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Jf Med Genet 1994;31:89-98 89 Review article J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from

The molecular basis of genetic

Andrew 0 M Wilkie

Abstract the of three : an Studies of mutagenesis in many organ- may behave as a dominant, semidominant, or isms indicate that the majority (over recessive depending both on its partner allele, 90%) of are recessive to wild and the character under consideration.3 Con- type. If recessiveness represents the sider A and B, with genotypes AA, AB, 'default' state, what are the distinguish- and BB. If a particular phenotypic character is ing features that make a minority of observed in the AA and AB genotypes, but mutations give rise to dominant or semi- differs from BB, then allele A is dominant to dominant characters? This review draws allele B. When the AB is inter- on the rapid expansion in knowledge of mediate between or combines characters from molecular and cellular to classify both the AA and BB phenotypes, alleles A and the molecular mechanisms of dominant B are semi- or codominant. Most wild type . The categories discussed in- alleles are dominant over other alleles, as the clude (1) reduced dosage, expres- wild type and heterozygote phenotypes are sion, or activity (haploinsuffi- usually indistinguishable; thus most genetic ciency); (2) increased gene dosage; (3) diseases are recessive (fig 1). ectopic or temporally altered mRNA A potential source of confusion when consi- expression; (4) increased or constitutive dering dominance phenomena in human gen- protein activity; (5) dominant negative etic disease, is that only the wild type and effects; (6) altered structural ; (7) heterozygous mutant phenotypes are generally toxic protein alterations; and (8) new encountered. Examples of homozygous mu- protein functions. This provides a frame- tants both for relatively common disorders work for understanding the basis of dom- (thalassaemia, familial hypercholesterolaemia) inant genetic phenomena in humans and and rarer conditions (, pie- other organisms. baldism) indicate that the phenotype of the homozygote usually tends to be more severe http://jmg.bmj.com/ (J Med Genet 1994;31:89-98) than the heterozygote, hence the wild type and mutant alleles are, strictly speaking, semidom- inant.6 The Huntington's disease mutation The concepts of dominance and recessiveness provides an unusual instance of a mutant allele (or recessivity), originally formulated by Men- that is truly dominant to wild type in that del,' are so fundamental to that they homozygotes appear no more severely affected

are often taken for granted. But why are some than heterozygotes7-9 (fig 1). Although it is on October 7, 2021 by guest. Protected copyright. diseases dominant and others recessive? This interesting to speculate on the differences in question is frequently ignored in textbooks of mechanism giving rise to semidominance and genetics, and it is surprisingly difficult to find complete dominance, there are insufficient much written on the subject. With the rapid molecular data to attempt a synthesis. The accumulation of molecular data on diploid more simple, but perhaps more fundamental, organisms as diverse as yeasts and humans, question addressed in this review may be sum- unifying themes are beginning to emerge.2 marised as follows: what aspects of a mutant This review attempts to classify and elaborate allele'sfunction cause it to affect the phenotype in these ideas, and collates some of the more the presence of a wild type allele? For simplicity useful references. I will first outline some I will use the term 'dominant mutation' to definitions and concepts, and then give describe a mutant allele in this context. illustrative examples of different molecular mechanisms of dominance. Although I have focused on human disorders where possible, Dominant mutations are much rarer Institute of Medical many additional lessons can be learned from Genetics, University than recessive ones Hospital of Wales, the study of non-human systems. Although dominant disorders outnumber re- Heath Park, Cardiff cessives by a ratio of nearly 4:1 in McKusick's CF4 4XW, UK A 0 M Wilkie 1992 compilation,'0 ascertainment in the Dominance, semidominance, and human is undoubtedly biased in favour of mild Correspondence to Dr Wilkie, Institute of recessiveness dominantly inherited phenotypes. By contrast, Molecular Medicine, John It should first be remembered that dominance it has long been known from systematic muta- Radcliffe Hospital, Headington, Oxford OX3 is not an intrinsic property of a gene or mutant genesis of a variety of diploid organisms that 9DU, UK. allele, but describes the relationship between the majority of mutations are recessive to wild 90 Wilkie

