TYPES OF MUTATION CAUSING HUMAN GENETIC DISEASE

Nucleotide substitutions (point mutations)

Missense mutationsNonsense mutationsSpice site mutationsFrame shift mutations

Rearrangements

DeletionsInsertionsDuplicationsUnstable repeat sequences

Small, a few base pairs

Large, can be many kilobases

MUTATION

Mutations in Coding regions

Mutations in regulatorydomains

Protein abnormal Expression abnormal

Loss of function - commonGain of function - rare

GENETIC TESTING IN HEMOPHILIA A

X-linked recessive, 1/10000 malesvariable in severitysevere cases, spontaneous and life threatening bleedingrepeated episodes can cause joint deformity and cripplingtreatable

Gene is large, many mutations, can look for specific common inversion with a PCR based test.

• 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.

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 todistinguish 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

• 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

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

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 sizeExons 12-24• As exons 2-12Exon 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.

Fragile X Syndrome

1/1200 males, 1/2500 females - most common form of X-linked mental retardation

Varying degrees of developmental delayHypermobile jointsHigh arched palateLong faceLarge 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.

FMR-1 CGG Repeat Southern blot

Affected>200 repeats

Premutation60-200repeats

EcoRI BssHII EcoRI

* CGGn