1. TYPES OF MUTATION CAUSING HUMAN GENETIC DISEASE Nucleotide substitutions (point mutations) Missense mutations Nonsense mutations Spice site mutations Frame shift mutations Rearrangements Deletions Insertions Duplications Unstable repeat sequences Small, a few base pairs Large, can be many kilobases

2. MUTATION Mutations in Coding regions Mutations in regulatory domains Protein abnormalExpression abnormal Loss of function - common Gain of function - rare

3. GENETIC TESTING IN HEMOPHILIA A X-linked recessive, 1/10000 males variable in severity severe cases, spontaneous and life threatening bleeding repeated episodes can cause joint deformity and crippling treatable Gene is large, many mutations, can look for specific common inversion with a PCR based test.

5. e.g. Alpha anti trypsin deficiency Disease leads to increased probability of developing pulmonary emphysema Results from single base pair change at a known nucleotide position Synthetic oligonucleotide probe that contains the wild type sequence in the relevant region of the gene can be used in a Southern blot analysis to determine whether the DNA contains wild type or mutant sequence Using the appropriate temperature the complimentary sequence will hybridize but a sequence with even a single mismatched base will not.

6. E.g. Oligoligation assay Normal sequence pair at site AT, Mutant is GC 2 short oligonucleotides that are complimentary to one of the 2 native DNA strands are synthesised Probe X has as its last base at the 3’ end the nucleotide that is complimentary to the normal sequence. It does not hybridize well to the mutant sequence as there is a mismatch Run test sample next to normal control Oligos hybridize When ligase is added the oligos bound to the mutant can’t ligate as have wobbley base that is misaligned In order to see whether the single base mutation is present need to be able to distinguish between ligated and non ligated (containing mutation) Probe X has a biotin residue or fluorescent molecule at the 5’ end, Probe Y has a dioxygenin residue at the 3’ end (called PEO in diagram) After hybridization and ligation, DNA is denatured to release hybridization probes and mix is transferred to wells coated with streptavidin. Biotin binds Unbound material washed away Anti dioxygenin antibodies coupled to alkaline phosphatase added to the well Substrate for AP added If oligos ligated get colour in well If oligos not ligated no colour

7. Some genetic diseases can be attributed to multiple mutations (together or separately) at multiple different sites. PCR has played a tremendous role in making diagnosis possible in a timely fashion. E.g. BRCA 1 Plays a role in hereditary breast cancer Very long gene Analysis is PCR based Don’t know specifically what you are looking for e.g. can be any of many mutations in BRCA 1, not all yet identified BRCA 1 has 24 exons that span a huge number of bases Most mutations have been found in Exon 11

8. BRCA 1 Exon 11 3500bases long Too long for convenient PCR, split into 3 pieces for analysis Oligonucleotides incorporate a promoter so amplified products end up with a promoter on the front 3 pieces are amplified and each used separately in a transcription translation system Protein products are produced Run on PAGE to see if are the correct size (next to normal controls) If incorrect size know there was a mutation Screening of the protein product allows screening of a very large pieceof DNA when you don’t know specifically what you are looking for

9. Exons 2-10 ReverseTranscriptase PCR is used 2-10 includes many introns and far too long to amplify. If start with RNA there are nointrons Use RT to get DNA, amplify with special oligo with promoter sequences Use transcription translation system as before, look at protein product size Exons 12-24 As exons 2-12 Exon 2 Mutation found that is common in Ashkenazi Jews If know source sometimes just screen directly from genomic DNA, and look at size on gel to see if different from normal control.

11. Fragile X Syndrome 1/1200 males, 1/2500 females - most common form of X-linked mental retardation Varying degrees of developmental delay Hypermobile joints High arched palate Long face Large protruding ears Known to be caused by expansion of a CGG repeat in the 5’ end of the FMR-1 gene. The repeat is polymorphic in the general population 6-45 CGG repeats Carriers, male and female, unaffected carry 50 to 200 CGGrepeats Affected individuals carry greater than 200 CGG repeats When the repeat size is great than 200, expression of the FMR-1 gene is turned off The presence of more than 200 CGG repeats in the FMR-1 gene contributes to the fragility of the X chromosome and this can be observed cytogenetically.

12. FMR-1 CGG Repeat Southern blot Affected >200 repeats Premutation 60-200 repeats EcoRIBssHIIEcoRI *CGGn