Patologia Molecular

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<p>Harvy Mauricio Velasco P Gentica Humana UNAL</p> <p>1* 1015 clulas adultas 2*1017 divisiones celulares 6 *109 nucletidos para cada divisin l l celular 1*10 -6 -8 Eficiencia de la l DNA polimerasa li</p> <p>Proliferacin, Divisin y apoptosis</p> <p>Noxas externas</p> <p>Oncogenes y Genes supresores</p> <p>Reparacin del DNA</p> <p>UN GEN SE ENCUENTRA AFECTADO</p> <p>GENERADAS POR LA EVOLUCIONNOVEDAD ADAPTACION ENFERMEDAD MUERTE</p> <p>ERRORES EN LA REPLICACIONDNA POLIMERASA</p> <p>TAMAO DE DNA3.000 3 000 MILLONES DE pb</p> <p>AGRESION DIRECTA A DNA DEGRADACION INTERNA DE NUCLEOTIDOS</p> <p>MUTACION Cualquier cambio en el material gentico(DNA nuclear y mitocondrial), que se herede a las generaciones siguientes.</p> <p>POLIMORFISMO Variacin V i i gentica ti que tiene ms de una variante i t allica en una i secuencia DNA Frecuencia &gt;1%</p> <p>MUTACIONES GERMINALESEl cambio en el DNA se da en l lnea parental en t l celulas germinales y esta mutacin se HEREDA</p> <p>MUTACIONES SOMATICASMutacin M t i que no compromete lneas germinales En un tejido especifico</p> <p>GERMINALES</p> <p>SOMATICAS</p> <p>ESPONTANEAS</p> <p>INDUCIDAS POR EL AMBIENTE O EN EL LABORATORIO</p> <p>A GRAN ESCALA O MUTACIONES CITOGENETICAMENTE VISIBLES</p> <p>A PEQUEA ESCALA O CITOGENETICAMENTE INVISIBLES</p> <p>EXISTE ALTERACION DE ALGUNOS DE LOS COMPONENTES DEL MATERIAL GENETICO O EN EL FLUJO DE INFORMACION DEL DNA HACIA PROTEINAS ERROR EN LA EXPRESION DE LA INFORMACION GENETICA</p> <p>TIPO SUSTITUCION DELECION INSERCION</p> <p>DEFINICION Reemplazo de una(s) base(s) por otra(s) Perdida P did o eliminacin d uno o li i i de ms nucletidos Uno o ms nucletidos estn adicionados en una secuenciaTransposicin con duplicacin de material gentico Transposicin sin duplicacin (translocacin)</p> <p>MUTACIONES</p> <p>Sustitucin de bases</p> <p>Inserciones</p> <p>Deleciones</p> <p>Transiciones Transversiones</p> <p>1 o pocos nucletidos</p> <p>1 o pocos nucletidos</p> <p>Sustitucin sinnima (neutra) i i ( t ) y no sinnima Conversin gnica, mltiple sustitucin de bases</p> <p>Expansin de i l tripletas</p> <p>Grandes deleciones</p> <p>Otras grandes inserciones: elementos transponibles ibl</p> <p>En el marco de lectura</p> <p>Transiciones: Pirimidina por p pirimidina (C-T)Ms comunes (ISLAS ( CpG)</p> <p>Transversiones: p p Purina por pirimidina o viceversa</p> <p>Purina por purina (AG)</p> <p>A</p> <p>C</p> <p>G</p> <p>T</p> <p>SINNIMAS (SILENCIOSAS): Neutral No cambia secuencia a.a. En DNA no codificante Ms comn en DNA codificante En DNA codificante cambia el A.A. en posicin 3 de codn</p> <p>NO SINNIMAS Altera secuencia TIPOSDeletreas Sin efecto Efecto benfico</p> <p>Mayor frecuencia Transversiones que transiciones Mayor frecuencia en secuencias no codificantes Cualquier secuencia puede p tener sustituciones</p> <p>No sinnimas Sinonimias Sinnimas</p> <p>Algunas sustituciones en secuencias no codificantes afectan la expresin</p> <p>p e q n r u s a a e z c r m a M a c M d U a c U o T n a N T S A m O d A q U d C n e C u S e O n C T O d N O m N o S a e N T E S E c U S c V a r E f S a C o A R u m M d T V n N e O o A c O n N S n V T e N E S m A S e S E d p V n E N e A S s S d a o E E p z m f e a r e d x a r o p a e d r n a e 3 a s e a y n p 2 o d g s a n c p o c s a n c c o d n o n c o d o n</p> <p>SUSTITUCIONES</p> <p>Gene Histone H3 Histone H4 Actin Aldolase A HPRT Insulin -Globin -Globin Albumin Ig VH Growth hormone Ig Interferon-1 Interferon-</p> <p>No. of codons 135 101 376 363 217 51 141 144 590 100 189 106 159 136</p> <p>Nonsynonymous 0.00 0.00 0 00 0.01 0.07 0.13 0.13 0.55 0.80 0.91 