Genetic Variation in Individuals and Populations: Mutation and Polymorphism
INHERITED VARIATION AND POLYMORPHISM IN PROTEINS Although all polymorphism is ultimately the result of differences in DNA sequence, some polymorphic loci have been studied by examining the variation in the proteins encoded by the alleles rather than by examining the differences in DNA sequence of the alleles themselves. Any one individual is likely to be heterozygous for alleles determining structurally different polypeptides at approximately 20% of all loci; when individuals from different ethnic groups are compared, an even greater fraction of proteins has been found to exhibit detectable polymorphism.
A striking degree of biochemical individuality exists within the human species in its makeup of enzymes and other gene products. As the products of many of the encoded biochemical pathways interact, each individual, regardless of state of health, has a unique, genetically determined chemical makeup and thus responds in a unique manner to environmental, dietary, and pharmacological influences.
Examples of polymorphisms of medical significance: the ABO and Rh blood groups, the major histocompatibility complex (MHC). Studying variation in proteins has real utility, the variant protein products of various polymorphic alleles are often what is responsible for different phenotypes and therefore are likely to dictate how genetic variation at a locus affects the interaction between an individual and the environment.
Blood Groups and Their Polymorphisms Genetically determined protein variation detected as antigens found in blood, the so-called blood group antigens. Numerous polymorphisms are known to exist in the components of human blood, especially in the ABO and Rh antigens of red blood cells. In particular, the ABO and Rh systems are important in blood transfusion, tissue and organ transplantation, and hemolytic disease of the newborn.
The ABO System Human blood can be assigned to one of four types according to the presence on the surface of red blood cells of two antigens, A and B, and the presence in the plasma of the two corresponding antibodies, anti-A and anti-B.
One feature of the ABO groups not shared by other blood group systems is the reciprocal relationship, in an individual, between the antigens present on the red blood cells and the antibodies in the serum. When the RBCs lack antigen A, the serum contains anti-A; when the cells lack antigen B, the serum contains anti-B. The reason for this reciprocal relationship is uncertain, but formation of anti-A and anti-B is believed to be a response to the natural occurrence of A-like and B-like antigens in the environment (e.g., in bacteria).
The ABO blood groups are determined by a locus on chromosome 9. The A, B, and O alleles at this locus are a classic example of multiallelism in which three alleles, two of which (A and B) are inherited as a codominant trait and the third of which (O) is inherited as a recessive trait, determine four phenotypes. The A and B antigens are made by the action of the A and B alleles on a red blood cell surface glycoprotein called H antigen.
The antigenic specificity is conferred by the specific terminal sugars, added to the H substance. The B allele codes for a glycosyltransferase adds d-galactose to the end H antigen B antigen. The A allele codes for an enzyme that adds N-acetylgalactosamine to the precursor H A antigen. The O allele codes for a mutant version of the transferase that lacks transferase activity, does not detectably affect H substance at all.
The molecular differences in the glycosyltransferase gene Four nucleotide differences between the A and B alleles result in amino acid changes that alter specificity of the glycosyltransferase. The O allele has a single-base pair deletion in the coding region, a frameshift mutation that eliminates the transferase activity in type O individuals. Now ABO blood group typing can be performed genotype level, especially when there are technical difficulties in serological analysis, as is often the case in forensic investigations or paternity testing.
ABO blood group is medically important in blood transfusion and tissue or organ transplantation. A compatible combination is one in which the RBCs of a donor do not carry an antigen that corresponds to the antibodies in the recipient's serum. Although theoretically there are universal donors (group O) and universal recipients (group AB), a patient is given blood of his or her own ABO group, except in emergencies.
In case of incompatibility antibodies can cause immediate destruction of ABO-incompatible cells. In tissue and organ transplantation, ABO compatibility of donor and recipient, human leukocyte antigen (HLA) compatibility, is essential to graft survival.
The Rh System The Rh system is clinically important because of its role in hemolytic disease of the newborn and in transfusion incompatibilities. The name Rh comes from Rhesus monkeys that led to the discovery of the system. In simplest terms, the population is separated into Rh-positive who express on their RBCs the antigen Rh D, a polypeptide encoded by a gene (RHD) on chromosome 1, and Rh-negative individuals, who do not express this antigen.
The Rh-negative phenotype usually originates from homozygosity for a nonfunctional allele of the RHD gene. The frequency of Rh-negative individuals varies in different ethnic groups. For example, 17% of whites and 7% of African Americans are Rh-negative, whereas the frequency among Japanese is 0.5%.