Genotype Descri ptiorn Examples ciency), or only in the homozygote (recessive- AA AB BB ness). In 1981 Kacser and Burns'8 proposed a most A is dominant to B Recessive 1isetnes theory of metabolic fluxes to explain why C,r errors of metabolism are recessive. inborn J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from Assuming that a metabolic pathway has many ° ___ A a rl di B a se r-incm o rn n a riI 3-tiha a ssa na a C' :farmlial hvyerchoestwo ann- non-rate limiting steps, control of flux at any particular point in a pathway will be small. B 's d orniT riai A: Rare: HUntngtongA s isease Hence, many pathways show a saturable rela- be an exampie tionship between level and metabolic _ 1 1 ? 0BB pher otue: irkiowr Most sn callec: o;'ar- flux, with fluxes fully saturated at wild type cdiseases enzyme level; a 50% reduction in enzyme Figure 1 Relationship between and clinical phenotype. A is a wild type activity would therefore cause little reduction allele and B a mutant allele; different phenotypes are represented by different shaded in flux below its saturation level. blocks. Although this theory fits metabolic path- ways well, it is not applicable to critical rate type. " For example, insertional inactivation by limiting steps of such pathways, nor to muta- random integration of retroviral DNA into the tions causing qualitatively altered function, mouse genome produces recessive and domin- especially when structural or controlling/sig- ant phenotypes with a ratio of about 10- nalling proteins are involved. It is perhaps not 20:1.12 13 surprising that most dominant mutants belong The search for an explanation of the reces- to one of these latter categories, and frequently sive behaviour of most mutations generated a involve developmental malformations. lively debate in the 1930s between Sewall An additional explanation for the rarity of Wright, who believed that it arose intrinsically dominant mutations is suggested by work on from the physiology of gene action, and RA the nematode Caenorhabditis elegans. Reces- Fisher, who proposed that the accumulation of sive mutations at a series of loci termed smg modifier alleles at other loci was responsible. may alter the behaviour of mutations at other Fisher's theory has now generally lost favour, loci from recessive to dominant (cryptic dom- and Orr'4 showed that in the alga Chlamydomo- inance). It seems that the wild type smg loci nas, which is usually haploid (so that Fisherian encode proteins that can recognise and select- selection cannot apply), most mutations never- ively degrade many mutant mRNA species, theless showed recessive behaviour when ex- forming part of a mutant surveillance sys- amined in a transiently diploid background, tem.'920 The relevance of this finding to supporting Wright's theory. Indeed, diploidy humans is not yet clear. may have evolved because it protects against Finally, note that although the number of recessive mutations. '5'7 Thus it is dominance, known recessive conditions in the human may rather than recessiveness, that demands special considerably underestimate the total, the true explanation; but why should the 'default' state figure is unlikely to approach the total number of mutations be recessive? of . There is a growing list of murine The usual explanation is as follows. The genes for which targeted disruption is not http://jmg.bmj.com/ most likely effects of a random gene mutation associated with any phenotypic abnormality in are that it will either be neutral (normal pheno- transgenic mice.21 A similar situation applies to type) or inactivating. If the latter, the question the mutational spectrum in C elegans, and it is is whether the inactivation would be clinically noteworthy that dominant "gain of function" manifest in the heterozygote (dominance or mutants exist at several loci for which the semidominance, specifically, haploinsuffi- homozygous null phenotype is entirely nor-

mal.22 on October 7, 2021 by guest. Protected copyright. Muller's classification Molecular classification Types of dominant mutation I In 1932, Muller23 suggested a classification of dominant mutations that is still widely quoted. I He coined the terms amorph, hypomorph, and + Gene dosage hypermorph to reflect quantitative changes to a Z pre-existing wild type character; antimorph to +/ Ectopic mRNA expression I describe mutual antagonistic interaction with -j wild type; and neomorph for a new phenotype, +/Constitutive protein acti not fully antagonised by wild type. His propo- I sal, made when the molecular nature of muta- i Dominant negative tion was still uncertain (and predating the identification of DNA as the genetic material dStructural function by 12 years),24 was remarkable for its pres- cience. Unfortunately, later authors have i Toxic protein I sometimes tended to assume a one to one relationship between this classification, based New protein I on , and underlying molecular i mechanism. While clear parallels exist, these Figure 2 Relationship between genetic and molecular mechanisms of dominance. The are inexact (fig 2). As this review focuses on genetic classification is that formulated by Muller23; the relationship between wild type of I have (A) and mutant (B) alleles is indicated diagrammatically. Thick lines join categ, aormesmolecular mechanisms dominance, that commonly show equivalence; dashed lines connect less frequent groupings. avoided using Muller's terms to highlight the The molecular basis ofgenetic dominance 91

