II. Mutation rates.

III. Molecular consequences of mutations.

IV. Unstable genes.

Terms.

Erratum Page 276, margin: The definition of frameshift mutation should read, "Mutational events in which one to two bases are added to or removed from DNA, causing a shift in the codon reading frame."

I. A mutation can be defined as any heritable change.

A. A mutant allele may be newly mutant if one is concerned about the process of mutation; or it may have mutated generations ago and the term is used merely as a synonym for variant allele. Don't be confused. The context should make clear which meaning is intended.

B. Historically, point mutations are those changes in DNA that are invisible microscopically. They may involve a single nucleotide or hundreds of thousands of nucleotides. With development of methods of analyzing DNA, the term point mutation is often equated with changes in a single nucleotide. Deletion or insertion of a small number of nucleotides are described by these more informative terms.

C. Mutations can also be distinguished by the types of cells in which they occur.

1. Germinal mutations occur in germ cells and can be passed on to future generations.

2. Somatic mutations occur in somatic cells and cannot be transmitted to offspring.

D. Mutations can occur at any site in a gene or in the regulatory regions. In genes that have been studied extensively, hundreds of different mutations are known. These can range from no phenotypic effect to complete nonfunction of the gene, which is lethal in some instances.

E. Inherited disorders that are quite distinct phenotypically may be due to different mutations in the same gene. E.g. Lesch-Nyhan syndrome and a rare form of gout are both mutations in the HPRT locus.

1. In Lesch-Nyhan syndrome, the X-linked gene that codes for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) produces no or very little functional enzyme. Uric acid in the blood rises to very high levels in hemizygous males, producing mental retardation and a remarkable compulsion for self-mutilation.

2. If the mutation in HPRT permits some enzymatic activity, hemizygous males have elevated uric acid in the blood but not nearly so high as in Lesch-Nyhan syndrome. This causes a rare, mild form of gout. It is not the common cause of gout.

A. Mutation rates are based almost exclusively on rare autosomal dominant or X-linked recessive traits. It is virtually impossible to measure autosomal recessive traits accurately.

B. The range of known mutation rates varies from 1 in 10,000 for Duchenne muscular dystrophy and neurofibromatosis type-1 (the largest genes known) to several genes in the range of 1 in 10,000,000.

C. Mutation rate studies never measure all the possible mutations at a locus. Many of the mutations cause no obvious phenotypic effect and could only be recognized by direct analysis of DNA sequences.

D. The rate of nucleotide substitutions is on the order of 1 per 100,000,000 nucleotides. Since there are 3 billion nucleotides per genome, that means that every gamete has about 30 new mutations involving nucleotide substitutions.

A. A nucleotide substitution in a coding region may be any of the following:
1. Silent or synonymous mutations consist in changing one codon for another that codes for the same amino acid; eg TTT (Leu) —> TTC (Leu).

2. Missense mutations involve a change of codons that cause substitution of one amino acid for another. E.g. Sickle cell hemoglobin: beta codon 6 GAG (Glu) —> GTG (Val).

3. Nonsense mutations involve change from one of the 61 codons that specify amino acids to one of the three termination codons. E.g. In the form of thalassemia common in the Mediterranean region, Hb beta thal-1, codon 17 AAG (Tyr) —> TAG (Ter). This causes premature termination of the protein chain.

4. Sense mutations involve change from a termination codon to one that codes for amino acids. E.g. Hemoglobin Constant Spring alpha 142 TAA (Ter) —> CAA (Gln). Translation continues beyond the normal termination until another termination codon is encountered.

B. Nucleotide substitutions can also affect the rate of transcription.
1. A change in the regulatory regions can abolish transcription.

2. In some instances, such as a mutation that causes persistence of fetal hemoglobin beyond the fetal period, nucleotide substitution in the 5' regulatory region causes the gene to be transcribed when it should be shut off.

C. Nucleotide substitutions can affect RNA processing.
1. Substitutions in the exon/intron boundaries can prevent normal splicing.

2. Substitutions within an intron or exon can create new splice sites.

3. Changes in the 3' direction (downstream) from the coding regions can interfere with addition of poly-A tail.

D. Insertion or deletion of one or two nucleotides destroys the reading frame. Such frameshift mutations cause absence of any functional gene product. The most common mutation that causes cystic fibrosis involves loss of three nucleotides (codon 508), which leaves the reading frame intact but the protein is nonfunctional.

E. Many mutations that cause loss of gene function are deletions of larger numbers of nucleotides. E.g. Duchenne muscular dystrophy.

F. The mutations that occur at a particular locus may be quite heterogeneous. Some may knock out gene function completely, others may reduce the function, and still others may have no effect on the phenotype, i.e., they are normal variants. As a result of this heterogeneity, many patients described as "homozygous" for a recessive allele may actually be heterozygous for two different defective alleles. Such a person is often called a compound heterozygote.

A. A number of repeated nucleotide triplets are scattered throughout the genome. For example, at the fragile X site, normal persons have 2 to 50 tandem copies of the triplet CGG. The number of copies on a particular chromosome is very stable. Some persons have more than 50 copies, in which case the number is unstable, with a strong tendency to increase each generation. If the number increases to greater than 200, the person develops the fragile-X syndrome, a major cause of mental retardation in males, who are hemizygous. Heterozygous females are less severely affected. If the number of copies is 50-200, that allele is described as a premutation.

B. Since the number of triplets (nearly) always increases, the condition is more severe in succeeding generations and also appears earlier. This phenomenon is known as anticipation.

C. Some ten diseases are known to result from trinucleotide expansion. In addition to the fragile-X syndrome, these include Huntington disease. HD is a late onset disease, usually first appearing in persons in their 30's and 40's.

D. The mechanism for trinucleotide expansion is unknown. Also, for unknown reasons, expansion occurs only in females in the case of the fragile-X site, only in males in the case of Huntington disease, and in both sexes in some other conditions.

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