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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. 9 10 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 11 12 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 14 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. 15 16 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 18 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 19 20 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. 27 28 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. 29 30 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 31 32 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 33 34 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. 35 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 54 REFERENCES REFERENCES 1. Delves PJ, Roitt IM. The immune system. First of two parts. N Engl J Med 2000;343:37-49. 2. Delves PJ, Roitt IM. The immune system. Second of two parts. N Engl J Med 2000;343:108-117. 3. Medzhitov R, Janeway CJ. Innate immunity. N Engl J Med 2000;343:338-344. 4. Buchner H. Über die nähere Natur der bakterientötenden Substanz in Blutserum. Zentralbl Bakteriol 1889;6:561-572. 5. Ehrlich P. Studies on immunity. 2nd ed. New York: J. Wiley and Sons, 1910. 6. Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:1058-1066. 7. 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