Immunogenetics of Disease Part 1

Differences in genetic makeup from individual to individual have long been recognized to have physiologic consequences in both health and disease. The recent ability to do high-throughput sequencing of genes has revealed that many genes have variants that are present in a significant proportion of the population. Inherited variants of specific genes, either alone or in combination with other genes, may confer a differential risk of disease or of rejection of transplanted tissue.

Genetic Polymorphism

The fundamental basis of genetic polymorphism in a population is variation of the nucleotide sequence of DNA at homologous locations in the genome. These differences in sequence can result from mutations involving a single nucleotide or from deletions or insertions of variable numbers of contiguous nu-cleotides. Each of these variants presumably occurred in a single ancestor in the distant past. Most new mutations are extinguished through random genetic drift and never become established in the population at any significant frequency. When the gene frequency of a mutation becomes established at more than 1% to 2%, it is often given the more dignified appellation of allele.

Allelic variants can occur anywhere in the genome. Some are found within coding regions of genes, and others are located in introns or gene regulatory regions. However, still others are found in areas that are not closely linked to any known expressed gene.


Equilibrium, disequilibrium, genotypes, and haplotypes

There can be multiple polymorphic nucleotide positions in or near an expressed gene on the same chromosome. In such cases, it is desirable to know whether specific variants at each of the polymorphic positions are independent of the variants at the other positions. If examination of a population shows that the variants at the different positions occur independently of one another, the system is said to be in Hardy-Weinberg equi-librium.1 If certain variants at one of the positions are statistically associated with specific variants at another of the linked positions, the system is said to exhibit linkage disequilibrium.

Hardy-Weinberg equilibrium can be reestablished over many generations through recombination events. The closer the polymorphic loci are to each other on the chromosome, the less likelihood there is of a recombination and the more likely it is for the specific alleles at the two linked loci to be inherited en bloc as a haplotype. For example, if there are two polymorphic positions within a gene, each of which has two alleles, a given individual will have up to four definable alleles. These alleles are inherited as two parental haplotypes, each of which carries one allele from each of the two loci. Most methods used to type individuals cannot organize the genotype into haplotypes without additional information. The common assays simply define the genotype at each of the two polymorphic positions. Extensive population studies permit sophisticated maximum-likelihood estimates of haplotype frequencies within the population.2 These studies, combined with confirmatory cloning and sequencing studies of individual DNA strands, often reveal that some theoretically possible haplotypes never occur, whereas others can be assumed when a specific allele is present (because of linkage disequilibrium) [see Figure 1]. The ability to deduce haplotypes provides a much higher degree of specificity to the analysis of genetic polymorphism, because the haplo-type more accurately defines a larger inherited region of DNA.

Types of genetic polymorphism

Single-nucleotide polymorphisms (SNPs) are allelic variants that have been generated as the result of conversion of one nu-cleotide to another at a homologous position. When present within a coding region (exon) of a gene, the expressed product may or may not have a single amino acid difference, depending on the resulting codon change. In some cases, the change can lead to either a nonsense codon or a stop codon, which halts the transcription process and results in the production of a truncated peptide. SNPs that are located in regulatory regions of an expressed gene can alter the transcription efficiency of that gene but not the protein sequence [see Figure 2].

Single-nucleotide variants occur at positions -1082, -819, and -592 in the promoter region of the interleukin-10 (IL-10) gene. Although eight variants are theoretically possible, only three of these potential IL-10 variants (in purple) are actually observed in large population studies. This is a consequence of strong linkage disequilibrium between the variants at those three positions.

Figure 1 Single-nucleotide variants occur at positions -1082, -819, and -592 in the promoter region of the interleukin-10 (IL-10) gene. Although eight variants are theoretically possible, only three of these potential IL-10 variants (in purple) are actually observed in large population studies. This is a consequence of strong linkage disequilibrium between the variants at those three positions.

Deletion or insertion mutants have also been found in functional genes, sometimes at frequencies that merit their inclusion as alleles. Again, the consequence of a deletion depends on the precise location of the deletion; whether it produces a nonsense frameshift; and whether it alters the function of the expressed product. Angiotensin-converting enzyme (ACE) represents a gene that has a deletion variant in which a 278-base-pair segment of intron 16 is excised. This deletion variant is associated with increased ACE levels.