Hemolytic Disease of the Newborn (HDN) Rh-negative persons can readily form anti-Rh antibodies after exposure to Rh-positive RBCs. A problem when an Rh-negative pregnant woman is carrying an Rh-positive fetus. Normally during pregnancy, small amounts of fetal blood cross the placental barrier and reach the maternal blood stream. If the mother is Rh-negative and the fetus Rh-positive, the mother will form antibodies that return to the fetal circulation and damage the fetal RBCs, causing HDN with consequences that can be severe if not treated.
The risk of immunization by Rh-positive fetal RBCs can be minimized with an injection of Rh immune globulin at 28 to 32 weeks of gestation and again after pregnancy. Rh immune globulin serves to clear any Rh-positive fetal cells from the mother's circulation before she is sensitized. Rh immune globulin is also given after miscarriage, termination of pregnancy, or invasive procedures such as chorionic villus sampling or amniocentesis, in case any Rh-positive cells gained access to the mother's circulation.
The discovery of the Rh system and its role in hemolytic disease of the newborn has been a major contribution of genetics to medicine. At one time ranking as the most common human genetic disease, hemolytic disease of the newborn is now relatively rare because of preventive measures that have become routine practice in obstetrical medicine.
The MHC is composed of a large cluster of genes located on the short arm of chromosome 6. On the basis of structural and functional differences, these genes are categorized into three classes, two of which, the class I and class II genes, correspond to the human leukocyte antigen (HLA) genes, originally discovered by virtue of their importance in tissue transplantation between unrelated individuals. The Major Histocompatibility Complex
The HLA class I and class II genes encode cell surface proteins that play a critical role in the initiation of an immune response and specifically in the "presentation" of antigen to lymphocytes, which cannot recognize and respond to an antigen unless it is complexed with an HLA molecule on the surface of an antigen-presenting cell. Many hundreds of different alleles of the HLA class I and class II genes are known and more are being discovered, making them by far the most highly polymorphic loci in the human genome.
Figure 9-7 A schematic of the MHC complex on chromosome 6p. DP, DQ, and DR, class II antigen genes; B, C, and A, class I antigen genes; LMP, genes encoding components of large multifunctional protease; DM, heterodimer of DMA and DMB genes encoding the antigen-processing molecule required for binding peptide to MHC class II antigens; other genes encode TAP, transporter associated with antigen processing; TNF, tumor necrosis factor; Bf, properdin factor B; C2, C4A, C4B, complement components; 21-OH, 21-hydroxylase. (One of the 21-OH loci is a pseudogene.)
The class I genes (HLA-A, HLA-B, and HLA-C) encode proteins that are an integral part of the plasma membrane of all nucleated cells. A class I protein consists of two polypeptide subunits, a variable heavy chain encoded within the MHC and a nonpolymorphic polypeptide, β2-microglobulin, that is encoded by a gene outside the MHC, mapping to chromosome 15. Peptides derived from intracellular proteins are generated by proteolytic degradation by a large multifunctional protease; the peptides are then transported to the cell surface and held in a cleft formed in the class I molecule to display the peptide antigen to cytotoxic T cells.
The Major Histocompatibility Complex Figure 9-8 The interaction between MHC class I and class II molecules, foreign proteins, and T-cell receptors. LMP, large multifunctional protease; TAP, transporter associated with antigen processing; Ii, invariant chain; DM, heterodimer encoded by the DMA and DMB genes; CD8+, cytotoxic T cells; CD4+, helper T cells.
The class II region is composed of several loci, such as HLA-DP, HLA-DQ, and HLA-DR, that encode integral membrane cell surface proteins. Each class II molecule is a heterodimer, composed of α and β subunits, both of which are encoded by the MHC. Class II molecules present peptides derived from extracellular proteins that had been taken up into lysosomes and processed into peptides for presentation to T cells.
Other gene loci are present within the MHC but are functionally unrelated to the HLA class I and class II genes and do not function to determine histocompatibility or immune responsiveness. Some of these genes are, however, associated with diseases, such as congenital adrenal hyperplasia, caused by deficiency of 21-hydroxylase, and hemochromatosis, a liver disease caused by iron overload.
HLA Alleles and Haplotypes According to the older, traditional system of HLA nomenclature, the different alleles were distinguished from one another serologically. An individual's HLA type was determined by seeing how a panel of different antisera or reactive lymphocytes reacted to his or her cells. These antisera and cells were obtained from hundreds of multiparous women who developed immune reactivity against the paternal type I and type II antigens expressed by their fetuses during the course of their pregnancies.