1.07 1.23 1.87 2.21 2.79</p> <p>Synonymous 6.38 6.12 6 12 3.68 3.59 2.13 4.02 5.14 3.05 6.63 5.66 4.95 5.66 5.88 8.59</p> <p>MUTACIONES QUE GENERAN GANANCIA O PERDIDA DE MATERIAL GENETICO DNA CODIFICANTE Y NO CODIFICANTE INVOLUCRA GENERALMENTE REPETICIONES CORTAS EN TANDEM FENOMENOS DE RECOMBINACION</p> <p>Short Tandem Repeats (STRs)AATG</p> <p>7 repeats 8 repeats</p> <p>Homologous equal crossover can result in fusion genes. The example shows how intragenic equal crossover occurring between alleles on nonsister chromatids can generate novel fusion genes composed of adjacent segments f t from the t th two alleles. Note that similar exchanges between genes on sister chromatids do not l i i l result in genetic novelty because the gene sequences on the interacting sister chromatids would be expected to be identical.</p> <p>Unequal crossover and unequal sister chromatid exchange cause insertions and deletions. The examples illustrate unequal pairing of chromatids within a tandemly repeated array. U d Unequal crossover l involves unequal pairing of nonsister chromatids followed by chromatid breakage and rejoining. Unequal sister chromatid exchange involves unequal pairing of sister chromatids followed by chromatid breakage and rejoining. For the sake of simplicity, the breakages of the chromatids are shown to occur between repeats, but of course breaks can occur within repeats. Note that both types of exchange are reciprocal - one of the participating chromatids l ti i ti h tid loses some DNA, while the other gains some.</p> <p>Unequal crossover in a tandem repeat array can result in sequence homogenization. Note that the initial spread of the novel sequence variant to the same position in the chromosomes of other members of a sexual population p p can result by random genetic drift. Once the mutation has achieved a reasonable population frequency (left panel) it can spread to other positions within the array (right panel). This can occur by successive gain of mutant repeats as a result of unequal crossover (or unequal sister chromatid exchanges) and occasional loss of normal repeats. Eventually the mutant repeat can replace the original repeat sequence at all positions within the array, leading to sequence homogenization for the mutant repeat. Such sequence homogenization is thought to result in species-specific concerted evolution for repetitive DNA sequences. UEC unequal crossover. UEC, l</p> <p>Tandem gene duplication can result from unequal crossover or unequal sister chromatid exchange, facilitated by short interspersed repeats. The double arrow indicates the extent of the tandem gene duplication f d li ti of a segment t containing gene A and flanking sequences. Original mispairing of chromatids ld b f ili db hi h could be facilitated by a high degree of sequence homology between nonallelic short repeats ( 1, R2). Note that p (R ) the same mechanism can result in large-scale deletions.</p> <p>Gene conversion involves a nonreciprocal sequence exchange between allelic or nonallelic genes. (A) Interallelic gene conversion. Note the nonreciprocal nature of the sequence exchange - the donor sequence is not altered but the acceptor q p sequence is altered by incorporating sequence copied from the donor sequence. (B) Interlocus gene conversion. This is facilitated by a high degree of sequence homology between nonallelic sequences, as in the case of tandem repeats (C) Mismatch repeats. repair of a heteroduplex. This is one of several possible models to explain gene conversion. The model envisages invasion by one strand of the donor sequence (-) to form a heteroduplex with the complementary (+) ( ) strand of the acceptor sequence, thereby displacing the other strand of the acceptor. Mismatch repair enzymes recognize the mispaired bases in the heteroduplex and correct' the mismatches so that the (+) acceptor sequence i is converted' to b d' be perfectly complementary in sequence to the (-) donor strand. Subsequent replication of the (-) acceptor strand and sealing of nicks results in completion of the conversion.