Another class of allelic variance in association with a particular gene is short tandem repeat (STR) polymorphism. Short sequences of two to four base pairs at a given location can be duplicated back-to-back a specific number of times and inherited as a genetic variant. Because such variation would usually result in a nonsense codon, these STRs are almost always located in noncoding regions. The interferon gamma (IFN-y) gene has such an STR within intron 1, in which the (CA) dinu-cleotide motif is repeated a variable number of times. The allele with (CA)12—that is, with 12 repeats of the CA motif—is associated with high IFN-y production [see Figure 3].

Methods of detection of genetic polymorphism

DNA-based genotyping methods are rapid, accurate, and economical. SNPs can easily be detected, with a high degree of specificity and sensitivity. The assays depend on amplification of the polymorphic locus in question to produce sensitivity in the setting of a background of sample genomic DNA. Specificity is ensured by using tailored oligonucleotides that are complementary to the DNA sequence of the allele one wants to detect.

One strategy for typing is to use polymerase chain reaction to amplify a segment of DNA that includes the polymorphic position and a moderate amount of flanking DNA on both the 3′ and 5′ sides of the polymorphic position. This is done with primers that are complementary to conserved sequences in either side of the desired segment to be amplified. This yields an amplicon of known size that contains inherited alleles and is present in an amount that can be tested for the presence of specific alleles without significant interference from genomic DNA. The amplicon can then be probed, using a set of fluoresceinated or radiolabeled oligonucleotides, each of which is complementary to the DNA sequence of one of the possible alleles. This method is often referred to as site-specific oligonucleotide probe (SSOP) testing.

Single-nucleotide polymorphisms have been identified in the gene for transforming growth factor-f>1 (TGF-f>1). Each polymorphism involves two alleles in the leader sequence of the gene. These biallelic nucleotide substitutions produce codon changes that result in alternative amino acids. Leu10 is in linkage disequilibrium with Arg25, and Pro10 is in linkage disequilibrium with Pro25. The Leu10Arg25 variant is associated with high TGF-|31 production, whereas the Pro10Pro25 variant is associated with lower production. This may be the consequence of different efficiency of posttransla-tional modification for the two variants, which differ only in the leader amino acid sequence.

Figure 2 Single-nucleotide polymorphisms have been identified in the gene for transforming growth factor-f>1 (TGF-f>1). Each polymorphism involves two alleles in the leader sequence of the gene. These biallelic nucleotide substitutions produce codon changes that result in alternative amino acids. Leu10 is in linkage disequilibrium with Arg25, and Pro10 is in linkage disequilibrium with Pro25. The Leu10Arg25 variant is associated with high TGF-|31 production, whereas the Pro10Pro25 variant is associated with lower production. This may be the consequence of different efficiency of posttransla-tional modification for the two variants, which differ only in the leader amino acid sequence.

 Illustration of a short tandem repeat (STR) polymorphism within intron 1 of the interferon gamma (IFN-y) gene. STR polymorphisms in this intron differ according to the number of repetitions of the cytosine-arginine (CA) motif. The allelic variant with 12 tandem repeats [(CA^)] is associated with higher IFN-y production.

Figure 3 Illustration of a short tandem repeat (STR) polymorphism within intron 1 of the interferon gamma (IFN-y) gene. STR polymorphisms in this intron differ according to the number of repetitions of the cytosine-arginine (CA) motif. The allelic variant with 12 tandem repeats [(CA^)] is associated with higher IFN-y production.

Another strategy for SNP typing, which does not require two steps, is called site-specific priming (SSP). This method takes advantage of the fact that the 3′ terminal base of a primer is where DNA synthesis commences during each cycle of PCR. For synthesis to proceed, the 3′ base must be closely bonded to its complementary base on the template DNA. Therefore, the terminal 3′ base of the primer can be used to render the PCR reaction itself exquisitely sensitive to the identity of the base that is on the template. For detection of SNPs, one can craft a set of PCR primers that are complementary to the alleles to be detected, with the terminal 3′ base of one of the primers located at the polymorphic position. The second PCR primer is usually complementary to a conserved segment of DNA and positioned to yield a product of a convenient size. If an allele is present, use of the appropriate set of primers will produce an amplicon. The amplicon can be separated from genomic DNA by simple agarose gel electrophoresis and identified by ethidium bromide staining under ultraviolet light, and the expected size can be confirmed.

