L14 Mutagenesis And Its Affects. Detec Dis Causing Mut Flashcards
LO2: desc diff types mut changes.
-pt mut 1bp.
-insert/delet of seqs.
-rearr of seqs.
Many spont, can be induced.
LO3: predic and expl eff diff muts may have eg silent, missense, nonsense, frame shift.
-mut not good/bad per se- source genet var, cause mut phenotype, mut in gene=mut allele.
Pt mut-sing base sub- transit= purine to urine or prim to prim. Transversion diff. Pt mut in coding regs of prot can be:
-silent- 1 base subst not alt AA eg GGA to GGG still gly. Some can disrup RNA splicing by eg intro slice site into exon=still cause heritab dis.
-missense- 1 AA repl by other, norm 1 base change. Eg GGC gly to TGC cys.
-nonsense- an AA codon mut to STOP codon=transl termin. Eg GGA to TGA STOP.
-pt mut in non cod regs or outside genes can be detrimental as can a change prot BS, prom seq, splice sites etc.
-in insert/delet seq add/rem. Sing base to triplet rep to millions nt eg rand duplics.
For insert/delet in COD reg of prot:
addit/substrac nt other than multip of 3= frame shift mut- RF of mRNA alt eg insert/delet/splice site mut. STOP codons comm fnd in alt RF=ribos termin transl premat.
Addit/subtract multip of 3 nt: no change RF but still change AA.
-conservation missense- some AA substitut better tol than others. Eg val to ala tol in non critic regs prot eg struc outs reg, non func. NOT in eg AS.
Val and ala adjac in genet code table , by evol. Substitut 2nd posit val=ala simil props.
-mRNA with PTC nonsense STOP (eg alt RF) are deg by nonsense med decay= little or no prot prod= protec. If not have STOP in expec place better to prod les norm prot as abn is disruptive.
-other mut types- in prom prev transc, in start/stop codon prev transl. All fairly comm.
mut at intron splice site v comm, us= skip exon immed adj to mut. If skipped exon is multip 3=mRNA short but in frame, prot prod truncated. Prot with interstit delet in gene= dam. If not multip 3 mRNA shortened and frameshift and PTC=NMD. Often better than maint RF.
LO1: expl rel btw changes nt and AA seq.
-22K prot COD genes-1-2% DNA, mut in in lot of genome no conseq. Mut in/close to gene most likely=dis. Type mut and locat in gene determ conseqs.
Sing base sub most comm seq var- 1 in ev 300 BP=SNPs. 2/3 SNPs are C to T transit- more comm than transversion.
1,2,3,4, or 6 condon triplets per AA. 3 stop codons. Sing base alt at 3rd codon posit often not change AA=silent substitut.
LO4: desc how spont and induced mut can occ.
-Inherent in DNA-
seq changes dur DNA replic- rare tautomeric forms with alt base pairing. DNA str slip dur replic.
chem induced mut- direc alt DNA bases. Disrup base stack indirect.
expos diff types rad- UV, radioac subst.
-tautomeric shift- 4 DNA bases, H+ briefly changes posit=rare forms have alt BP props. Behave as alt templ base dur replic= anom bp if tautomeric at time replic: CA, TG. Eg if C in taut form DNAP recog as T and insert an A opp.
-slipp dur replic- sev same base adj= new str loops about cause addit 1nt on new str. Or temp, str loops out=new str 1 nt short. Us 1 BP insert/delet dur replic. Alt no rep bases, comm.
-chem mutagen base changes-
Nitrous act repl amino grp with keto grps=alt base props and pair- C changed to U so pair A. A to hypoxanthine so pairs C. G to xanthine so pairs C.
Ethyl methyl sulphonate (EMS)env mutagen = rem purine rings, not predictable outc. Purine rem w/o disrup backb= apurinic sites can be paired with any base dur replic.
-base stacking mutagens-
IQ= heterocyclic aromatic amine food mutagen in cooked meat and cig smoke. Disrup packing of DNA bases= mostly 1 b delet at GC pairs. Planar, enters DNA helix, interf with Sep of bases and how DNAP inserts which base.
struc and action- intercal of molec forces bases furth apart on 1 DNA str= misread by DNAP= delet 1b. Ethid bromide also intercals DNA. Stain in elecphor. Also mutagenic.
-elecmag spectrum- non ionising state. Tow blue visib light starts aff DNA integ. Shorter w/l higher energ, more dam.