</p> <p>LUGARES DE DNASECUENCIAS CODIFICANTES DEL GEN</p> <p>CARACTERISTICA</p> <p>SECUENCIAS INTRAGENIAS NO CODIFICANTES</p> <p>SECUENCIAS REGULADORAS FUERA DE EXONES</p> <p>La mayora patolgicas (sustituciones) (1ra y 2da posicn) Sitios mas susceptibles : islas CpG cerca a hotspots Menor porcentaje mutacional (10 15%) (10-15%) Afecta regiones intronicas En las regiones promotoras de los g genes ( (antes del Exon 1) ) Alteracin en la metilacin</p> <p>Mutations at conserved splice donor (SD) or splice acceptor (SA) sequences (see Figure 1.15 for consensus sequences) result in (A) intron retention where there is failure of splicing and an intervening intron sequence is not g q excised; or in exon skipping where the spliceosome brings together the splice donor and splice acceptor sites of nonneighbouring exons. (B) Sequences that are very similar to the consensus splice donor or splice acceptor sequences may coincidentally exist in introns and exons (sd and sa). These sequences are not normally used in splicing and so are known as cryptic splice sites. A mutation can activate a cryptic splice site by making the sequence more like the consensus splice donor or acceptor sequence and the cryptic splice site can now be recognized and used by the spliceosome (activation of the cryptic splice site). S Fi li i ) See Figures 9.12 and 9 13 9 12 d 9.13 for examples of activation of an exonic and an intronic cryptic splice site, respectively</p> <p>GANANCIA DE INTRON</p> <p>PERDIDA DE EXON</p> <p>GENERACION DE INSERCIONES O DELECIONES EN EL DNA PRODUCIENDO MUTACIONES TIPO FRAMESHIFT</p> <p>Location and nature of mutation Effect on gene function Extragenic mutation Normally none</p> <p>Comments Rare mutations may result in inactivation of distant regulatory elements required for normal gene expression (see Figure 8.23)</p> <p>Multigene deletion Whole gene deletion Whole gene duplication</p> <p>Abolition</p> <p>Associated with contiguous gene syndromes (see Figure 16.9)</p> <p>Abolition Can have effect due to altered gene Large duplications including the peripheral myelin protein 22 gene can cause Charcot-Mariedosage Tooth syndrome (see Figure 16.7)</p> <p>Whole exon deletion Within exon</p> <p>Abolition or modification Abolition</p> <p>May cause shift in reading frame; protein often unstable If loss/change of key amino acids, shift of the reading frame or introduction of premature stop codon If nonconservative substitutions, small in-frame insertions or other mutations at some locations If conservative/silent substitutions or mutation at nonessential sites</p> <p>Modification</p> <p>None Whole intron deletion Splice site mutation</p> <p>None Abolition or modulation of expression Conserved GT and AG signals are critically important for normal gene expression. Mutations ki i t ti may i d induce exon skipping or i t intron retention</p> <p>Promoter mutation</p> <p>Abolition or modulation of expression Deletion, insertion or substitution of nucleotides within promoter may alter expression. Complete deletion abolishes function</p> <p>Mutation of termination codon</p> <p>Modification</p> <p>Additional amino acids are included at the end of the protein until another stop codon is reached</p> <p>Mutation of poly(A) signal</p> <p>Abolition