Mutations M.Dianatpour MLD, PhD

Mutations A mutation is defined as a heritable alteration or change in the genetic material. Mutations drive evolution but can also be pathogenic.

Mutations can arise through exposure to mutagenic agents, but the vast majority occur spontaneously through errors in DNA replication and repair. Sequence variants with no obvious effect upon phenotype may be termed polymorphisms.

Mutations classification Mutations can be classified into three categories: Mutations that affect the number of chromosomes in the cell (genome mutations), Mutations that alter the structure of individual chromosomes (chromosome mutations), Mutations that alter individual genes (gene mutations)

Genome mutations are alterations in the number of intact chromosomes (called aneuploidy) arising from errors in chromosome segregation during meiosis or mitosis

Chromosome mutations are changes involving only a part of a chromosome, such as partial duplications or triplications, deletions, inversions, and translocations, which can occur spontaneously or may result from abnormal segregation of translocated chromosomes during meiosis

Gene mutations are changes in DNA sequence of the nuclear or mitochondrial genomes, ranging from a change in as little as a single nucleotide to changes that may affect many millions of base pairs.

Aims of the study of Mutations Detection and screening of genetic disease in particular families at risk as well as for some diseases in the population at large.

Even a small gene mutation can have large effects, depending on which gene has been altered and what effect the alteration has on expression of the gene. A gene mutation consisting of a change in a single nucleotide in the coding sequence may lead to complete loss of expression of the gene or to the formation of a variant protein with altered properties.

Some DNA changes, however, have no phenotypic effect. A mutation within a gene may have no effect either because the change does not alter the primary amino acid sequence of a polypeptide (Silent Mutation) or because, even if it does, the resulting change in the encoded amino acid sequence does not alter the functional properties of the protein. Not all mutations, therefore, have clinical consequences. Conservative mutations

All three types of mutation occur at appreciable frequencies in many different cells. If a mutation occurs in the DNA of cells that will populate the germ line, the mutation may be passed on to future generations. In contrast, somatic mutations occur by chance in only a subset of cells in certain tissues and result in somatic mosaicism as seen, for example, in many instances of cancer. Somatic mutations cannot be transmitted to the next generation.

The Origin of Mutations

Gene Mutations Gene mutations, including : base pair substitutions, Insertions and deletions Gene mutations can originate by either of two basic mechanisms: errors introduced during the normal process of DNA. replication, mutations arising from a failure to repair DNA after. damage

Some mutations are spontaneous, whereas others are induced by physical or chemical agents called mutagens, because they greatly enhance the frequency of mutations.

TYPES OF MUTATIONS AND THEIR CONSEQUENCES

Nucleotide Substitutions Missense Mutations A single nucleotide substitution (or point mutation) in a DNA sequence can alter the code in a triplet of bases and cause the replacement of one amino acid by another in the gene product. In many disorders, such as the hemoglobinopathies, most of the detected mutations are missense mutations.

Nonsense Mutation (Chain Termination Mutations) Point mutations in a DNA sequence that cause the replacement of the normal codon for an amino acid by one of the three termination codons. No expression or truncated protein. nonsensemediated mRNA decay

A point mutation not only may create a premature termination codon, it also may destroy a termination codon and allow translation to continue until the next termination codon is reached.

RNA Processing- Mutations RNA Processing capping, polyadenylation, and splicing.

Mutations of the highly conserved splice donor (GT) and splice acceptor (AG) sites usually result in aberrant splicing. This can result in the loss of coding sequence (exon skipping) or retention of intronic sequence, and may lead to frame shift mutations. Cryptic splice sites, which resemble the sequence of an authentic splice site, may be activated when the conserved splice sites are mutated. In addition, base substitutions resulting in apparent silent, missense and nonsense mutations can cause aberrant splicing through mutation of exon splicing enhancer sequences.

''Hotspots" of Mutation Nucleotide changes that involve the substitution of one purine for the other (A for G or G for A) or one pyrimidine for the other (C for T or T for C) are called transitions. The replacement of a purine for a pyrimidine (or vice versa) is called a transversion. If nucleotide substitutions were random, there should be twice as many transversions as transitions because every base can undergo two transversions but only one transition. But the rate of transition is quite more than transversion.

