- There are approximately 20,000 genes in the human genome.
- The human genome possesses about 3 x 109 nucleotides.
- Genes are 1-200,000 kb in size (average 10 kb).
- Genes have exons (coding sequences) and introns (noncoding sequence found between exons).
- It includes 22 autosomes plus X and Y (sex) chromosomes.
- Genes are arranged linearly on chromosomes.
| Table I Perspectives on the Number of Genes and the Genetic Distance in the Human Genome |
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Figure 15.1 |
Genetic and physical map of the X chromosome. Association of a trait with a position on a given chromosome is a critical step in gene identification. The relationship between genetic loci is measured on a genetic map as the frequency of recombination events between the loci. On physical maps relationships between loci are measured in nucleotides. The genetic and physical maps have the same linear sequence although the distances are not correlated. After narrowing the chromosomal localization, a candidate gene can be localized on a genetic map that contains marker genes known to map to that chromosomal region. Subsequently, the gene can be localized on a physical map that allows delineation of its position with a higher degree of resolution.
| Table II Gene Identification Enables Therapeutic and Diagnostic Possibilities by Increased Understanding of Protein Function, Mutation Identification, and Expression Patterns |
15.2 There are Four Major Routes to Gene Identification
- Functional Cloning - know the function of the gene, purify protein, clone
- Candidate Gene - identify a potential candidate gene based on mechanism of the primary pathology
- Positional Cloning - identify gene without prior knowledge of gene function, solely based on mapping
- Positional Candidate - identify Expressed Sequence Tag (EST) or cDNA and compare its chromosomal localization with a disease locus map
15.3 Homologous Recombination
- Two homologous chromosomes pair and crossover events (recombinations) occur between homologous chromatids.
- Homologous chromosomes segregate.
- Duplicate chromosomes segregate yielding cells containing only one copy of each homologous chromosome.
Figure 15.2 |
Figure 15.3 |
A recombination frequency of 1% (1 cM or 1 centimorgan) means that two genes recombine on average once in every 100 meiotic events. This is equal to about 1000kb (1 Mb) of DNA sequence.
Analysis of recombination frequencies among genes establishes their linear order and the distance between them.
Genes that are close to each other on a chromosome tend to be inherited together. This allows for the use of polymorphic loci near a candidate gene to be used as markers to examine whether they cosegregate with a phenotype (see linkage analysis below).
Association of a marker allele with a disease gene. A mutation (blue line) takes place that produces a disease gene. This mutation occurs in a DNA background that contains other polymorphisms. During subsequent meiotic events allelic markers that are further away from the mutation will segregate away from it, while those that are close by will cosegregate, allowing an association between the disease gene and the cosegregating polymorphic markers. The associated region will depend on the number of meiotic events occurring after the mutation and the probability of recombination events in this stretch of DNA.
15.4 Patterns of Mendelian Inheritance
- Autosomal dominant-phenotype with one mutant gene copy
- Autosomal recessive-phenotype with two mutant gene copies
- X-linked e.g., disease affects males and is not passed from father to son
15.5 Genetic (linkage) Mapping
A procedure by which any genetic trait is localized in the genome based on its segregation pattern with another marker or set of markers.
Recombination of homologous chromosomal segments
- Frequency of recombination events depends on length of segments, the specific nucleotide sequence and its genomic location, and whether the event takes place in the male or female.
- The consequence of recombination is that different offspring receive equal, but not identical, genetic information from each parent.
The genetic mapping of an unknown locus is established by examining the frequency with which it cosegregates with other previously mapped genetic markers. Mapping of anonymous markers and genes has allowed the construction of a fairly complete human genetic map. A number of disease genes with Mendelian phenotypes have been localized on the human linkage map to allow positional cloning. This has been helpful for linkage studies with traits that do not obey simple Mendelian patterns of inheritance.
Linkage analysis is a sequential procedure where data is collected until linkage is detected or refuted. Linkage is based on a LOD score-a statistical evaluation of a set of data that examines the probability of joint segregation of two markers with a given recombination distance under the assumption of linkage or no linkage.
Figure 15.4 |
15.6 Applications of a Linkage Map
- establish the genetic basis of traits
- predict risk for disease
- localize hereditary disorders to specific regions of the genome
- clone genes by positional cloning
- study gene conservation across species and chromosomal evolution
15.7 Factors Affecting Linkage Mapping of a Disease Trait
- penetrance-probability that a carrier will have the phenotype
- frequency of the disease gene in the population (Mendelian < 0.1%)
- age of onset variability
The large majority of human diseases are NOT Mendelian (multigenic).