or modulation of expression Deletion, insertion or substitution of nucleotides within poly(A) site may alter expression. Complete deletion abolishes function</p> <p>Elsewhere in introns/UTS</p> <p>Usually none</p> <p>Change Delete: (i)the (i) the entire gene (ii)partofthegene Insertasequenceintothegene Disrupt the genestructure: (i)by atranslocation (ii)byaninversion (ii) by an inversion Preventthepromoterworking: (i)bymutation (ii)bymethylation Destabilize the mRNA: (i)byapolyadenylationsitemutation (ii)bynonsensemediatedRNAdecay Prevent correct splicing : (i)byinactivatingdonorsplicesite (ii)byinactivatingacceptorsplicesite (iii)byactivatingacrypticsplicesite Introduceaframeshift intranslation Convertacodon intoastopcodon Replace an essential aminoacid Preventpost transcriptionalprocessing Prevent posttranscriptional processing Preventcorrectcellularlocalizationofproduct</p> <p>Example Mostthalassemia mutations Most thalassemia mutations 60%ofDuchenne musculardystrophy InsertionofLINE1repetitivesequence intoF8C genein hemophiliaA Xautosome translocationsinwomenwithDuchenne musculardystrophy InversioninF8C gene Inversion in F8C gene Globin 29AGmutation FragileXfullmutation(FMR1) globinAATAAAAATAGAmutation Fibrillinmutations(FBN1) PAX3 451+1GTmutation PAX3 4522AGmutation Globin intron 1110GAmutation PAX3 874875insGmutation PAX3 Q254Xmutation PAX3 R271Cmutation Cleavage resistantcollagenN terminalpropeptide in Cleavageresistant collagen Nterminal propeptide in EhlersDanlos VIIsyndrome F508delmutationincysticfibrosis</p> <p>NOMENCLATURA</p> <p>EFECTO EN EL ALELO</p> <p>ALELO NULO</p> <p>El alelo no genera prod cto producto proteico A. HIPOMORFO Genera una reducida cantidad del producto A.HIPERMORFO Genera una excesiva cantidad del producto p A. NEOMORFO Genera un nuevo producto A. A ANTIMORFO Genera un producto que G d t antagoniza la actividad o funcin d un producto f i de d t normal</p> <p>Sustitucin a.a. R117H: arginina por histidina hi tidi en 117 (Arg117 (A 117 His) G542X: glicina por Stop (Gly Stop) Sustitucin nucletidos1162(G&gt;A) 621(G&gt;T) ( )</p> <p>Deleciones inserciones i i Delta - F508 o F508 nt6232(del5) o nt6232(del ACCTG) ( ) nt409(insC)</p> <p>IVS4+1G&gt;T :Cambio de G por T en la primera base del intron 4</p> <p>409-410insC :Insercin d C entre l nucleotidos 409 y 410 I i de los l id</p> <p>PERDIDA DE FUNCION</p> <p>Enfermedades A.R.</p> <p>Haploinsuficiencia A.D.</p> <p>Efecto dominante negativo A.D.</p> <p>Toxicidad celular</p> <p>&lt; 50% de la actividad enzimatica</p> <p>Expansin de tripletas (GRANDES)</p> <p>GANANCIA DE FUNCION</p> <p>Protenas hiperfuncionantes o autoestimuladas</p> <p>Protenas neoformadas</p> <p>Enfermedades A.D.</p> <p>Repeticin de tripletas con presencia de protenas con poli glutamina (MEDIANA)</p> <p>ABL -BCL</p> <p>Loss of function mutations in the PAX3 gene. The 10 exons of the gene are shown as boxes, with the connecting introns not to scale. The shaded areas show the sequences encoding the two DNA-binding domains of the PAX3 protein. Note that mutations that completely destroy the structure of the PAX3 protein (drawn above the gene diagram) are scattered over at least the first six exons of the gene, but missense mutations (shown below the gene diagram) are concentrated in two regions, the 5 part of the paired domain and the third helix of the homeodomain. A196T is believed to affect splicing. The 874875insG mutation introduces a seventh G into a run of six Gs; it has arisen independently several times and illustrates the relatively high frequency of slipped-strand mispairing</p> <p>HPRT activity (% of normal) Phenotype &gt;60 860 1.68 1.41.6 14 16 </p>