, BFS
RP1--C4A--CYP21A--RP2--C4B/S--CYP21B
c
A19(32), B15(62), Cw3, DR5(11); C4A3aB1b, BFS
RP1--C4A--CYP21A--RP2--C4B/L--CYP21B
b A1, B40, Cw6, DR6(13); C4AQ0B2, BFS
RP1--C4A--CYP21A--RP2--C4B/S--CYP21B
d A2, B5, DR4; C4A3aB1b, BFS
RP1--C4A--CYP21A--RP2--C4B/L--CYP21B
a
A10(26), B40, Cw3, DR8; C4A3aB2, BFS
RP1--C4A--CYP21A--RP2--C4B/S--CYP21B
b A1, B40, Cw6, DR6(13); C4AQ0B2, BFS
RP1--C4A--CYP21A--RP2--C4B/S--CYP21B
c
A19(32), B15(62), Cw3, DR5(11); C4A3aB1b, BFS
RP1--C4A--CYP21A--RP2--C4B/L--CYP21B
c
A19(32), B15(62), Cw3, DR5(11); C4A3aB1b, BFS
RP1--C4A--CYP21A--RP2--C4B/L--CYP21B
a
A10(26), B40, Cw3, DR8; C4A3aB2, BFS
RP1--C4A--CYP21A--RP2--C4B/S--CYP21B
c
A19(32), B15(62), Cw3, DR5(11); C4AQ0B1b,5-like, BFS
RP1--C4/L--CYP21A--RP2--C4B/L--CYP21B
Figure 9. Pedigree of family III showing the structure of the C4 gene region and MHC
haplotypes. The converted C4 gene with the de novo conversion is marked in bold. Paternal
C4A gene carrying the mutation in exon 29 is outlined with a rectangle.
polymorphic C4d region was studied. Clones corresponding to C4B2
and C4B1b were found as expected. Interestingly, there were clones
carrying C4B specific sequences in exon 26 and C4A3a specific sequences
in exon 28 (Figure 10). Thus, the site in exon 26 determining the isotype
of the converted gene originates from C4B and the sequence
downstream is from C4A3a.
The 3' break point region was determined between His1106 in exon 26
and Asn1157 in exon 28 (Figure 10). A comparison between the converted
gene and the parental C4 genes revealed a polymorphism in intron 28
that was shared by the proband’s converted gene and the maternal
C4A3a gene, confirming the maternal origin of the C4A3a. The site
Asp1054 corresponds to C4A3a suggesting that the 5' break point region
lies between Asp1054 in exon 25 and Leu1101 in exon 26. However, the
site Asp1054 was not very informative in determining the 5' break point as
it is also seen in C4B2, indicating that the origin of the sequence upstream
the isotype specific site is not quite unambiguous. Previously reported
de novo mutations are of maternal origin133,199,200. This is convergent with
45
RESULTS AND DISCUSSION
the maternal sexual preference in conversions reported in H-2 genes,
the mouse counterpart for human MHC201. Thus, the current de novo
conversion probably results from the exchange of genetic information
between maternal C4A and C4B genes. Thus, the hybrid protein
possessed characteristics of both C4A and C4B. The different
polymorphic forms of C4 proteins may have varying effectiveness in
complement activation202. Distinction between intrachromosomal or
interchromosomal conversion could not be made due to the homology
of maternal C4B alleles.
A VL
Exon 26
Exon 27
Ch4
25
N
207
1186
LS--IH
14-19
1157
D
Exon 25
1188
1191
1101
1102
1105
1106
C4B5-like
C4B5-like/L
/L
1054
46
c----c t
g
Exon 28
Rg3
Exon 29
Rg1
Rg2
5' break point region
Origin:
A3a(B2)
3' break point region
C4B
A3a
Maternal A3a
Figure 10. Schematic representation of the long C4 gene with the de novo conversion.
Polymorphic amino acids used to determine the nature and origin of the converted gene
are indicated with single letter codes with the respective amino acid position numbers.
Polymorphic motif at positions 14 and 19 in intron 28 corresponded to C4A3a. The
nucleotide at position 207 in intron 28 was identical in the converted gene and in the
C4A3a of maternal origin, but was different in paternal C4A3a and all parental C4B genes.
At position 25 in intron 29, the converted gene shared the same nucleotide with maternal
C4A3a genes, whereas the paternal genes were heterozygous at that position. Deduced
Rg/Ch epitopes are shown underneath the corresponding amino acid positions in exon 26
and 28. The 3' break point region and the postulated 5' break point region are marked with
horizontal lines. The putative origin of the polymorphic sites is indicated below.