Both SSOP and SSP can be modified to detect deletion or insertion variants. With SSP, using primers that flank the deletions or insertions, amplicons of characteristic sizes are produced. SSOP can confirm the presence or absence of the deletions or insertions through the use of probes that include the junctions of the deleted or inserted regions.

Relevance of genetic polymorphism in humans

Historically, polymorphisms in several genetic systems have been recognized as a barrier to transfusion and transplantation. The ABO blood group antigens were among the earliest genetically determined glycoproteins that exhibited mendelian inheritance and had biologic relevance in humans.3 Mismatch for the ABO antigens is a risk factor not only for transfusion reactions but also for solid-organ transplantation because of the prominent expression of these antigens on the vascular endothelium.

The major histocompatibility complex (MHC)—so called because of its prominent role in rejection of allogeneic tissue—is a primary barrier to transplantation of solid organs, tissue, and hematopoietic stem cells. This closely linked cluster of highly polymorphic genes, grouped on the short arm of chromosome 6, encodes cell surface molecules (human leukocyte antigens [HLA]). The normal role of the MHC is presentation of endogenous and exogenous peptide antigen fragments to T cells, thereby initiating an immune response against the molecule (or pathogenic organism) from which the peptide was derived.4 The extreme variability of molecular structure in the MHC antigens permits a wide range of different peptides to be presented by autologous human antigen-presenting cells, although some persons may have a specific repertoire of MHC antigens that do not present certain antigens effectively. The focused immunogenicity of MHC molecules and the variability of these molecules from person to person render them prominent targets for the immune response in the context of solid-organ and bone marrow transplantation. In cases in which live allogeneic cells are the target of the immune response, the apparent target is the nonself MHC molecule itself. Freedom from rejection and, in the case of bone marrow transplantation, graft versus host disease (GVHD) is improved with HLA matching of donor and recipient.

Innate and Adaptive Responses

It has become abundantly clear that the selective (adaptive) immunologic response, which is important in organ transplantation, tissue transplantation, and defense against certain microorganisms, is closely associated with the innate cellular and humoral pathways of nonspecific tissue injury, inflammation, hypoxia, and healing. Macrophages, for example, play a central role in the response to hypoxia, trauma, bacterial invasion, and inflammation caused by exogenous toxins, but they are also important in the processing and presentation of antigen to the specific immune system. Natural killer (NK) cells, which constitute approximately 10% of human mononuclear cells, are thought to be important mediators of innate immunity. Their cytolytic activity is regulated by inhibitory receptors, called killer im-munoglobulin-like receptors (KIRs).5 Class I MHC molecules are ligands for the KIRs—in particular, genetically determined epitopes on HLA-B and HLA-C molecules that have limited polymorphism.5 In bone marrow transplantation, recipients who present the appropriate class I ligands to donor NK cells will downregulate the NK response. This is thought to decrease both GVHD and graft versus tumor activity.

Humans also have innate humoral immunity against a number of glycoprotein antigens. This so-called natural antibody is thought to have protective effects against a wide range of bacterial products. At the same time, the humoral immune system is able to mount a robust adaptive response to an astonishingly broad spectrum of specific antigens, if challenged to do so. The genes responsible for the adaptive immune response are highly polymorphic, but they are found only in specific subsets of T cells with antigen receptor genes that are rearranged during thymic development and in B cells that undergo somatic mutation in response to antigenic challenge. Specific germline variant alleles of the T cell receptor for antigen (before somatic mutation) are also associated with differential susceptibility to a number of immunologically mediated conditions, including renal allograft rejection and several rheumatic diseases, such as rheumatoid arthritis.6

