Question

Many human genetic diseases are caused by mutations that occur at splice sites. Propose some ways that mutations at the 5’ splice site, 3’ splice site, and branch point might disrupt splicing and alter the phenotype.

Answer 1

Splice site mutation:

Splice site is the site on the pre mature mRNA where the splicing takes place to remove the introns and join the exons to form mature mRNA.

When a mutation such as deletion or insertion takes place at these splice sites the site becomes un recognizable by the spliceosomes to carry out splicing and it remains between the coding regions.

When this mRNA undergoes translation it codes for the mutant protein, which causes disease.

Disease caused by splice site mutation:

1. Familial dysautonomia:

FD is the rare disease that affects autonomic nervous system and somatic sensory neurones.

• It is caused by mutation in the IKBKAP gene, it encodes a transcription factor – elongation complex known as IKAP.
• In 99.5% of this disease is caused by the T>C substitution in the intronic region at position 6 of intron 20.
• This mutation disrupts binding of U1 to the 5’ splice site of exon 20 and cause exon skipping, which results in frame shift and pre mature termination codon.

2. Chain acyl-coA-dehydrogenase deficiency (MCAD):

This is required for the degradation of medium chain length fatty acids.

MCAD difficiency causes defect in mitochondrial ?-oxidation.

Medium chain acylcarnitines accumulate in the urine which can be detected diagnostically.

Missense mutation at exon 5 of MCAD causes exon skipping and degradation by non-sense mediated decay.

3. Hutchinson- Gilford progeria syndrome:

This is the disease which cause the premature aging.

• HGPS is caused by the mutation in the lamin A and lamin C gene (LMNA).
• LMNA gene codes for 2 protein which depend on alternative splicing of the transcript.
• Lamin A and C are the members of nuclear lamin family of structural proteins, that forms intermediate filaments and constitute the nuclear lamina. Forms the meshwork structure that supports the inner nuclear membrane in the eukaryotic cells.
• HGPS is caused by the mutation in the exon 11 which alters the splice site 5nt upstream where GGTGGGC becomes GGTGGGT which is a splice donor site and the protein lacks last 50 amino acid of exon 11.
• The mutant protein is known as progerin.

4. Menkes disease:

Menkes disease is the X-linked disorder which causes defect in the copper metabolism.

• This disease is caused by the mutation in the ATP7A.
• This encodes for the ATPase that transports copper across intestinal mucosa into blood.
• This is caused by the mutation in the conserved regions of 5’ and 3’ splice sites which disrupts normal splicing.
• The symptoms include neurological impairment, kinked brittle hair, dysmorphic features, death usually occurs before the age of 3.

5. Myotonic dystrophy:

Myotonic dystrophy is an autosomal dominant disease which causes progressive myopathy, delayed relaxation of muscle contractions (myotonia), cardiac conduction defects, cataracts etc.

• 2 forms of Myotonic dystrophy occurs, known as type1 DM1 and type2 DM2.
• DM1 is caused by the CTG expansion at 3’untranslated region of DMPK gene.
• DM2 caused by the expansion of CCTG in intron 1 of ZNF9 gene.
• This mutation causes MBNL1 depletion and CUGBP1 up regulation which causes the disruption of alternative splicing.

6. Cancer:

Splice site mutation also cause cancer.

• Mutation in the tumour suppresser gene splice sites results in the cancer.
• KLF6: is the tumour suppresser gene, which inhibits the cell growth through various mechanisms, which include activation of p21, a cyclin-dependent kinase inhibitor.
• Single nucleotide polymorphism near the exon 2 – intron exon boundary 5’ splice site cause upregulation of KLF6-SV1 isoform which causes prostate cancer.