The protein produced by the converted gene possessed partially
reversed antigenicity being Rg1,2,3 and Ch4 positive (Figure 10).
According to the proposed C4 designation that covers all possible
combinations of Rg/Ch antigens, the allotype described here would be
RESULTS AND DISCUSSION
called C4B*0508115. The identification and nomenclature of C4 subtypes
is variable by reason of the different level of methodology used in the
studies. Most of the studies on allotypes with partially or totally reversed
Rg/Ch antigenicity are based on serological analysis and comparison with
known allotypes, that are not always definite. The exact recognition of
rare C4 alleles involves analysis at sequence level. However, the conclusive
understanding of typing results requires that the DNA sequence analysis
is combined with allotyping and immunoblotting studies at protein level.
The function of Rg/Ch is still obscure. The number of Rg/Ch determinants,
i.e. C4b or C4d on erythrocytes, has been shown to increase as the
cells become older203, suggesting a function in removal of aging cells. Rg/Ch
may also mediate the clearance of immune complexes through the
attachment to complement receptors on erythrocytes204. Thus, Rg/Ch
can play a role in regulating the location, efficiency and safety of immune
clearance. The effect of reversed Rg/Ch antigenicity on these postulated
functions is not yet known.
Gene conversion can act in a homogenizing mode by transferring genetic
information between homologous loci. This mechanism to maintain the
homogeneity of tandem genes has been recognized to operate in the
MHC class III 205,206 . Conversion may lead to the generation of
homoexpression haplotypes. The proband carried two C4B genes on
the maternal chromosome. C4B homoexpression haplotypes with a
bimodular RCCX structure are exceptionally rare with a frequency of
0.67% in the North American Caucasian population50. Homoexpression
may lead to overexpression of a certain C4 isotype, yet its effect on
complement activation remains to be unraveled. The homogenizing
process, increasing the frequency of C4B genes, may be beneficial in the
defense against microbes. There is evolutionary support for the
importance of C4B in this context as C4A is apparently not required in
some mammals207,208.
47
48
RESULTS AND DISCUSSION
Genetic basis of C4 null alleles
Altogether, 32 individuals carrying C4 null alleles were studied. Of these,
twelve were members of families 1-3 (studies I-IV), and 20 were other
subjects. The C4AQ0 phenotype was seen in 17 individuals, and C4BQ0
in 20 individuals. Five individuals had both C4AQ0 and C4BQ0.
The most common cause of C4AQ0 was the exon 29 mutation, which
was detected in eight subjects of the studied group of 32 individuals.
Gene deletion was seen in seven and conversion in one individual with
C4AQ0. C4BQ0 was due to gene deletion in nine individuals, and
conversion was detected in eight subjects. The exon 29 mutation of the
C4B gene was detected in two individuals. Interestingly, this two base
pair insertion, linked to the [HLA-B40, DRB1*13] haplotype, was the
only previously known C4 mutation found.
The homozygous phenotype C4AQ0,Q0 was established in ten
individuals, whereas 12 subjects were C4BQ0,Q0 homozygotes. Of
these, seven were true homozygotes carrying identical defects in their
genome (deletion-deletion or conversion-conversion). Four individuals
were compound heterozygotes, in other words they were heterozygous
for two different alleles, neither of which were functional. In the remaining
11 homozygous individuals, the nature of the C4 double null phenotype
could not be determined conclusively. No specific genetic pattern could
be identified as the cause of the homozygous phenotype C4AQ0,Q0 or
C4BQ0,Q0, as different combinations of deletion, conversion and exon
29 mutation were observed.
Taken as a whole, gene deletion was the major cause of C4 null alleles
among the individuals studied (12/32). In addition, isotype specific PCR
analysis suggested a lack of C4A genes in two individuals and a lack of
C4B genes in two individuals. Assuming that this is due to gene deletion,
it would increase the putative deletion frequency to 50%. However,
this was a selected study group, in which solely the genetic basis of C4
null alleles was studied, so the results do not depict factual frequencies.
Three individuals with a complete lack of C4 proteins were also studied.
They had no apparent defect in their C4 genes based on isotype or
mutation specific PCR analysis, indicating for instance a condition leading
to excessive consumption of C4, such as an immune complex disease.
RESULTS AND DISCUSSION
Moreover, 25 different MHC haplotypes were studied comprehensively.
