MUTAGENESIS & MUTATIONS

MUTAGENESIS & MUTATIONS http://biolmolgen.slam.katowice.pl http://www.elearning.sum.edu.pl/www/professorpage.php Aleksander L. SierońKatedra BiologiiMolekularnej i Genetyki, ŚUM Katowice

MUTAGEN • A MUTAGEN is a substance or agent that causes an increase in the rate of change in genes. • These mutations can be passed along as the cell reproduces, sometimes leading to defective cells or cancer. • Examples of MUTAGENS include but are not limited to: • biological agents • chemicalagents • exposure to ultraviolet light • exposure to ionizing radiation.

There are many types of mutations, • some of which are harmful • and others that have little or no effect on a body's function.

Mutagens can be identified using the Ames Test and other biochemical testing methods. The Ames Test is a way of determining whether a compound causes genetic mutations. Animal liver cell extracts are combined with a special form of salmonella bacteria. The mixture is then exposed to the test substance and examined for signs that the bacteria have mutated (a process called MUTAGENESIS). The Ames test does not directly indicate the carcinogenic (cancer-causing) potential of the substance, however, there is a good correlation between mutagen strength and carcinogen strength in animal studies.

DO NOT CONFUSE A MUTAGEN WITH A CARCINOGEN (A SUBSTANCE THAT CAUSES CANCER) MUTAGENS MAY CAUSE CANCER BUT NOT ALWAYS A carcinogen is a substance that causes cancer (or is believed to cause cancer). A material that is carcinogenic is one that is believed to cause cancer. The process of forming cancer cells from normal cells or carcinomas is called CARCINOGENESIS.

DO NOT CONFUSE A MUTAGEN WITH A TERATOGEN (A SUBSTANCE THAT CAUSES CHANGE OR HARM TO A FETUS OR EMBRYO) A teratogen is an agent that can cause malformations of an embryo or fetus. This can be a chemical substance, a virus or ionizing radiation. This is closely related to a fetotoxin, an agent that causes poisoning effects on a developing fetus. Both fetotoxins and teratogens are reproductive toxins, substances which cause damage to one's reproductive and/or endocrine system and/or a developing fetus.

MUTATIONS MUTATION (adj. mutant) A heritable change in DNA sequence resulting from mutagens. Various types of mutations include frame-shift mutations, missense mutations, and nonsense mutations.

TYPES OF MUTATIONS • CHROMOSOMAL • Can refer to any of a number of DNA mutations that result in a change in the protein encoded by the mutated gene, such as point mutations, insertion or deletion mutations(frameshift mutations), or nonsense mutations. • More often this refers to mutations involving chromosomes, such as the inversion of part of one chromosome such that the inverted part no longer matches with its homologous pair, a translocation of one part of a chromosome to a different chromosome, deletions of parts of chromosomes, or accidents, which happen during the division of the nucleus like the unequal portioning of chromosomes between the daughter cells.

TYPES OF MUTATIONS by single basechange • AMBER • A mutation from a codon which codes for an amino acid into the AMBER CODON UAG, which normally signals that the translation of mRNAinto an amino acid chain should stop. The mutation causes the amino acid chain to stop forming before it is actually completed. • (NONSENSE - A mutation that causes a polypeptide chain to be ended prematurely.) • 2. BACK • A mutation that causes a mutant gene to revert to its original wild-typebase sequence. • 3. FORWARD • This is the opposite of a back mutation.

TYPES OF MUTATIONS • CONDITIONAL • A mutation, which makes itself apparent only under certain conditions, such as low or high temperatures.

TYPES OF MUTATIONS • DOWN PROMOTER • A mutation (a change in base pair sequence) in a promoter region; this results in lower gene expression (less transcription of the gene occurs).

TYPES OF MUTATIONS • POINT • A change in a single base pair of a DNA sequence in a • gene. • POLAR (polarity mutation) • A mutation in a single gene, which affects the rate of • expression of other genes that are near it on a • chromosome.