-ionising rad- prods ions dur interac with cellul molecs. Solar UV, X Ray’s, nuc plant as, env sources eg radon gas and granite.
Expos somet unavoidable eg in env, foods, air. Sun cream, stop air travel, avoid X Ray’s only prevention.
-UV light effs- UVB (280-315nm) induces prod active vitD in skin. Overexpos =sunburn and CA. UVA (315-400nm), UVB, UVC (100-280nm) cam all dam Coll fibres=aging skin. UVA+B destr vitA in skin.
-thymine dimmer format- UV photons= adj thymine on same str BP. Often resolve spont by photo reactiv.
LO5: desc proc and role DNA rep.
-cell sev v cons mechs check and rep genet code. Muts freq but us recog and rep. Eg Mism rep post replic. 2 types excis rep- base excis rep and nt excis- rep ssDNA dam by ext agent eg chem mutagen , UV or endog facs eg ROS. 2 types in diff enzs.
Also double str break rep- break can= chrom rearr.
Fail rep serious dis conseqs.
-DNAP incorp err rate 1/100K nt. 120K ev cell replic would be intol load. Most corr by proofread-DNAP detec miss paired 3’ base in new str and corr err 99% of time. Need addit procs.
-nt Mism rep- post replic. Repl most Mism but are other faults. Enzs detec Mism in new str, excis and repl. Often patch DNA repl. Unkn how know which base is corr.
-excis rep- can accum dam bases- oxidised, alkylated, de aminated, uracil. For bases alt by eg rad/chem. sing base or longer patch repl.
LO6: expl rel btw DNA dam and CA.
-DNA rep and CA- fail rep. Hum MLH1 and MSH 2 and 6 genes encode Mism rep enzs. Comm mut in hered non polyposis colorectal CA- MSH2 mut in 60% fams, MLH1 30%, MSH 6 10%.
-nat selec and evol- form tum from mut=growth adv conferred on cells which acq 6 new chars-
Div indep of ext growth sigs, norm cells sequesce w/o. Ignore ext antigrowth sigs. Avoid apop which is imp in dev. Div indefin w/o sequescance. Stim sust angiogenesis- synth compons to make capills. Invade tiss and est secondary tums.
-successive adv and selec- assume rate mut 10^-6 per gen, chance six successive mut 10^-36. Only 10^14 hum cells. Early mut affs func which incr probab of successive mut occ- not indep. All CA cells show chrom in stabil and micros at instabil. CA cells concurr delet/duplic etc of chroms.
-inherited breast CA- 1/8 UK dev. 5-10% pop familial and in to map to BRCA1 gene and BRCA2. Early onset. Both prots inv in detec DNA dam and sig to cell cyc check pts- check replic Orr, ev of dam to cause uncontr fut expans would= apop. Sporadic tum rarely BRCA1/2 mut. Aff males comm in familial. 100s mut id’d on both genes. Some more comm in specif pops. Eg 3/1000 fnd in Ashkenazi Jewish wom= screen more cost effec.
-oncogenes- some CA in assoc specif Retrovir eg cervic, anal, penile with HPV esp 16 and 18. Retrovirus cont genes able to transform cells to CA phenot. Sev hum genes subseq ID exhibit seq sim to vir oncogenes- Proto oncogenes. Have norm wt funcs, not oncogenes.
-hum proto oncogenes- range cell cyc contr funcs. SRC and ABL- tyrosine kinase. HRAS small GTPase. MYB nuc prot. SIS PDGF B chain. Key AA subs can activ into domin acting CA causing oncogenes.
-heritab or sporadic- Inher CA genes tend recess muts. But CA dev displ domin patt Inher. Init of tum format reqs both copies mut or that func at copy deleted=recess. BUT pedigree domin. Knudsons 2 hit theory.
-mechs of homoz- (=patt domin but gene recess)
Loss at chrom- v rare. Only req in 1 cell retina for tum est.
delet- interstit, rare. We copy deleted prev protec.
Pt mut- another, poss diff, inactivs prot encoded.
Mitot recomb- v rare. No loss genet mater and no new pt mut, no loss chrom. For reg of RBC gene, indiv becomes homoz. CO.
-heritability mechs- inherited CA- all cells carry 1 mut. 2nd=tum founder cell.
Sporadic CA- somat mut in 1 cell. Need acq 2nd mut in same cell=Lower probab.