distinction between the genetic and molecular ant than their rarity might suggest, for two levels of analysis. The following classification reasons. First, mutation may arise from any seems to accommodate most situations, al- mechanism producing loss of function: dele- though some ambiguities and overlaps are tion, translocation, truncation J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from inevitable. caused by nonsense and frameshift mutation, (1) Reduced gene dosage, expression, or pro- and some promoter and splice site mutations tein activity: haploinsufficiency. and amino acid substitutions may all be re- (2) Increased gene dosage. sponsible. Such variety will tend to increase (3) Ectopic or temporally altered mRNA the frequency with which the disease is expression. observed. Second, dosage sensitive genes seem (4) Increased or constitutive protein activity. to be an intrinsically interesting group.26 (5) Dominant negative effects. Genes showing haploinsufficiency fall into (6) Altered structural proteins. two broad categories. A few code for tissue (7) Toxic protein alterations, not covered in specific proteins synthesised in large quantit- other categories. ies, for instance, type 1 collagen27 (but see also (8) New protein functions. the section on structural mutations), globins, Some examples of these mechanisms are low density lipoprotein ,28 haem syn- shown in the table and are further discussed thesis (),2 and Cl esterase inhibitor below. A general distinction can be made (hereditary angio-oedema).29 In the first two between category (1), which involves loss of cases, the abnormal heterozygous phenotype function, and categories (2) to (8), which rep- may be because of the resulting imbalance with resent gain of function. Note that the latter, a matched component protein; in the latter frequently used term encompasses a wide three, because of interference with a rate limit- range of mechanisms, and is thus only appli- ing step of a metabolic pathway. Of particular cable in a broad context. The table includes note, levels of Cl esterase inhibitor associated two other mechanisms that may give rise to a with heterozygous deficiency are only 15 to "dominant" pattern of inheritance, but in 20% of normal, even during remission. This is which the inherited mutation is not dominant because the normal inhibitor is "mopped up" at a cellular level. These involve recessive anti- relatively rapidly by complexing with plasma oncogenes and genomic imprinting, and are , and the rate at which this occurs is discussed briefly in a later section. largely independent of inhibitor concentration (zero order kinetics).29 The quantitative defi- ciency is hence greater than the expected value REDUCED GENE DOSAGE, EXPRESSION, OR of 50%. PROTEIN ACTIVITY: HAPLOINSUFFICIENCY A second category comprises regulatory For the minority of cases in which the abnor- genes working close to a threshold level for mal phenotype results from inactivation of one different actions. Examples in humans include of a pair of alleles, the term "haploinsuffi- PAX3 (),303' PAX6 ciency" is used ("haplolethality" if early (),32 GLI3 (Greig cephalopolysyndactyly, embryonic death occurs). Haploinsufficient GCPS),33 34 WT1 (Wilms's tumour/genito- http://jmg.bmj.com/ loci are relatively unusual: a careful survey of urinary abnormalities),35 36 RD S/peripherin the Drosophila genome showed only 56 loci (),37 and KIT (pie- associated with an altered phenotype when baldism).38 Such threshold dosage effects may present as a single copy, of which four were be clinically manifest in only a subset of the lethal.25 However, such loci are more import- tissues in which the gene is expressed (aniridia on October 7, 2021 by guest. Protected copyright.

Major categories and mechanisms ofgenetic dominance, with the types of mutation commonly responsible. See text for further examples and references. D = large deletion, T truncation (nonsense or frameshift mutation), M = missense mutation or small in frame deletion, S = splice site mutation, P = promoter mutation, Tr = translocation or other rearrangement, Dup = duplication, A = amplification, () = inconsistent association. Category of mutation Mechanism Types of mutation Examples Loss offunction Haploinsufficiency Subunit imbalance D, T, S, (M) a and ( globins Metabolic rate determining step D, T, S, (M) LDL receptor Developmental regulator D. T, S, (M), (Tr) PAX3, PAX6 Gain offunction tGene dosage Duplication Dup PMP-22 Amplification A MDM2 T/Ectopic mRNA expression Altered temporal pattern P,Tr,(D) y globin, MYC Altered tissue distribution P, Tr Ubx, Antp, MYC TmRNA stability D lin-14 T/Constitutive protein TStability (PEST deletion) T CLN3, glp-l activity Constitutive activation M RAS, Gso, SCN4A Dominant negative Disruption of dimer M, (T) KIT, Competition for substrate M, (T) RAS Structural protein Disruption of structure M, S, (T) Collagen, fibrillin Toxic protein Disruptive interaction M Rhodopsin, amyloidoses New protein Altered substrate specificity M a, antitrypsin Exon shuffling Tr BCR/ABL Other mechanisms Recessive antioncogene - Retinoblastoma Genomic imprinting - Beckwith-Wiedemann syndrome 92 Wilkie