If cells from two unrelated individuals evoked the same pattern of reaction in a typing panel of antibodies and cells, they would be considered to have the same HLA types and the allele they represented would be given a number, such as B27 in the class I HLA-B locus or DR3 in the class II DR locus. However, as the genes responsible for encoding the class I and class II MHC chains were identified and sequenced, single HLA alleles initially defined serologically were shown to consist of multiple alleles defined by different DNA sequence variants even within the same serological allele.
The 100 serological specificities at HLA-A, B, C, DR, DQ, and DP now comprise more than 1300 alleles defined at the DNA sequence level. For example, more than 24 different nucleic acid sequence variants of the HLA-B gene exist in what was previously defined as "the" B27 allele by serological testing. Most but not all of the DNA variants change a triplet codon and therefore an amino acid in the peptide encoded by that allele. Each allele that changes an amino acid in the HLA-B peptide is given its own number, so allele number 1, number 2, and so on in the group of alleles corresponding to what used to be a single B27 allele defined serologically, is now referred to as HLA-B*2701, HLA-B*2702, and so on.
The set of HLA alleles at the different class I and class II loci on a given chromosome together form a haplotype. The alleles are codominant; each parent has two haplotypes and expresses both. These loci are located close enough to each other that, in an individual family, the entire haplotype can be transmitted as a single block to a child. As a result, parent and child share only one haplotype, and there is a 25% chance that two sibs inherit matching HLA haplotypes. Because acceptance of transplanted tissues largely correlates with the degree of similarity between donor and recipient HLA haplotypes (and ABO blood groups), the favored donor for bone marrow or organ transplantation is an ABO-compatible and HLA-identical sibling of the recipient.
Figure 9-9 The inheritance of HLA haplotypes. A haplotype is usually transmitted, as a unit. In extremely rare instances, a parent will transmit a recombinant haplotype to the child, as seen in individual II-5, who received a haplotype that is recombinant between the class I and class II loci.
Within any one ethnic group, some HLA alleles are found commonly; others are rare or never seen. Similarly, some haplotypes are much more frequent than expected, whereas others are exceptionally rare or nonexistent. For example, most of the 3 × 107 allelic combinations that could theoretically occur to make a haplotype among white individuals have never been observed. This restriction in the diversity of haplotypes possible in a population results from a situation referred to as linkage disequilibrium and may be explained by a complex interaction between a number of factors:
These factors include: low rates of meiotic recombination in the small physical distance between HLA loci; environmental influences that provide positive selection for particular combinations of HLA alleles forming a haplotype; and historical factors, such as how long ago the population was founded, how many founders there were, and how much immigration has occurred.
Major differences in allele and haplotype frequencies exist between populations as well. What may be a common allele or haplotype in one population may be very rare in another. Once again, the differences in the distribution and frequency of the alleles and haplotypes within the MHC are the result of complex genetic, environmental, and historical factors at play in each of the different populations.
HLA and Disease Association With the increasing delineation of HLA alleles has come an appreciation of the association between certain diseases and specific HLA alleles and haplotypes. The etiological basis for most of the HLA-disease associations remains obscure. Most but not all of these disorders are autoimmune, that is, associated with an abnormal immune response apparently directed against one or more self antigens that is thought to be related to variation in the immune response resulting from polymorphism in immune response genes.
Ankylosing spondylitis, a chronic inflammatory disease of the spine and sacroiliac joints, is one example. In older studies that relied on serologically defined B27 alleles, only 9% of Norwegians, for example, are B27-positive, whereas more than 95% of those with ankylosing spondylitis are B27-positive. Thus, the risk of developing ankylosing spondylitis is at least 150 times higher for people who have HLA-B27 than for those who do not. Although less than 5% of B27-positive individuals develop the disease, as many as 20% of B27-positive individuals may have subtle, subclinical manifestations of the disease without any symptoms or disability. Ankylosing Spondylitis
One explanation for why some B27-positive individuals do not develop disease rests in part on that fact that DNA sequencing has revealed more than two dozen different alleles within "the" HLA-B27 allele originally defined serologically. The frequency of each of these different alleles varies within a given ethnic group and between ethnic groups. *If only certain of these B27 alleles predispose to disease, while others may actually be protective, studies in different ethnic groups that lump all the B27 alleles into a single allele will find quite different rates of disease in B27-positive individuals.
In other cases, the association between a particular HLA allele or haplotype and a disease is not due to functional differences in immune response genes encoded by the HLA alleles. Instead, the association is due to a particular MHC allele being present at a very high frequency on chromosomes that also happen to contain disease-causing mutations in another gene within the MHC, because of linkage disequilibrium. As mentioned earlier, the autosomal recessive disorders congenital adrenal hyperplasia due to 21-hydroxylase deficiency and primary hemochromatosis result from mutations in genes that lie within the MHC.