Twelve haplotypes carried C4 null alleles. Gene deletion, point mutation
or gene conversion leading to C4AQ0 was seen in two, two and one of
the haplotypes, respectively (Table 4). Point mutation was not detected
in any of the haplotypes carrying C4BQ0 alleles, whereas gene deletion
accounted for the C4BQ0 in four haplotypes. Gene conversion was the
cause of C4BQ0 alleles in three haplotypes. Eighteen bimodular RCCX
haplotypes were detected, and six had monomodular RCCX structures.
Only one trimodular haplotype was found. In this trimodular haplotype,
both short and long RP2-C4 fragments were detected by Southern
blotting. One of the genes produced a C4B1 protein and the other
produced a novel protein electrophoretically anodal to C4A3. Conversely,
the novel protein was detected with monoclonal C4A antiserum indicating
a gene conversion from C4B to C4A. The genetic diversity generated
by the RCCX variation may increase the degree of linkage disequilibrium
in the MHC209. In chromosomes with subsequent mutated alleles or
genetic modules, this would maintain disadvantageous coupling by
preventing recombination and the exchange of genetic information.
Table 4. Genetic basis of C4 null alleles in 12 MHC haplotypes.
Gene deletion
Point mutation
Gene conversion
Total
C4AQ0
C4BQ0
Total
2 (8%)
2 (8%)
1 (4%)
5 (20%)
4 (16%)
0 (0%)
3 (12%)
7 (28%)
6 (24%)
2 (8%)
4 (16%)
12 (48%)
The genes in MHC class III are characterized by deletions, truncated
versions, duplications and nonfunctional copies resulting from conversions
and unequal crossovers. Many of these genetic rearrangements are HLA
haplotype specific. Certain HLA haplotypes are associated with
immunological disorders. The factors predisposing to these conditions
are probably due to the combination of specific classical HLA alleles and
particular MHC class III genes. There may also be unrevealed genes or
environmental factors that increase the disease susceptibility.
49
50
SUMMARY AND CONCLUSIONS
SUMMARY AND CONCLUSIONS
C4 null alleles are frequently observed in individuals with recurrent
infections, autoimmune diseases or other immunological disorders. In
the majority of the cases, the C4 deficiency is caused by genetic
rearrangements encompassing a larger gene segment round the C4 genes.
Certainly the lack of protein production is easily explained by a missing
gene, but the overall view is more complex.
The binding properties of C4B render it a significant molecule in the
defense against enveloped viruses and bacteria covered with capsular
polysaccharides. The association between C4B deficiency and bacterial
infections has been studied earlier, but the issue is controversial. The
comparison of the previous studies is difficult due to the heterogeneity
of study populations. A coexisting deficiency of another complement
component or a defect in another part of the immune system further
increases the susceptibility to infections. Ubiquitous environmental factors
are also likely to be involved.
The present studies suggest that complete C4B deficiency is a risk factor
for both bacterial and viral infections. However, the infectious
consequences are probably not a result of the C4B defect only, but are
due to interactions between genes within and outside the MHC. The
impact of a missing complement component on the function of the
immune system as a whole may be of great significance. The genetically
heterozygous state of MBL is indicated as an additional risk factor for
infections. The future aspects of treatment for individuals with severe
complement deficiencies could include stem cell transplantation or
replacement therapy in the form of purified complement components.
Deletion of a gene or a larger gene segment has long-range effects and
influences the expression of nearby genes. The presence of monomodular
and trimodular RCCX haplotypes increases the frequency of
recombinations and unequal crossovers in the MHC. Thus, the structural
variation of RCCX is both the cause and the consequence of these
rearrangements. The examples shown in the present studies demonstrate
SUMMARY AND CONCLUSIONS
that conversions and unequal crossovers are ongoing processes in the
C4 gene region leading to qualitative and quantitative variation. During
the evolutionary process, besides the beneficial consequences creating
variety, some harmful events take place, leading to deficiency states.
The purpose of a vast genetic diversity by length polymorphism remains
to be appraised.
Further studies are needed to examine the variation of the C4 gene
region using wider research material. Also, a larger patient cohort will
be necessary to conclusively evaluate the role of C4B deficiency in
infections. Moreover, the role of genetic rearrangements in different
clinical associations is yet to be elucidated. To conclude, these studies
add to our knowledge of the polymorphism of the C4 genes, the
complexity of the rearrangements in the C4 gene region and the unstable
nature of MHC class III.
51
52
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
This study was carried out at the Department of Tissue Typing, Division of Stem Cell
and Transplantation Services, Finnish Red Cross Blood Transfusion Service, Helsinki.