and GCPS are examples) and the phenotype (discussed further below), leading to escape may be sensitive to the genetic background. from normal p53 regulated cellular growth To understand the mechanisms of dosage control.47 sensitivity in this "regulatory" group requires a detailed knowledge of the molecular interac- J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from tions involved, something not yet achieved for ECTOPIC OR TEMPORALLY ALTERED mRNA any human gene. However, simpler organisms EXPRESSION provide some excellent model systems. For This group is characterised by disturbance of instance, sex determination in Drosophila re- the exquisite controls of mRNA expression quires the ability to distinguish between X:au- that dictate the normal cellular distribution, tosomal ratios of 1 in females and 0 5 in males. temporal restriction, and absolute levels of This may be achieved by titration of "numer- mRNA. In principle, altered gene expression ator" genes against "denomina- can arise in any gene or message that contains a tor" autosomal ones, possibly by competition regulatory domain, and the molecular patho- of the cognate proteins for binding to a regula- logy of such mutants is correspondingly di- tory DNA sequence.'9 Further insight may be verse.48 gained by studies of morphogenic proteins, for A fairly specific illustration of loss of tem- example the Drosophila factor poral regulation is provided by hereditary per- dorsal (dl), which is distributed in a nuclear sistence of fetal haemoglobin (HPFH). Known concentration gradient along the dorsoventral causes include point mutation of the y globin axis of the early embryo. Dosage dependent promoter, which alters binding of the eryth- activation of different sets of downstream roid GATA-1,49 certain 3' genes by dl correlates with the strength of the deletions encompassing the 6 and P globin dl binding sites in their promoters: genes with genes,50 and alterations of unidentified trans high affinity dl binding are activated or acting factors. The effect of all these mutations repressed by dl at lower threshold levels.4' is to abrogate the normal switch from expres- Correspondingly, female flies heterozygous for sion of y to 6 and 1 globin, which occurs dl null mutations produce abnormal embryos around the time of birth. The resulting HPFH that fail to develop mesoderm, which requires dominantly ameliorates the effects of 1 thalas- the highest level of dl activity.42 saemia mutations. An example of ectopic expression is pro- vided by the contrabithorax (Cbx) mutations INCREASED GENE DOSAGE Application of Kacser and Burns' principles'8 of Drosophila, which involve the ultrabithorax predicts that an increase in gene dosage to (Ubx) gene, normally expressed in the poster- three copies should affect the phenotype even ior part of the embryo with an anterior bound- less often than a reduction to one copy. Experi- ary in the third thoracic segment (T3). In Cbx mental analysis supports this: for example, the mutants, which comprise insertions, inver- survey of in Drosophila previously sions, and other chromosomal rearrange- Ubx is also expressed in T2 and this mentioned25 identified only one triplo-lethal ments,5' http://jmg.bmj.com/ and one triplo-abnormal . Nevertheless, results in the homeotic transformation of T2 cytogenetically visible trisomy in humans into a T3 like structure. Similarly, dominant (which will usually encompass at least 40 to 50 homeotic mutations of the Antennapedia gene genes) is usually associated with phenotypic occur because of ectopic expression: in one abnormality, indicating that a significant case studied in detail (Antp73b), a chromosomal minority of loci must be sensitive to 3 versus 2 inversion results in the entire Antp coding region being placed under a new promoter.52

dosage. It may be relevant that the increase in on October 7, 2021 by guest. Protected copyright. dosage at the mRNA and protein level can More commonly, the disease phenotype may exceed the expected factor of 1-5; considerably reflect a combination of alterations in the tem- greater rises are observed for some genes poral specificity, tissue distribution, and abso- on chromosome 21 in Down's syndrome.4' lute level of mRNA expression. The primary Although the distinctive phenotypes associ- abnormality usually lies at the level of tran- ated with certain trisomies may therefore be scription, but sometimes mRNA processing attributable to a small number of critical genes, may be affected. Examples of transcriptional few of these have been specifically identified. alterations include the following. Chromoso- An exception is PMP-22, duplication of which mal translocations resulting from errors in is likely to be the principal cause of type I recombinase mediated gene rearrangement in Charcot-Marie-Tooth disease.44 The PMP-22 lymphoid cells activate expression of tran- region is also haploinsufficient, giving the scription factors like MYC, causing B and T different phenotype of dominant pressure cell neoplasms.5'54 Promoter mutations in the palsies45; however, the cellular mechanisms Caenorhabditis sex determining gene her-1 of these contrasting dosage effects are not (the only member of this pathway subject to understood. transcriptional control) increase expression Gene amplification in somatic cells to much levels and result in partial transformation of higher copy numbers frequently occurs in cer- XX worms into phenotypic males.55 Increased, tain neoplasias.46 A particularly clear example ectopic expression of a chimeric mRNA of how this causes a dominant phenotype is encoding a normal protein accounts for the provided by the amplification of the MDM2 lethal yellow mutant at the mouse agouti gene in sarcomas. MDM2 protein binds to and locus.5657 inactivates the tumour suppressor gene P53 Control of expression at the level of mRNA The molecular basis of genetic dominance 93