Analysis of 21-hydroxylase mutations responsible for adrenal hyperplasia has revealed that certain of the mutations at this locus originally occurred on chromosomes with particular haplotypes and were subsequently inherited through multiple generations along with these specific haplotype markers as a block. Another example is hemochromatosis, a common autosomal recessive disorder of iron overload. More than 80% of patients with hemochromatosis are homozygous for a common mutation, Cys282Tyr, in the hemochromatosis gene (HFE) and have HLA-A*0301 alleles at their HLA-A locus. The association is not the result of HLA-A*0301 somehow causing hemochromatosis.
HFE is involved with iron transport or metabolism in the intestine; HLA-A, as a class I immune response gene, has no effect on iron transport. The association is due to proximity of the two loci and the linkage disequilibrium between the Cys282Tyr mutation in HFE and the A*0301 allele at HLA-A.
The functional basis of most HLA-disease associations is unknown. HLA molecules are integral to T-cell recognition of antigens. Perhaps different polymorphic alleles result in structural variation in these cell surface molecules, leading to differences in the capacity of the proteins to interact with antigen and the T-cell receptor in the initiation of an immune response, thereby affecting such critical processes as immunity against infections and self-tolerance to prevent autoimmunity.
HLA and Tissue Transplantation As the name major histocompatibility complex implies, the HLA loci are the primary determinants of transplant tolerance and graft rejection and therefore play an important role in transplantation medicine. Despite the impressive progress in the design of powerful immunosuppressive drugs to suppress rejection of organ transplants, only an absolutely perfect match for all HLA and blood group alleles, such as occurs between monozygotic twins, can provide a 100% transplantation success rate without immunosuppressive therapy.
For the transplantation of solid organs, such as kidneys, the percentage of grafts surviving after 10 years when the recipient and the donor are HLA-identical siblings is 72% but falls to 56% when the donor is a sibling who has only one HLA haplotype in common with the recipient. Bone marrow transplantation is a greater challenge than solid organ transplantation; not only can the host reject the graft, but also the graft, which contains immunocompetent lymphocytes, can attack the host in what is known as graft-versus-host disease (GVHD).
Survival to 8 years after BMT for patients with chronic myelogenous leukemia following chemotherapy is 60% if graft and host mismatch at no more than one class I or class II locus but falls to 25% when there are both class I and class II mismatches. GVHD is also less frequent and severe the better the class I match. Given the obvious improvement in the success of BMT with the greater number of matches, and the tremendous diversity of HLA haplotypes within a population and between different ethnic groups, millions of HLA-typed unrelated BM donors have been registered in databases that can be searched to look for the best possible match for a patient needing a BMT.
GENOTYPES AND PHENOTYPES IN POPULATIONS Genetic Variation in Populations Population genetics is the quantitative study of the distribution of genetic variation in populations and of how the frequencies of genes and genotypes are maintained or change. Population genetics is concerned both with genetic factors, such as mutation and reproduction, and with environmental and societal factors, such as selection and migration, which together determine the frequency and distribution of alleles and genotypes in families and communities.
At present, human geneticists are using the principles and methods of population genetics to address the history and genetic structure of human populations, the flow of genes between populations and between generations, and, very importantly, the optimal methods for identifying genetic susceptibilities to common disease. In medical genetic practice, population genetics provides the knowledge about different disease genes that are common in different populations, information that is needed for clinical diagnosis and genetic counseling, including determining the allele frequencies required for risk calculations.
Genetic Factors in Human Immunodeficiency Virus Resistance An important example of a common autosomal trait governed by a single pair of alleles can be used to illustrate the basic principles that determine allele and genotype frequencies in populations. Consider the gene CCR5, which encodes a cell surface cytokine receptor that serves as an entry point for certain strains of the HIV that causes AIDS. A 32-base pair deletion in this gene results in an allele (ΔCCR5) that encodes a nonfunctional protein due to a frameshift and premature termination.
Individuals homozygous for the ΔCCR5 allele do not express the receptor on their cell surface and, as a consequence, are resistant to HIV infection. Loss of function of CCR5 appears to be a benign trait, and its only known phenotypic consequence is resistance to HIV infection. The normal allele and the 32-base pair deletion allele, ΔCCR5, are easily distinguished by PCR analysis of the gene. A sampling of 788 individuals from Europe provides absolute numbers of individuals who were homozygous for either allele or heterozygous.