Clinical Molecule Genetics

23.2.07

Molecular genetics laboratory
• Sample (blood, buccal cells, muscle biopsy, fixed tissue, CVS, amniotic fluid, hair roots).


• DNA.


• Analysis (PCR, DNA hybridisation, DNA sequencing).


• Result (interpretation - allele sizing, risk calculation, unknown mutation).


• Report.

Methods of DNA analysis
• DNA amplification (PCR).
• DNA hybridisation.
-Genomic Southern blotting.
-DNA Microarrays (DNA chips).
• DNA sequencing.

DNA Microarrays
• 100,000+ oligonucleotide "probes" on microchip.
• Uses include:
-Expression studies.
-Rapid linkage analysis - SNPs.
-SNP analysis for pharmacogenetics.
-DNA sequencing.

Types of mutation
• Large-scale rearrangements.
-Deletions, duplications.
-Inversions.
• Point mutations.
-Base substitution.
-Small deletion/insertion.
• Expanding Trinucleotide repeats.
• Imprinting.

Deletions
• Part/whole of gene.
-DMD - ~66% cases.
-SMA - 95% cases = homozygous deletion.
• Large deletions of several genes.

Duplications
• Part/whole of gene.
-HMSN Ia - ~70% cases.
• Often reciprocal to deletion.
-HMSN/HNPP.

Inversions
• Part/whole of gene.
-Haemophilia A.
• Often many genes.

Point mutations
• Base substitution (SNPs).
-Nonsense mutation (creates stop codon).
-Misssense mutation (changes one amino acid).
-Silent mutation (no effect on protein).
-Splice site mutation (leads to exon slipping/translation of intronic sequence).
-Regulatory region mutations.
-Frameshift mutation.
• Small deletion/insertion.
• Single base change in promoter.
• Can lead to increase/decrease in protein product.
• Leads to milder forms of genetic disease.
• Likely to be involved in polygenic disorders.

Pathogenesis: CF mutation types
Expanding trinucleotide repeats
• Usually polymorphic - tend to be neurological consequences.
• CAG = HD/SCAs/SBMA (affected range 40-120 repeats).
• CTG - Myotonic dystrophy.
• CGG - Fragile X.
• Inheritance:
-Autosomal dominant.
-Autosomal recessive.
X-linked recessive.
-X-linked - Fragile X.
• Location:
-Exonic.
-Intronic.
-Close to gene.

Imprinting
• Maternal and paternal copies of gene differentially imprinted.
• Very few genes imprinted.
• Mechanism is methylation - silences gene.
• Imprint lost in germ cells.
• Imprinting-related disorders include Prader-Willi and Angelman syndromes (15q 1.4).
• Uniparental disomy.

Molecular genetics - types of analysis
• Known mutation.
-E.g. common point mutation, deletion/duplication, triplet repeat expansion.
-Set up mutation-specific assay. Relatively easy.
• Unknown mutation.
-Screen for changes.
-DNA sequencing.
• Linkage analysis.
-Family studies using polymorphic markers linked to gene of interest.

DNA polymorphic markers
• Identity testing.
• Rapid chromosome aneuploidy screening.
• Linkage analysis.

Potential problems for DNA analysis
• Germ line and somatic mosaicism.
• Genetic heterogeity.
-E.g. retinitis pigmentosa >20 genes to date.
• Sensitivity of mutation detection.
-Size of gene, limits of current technology.
-Cryptic splice sites.
• Pathogenicity of missense mutations.
• Linkage: recombination/new mutation.

Future developments
• Proteomics/metabolomics.
• Genetics of common disease.
-Susceptibility/risk factors.
• Pharmacogenetics.
-Adverse reactions/tailored drug therapy.
• Oncology.
-Acquired mutations.
-Tumour expression profiling.
• Gene therapy.

New technologies
• Electronic hybridisation.
• Microarrays.
• Comparative Genome Hybridisation (CGH).
• Mass spectrometry (1 million SNPs per day).
• Individual genome sequence.