processing is illustrated by the heterochronic lethality) phenotypes. The former is attribu- (defining developmental time) C elegans gene table to stabilisation of the truncated protein lin-14. Dominant mutants, which cause the re- owing to the PEST deletion, while the latter expression of early cell lineages at later de- may result from counteracting destabilisation velopmental stages, delete the 3' untranslated of the mutant mRNA.62 J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from region (UTR) of the mRNA and lead to raised A paradigmatic example of constitutive pro- protein levels. This 3'UTR may regulate tein activation is provided by the RAS genes: export of the transcript from the nucleus, oncogenic point mutations prevent GTP hy- transcript stability, or translation.48 Splice site drolysis, thus maintaining the protein in an mutations of mRNA subject to differential activated state6'65 (fig 3A). Similarly, activat- splicing will alter the pattern of mature mRNA ing missense mutations at the 201Arg residue isoforms: this is observed at the WT1 of the Gsot protein (which stimulates adenylyl locus.35 58 59 cyclase) have been documented (as somatic mosaics) in five cases of McCune-Albright syndrome.707' Different point mutations in the adult skeletal muscle sodium channel a subu- INCREASED OR CONSTITUTIVE PROTEIN ACTIVITY nit gene SCN4A cause hyperkalaemic periodic At the protein level, increased activity may be paralysis7273 and paramyotonia congenita,74 by caused by increased half life or by loss of interfering with normal voltage sensitive inac- normal inhibitory regulation (constitutive tivation of the sodium current. In view of the activity). One class of mutations conferring differing effects of single and triple dosage of increased half life are those occurring in PEST the PMP-22 gene described above, it is inter- sequences (rich in proline, glutamic acid, ser- esting that the phenotype associated with a ine, and threonine),6' which act as recognition heterozygous missense mutation (1 6Leu -. signals for proteolytic degradation: loss of Pro) resembles that of triple dosage, that is, these sequences by C-terminal truncation sta- Charcot-Marie-Tooth disease.75 This suggests bilises the protein. Examples of PEST dele- that the missense mutation may increase PMP- tions include mutations of the CLN3 gene of 22 activity, but this has not yet been shown. Schizosaccharomyces pombe (WHI-l/DAF-1 A particularly complex spectrum of muta- cell mutants)6' and the glp-1 gene of C tions is encountered at the Drosophila locus elegans.62 glp-1is required for induction of Notch, which encodes a transmembrane germline proliferation and embryogenesis, and receptor protein that transduces a variety of the glp-l(q35) point mutation is particularly cellular signals, and includes an extracellular instructive, as it causes both semidominant domain rich in epidermal growth factor (EGF) (multivulva) and recessive (sterility/embryonic like repeats. Increased intrinsic activity is as- sociated with some of the so called Abruptex mutations: these are missense, clustered in the EGF domain, and are thought to perturb the normal balance of homo- and heterodimeric A let-60 ras protein interactions.7677 Heterozygous null http://jmg.bmj.com/ Recessive (loss of function) mutations of Notch give a *) Constitutive activation Dominant negative different phenotype, and yet other mutants exist that have recessive or dominant negative 77 GTP/GDP effects76 (see below). Another interesting binding Drosophila locus is Toll. This encodes a trans- B Toll membrane protein that provides an unusual

Recessive example of two distinct activation mechanisms on October 7, 2021 by guest. Protected copyright. f f + 4 'Class lI' dominant (fig 3B).66 "Class I" mutants are missense and Constitutive activation act constitutively, possibly owing to direct structural modulation of the protein. "Class Transmembrane II" mutants are truncations that retain the C KIT extracellular component: this activates wild Haploinsufficient type Toll by an undefined mechanism. Mu- Dominant negative tants in the class II group differ genetically EI aeuar *)(*) from other categories of active protein mutants Extracellular Transmembrane Tyrosine in that they are non-functional when heterozy- (Oligomerisation) kinase gous to a null.66 Such truncation mutants more D P53 commonly cause dominant negative effects, as described below. + +# +* ++ + Recessive * * * Dominant negative DOMINANT NEGATIVE MUTATIONS Mutational hotspots Oligomerisati on In the heterozygous state these mutants anta- Figure 3 Diagrammatic representation offour gene products, illustrating the complex gonise the activity of the remaining wild type relationship of point mutation to phenotype. The N-terminus is on the left. *indicates a allele, giving a phenotype approaching a null; missense alteration, I a frameshift or nonsense mutation leading to truncation. (A) let-60 ras.6465 Additional mutations documented in human tumours63 are denoted (*). when homozygous, or heterozygous to a null (B) Toll.66 (C) KIT.38 Truncations of the tyrosine kinase domain may have mixed mutation, they are non-functional. Hersko- haploinsufficient and dominant negative effects. Murine W mutations3 are shown in witz78 drew attention to the value of these brackets. (D) P53.67-9 Clusters of missense mutations are represented by black boxes. Four specific missense mutations tested in vitro for presence or absence of dominant mutations in experimental studies and pro- negative effects69 are shown individually. posed a classification. The major group com- 94 Wilkie

