Patterns of Inheritance - Session 2 In dominant disorders, dominant-negative mutations, gain of function mutations and haploinsufficiency have been described. Explain what these terms mean with examples. How could one distinguish experimentally between them. Roy Poh

Loss of function mutation A mutation that causes in a reduction in product or an abolishment of product function. Phenotype arising from loss of function mutation of a gene is less specific and can be due to any change that inactivates the gene. Eg. In Waardenburg syndrome Type I, causative mutations in the PAX3 gene include missense, nonsense, frameshifts, splicing and complete deletion of the gene. Loss of function mutations most often produce recessive phenotypes. Most genes, the precise quantity of products are not crucial and half of normal amount does not cause problems. However for some gene products, 50% of normal products is not sufficient for normal function. Haploinsufficiency refers to a condition that arises when the normal phenotype requires the protein product of both alleles, and reduction of 50% of gene function results in an abnormal phenotype and therefore inherited in a dominant manner.

Phenotypes probably caused by Haploinsufficiency Condition Gene Notes Alagille syndrome JAG1 Patients are seen with either microdeletion at 20p11 including the JAG1 gene or point mutations in the JAG1 gene (seen in >88% patients). Tomaculous neuropathy PMP22 80% patients with single copy and remaining 20% patients have point mutations that leading to frameshifts or other loss of function mutation. (While individuals with 3 copies have clinically different Charcot-Marie-Tooth disease.) Waardenburg syndrome PAX3 Causative mutations in the PAX3 gene include missense, frameshifts, nonsense, splicing mutations and deletion of the gene. All these events produce the same clinical results and indicating loss of function disorder.

Dominant negative effect A dominant negative mutation occurs when the gene product not only loses its own function but adversely affects the normal, wild-type gene product within the same cell. That is the product can still interact with the same cellular components as the wild-type product, but block (loss) some aspect of its function. Due to the partial loss of function and a novel gain of function as a whole, some people argue between the two types. Such mutant products may act as competitors for the wild type protein functions. Dominant negative mutations cause more severe effects than simple null alleles of the same gene. Multimeric proteins that are particularly vulnerable to dominant negative effects. Example: Non-structural protein that dimerize or oligomerize. Transcription factors of the b-HLH-Zip family bind DNA as dimers. Mutants cannot dimerize often cause recessive phenotype but mutants that are able to sequester functioning molecules into active dimers give dominant phenotypes.

Dominant negative effect (2) Fibrillar collagens, the major structural proteins of connective tissue, are build of triple helices procollagen units (homotrimers or heterotrimers) that are assembled into close-packed cross-linked arrays to form rigid fibrils. Type 1 procollagen consists of 2 chains encoded by COL1A1 gene and 1 encoded by COL1A2. In newly synthesized polypeptide chains (preprocollogen), N- and C-terminal propeptides are flanked by a regular repeat sequence. The 3 chains associate and wind into a triple helix under control of the C-terminal propeptide. After triple helix formation, both terminus are cleaved and followed by further assembly. Missense mutations at the N-terminal part of the triple helix cause minor disturbance of packing (seen in Brittle bone disease/Osteogenesis imperfecta Type 1, 3 or 4) while mutations at the C-terminal part result in major disturbance (most severe OI type 2 or 4). Null mutations of either genes cause a reduction of normal gene products and have a less severe effect (OI type 1).

Gain of Function mutation A mutation that give rise to a product that does something new or positively abnormal. This kind of mutations usually cause dominant phenotypes because the presence of normal allele does not prevent the mutant allele from behaving abnormally. The Mutational spectrum in gain of function conditions are more specific and restricted. The same condition is not likely due to random deletion or disruption of the gene. For example in Achodroplasia (Short-limbed dwarfism) virtually all cases have the same missense mutation of Gly380Arg in the FGFR3 Fibroblast growth factor receptor gene. Biochemical studies combined with knockout experiments in mice showed the receptor to be overly active. Other substitutions in the same gene cause other syndromes. In Huntington disease, only repeat expansion mutation and no other type of mutations being seen suggesting a gain of function mutation. Gain of Function mutation is often found in genes that involve control or signalling system behaving inappropriately.

Gain of Function mutation 2 Example: Mutation that give rises to a product that do something novel Myotonic dystrophy type 2 is caused by a CCTG expansion in the intron 1 of the zinc finger protein 9 (ZNF9) gene. Experiments carried out with myoblast cell lines and DM2 skeletal muscle biopsy tissue show that large DM2 CCTG expansions (i) do not prevent pre-mRNA splicing, (ii) do not affect expression ratios of alternatively splicing transcripts, (iii) do not change levels of transcripts or protein and (iv) result in accumulation of CCUG ribonuclear inclusions. These data show gain of abnormal activity and therefore support a gain of function RNA model. Mutation give rises to a product that increase its activity. The PTPN11 gene, encodes for protein tyrosine phosphatase and is responsible for Noonan Syndrome. The protein contains 2 SHP2- binding domain and a tyrosine phosphatase domain. Only missense mutations are found in these domain and no deletion or duplication for the gene is seen in Noonan individuals. Immune complex phosphatase assays of mutants in COS7 cells showed increased phosphatase activation compared to wild-type SHP-2 and therefore supports gain of function mechanism.

Gain of Function mutation 3 Disease Gene Malfunction Charcot-Marie-Tooth Disease PMP22 Over-expression McCune-Albright Disease GNAS Receptor permanently ‘On’ Huntington Disease HD Protein Aggregation Paramyotonia Congenita SCN4A Ion Channel inappropriately open Chronic Myeloid Leukemia BCR-ABL Chimeric gene

Experimental Evidence Biochemical studies – to determine if mutant product is still functional (enzymatic activity, binding properties, Yeast-2-Hybrid / Protein-Protein interaction assay, Chromatin / immuno-precipitation, Native PAGE). Transcript alterations and Levels – to determine if mutations affect expression and length of transcripts (RT-PCR), accumulation of transcripts (Quantitative PCR), formation of aggregations/inclusion bodies. Protein Structure and level – (ELISA, Western blotting, Immuno-histochemistry, Mass Spectrometry, X-ray Crystallography, Molecular Dynamics Simulation assay, Energetics-based Structural Analysis) Gene Knock-in / Knock-out in animal model or cell line – Investigate the phenotype and cellular response by introducing a functional gene. Interpretation must take into consideration of other forms of dose sensitivity. If phenotype is due to haploinsufficiency, by increasing gene product might rescue the phenotype. In the case where introducing a normal gene, the mutant still retains abnormal activity then likely due to gain of function.

Experimental Evidence (2) In most cases due to the involvement of complex biochemical pathway, evidence must be collected from all levels (types of mutation found, biochemical and structure studies and studies carry out in biological systems) to elucidate the true mechanism.

Source Human Molecular Genetics 3. Tom Strachan & Andrew P. Read. www.Geneclinics.org Recent milestones in achondroplasia research. Am J Med Genet (2006) 140, 166-169. Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood Leukemia. J Hum Genet (2005) 50, 192-202. DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression. Hum Molecular Genet (2006) 15, 1808-1815.