Resistance to drugs in tuberculosis develops by: March 2011, March 2013

Ans. D: Mutation MDR-TB strains could arise as a consequence of sequential accumulation of mutations conferring resistance to single agents, or by a single step process such as acquisition of an MDR element. Resistance to common anti-TB drugs The most common mechanism of INH resistance is by mutation of the catalas-peroxidase gene so that the bacilli do not generate the active metabolite of 1NH. Rifampicin resistance is nearly always due to mutation in the repoB gene (for the beta subunit of RNA polymerase-the target of rifampin action) reducing its affinity for the drug Resistance to Pyrazinamide develops due to mutation in the pncA gene which encodes for the enzyme generating the active metabolite of pyrazinamide Classification of mutation A. By effect on structure: Small-scale mutations, such as those affecting a small gene in one or a few nucleotides, including: - Point mutations, often caused by chemicals or malfunction of DNA replication, exchange a single nucleotide for another. - These changes are classified as transitions or transversions. - Most common is the transition that exchanges a purine for a purine (A 4-, G) or a pyrimidine for a pyrimidine, (C T). - A transition can be caused by nitrous acid, base mis-pairing, or mutagenic base analogs such as 5-bromo-2 deoxyuridine (BrdU). - Less common is a transversion, which exchanges a purine for a pyrimidine or a pyrimidine for a purine (C/T A/G). - An example of a transversion is adenine (A) being conveed into a cytosine (C). - A point mutation can be reversed by another point mutation, in which the nucleotide is changed back to its original state (true reversion) or by second-site reversion (a complementary mutation elsewhere that results in regained gene functionality). - Point mutations that occur within the protein coding region of a gene may be classified into three kinds, depending upon what the erroneous codon codes for: Silent mutations: which code for the same amino acid. Missense mutations: which code for a different amino acid. Nonsense mutations: which code for a stop and can truncate the protein. - Inseions add one or more extra nucleotides into the DNA. They are usually caused by transposable elements, or errors during replication of repeating elements. - Inseions in the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product. - Inseions can be reveed by excision of the transposable element. - Deletions remove one or more nucleotides from the DNA. - Like inseions, these mutations can alter the reading frame of the gene. - They are generally irreversible Note that a deletion is not the exact opposite of an inseion: the former is quite random while the latter consists of a specific sequence inseing at locations that are not entirely random or even quite narrowly defined. Large-scale mutations in chromosomal structure, including: Amplifications (or gene duplications) leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them. - Deletions of large chromosomal regions, leading to loss of the genes within those regions. - Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially bringing together separate genes to form functionally distinct fusion genes (e.g. bcr-abl). These include: Chromosomal translocations: Interchange of genetic pas from nonhomologous chromosomes. Interstitial deletions: An intra-chromosomal deletion that removes a segment of DNA from a single chromosome, thereby apposing previously distant genes. For example, cells isolated from a human astrocytoma, a type of brain tumor, were found to have a chromosomal deletion removing sequences between the "fused in glioblastoma" (fig) gene and the receptor tyrosine kinase "ros", producing a fusion protein (FIG-ROS). The abnormal FIG-ROS fusion protein has constitutively active kinase activity that causes oncogenic transformation (a transformation from normal cells to cancer cells). Chromosomal inversions: Reversing the orientation of a chromosomal segment. - Loss of heterozygosity: Loss of one allele, either by a deletion or recombination event, in an organism that previously had two different alleles. B. By effect on function Loss-of-function mutations are the result of gene product having less or no function. When the allele has a complete loss of function (null allele) it is often called an amorphic mutation. Phenotypes associated with such mutations are most often recessive. Exceptions are when the organism is haploid, or when the reduced dosage of a normal gene product is not enough for a normal phenotype (this is called haploinsufficiency). Gain-of-function mutations change the gene product such that it gains a new and abnormal function. These mutations usually have dominant phenotypes. Often called a neomorphic mutation. Dominant negative mutations (also called antimorphic mutations) have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually result in an altered molecular function (often inactive) and are characterised by a dominant or semi-dominant phenotype. In humans, Marfan syndrome is an example of a dominant negative mutation occurring in an autosomal dominant disease. In this condition, the defective glycoprotein product of the fibrillin gene (FBN1) antagonizes the product of the normal allele. Lethal mutations are mutations that lead to the death of the organisms which carry the mutations. A back mutation or reversion is a point mutation that restores the original sequence and hence the original phenotype. C. By effect on fitness In applied genetics it is usual to speak of mutations as either harmful or beneficial A harmful mutation is a mutation that decreases the fitness of the organism. A beneficial mutation is a mutation that increases fitness of the organism, or which promotes traits that are desirable. In theoretical population genetics, it is more usual to speak of such mutations as deleterious or advantageous. A neutral mutation has no harmful or beneficial effect on the organism. Such mutations occur at a steady rate, forming the basis for the molecular clock. A deleterious mutation has a negative effect on the phenotype, and thus decreases the fitness of the organism. An advantageous mutation has a positive effect on the phenotype, and thus increases the fitness of the organism. A nearly neutral mutation is a mutation that may be slightly deleterious or advantageous, although most nearly neutral mutations are slightly deleterious. D. By pattern of inheritance: The human genome contains two copies of each gene - a paternal and a maternal allele. A heterozygous mutation is a mutation of only one allele. A homozygous mutation is an identical mutation of both the paternal and maternal alleles. Compound heterozygous mutations or a genetic compound comprises two different mutations in the paternal and maternal alleles. A wildtype or homozygous non-mutated organism is one in which neither allele is mutated. (Just not a mutation) E. By impact on protein sequence A frameshift mutation is a mutation caused by inseion or deletion of a number of nucleotides that is not evenly divisible by three from a DNA sequence. Due to the triplet nature of gene expression by codons, the inseion or deletion can disrupt the reading frame, or the grouping of the codons, resulting in a completely different translation from the original. The earlier in the sequence the deletion or inseion occurs, the more altered the protein produced is. In contrast, any inseion or deletion that is evenly divisible by three is termed an in-frame mutation A nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and possibly a truncated, and often nonfunctional protein product Missense mutations or nonsynonymous mutations are types of point mutations where a single nucleotide is changed to cause substitution of a different amino acid. This in turn can render the resulting protein nonfunctional. Such mutations are responsible for diseases such as Epidermolysis bullosa, sickle-cell disease. A neutral mutation is a mutation that occurs in an amino acid codon which results in the use of a different, but chemically similar, amino acid. The similarity between the two is enough that little or no change is often rendered in the protein. For example, a change from AAA to AGA will encode arginine, a chemically similar molecule to the intended lysine. Silent mutations are mutations that do not result in a change to the amino acid sequence of a protein. They may occur in a region that does not code for a protein, or they may occur within a codon in a manner that does not alter the final amino acid sequence. The phrase silent mutation is often used interchangeably with the phrase synonymous mutation; however, synonymous mutations are a subcategory of the former, occurring only within exons. The name silent could be a misnomer. For example, a silent mutation in the exon/intron border may lead to alternative splicing by changing the splice site, thereby leading to a changed protein. Silent mutations occur because of the degenerate nature of the genetic code. F. Special classes Conditional mutation is a mutation that has wild-type (or less severe) phenotype under ceain "permissive" environmental conditions and a mutant phenotype under ceain "restrictive" conditions. For example, a temperature-sensitive mutation can cause cell death at high temperature (restrictive condition), but might have no deleterious consequences at a lower temperature (permissive condition).