TYPES OF MUTATIONS • SILENT • A mutation that causes NO noticeable change in the • biological activity of the proteins the gene codes for. • SPONTANEOUS • A mutation which occurs by itself without first being • affected by a mutagen, for example during the • process of DNA replication. • Spontaneous mutations arise at a remarkably constant • rate. The rate that spontaneous mutations arise has • been used as an evolutionary clock to estimate how • closely related two (or more) separate species are to • each other.

TYPES OF MUTATIONS • SUBSTITUTION • A mutation caused by a nucleotide base being replaced • by a different one. • SUPPRESSOR • A mutation that restores, at least to some degree, a • function lost as the result of a primary mutation; the • suppressor mutation is at a different site from the • primary mutation. • UNSTABLE • A mutation that has a high likelihood of reverting to • its original form.

TYPES OF MUTATIONS • UP • Refers to any mutation in the promoter region of • agene, which can trigger transcription initiation.

CODON MUTATIONS • 1. CODON IS • the basic unit of the genetic code, comprising three • nucleotide sequences of messenger ribonucleic acid • (mRNA), each of which is translated into one amino • acid in protein synthesis. • 2. INITIATION • A nucleotide sequence that codes for the first amino • acid in a polypeptide sequence; the codon is typically • AUG in eukaryotes and sometimes GUG in prokaryotes. • 3. NONSENSE • A codon that signals the end of a polypeptide chain; it • doesn't code for any amino acids.

CODON MUTATIONS • 4. TERMINATION (stop codon) • The codons UAA, UAG and UGA, which signal the end • of a polypeptide chain. • 5. AMBER CODON • The nonsense codon UAG, one of three codons which, • instead of coding for an amino acid, signals that the • translation of mRNA into a chain of amino acids • should stop. • 6. AMBIGUOUS CODON • A codon that codes for more than one amino acid.

CODON MUTATIONS • 7. ANTICODON • A specific sequence of three nucleotides in transfer • RNA which are complimentary to a codon for a • specific amino acid in a messenger RNA.

DNA Repair Disorders. Patients with DNA repair disorders. Clinical diagnosis of patients with DNA repair disorders is developed in many laboratories. E.g. the lab of David Busch (Armed Forces Institute of Pathology - AFIP) has emphasized clinical diagnosis of Xeroderma Pigmentosum (XP) and Cockayne's syndrome (CS), two genetic diseases in which there is abnormal cellular UV sensitivity associated with abnormal excision repair of UV damaged DNA.

XERODERMA PIGMENTOSUM (XP) Is a rare genetic defect in ultraviolet radiation induced DNA repair mechanisms; characterized by severe sensitivity to all sources of UV radiation (especially sunlight). XP is categorized in complementation groups according to the capacity of the body to repair DNA. Groups A, C, D, and Variant make up over 90% of XP cases. Group A, for example, has the lowest level of DNA repair and the most neurological manifestations.

XERODERMA PIGMENTOSUM • Is characterized by wide range of symptoms: • blindness and deafness • blistering or freckling on minimum sun exposure • developmental disabilities • dwarfism and hypergonadism • increased skin and eye cancers • mental retardation

INHERITANCE of XP Recessive autosomal; Occurrence is favored by consanguinity; Frequency is 0.3/105 with large geographical variations; Higher frequency is observed in Tunisia (10/105, role of consanguinity) and in Japan (1/105); It is rare in black people.

XP CYTOGENETICS INBORN CONDITIONS Hypermutability after UV irradiation in cell cultures; No increase of spontaneous chromosome abnormalities in lymphocytes or fibroblasts; After UV-exposure an increased number of sister chromatids exchanges (SCE) and chromosome aberrations are observed (mainly chromatids-type abnormalities); Fibroblasts express an increased sensitivity to chemical mutagens; No cytogenetic feature useful for XP diagnosis

Genes Involved and Proteins IN XP XP heterogeneity is the consequence of the genetic heterogeneity: 7 complementation groups (XPA to G) plus an additional variant form, (evidenced by somatic cell fusion experiments)