prises multimeric proteins dependent on oligo- tion.6" Thus, although P53 is conventionally merisation for activity: the presence in a mul- viewed as a "recessive" tumour suppressor timer of a mutant subunit with intact binding gene, some mutants can deregulate p53 func- but altered catalytic domains can abrogate the tion in a dominant negative fashion. In con- function of the entire multimer. For example, trast, no alteration in wild type activity is J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from if the protein normally dimerises, admixture of induced by a missense mutant associated with equal numbers of normal and mutant subunits the Li-Fraumeni syndrome, suggesting that will result in only 25% normal dimers, poten- Li-Fraumeni p53 mutants may be relatively tially causing a 75% reduction in activity. For "weak" ones.9 Note that the p53 oligomerisa- monomeric proteins, dominant negative muta- tion domain lies at the extreme C-terminus (fig tions could occur if substrate was limiting: a 3D); prematurely truncated forms cannot bind mutant able to bind the substrate, but not wild type and therefore do not act in a domin- metabolise it, would have this effect. Muta- ant negative fashion. tions of polymeric structural proteins, some- A possible example of Herskowitz's second times classed as dominant negative, are dis- class of dominant negative effect, involving a cussed separately (next section). monomeric protein, is provided by certain Dominant negative effects have been de- point mutations in the RAS gene (fig 3A).6365 A scribed in many types of protein with signal- mutant protein able to bind the guanine nuc- ling or transcriptional functions. A specific leotide exchange factor, but not be activated by example is provided by the DNA binding it, will deplete the pool of this limiting factor activity of Drosophila dorsal, mentioned pre- available for activation of normal RAS. viously, which depends on dimerisation: most Intriguingly, the dominant negative prin- mutations are true recessives, but one particu- ciple seems to have been exploited by certain lar mutant exerts a dominant negative effect. naturally occurring regulatory systems, and This is an Arg-*Cys substitution that maps to representatives of both Herskowitz's classes the DNA binding domain but does not affect are known. Belonging to the first class is the oligomerisation: it appears to act by abolishing negative regulation, by formation of inactive the DNA binding of normal/mutant hetero- heterodimers, of the transcription factors dimers.40 Similarly the more severe phenotype MyoD and c-Jun by the proteins Id and JunB associated with WT1 mutations in Denys- respectively. Id is a truncated helix loop helix Drash syndrome, as compared with Wilms's protein that forms dimers with MyoD, but tumour/genitourinary abnormalities, may be lacks the adjacent basic region required for explained by the dominant negative behaviour DNA binding.8' Similarly, critical amino acid of specific zinc finger mutations in the former substitutions in JunB abolish its homodimeri- condition: it is not yet certain whether this is sation and DNA binding, but favour forma- mediated by WT1 dimers.3536 Specific Abrup- tion of inactive JunB/c-Jun heterodimers.82 In tex missense mutations at the Drosophila Herskowitz's second class is the interferon Notch locus are dominant negative, as men- activator IRF1 and its antagonist IRF2; IRF2 tioned above.767 has enhanced DNA binding and displaces A wider variety of mutations may cause IRF1 from the interferon promoter, but is http://jmg.bmj.com/ dominant negative effects in the KIT proto- only weakly activating.83 oncogene, manifested as white spotting (W) in the mouse and in the human. KIT encodes a and dimeri- ALTERED STRUCTURAL PROTEINS sation, which occurs in response to At a simplistic level it is easy to understand binding, is essential for activity. Whereas the why mutations of structural proteins are fre-

phenotype associated with complete quently dominant: admixture of normal and on October 7, 2021 by guest. Protected copyright. KIT deletion is relatively mild in the hetero- abnormal structural components will disrupt (haploinsufficiency), point mutations the integrity of the overall structure on a involving the intracellular tyrosine kinase do- "weak links in a chain" principle. Careful cell main cause severe disease338 (fig 3C). Trunca- biochemical analysis shows a more complex tions in the same domain tend to have a vari- picture: additional modulators of the abnormal able, intermediate phenotype: although partly phenotype will include mRNA stability, and the result of haploinsufficiency, a dominant the degree of abnormality in cellular process- negative effect is probably also contributing, as ing, secretion, and extracellular incorporation seen in an analogous truncation of the fibro- into mature fibrils, of the nascent protein. blast (FGF-R).79 The Mutations of type I collagen in osteogenesis reovirus cell attachment protein provides a imperfecta (OI)8485 and of fibrillin in Marfan further example.80 syndrome" 87 provide the best studied ex- Dominant negative effects may be very im- amples; in OI, there is a reasonable correlation portant in neoplasia, a paradigm being the between predicted disruption of structure tumour suppressor P53: a wide variety of caused by point mutations and exon skips and acquired mutations has been described, the severity of phenotype.8485 By contrast, loss of many missense mutants being concentrated in function mutations have milder effects (see four clusters6768 (fig 3D). p53 oligomerises in section on haploinsufficiency).27 Other struc- vitro and can adopt two conformations, one tural proteins showing dominant mutations active and the other inactive; wild type protein include myosin heavy chains (unc-54(d) muta- is normally in the active state. Cotranslation tions in C elegans88 and hypertrophic cardio- with certain missense mutants results in mixed myopathy in humans89), and keratins 5 and 14 oligomers that adopt the inactive conforma- (Dowling-Meara ).90 The molecular basis ofgenetic dominance 95