Other Polymorphic Genes Involved in Organ and Tissue Injury

Variants of genes can influence organ and tissue physiology, directly induce diseases, or render the person more susceptible or resistant to a pathologic state. Variants that directly induce a profound disease state are usually rare in the population, because the disease may cause death before the person can reproduce. Variants or mutations that cause severe early disease are not discussed in this subsection. Polymorphic variants of loci that have a more subtle effect on disease susceptibility are more likely to become established in the population at frequencies of 1% or more (i.e., to become alleles). Several patterns can be appreciated with these alleles. Variant alleles may exhibit a gene-dose effect, with heterozygotes having an intermediate influence, between that of the normal genotype (the so-called wild type) and the homozygous variant genotype. In other cases, a variant allele appears to have a dominant influence; presumably, these variants are able to achieve significant frequency in the population because the condition they produce does not substantially decrease reproduction. The disease phenotype that is a measurable physiologic consequence of a particular genotype may be a downstream effect that depends on multiple influences, including the genotype in question, interaction with other genes, and environmental exposure.

Loci that encode cytokines, chemokine receptors, costimulatory molecules, and components of physiologically important pathways such as the angiotensin system are all concrete examples in which genetic polymorphism influences pathophysiology. These examples can be used to highlight some ways in which determination of individual genotype can assist in assessing risk of disease.

Cytokines and chemokines are secreted proteins and glyco-proteins that act as important signaling devices in both the innate and the adaptive responses. They serve variously as che-moattractants and as inducers or suppressors of leukocyte, en-dothelial cell, platelet, fibroblast, and myocyte function. They have a particularly notable effect on cells that bear the appropriate receptors. Cytokines and chemokines often represent a common pathway that links the classical immune pathway and other pathways of tissue injury and repair, such as those involved in ischemia, trauma, and toxic damage.

Costimulatory molecules such as CTLA-4 are expressed on the cell membranes of T cells and serve as ligands for complementary molecules on antigen-presenting cells [see 6:IX Immunologic Tolerance and Autoimmunity]. The engagement of costimula-tory molecules with their ligands can augment or suppress the magnitude of the immune response induced by the recognition of antigen via the T cell receptor.8,9 Soluble CTLA-4 has been used to block antigen-dependent T cell activation by competitive blockade of normal cell membrane-bound interaction.

Functional Consequences of Specific Genetic Variants

Cytokine polymorphisms

Variants in the genes that govern the production of cy-tokines such as interleukin-10 (IL-10), tumor necrosis factor-a (TNF-a), and transforming growth factor-| (TGF-|) can help determine whether a person has high or low levels of these cy-tokines.10 The cytokine network is thought to play an important role both in rejection of allografts and in tolerance,11 and a number of clinical effects of these polymorphisms in cytokine genes have now been described [see Tables 1 and 2].

TGF-| has two well-studied dimorphic positions within the leader sequence of the gene [see Figure 2]. These polymorphisms are in linkage disequilibrium; only two variants of the TGF-| gene have been described, rather than the four theoretically possible combinatorial variants. TGF-| is considered to be a major mediator of fibrosis in kidney and lung allografts.

Table 1 Cytokine Genetic Polymorphisms and Their Pathophysiologic Effects931-59

Locus

Position

Genotype

Pathophysiologic Effect

CTLA4

Microsatellite

Allele 3 and allele 4

Increased rejection, liver/kidney transplants

IFN-y

Microsatellite

Allele 2 (12 CArepeat)

Increased production

IFN-y

Microsatellite

Allele 2 (12 CArepeat)

Increased production

IFN-y

Microsatellite

Allele 2 (12 CArepeat)