Genes Involved and Proteins • - XPA (Xeroderma Pigmentosum, complementation group A) , located in 9q22, • - XPB (also called ERCC3 (ERCC for Excision-Repair Cross Complementing • rodent repair deficiency), located in 2q21, • - XPC (Xeroderma Pigmentosum, complementation group C), located in 3p25, • - XPD, also called ERCC2 (Excision repair cross-complementing rodent repair deficiency, complementation group 2), located in 19q13, • XPE (Xeroderma Pigmentosum, complementation group E), located on chromosome 11, • XPF also called ERCC4 (Xeroderma Pigmentosum, complementation group F), located in 19q13, • XPG, also called ERCC5 (Xeroderma Pigmentosum, complementation group • G), located in 13q32, • XPV, also called Pol eta (polymerase (DNA direct), eta), and located in 6p12-21,

Genes Involved and Proteins All XP genes are implicated in various steps of the NER (nucleotide excision repair) system, except the XP variant that is mutated in a mutagenic DNA polymerase (POL H) able to bypass the UV-induced DNA lesions. Various alterations of the same gene may involve various phenotypes e.g. Cockayne syndrome , trichothiodystrophy

There is no cure for XP. The DNA damage is cumulative and irreversible. Management is limited to avoidance of exposure to damaging UV radiation by staying indoors with sunlight blocked out, and use of protective clothing, sunscreens and sunglasses. Also, other known carcinogens shall be avoided. Regular surveillance for and treatment of all neoplasms is very important.

Other disorders associated with defective DNA repair • - Ataxia-Telangiectasia • - Bloom Syndrome • - Cockayne Syndrome • - Fanconi Anemia • Another related disorder • - Trichothiodystrophy (TTD) • TTD is very rare - fewer than 1,000 cases known worldwide • Clinical diagnosis is available for TTD • TTD is life threatening condition - The DNA damage is cumulative and irreversible

Various alterations of the same gene may involve various phenotypes e.g. (CS) Cockayne Syndrome, (TTD) TrichoThioDystrophy

COCKAYNE SYNDROME INHERITANCE IS AUTOSOMAL RECESIVE

COCKAYNE SYNDROME Included disease(s) :Cockayne syndrome type 1 (A)Cockayne syndrome type 2 (B)Cockayne syndrome type 3 (C)

COCKAYNE SYNDROME Phenotype and clinics • normal newborn; • growth failure from the age of six months; • diagnosis from the age of two years on; • senile appearance of the skin (pigmentation, atrophy) with "mickey mouse" • aspect (microcephaly, large ears, large nose, deep set eyes); • "senile dwarf" aspect in contrast with long limbs, large hands and feet, cold • fingers with cyanosis, flexion contractures of joints; • sensitivity to sunlight; • severe encephalopathia with profound mental retardation and sensory disorders • (deafness, optic atrophy); • pigmentary retinitis leading to cecity; • other disorders: hypertension, early atherosclerosis, intracranial calcification, • glomerulosclerosis.

Cockayne Syndrome Cytogenetics Inborn conditions As in XP, the UV light-induced level of sister chromatid exchange (SCE) is increased as well as the rate of chromosome aberrations, mainly chromatids breaks.

COCKAYNE SYNDROME Neoplastic risk No increased susceptibility to skin tumors and other cancers, except for Cockayne Syndrome expressing Xeroderma Pigmentosum (XP) symptoms (association with XPG, XPD or XPB group) Evolution Clinical heterogeneity, but early death from cachexia and dementia, early cutaneous tumors and atherosclerosis.

Cockayne Syndrome Calcification of the brain. Here is a CT scan of a CS patient, showing calcium deposits in the cerebellum (white area next to + sign).

Cockayne Syndrome The amount of brain tissue also is likely to be unusually small. Here is a CT image of a CS patient showing hydrocephalus ex vacuo i.e., abnormally large amount of fluid in skull filling space normally occupied by brain tissue; the ventricles (large, dark, rounded, symmetrical areas near center) are abnormally dilated with cerebrospinal fluid instead of being compressed and slit.like. Also, there are symmetrical, wavy, white areas of dense calcium deposition in the basal ganglia of the brain (marked on left with +).