TOXIC PROTEIN ALTERATIONS example, distinct chimeric fusion proteins The common thread to these mutations, which being associated with chronic myeloid and are usually missense, is that they cause struc- acute lymphatic leukaemia.100 These proteins

tural alterations in mono- or oligomeric pro- have a higher tyrosine kinase activity than J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from teins. These disrupt normal function and lead normal c-ABL, and may also differ in sub- to toxic precursors or waste products from side strate specificity. The PAX3 gene provides reactions that poison the cell. Dominant nega- another example. Haploinsufficiency causes tive effects are excluded. There are clear paral- Waardenburg syndrome (see above), but lels with the mutational mechanisms in true translocation to a specific region of chromo- structural proteins, but the phenotypic effects some 13 is associated with alveolar rhabdo- of toxic mutants are more unpredictable. myosarcoma.'0' One commonplace example is the sickle mutation (haemoglobin S, P6Glu--Val). (Although this shows largely recessive be- OTHER MECHANISMS OF DOMINANCE haviour, coinheritance of a second mutation In this section are summarised briefly a variety in cis (haemoglobin S Antilles, P23Val-+Ile) of other more obscure, but nevertheless inter- causes sickling to manifest in the heterozy- esting mechanisms of dominance acting at a gote9'). Other examples include missense mu- cellular level. tations of C elegans degenerins, mec-4 and Position effect variegation in Drosophila is the deg-1, which cause specific neuronal cells to variable reduction in expression of a gene swell up, vacuolate, and lyse92; a point muta- juxtaposed to heterochromatin by chromo- tion in mouse -related protein-I some rearrangement. Variegating mutations (Light mutant) that disrupts melanosome are generally recessive in that they reduce structure93; and various point mutations in expression only from the rearranged (cis) chro- rhodopsin associated with slow degeneration mosome. The brown locus is unusual in that of rod photoreceptor outer segments.37 A par- expression is also reduced from the normal ticularly striking group comprises the domi- (trans) allele. This dominant effect seems to nantly inherited hereditary amyloidoses, a di- depend on somatic pairing between the homo- verse collection of diseases associated with logous , but the mechanisms of alterations in the structure of soluble proteins this and other 'trans sensing' effects are still that increase stability of the protein and uncertain. 102 103 predispose to multimerisation. Proteins impli- The phenomenon of nucleolar dominance in cated include transthyretin, ,B amyloid precur- wheat reflects the relative expression of tan- sor protein, gelsolin, , prion protein, dem ribosomal DNA from allelic loci. Expres- apolipoprotein AI, lysozyme, and fibrino- sion at an individual locus correlates with the gen.94-96 number of upstream regulatory sequences. These appear to compete for binding to limit- ing amounts of an activating protein, so that NEW PROTEIN FUNCTIONS the more repeats present, the greater the likeli- The creation of new, advantageous protein hood of activation.'04 http://jmg.bmj.com/ functions by mutation is the life blood of Segregation distortion loci subvert the nor- evolution, but occurs over a protracted time mal pattern of 1:1 gametic segregation, leading scale. Proteins with truly new functions are to meiotic drive. This may occur either at only rarely encountered in natural human mu- , when some property of the general tation and are usually pathological. Two cat- structure or size of a chromosome gives it a egories may be recognised; missense mutations replication advantage on the spindle (chromo- with specific functional effects, and assortative somal drive), or postmeiotically, when direct on October 7, 2021 by guest. Protected copyright. shuffling of exons. In protein engineering, competition between the occurs which seeks to accelerate the evolutionary pro- (genic drive).'05 This may allow disadvanta- cess and develop proteins with new functions, geous mutations to spread through the popula- the same principles apply in the design of new tion, by virtue of close linkage to the drive mutants.97 locus. A well known example is the t complex The serine protease inhibitors (serpins), of mouse. popular targets for protein engineering, pro- Unlinked non-complementation occurs when vide perhaps the best natural example involv- heterozygous mutations occur at two genes ing a missense mutation. A (358Met-+Arg) coding for interacting proteins. Whereas the substitution in a l antitrypsin converts its ac- heterozygous state for either locus on its own is tivity to antithrombin, by altering the specifi- silent, concurrent mutations at both loci cause city of the active site.98 Another example, iden- the phenotypic threshold to be exceeded, and tified only in vitro, is a missense mutant the disease becomes manifest. Examples in- protein (mev) that facilitates cellular uptake of clude the interaction of a and 3 tubulin muta- mevalonate99; the wild type protein lacks this tions in Drosophila'06 and, more speculatively, activity, but its normal function is unknown. the enhanced severity of dystrophin mutations The juxtaposition of domains from different in trans to an abnormal allele for autosomal proteins to generate potentially new functions recessive Fukuyama congenital muscular dys- is best illustrated by the chimeric fusion pro- trophy. 107 teins produced by some oncogenic chromo- An allied phenomenon, called negative com- some translocations.5354 The c-ABL/BCR plementation or metabolic interference, occurs fusion products in the 9;22 Philadelphia trans- when two alleles at the same locus interact to location provide the most well characterised give a more severe phenotype in the compound 96 Wilkie

