Patterns of Inheritance: Dominant Disorders

1. Patterns of Inheritance: Dominant Disorders Helen Lord, September 2009

2. Overview • Discuss with examples: • Dominant-Negative mutations • Gain of function mutations • Haploinsufficiency • How to distinguish between these experimentally.

3. Dominant-negative mutations • Occurs when a mutant polypeptide not only loses it’s own function; it interferes with the product of the normal allele in a heterozygote. • Also known as an antimorph. • These mutants tend to be more severe than null alleles occurring in the same gene. • Tend to be dosage sensitive, therefore over-expression of the allele causes a stronger phenotype than a single copy, therefore a greater excess of mutant exacerbates the phenotype.

4. Dominant-negative mutations • Exert effects on the wild type in two ways: • By binding directly to and inactivating the wild type • By binding to and inactivating a second protein that is required for the wild types protein function. • These have been described in many types of proteins with signalling or transcriptional functions. • A major group of dominant-negative mutants occur in the multimeric proteins, which are dependant on oligomerisation for activity.

5. Example 1: Collagens • Fibrillar collagens • built of triple helices of polypeptide chains (homo or heterotrimers), assembled into close packed cross-linked arrays to form rigid fibrils. • In preprocollagen, N- and C-terminal propeptides flank a repeat sequence (Gly-X-Y)n. • 3 of these chains associate and wind into a triple helix (Type 1: 2x COL1A1 and 1x COL1A2), the N- and C- terminal propeptides are then cleaved off. • A polypeptide that complexes with normal chains but wrecks the triple helix can reduce functional collagen to <50% (due to substitution of a bulkier amino acid in the Gly-X-Y unit = disrupts close packaging of the triple helix)

6. Gain of function mutations • Gain of function mutations usually cause dominant phenotypes, as the presence of a normal allele doesn’t prevent the mutant allele from behaving abnormally. • GOF mutations either, • activate a process that is not normally active-McCune Albright disease • over activate a process which is tightly controlled in cells-Charcot-Marie-Tooth disease • lead to production of a novel function-Huntington disease • Require a more specific change than LOF – same phenotype NOT produced by deletion/ disruption of the gene.

7. Gain of function mutations • They normally affect: • Growth factors • Growth receptors • Signal transduction pathways • Transcription factors • Cell cycle proteins • This can therefore lead to increased dosage, altered gene expression and increased protein activity.

8. Example 2: Achondroplasia • An autosomal dominant condition that leads to dwarfism in affected individuals. • Due to mutations in the FGFR3 gene • A transmembrane tyrosine kinase receptor which negatively regulates bone growth by inhibiting the proliferation of chondrocytes. • In 98% of cases it is caused by c.1138G>A, and in ~1% by c.1138G>C. • Both lead to the same amino acid substitution, p.Gly380Arg. • The mutated form of the receptor is therefore constitutively active, leading to severely shortened bones. • Other variants in FGFR3 are also responsible for other syndromes, including hypochondroplasia, thanatophoric dysplasia, and Muenke syndrome.

9. Haploinsufficiency • This is where having 50% of the normal gene product isn’t sufficient to produce the correct function. • Only occurs for loss of function mutations, and relatively few genes show haploinsufficiency. • Itcan occur through a variety of types of mutation such as: • Deletion of one of the two copies of the gene • A mutation in the gene – which may either wipe out the production of the message, or the message of the protein may be unstable or degraded by the cell. • Variable expression can be seen with haploinsufficiency, therefore a larger reduction in gene product will lead to increased severity of symptoms.

10. Example 3: SVAS • Genes showing haploinsufficiency fall into two broad categories: • A few code for tissue specific proteins, which are required at specific levels, so even though one functional copy is present it isn’t capable of producing enough product. • ELN - Supravulvar aortic stenosis(SVAS) – • Individuals heterozygous for a deletion or LOF mutation in elastin. • Most of the elastic tissues function normally – skin, lung and blood vessels • The aorta however shows narrowing just above the heart – SVAS.

11. Example 4: Waardenburg Syndrome Type I • Regulatory genes working close to threshold levels for different actions. In these cases such threshold levels may only manifest in a limited number of tissues where the gene is expressed. • PAX3 - Waardenburg syndrome Type I: • PAX3 is a DNA-binding transcription factor • Normal role of PAX3 is to interact with SOX10 and MITF in the regulation of melanocyte development • Whole gene deletion or point mutations within the PAX3 gene, lead to problems in this pathway including a decreased ability for PAX3 to regulate reporter genes fused to MITF and TRP-1promotors.

12. GOF and LOF mutations in the same gene • Mutations in the same gene can produce 2 or more dominant conditions • The milder one tends to be haploinsufficiency, and the more severe due to dominant-negative effects. • Example 5: RET gene • RET encodes a receptor that straddles the cell membrane. • GDNF (ligand) binds to the extracellular domain- induces dimerisation of the receptors, which transmit the signal into the cell via tyrosine kinase modules. • A variety of LOF mutations (frameshift, nonsense and amino acid substitutions) that interfere with the post-translational maturation of the Ret protein = Hirschsprung’s disease. • Certain very specific missense mutations = Familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia type 2 (MEN2). • In some individuals missense mutations affecting Cys 618 or 620 can have both diseases.

13. Examples of genes responsible for more than one disease Table adapted from HMG2, p392.

14. Experimental techniques • Dominant-Negative mutations • An increase in the number of wild type alleles by duplication or up-regulation – should lead to a milder phenotype. • Use of a knockout model – the wild type/knock-out model should produce a milder phenotype than the wild type/ mutant. • Co-expression of mutant and wild type proteins by immunoblot.

15. Experimental techniques • Gain of Function mutations • Deletion/ disruption of the gene should produce a different/normal phenotype. • A knockout mouse model / transgenic miceshould lead to an opposing phenotype i.e. Achondropasia - the knock out mice have excessively long bones and elongated vertebrate

16. Experimental techniques • Haploinsufficiency • Loss of Function mutations in the same gene should lead to the same phenotype. • Knockout animal model – The phenotype of those heterozygous for the deletion should be the same as the phenotype of the mutant.