Familial aggregation of some genes (e.g., twins studies in schizophrenia) suggests both genetic and environmental factors contribute.
15.8 Proving a Candidate Gene is Causally Mutated
- DNA sequence comparisons of affected and unaffected individuals
- Gene expression patterns
- Multiple mutant alleles
- Phylogenetic (cross-species) comparisons
- Using a transgenic or knockout approach in the mouse
These are functional approaches but do not give much molecular insight into the mechanisms by which the mutated gene produces the aberrant phenotype. Dissection of the role of the candidate gene product and alterations in cellular function due to a given mutation must rely on examination of the function of the deduced protein using molecular and cell biological techniques.
15.9 Single Gene or Polygenic?
How can it be determined whether a disease results from mutations in a single gene or multiple genes (polygenic)? Likely single gene diseases are those in which a single biochemical step is altered (e.g., some lipid storage disorders in which the inactivity of an enzyme leads to the accumulation of a precursor). Highly complex disorders (e.g., schizophrenia) are likely to be polygenic because of the wide-ranging disease phenotype.
- Huntington's disease-single gene
- Parkinson's disease-single gene
- Spinocerebellar ataxia-single gene
- Lipid storage disorders e.g., Tay-Sachs, Battens, Metachromatic Leukodystrophy-single genes
- Alzheimer's disease-likely polygenic
- Schizophrenia-likely polygenic
15.10 Mapping Complex Traits
Most traits are complex (non-Mendelian). Therefore, in order to attempt to identify genes, two successful strategies have been employed:
- Define a subtype that shows near-Mendelian inheritance
Alzheimer's Disease (AD)
Individuals have been identified who possess early- or late-onset AD phenotype. The early-onset phenotype exhibits near-Mendelian patterns of inheritance. Three genes and one locus were isolated: Amyloid precursor protein (APP), Presenilin-1 (PS-1), Presenilin-2 (PS-2), and apolipoprotein E (ApoE). APP and PS-1 were isolated from early-onset linkage studies while ApoE was from late-onset studies. PS-2 was identified from an ethnic isolate.
- Study families found in an isolated population where the disease incidence is high (many recombination events)
Huntington's Disease
Huntington's disease follows an autosomal dominant pattern of inheritance. However, isolation of the gene underlying this disease was only possible by examining an isolated family in the Lake Maracaibo region of Venezuela. This family has 12,000 individuals, 258 have the disease, 1227 have 50% risk, 2885 have 25% risk and all are descendants from one woman who lived in the early 1800s. Linkage mapping enabled the localization of a single gene, the Huntington gene, that codes for the huntingtin protein. Mutations in the gene result in expanded triplet (CAG) repeats with the number of CAGs proportional to severity of symptoms which are motor and cognitive.
Figure 15.5 |
Disorders affecting midbrain dopamine neurons include Parkinson's disease and Huntington's disease. Huntington's disease is a single gene disease resulting from a mutation in the gene coding for huntingtin. In Huntington's disease, a population of neurons in the striatum degenerate presumably due to the presence of a mutated huntingtin protein. The mechanism by which this mutated protein produces cell death is being intensely studied but is not yet clear. The symptoms of Parkinson's disease result from the degeneration of midbrain dopamine neurons in the substantia nigra and ventral tegmental area that project to the striatum and nucleus accumbens. A candidate gene has been isolated but does not account for the entire phenotype.
15.11 Gene Therapy
Understanding that a particular gene underlies a phenotype is the first step in trying to provide therapeutic benefits to patients carrying a mutation. However, there are considerable obstacles to overcome between finding the gene and being able to "fix" the mutation. The strategy employed to functionally "fix" mutations leading to inactive or partially active proteins involves supplying the "correct" gene to the cells needing to produce the inactive protein.
Figure 15.6 |
Figure 15.7 |
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The general strategy involved in "fixing" a mutant gene. |
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The major problem is how to get the corrected gene into the appropriate cells. In many cases a defective viral vector is used that enables entry into cells but lacks the ability to self replicate (Figure 15.6). A minigene is constructed that contains the "correct" coding region of the wild type protein along with additional sequences necessary for its propagation. The minigene is packaged and modified cells are made that contain viral particles that make the corrected protein. These cells are then injected into the patient. Intracranial injection (Figure 15.7) can be used along with various delivery methods (e.g., viral vectors, liposomes) to enable targeting to neural tissues. Other injection sites can be used for appropriate tissue targeting.