The white matter of the brain may be deficient in myelin in CS patients. A photomicrograph of cerebral tissue appearing foamy due to loss of myelin in the white matter. Cockayne Syndrome

Cockayne Syndrome Neurons are likely to be scarcer than expected. The photomicrograph of cerebellar tissue, shows abnormally few, irregularly spaced large cerebellar neurons (Purkinje cells).

Cockayne Syndrome Genes involved and Proteins • There is genetic heterogeneity in CS, giving rise to • complementation groups. • The genes involved are: • CSA (Cockayne Syndrome A), also called ERCC8 (ERCC for • Excision-Repair Cross Complementing rodent repair deficiency) • located on chromosome 5, • CSB, also called ERCC6 , located in 10q11-21; outside CSA and CSB, • there is: • 3 patients who are XPB/CS, involving XPB, also called • ERCC3, located in 2q21; • 2 patients XPD/CS, involving XPD, also called ERCC2, • located in 19q13; • 6 patients XPG/CS, involving XPG, also called ERCC5, • located in 13q32 (note: the class of patients with both XP and CS were classified earlier as CS III, but not anymore).

Cockayne Syndrome CS children are spared the cancers and scarring seen in XP, They do suffer from: - developmental delay, - dwarfism, premature aging with greatly - shortened lifespan, - nerve deafness, - cataracts, - retinal degeneration (retinitis pigmentosum), - severe dental problems, - facial abnormalities.

Trichothiodystrophy (TTD) Other names PIBIDS syndrome or IBIDS syndrome Inheritance recessive autosomal

Trichothiodystrophy (TTD) Clinics Phenotype and clinics • Photosensitivity, • Ichtiosys, • Brittle hair, • Intellectual impairment, • Decreased fertility, • Short stature (PIBIDS syndrome). • Photosensitivity is absent in 50% of cases (therefore called • IBIDS syndrome).

Trichothiodystrophy (TTD) Clinics Neoplastic risk This familial disease IS NOT a cancer prone disease but it involves the same complementation groups as in Xeroderma Pigmentosum and Cockayne syndrome (XPD, XPB), and share defects in similar genes Prognosis depends on the DNA repair defect (photosensitivity: XPD-ERCC2, XPB-ERCC3, TTD-A) and on the transcription errors (other signs)

Trichothiodystrophy (TTD) Cytogenetics Inborn conditions no known chromosome abnormalities

Trichothiodystrophy (TTD) Genes involved and Proteins • The DNA repair defect is found in 3 classes: • patient with TTD-A group (low level of the TFIIH transcription factor), • patients mutated in the XPB gene (TTD/XPB), involving XPB, alsocalled ERCC3, located in 2q21; • all the other patients mutated in the XPD gene (TTD/XPD), involving XPD, also called ERCC2, located in 19q13

CYCLIN Cdk ADP pRB : E2F E2F ppRB : E2F CYCLIN : Cdk ATP pRB : E2F PHASE G1 PHASE S PHASE S PHASE G1 REPARE OR APOPTOSIS

CYCLIN Cdk ADP pRB : E2F E2F ppRB p53 MDM4 DNA damage MDM2 p53 stabilization p21WAF1/CIP CYCLIN : Cdk ATP pRB : E2F PHASE G1 PHASE S PHASE S PHASE G1 REPAIR OR APOPTOSIS

CYCLIN : Cdk ATP pRB : E2F CYCLIN Cdk ADP pRB : E2F PHASE G1 PHASE S E2F ppRB : E2F PHASE S PHASE G1 REPARE OR APOPTOSIS IF LACK OF p53 ACTIVITY

CLASSIFICATION OF MEDULLOBLASTOMAS Classic Medulloblastoma Neuroblastic Medulloblastoma Desmoplastic Medulloblastoma Krynska B., et al. (1999) PNAS, 96:11519-24

Arrows indicate cells with JCV T-antigen Arrowheads point to proliferating cells Magnification x400 (A & B); x200 (C); x1000 (inserts) IMMUNOHISTOCHEMICAL DETECTION OF JCV T-ANTIGEN IN MEDULLOBLASTOMAS Krynska B., et al. (1999) PNAS, 96:11519-24

MUTAGENESIS & MUTATIONS