C<T Transition The major form of DNA modification in the human genome involves methylation of cytosine residues (to form 5-methylcytosine), specifically when they are located immediately 5' to a guanine (i.e., as the dinucleotide 5'-CG- 3'). Spontaneous deamination of 5- methylcytosine to thymidine in the CG doublet gives rise to C > T or G > A transitions More than 30% of all single nucleotide substitutions are of this type, and they occur at a rate 25 times greater than does any other single nucleotide

mutation. Thus, the CG doublet represents a true "hotspot" for mutation in the human genome.

Deletions and Insertions Mutations can also be caused by the insertion, inversion, fusion, or deletion of DNA sequences. Some deletions and insertions involve only a few nucleotides and are generally most easily detected by nucleotide sequencing. In other cases, a substantial segment of a gene or an entire gene is deleted, inverted, duplicated, or translocated to create a novel arrangement of gene sequences. Such mutations are usually detected at the level of Southern blotting of a patient's DNA, or by polymerase chain reaction (PCR) analysis of the novel junction formed by the translocated segment

Deletions and Insertions In rare instances, deletions are large enough to be visible at the cytogenetic level. To be detected even with high-resolution prometaphase banding, these mutations generally must delete at least 2 to 4 million base pairs of DNA. In many instances, such deletions remove more than a single gene and are associated with a contiguous gene syndrome.

Small Deletions and Insertions Some deletions and insertions affect only a small number of base pairs. When the number of bases involved is not a multiple of three (i.e., is not an integral number of codons), and when it occurs in a coding sequence, the reading frame is altered beginning at the point of the insertion or deletion. The resulting mutations are called frameshift mutations. At the point of the insertion or deletion, a different sequence of codons is thereby generated that encodes a few abnormal amino acids followed by a termination codon in the shifted frame

If the number of base pairs inserted or deleted is a multiple of three, no frame shift occurs and there will be an insertion or deletion of the corresponding amino acids in the translated gene product.

Large Deletions and Insertions The frequency of such mutations differs markedly among different genetic diseases; some disorders are characterized by a high frequency of detectable deletions, whereas in others, deletion is a very rare cause of mutation. For example, deletions within the large dystrophin gene on the X chromosome in Duchenne muscular dystrophy (DMD) or large neurofibromin gene in neurofibromatosis type I are present in more than 60% of cases.

Many cases of α-thalassemia are due to deletion of one of the two α -globin genes on chromosome 16, whereas β-thalassemia is only rarely due to deletion of the β - globin gene

In some cases, the basis for gene deletion is well understood and is probably mediated by aberrant recombination between multiple copies of similar or identical DNA sequences. In other cases, the basis for deletion is unknown.

unequal crossing over

Examples α-Thalassemia Familial hyper cholestrolemia (Alu repeats)

Insertion of large DNA Insertion of large amounts of DNA is a cause of mutation that is much rarer than deletion. However, a novel mechanism of mutation, the insertion of LINE sequences, has been described in a few unrelated, sporadic patients with hemophilia A The LINE (Long INterspersed Elements) family of interspersed repetitive sequences represents a class of repeated DNA that may transcribed into an RNA that, when it is reverse transcribed, generates a DNA sequence that can insert itself into different sites in the genome (Retro transposons).

In a few patients with hemophilia A, LINE sequences several kilo base pairs long were found to be inserted into an exon in the factor VIII gene, interrupting the coding sequence and inactivating the gene. This finding suggests that at least some of the estimated 850,000 copies of the LINE family in the human genome are capable of causing disease by insertional mutagenesis.

Abnormal pairing and recombination between two similar sequences repeated on a single chromosome may also occur; depending on the orientation of these sequences, such recombination may lead to deletion or inversion. For example, nearly half of all severe hemophilia A is due to recombination that inverts a number of exons, thereby disrupting gene structure

Dynamic Mutations The mutations in disorders such as Huntington disease and fragile X syndrome involve expansion of trinucleotide repeat sequences. In these diseases, a simple trinucleotide repeat, located either in the coding region or in a transcribed but untranslated region of a gene (UTR), may expand during gametogenesis, in what is referred to as a dynamic mutation, and Interfere with normal gene expression.

A repeat in the coding region will generate an abnormal protein product, whereas repeat expansion in transcribed but untranslated parts of a gene may interfere with transcription, mRNA processing, or translation. How dynamic mutations occur is not completely understood. It is thought that during replication, errors can occur when the growing strand slips while the polymerase is attempting to extend the strand and subsequently sits back down on the template strand out of register with where it was when it lost contact with the template strand.

Functional Effects of Mutations on the Protein Loss of function mutation Gain of function mutation

Sex Differences in Mutation Rates

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