Increased acute rejection, kidney transplants

IFN-y

Microsatellite

Allele 3 homozygotes

Increased GVHD, bone marrow transplant patients

IFN-y

T+874A

T allele

Increased production

IL-10

-1082A

A allele

Low producer

IL-10

-1082A

A/A homozygotes

Increased frequency in Wegener granulomatosus

IL-10

-1064

Low producer

Increased GVHD, bone marrow transplant patients

IL-10

-1082, -819, -592

Low producer

Increased rejection, pediatric heart transplant patients

IL-10

-1064

High producer

Increased graft survival, renal transplants

IL-10

-1082A

High producer

Increased rejection, if high TNF genotype

IL-10

-1082A

High producer

Increased rejection episodes, renal transplants

IL-10

-1082A

Recipient high; donor low

Increased rejection, renal transplants

IL-4

-590T

Recipient and donor low

Decreased rejection, renal transplants

IL-6

-174C

G allele

Increased acute GVHD, bone marrow transplants

TGF-P1

Arg25Pro

A/A homozygotes

Increased production

TGF-P1

Arg25Pro

Arg

Increased in patients with fibrotic lung disease

TGF-P1

Arg25Pro

A/A homozygotes

Progression of renal insufficiency, heart transplant patients

TGF-P1

Arg25Pro

A/A homozygotes

Decreased gingival hyperplasia with cyclosporine

TGF-P1

Arg25Pro

A/A homozygotes

Increased coronary vasculopathy, heart transplants

TGF-P1

Arg25Pro

Arg

No correlation with renal transplant rejection

TGF-P1

Leu10Pro

Leu

Progression of renal insufficiency, heart transplant patients

TGF-P1

Leu10Pro

L/L homozygotes

Decreased renal dysfunction, heart transplant patients

TGF-P1

Leu10Pro

Pro

Association with dilated cardiomyopathy

TGF-P1

Leu10Pro

Pro/Pro

Increased gingival hyperplasia with cyclosporine

TNF-a

-308A

High A/Aor A/G

Increased rejection and creatinine, renal transplants

TNF-a

-308A

High A/Aor A/G

Increased GVHD and mortality, bone marrow transplant patients

TNF-a

-308A

High A/Aor A/G

Increased rejection, renal transplants

TNF-a

-308A

Low producer

Decreased acute rejection, pediatric heart transplants

TNF-a

-308A

A allele

Sixfold to sevenfold higher production

TNF-a

-308A

A allele

Risk factor renal transplants, if HLA-DR mismatch

TNF-a

-308A

A allele

Increased rejection, pediatric heart transplants

TNF-a

-308A

A allele

Increased frequency in primary sclerosing cholangitis

TNF-a

-308A

A allele

Increased mortality, heart transplant patients

TNF-a

-308A

A allele

Increased rejection, renal transplants

TNF-a

-308A

A allele

Increased hepatitis C recurrence after liver transplants

TNF-a

-308A

A allele

Decreased plasma TNF levels

TNF-a

-308A

A/A homozygotes

Increased rejection, liver transplants

TNF-a

-308A

A/A homozygotes

Increased acute rejection, liver transplants

TNF-a

Microsatellite

High producer

Increased rejection, cardiac transplants (low IL-10 subset)

TNF-a

Microsatellite

High producer

Increased acute GVHD, bone marrow transplant patients

TNF-a

Microsatellite

a9

Increased in rejection, renal transplants, in patients with HLA-B35

TNF-a

Microsatellite

d3/d3 homozygotes

Increased GVHD grade III/IV

TNF-a

Microsatellite

d4

Decreased in rejection, renal transplants, in patients with HLA-B44

TNF-a

Ncol

Low recipient

Increased infection, renal transplants

TNF-a

-308A

A allele

Nonischemic cardiac dysfunction

Note: Some of these gene variants appear to have paradoxical effects, depending on the investigator and the assay system.

GVHD—graft versus host disease

IFN—interferon

IL—interleukin

TGF—transforming growth factor

TNF—tumor necrosis factor

Specific variants of the TGF gene that result in high production of TGF-| (so-called high-producer genotypes) are associated with poor outcome in lung transplants: 98% of patients with chronic rejection are homozygous for the high-producer TGF-| genotype represented by Leu at position 10 and Arg at position 25. Moreover, fibrosis develops in the lung grafts of 93% of those with homozygous high-producer TGF-| genotype but only in 7% of those with heterozygous (high/low) producer genotype.12 TGF-| also mediates the gingival hypertrophy induced by the immunosuppressive agent cyclosporine. Increased gingival hypertrophy has been reported in patients with the low-producer TGF-| genotype, represented by Pro at both position 10 and position 25. Because the two variants differ only in the leader amino acid sequence, the different production levels may be the consequence of different efficiency of posttranslational modification.

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