I am grateful to former and present Directors of the Institute, Professor Juhani Leikola
and Docent Jukka Rautonen; former and present Directors of the Division, Docent Tom
Krusius and Docent Jarmo Laine; former and present Department Heads, Professor
Saija Koskimies and Docent Jukka Partanen, for providing excellent research facilities.
I also wish to thank Professor Carl G. Gahmberg, Head of the Division of Biochemistry,
Department of Biosciences, University of Helsinki, for encouraging me to finish my PhD
studies on a tight schedule.
My warmest thanks are due to my supervisor Docent Maisa Lokki for arousing my
interest in the complex field of C4 genetics, a subject I really was not supposed to get
involved with. Her ability to introduce wild scientific ideas has been an inspiration.
I am deeply grateful to Professor C. Yung Yu and Doctor Sakari Jokiranta for their
constructive criticism as reviewers of this thesis, and for their attempts to make me a
better complementologist. I sincerely acknowledge the long and detailed, yet quite fun
discussions with Sakari. I wish to thank The James Woolley for revising the language,
particularly for putting all the missing articles in place. I address warm thanks to Miikka
Haimila, who helped me edit this thesis. His patience and great sense of humor brought
relief to a stressful situation.
I am indebted to the families who participated in these studies. I express my gratitude to
clinicians Olli Ruuskanen and Tarja Laitinen for their great help with sample collection.
Their valuable advice and contribution in the original publications is also warmly
appreciated. Very special thanks go to undergraduate students Meri Lahti and Miia
Eholuoto, whose role in these studies cannot be excessively stressed. Additionally,
Lennart Truedsson, Erwin Chung, Riikka Kinos and Riitta Lahesmaa are kindly
acknowledged for fruitful collaboration.
I wish to thank all the personnel of the Department of Tissue Typing. Marjaana Mustonen
is acknowledged for her excellent technical assistance, and Pipsa Vartiainen for helping
me to solve numerous problems – sometimes not even very scientific in nature.
I address special thanks to Hannele Sareneva and Pertti Sistonen for sharing their views
on Rg/Ch ‘blood group antigens’. Riitta Kekomäki is warmly thanked for her interest
and encouragement during the final stage of my studies. Marja-Leena Hyvönen and
Maija Ekholm are gratefully acknowledged for their willingness to be of service in
ACKNOWLEDGMENTS
library matters. The secretarial assistance of Raija Holopainen and Päivi Ahola has been
valuable, but above all they are thanked for their friendly and understanding attitude.
I am grateful to Kaisa and Iiris for the friendship that developed despite the countless
hours spent together in the lab. The girls of our underground office and former roommates of Impivaara are thanked for all the nice moments shared together: Kati for her
optimism, Katri for her sincerity, Niina for her tranquility, Paula for her
straighforwardness and Päivi for her laughter. I especially want to thank Päivi for sharing
the problems with chromosome 6. Former and present members of the young scientists’
community: Anne, Antti, Elisa, Kristiina, Laura K, Laura W, Leena, Lotta, Marja-Kaisa,
Mervi, Mikko, Nina, Pekka, Pia, Rosa, Satu Ko, Satu Ke, and Tuuli, are thanked for
creating such a variable and enjoyable working atmosphere. I owe my sincere gratitude
to Riitta for teaching me who is who at the BTS, but most of all I thank her for true and
warm friendship. I am grateful to Riina for the therapeutic sessions and for being such an
admirable model of a brilliant female scientist. My thanks are due to Elina and Minna for
the many joyful moments spent together during my time at Minerva, and later on as well.
I am thankful to my godchildren Ida and Teemu for every now and then reminding me of
what life is really about. I feel privileged to have been able to be part of their families’
lives. Mutte and Keijo are sincerely thanked for their support throughout these years
and especially for providing me the best possible milieu to study for my finals. I express
my gratitude to my dear friend Guinevere for acting as my ‘personal language revisor’.
I owe special thanks to my friends, near and far, for always being there for me even
though these last months were hectic and I sometimes had very little time to
communicate.
I am ever so grateful to my parents Eva and Timo for their endless love and willingness
to help in every way, whether it was doing laundry for me or listening me practice a
presentation for a scientific meeting. That must have been a blast! I am indebted to
Pekka, who has made sure that I leave the lab for at least a couple of hours every day.
Through his love and care he has made my life more fulfilling than I dared to dream of.
I acknowledge the financial support of the Foundation for Paediatric Research, the
Finnish-Norwegian Medical Foundation, and the Medical Research Fund of Finnish Red
Cross Blood Transfusion Service.
53
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