heterozygote than in either homozygote. For different mutational spectrum may be antici- example, Abruptex (Abx) mutations of the pated in different diseases, according to their Drosophila Notch gene fall into two genetic cellular mechanism. A wide variety of muta- types, "enhancers" and "suppressors" of tions cause loss of function: disease genes with Notch. Homozygotes for either type are viable a high mutation rate will often be haploinsuffi- J Med Genet: first published as 10.1136/jmg.31.2.89 on 1 February 1994. Downloaded from (characterised by gapping of the wing cient and be involved in regulatory pathways veins), yet compound enhancer/suppressor Abx or act as tumour suppressors or both. A search heterozygotes are lethal.7677 Metabolic interfer- for constitutional chromosomal abnormalities ence may theoretically result in various patterns (deletions, translocations), which provide such of phenotypic segregation'08 and has been an invaluable resource for disease location and invoked to explain the atypical inheritance of positional cloning,' '4 is much more likely to be several human genetic diseases; none has yet successful in this group than in the "gain of been corroborated at the molecular level. function" categories. By contrast, acquired chromosomal abnormalities in neoplasia may often pinpoint specific oncogenes involved in "gain of function" transformation. The pheno- DOMINANT INHERITANCE, WITHOUT type associated with missense mutations will DOMINANCE AT A CELLULAR LEVEL usually be critically dependent on their exact Although the vertical transmission of an ab- position and nature, except in structural pro- normal character is usually assumed to imply teins; hence multiple, independent point mu- dominance of the mutation at the cellular level, tations as a cause of dominant disease are most this is not always the case. In humans, two commonly encountered in such proteins. exceptions are sufficiently important to have In understanding mechanisms of cancer, the been included in the table: recessive antionco- dominant negative effects illustrated for p53 genes and imprinted loci. may occur in other tumour suppressor genes. Retinoblastoma provides the paradigmatic For instance, germline mutations of the APC example of a phenotype that segregates in a gene cause familial adenomatous polyposis/ dominant pattern, yet is the result of a muta- Gardner's syndrome, and somatic mutations tion (in the RB1 gene) that is recessive at a occur in sporadic colon cancer. The amino acid cellular level. Cells carrying a heterozygous sequence of APC predicts that it will form RB 1 mutation are entirely normal, but a coiled coils, structural elements that permit "second hit" somatic mutation of the normal oligomerisation. 15 116 The majority of APC allele in at least one retinal cell (a relatively Ana- mutants, both germline and somatic, are mis- likely event) causes retinoblastoma.'09110 sense17 118 and some could disrupt normal oli- logous putative "antioncogenes" or "tumour gomers to give dominant negative effects. suppressors" have been cloned in several other Analysis of the particular mutations present dominantly inherited cancer syndromes, may therefore guide prognosis. including Li-Fraumeni syndrome (P53), neuro- The mechanisms of dominance in con- fibromatosis types 1 and 2, familial adenoma- ditions associated with unstable triplet repeats tous and Von Hippel-Lin- polyposis (APC), (for example, fragile X syndrome, myotonic http://jmg.bmj.com/ dau disease. At the cellular level, evidence for a dystrophy, and Huntington's disease) are not purely recessive mechanism of gene action is, yet clear, and probably heterogeneous, with however, less certain than with RB 1, and vary- effects owing to alterations in both mRNA ing contributions from haploinsufficient and expression and protein function. Although the dominant negative effects are possible, as dis- (CGG)n expansion in the fragile X syndrome is cussed for P53 and APC. associated with DNA methylation and absence Genomic imprinting may give rise to a com- 9 of FMR- 1 gene expression,' in myotonic dys- on October 7, 2021 by guest. Protected copyright. plex pattern of dominant inheritance. If a gene trophy, DMK alleles containing (CTG)n is transcribed only from the chromosome ori- expansions may actually be overexpressed'20 ginating from one of the two , the locus (although this is disputed'2' 122). Other poten- is effectively hemizygous. Mutation of the tial variables are whether the expanded triplet allele on the 'active' chromosome will com- lies in the coding or non-coding region of the pletely inactivate the locus, whereas mutation protein, and the sequence of the repeat itself.'23 of the allele on the other chromosome will have Complete elucidation of the mechanisms of no phenotypic effect. Apparent dominant with but this dominance associated triplet repeat transmission of the disorder can occur, expansion may well yield some surprises. will show dependence on the sex of the trans- Finally, an understanding of the molecular mitting . Representative pedigrees are mechanism of a disease is a prerequisite for provided by transgenic mutation of the mouse attempting . Nearly all diseases insulin-like growth factor-II gene,"1' and in currently targeted for gene therapy are reces- the human diseases Beckwith-Wiedemann sive,'24 in which the goal is simply to replace syndrome"2 and hereditary paraganglioma."3 the missing product. It should be evident that most categories of dominant disease pose a formidable challenge to gene therapy, but Perspectives on human genetic disease already the "molecular engineers" are contem- Although this classification may initially ap- plating strategies to overcome these problems. pear to be an academic exercise, appreciation Examples include antisense RNA therapy to of these various mechanisms is helpful for antagonise selectively the action of dominant thinking about disease processes. For example, negative mutants; or conversely, the introduc- perusal of the table and fig 3 indicates that a tion of such mutants to counteract the effects The molecular basis of genetic dominance 97

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