CF
-couple have kid- chest infec, poo bulky and smell, sweat test Na 87mM (60). =CF.
autos recess. Mut CFTR gene chrom 7- gene has 27 exon so, 189kb, 7q31.2. Both copies must have mut=CF. unlike SC mut can be anywhere in gene, over 1000 desc. Poss compound heteroz-2 diff mut-1 mat 1 pat. 1/4 risk other kid CF. pre natal diag req Id mut in 1st kid. Maj in muts rare.
-pF508 CF mut- most comm. Phe delet 3bp. Some pops= 60-70% mut alleles.
-other comm- 13 specif muts acc for 92% all mut alleles. 2nd comm pGly 551 Asp in 3.5% alleles. Lot rarer muts 6.5% remain. Unkn mut 1.5%.
LO7: recog fundam imp PCR in diag genet dis.
LO8: prov overview of genet tests avail for detec gene mut.
-UK test av for 29 most comm CF muts using multiplex PCR and expanded panel 97 tests. Baby heteroz for pF508 allele, no other mut detec foll test all 126. Need screen both her CFTR genes to find other allele mut. Seq expens in diag sett, becoming less, seq immed now- exon adn adj introns.
-SSCP mut scanning- decr use now as seq cheap.
1st Id mut red of gene, then targeted seq.
If heteroz mut- PCR=mix norm and mut seqs. Amplif each exon of CFTR. If amplif DNA heated then Coll= str adop seq specif ds forms- not re-pair.
DNA elec polyacrylamide and silver stain= sing str conf polymerise (SSCP). Wet and mut= diff 3D strucs=diff Mobil in gel.
Scan all exon so eg do with exon 3 of CFTR gene. Pt extra band to pts with diff muts so seq exon 3. Extra slow mig band= mut exon 3. Fast moving extra band exon 14b, seq too. Only 1 of the 2 addit variants is dis causing, other nat variant.
Seq-2 peaks same base= heteroz. If Rev str seq then act it is opp base.
PCR incl vases at end and start intron either side exon as can aff alt splicing. If v freq in norm pop then not dis causing. Pts dad had F508 delet and intron 14a mut so must be on same chrom as Joanne Inher both. So exon 3 other CF cause. Exon 3 G to A= tryp TGG to TAG STOP nonsense= mRNA deg.
LO8: obt fetal DNA
- par and sibling samps blood/saliva. Also samp fetus-DNA isol from amn fluid cells, chronic villus biop, or fetal DNA in mums blood (new).
- Amniocentesis- 15-20wk once can acc retrieve fluid. US guid. Amn sac cells recov and cult in mitot stim for 2wk. 0.5-1% risk misc.
- chronic villus biop- 10-13wk. US guid. Transcervic or trans abdo. Fetal villi Sep from mat placental tiss- direc tiss samp. 2% misc.
- fetal DNA in mums blood- isol along with mum DNA. If both par been typed for partic dis mut, fetal status der from seq isol DNA. Still mainly research only.
LO8: exon delet and duplic. MLPA.
-PCR exons and seq- but if delet ent exon would get prod from good copy, norm seq. Need screen both copies gene wrt ev exon.
Some Inher disord arise comm by whole exon dupli/delet- large segm or ent gene. Eg osteogen imperf brittle bone. Autos domin COLA1+2 delet exons and ent gene T1 Coll. DMD X linked recess, exon delet comm-diffic fig carrier status of fems. Multiplex ligat dep probe amplif (MLPA) count copy no of many exons in paral. Gene specif anal kits avail.
-MLPA- for each exon of gene. Pair primers that cover reg of exon. Anneal to gene v small gap btw. If anneal to exon of int, DNA ligase present in reac= 2 halves test DNA joined.
If 1 copy exon delet, only half expected amnt DNA lighted. Can only BP with 1 copy. Less targ DNA avail. Half prod can’t detec prod direc, tiny amnt prod init. Can PCR amplif- for end sites of PCR primers. Elec Sep.
Not paral anal all exons if PCR prods all same length. So stuffer at 1 end diff length for each exon. So anal eg 20 paral. Elec Sep look for amnt amplif prod decr by half for some exons= 1 allele has exon delet.
-MLPA- eg pt peaks half height of contr= has been delet. PCR amplif not eq effic for ev frag so run Ag contr.
-genet testing UK- NHS 30 labs. UKGTN.
