POLYALANINE TRACT DISORDERS AND NEUROCOGNITIVE PHENOTYPES

Shoubridge C, Gecz J.

Expansion of polyalanine tracts cause at least 9 inherited human diseases. Eight of these nine diseases are due to expansions in transcription factors and give rise to congenital disorders, many with neurocognitive phenotypes. Disease-causing expansions vary in length depending upon the gene in question, with the severity of the associated clinical phenotype generally increasing with length of the polyalanine tract. The past decade has seen considerable progress in the understanding on how these mutations may arise and the functional effect of expanded polyalanine tracts on the resulting protein. Despite this progress, the pathogenic mechanism of expanded polyalanine tracts contributing to the associated disease states remains poorly understood. Gaining insights into the mechanisms that underlie the pathogenesis of different expanded polyalanine tract mutations will be a necessary step on the path to the design of potential treatment strategies for the associated diseases.

INTRODUCTION

Expansions of tri-nucleotides sequence in the genome above a certain length have been linked to a growing number of human diseases. Unstable tri-nucleotide repeats can be located in either noncoding sequences, transcribed but not translated sequences or within translated sequences for homomeric stretch of either glutamine or alanine amino acids. There are nine hereditable disorders caused by the expansion of polyalanine tracts.1 In the human proteome approximately 500 proteins contain polyalanine tracts of greater than 4 residues, with a quarter of these containing a tract of seven or more uninterrupted alanines but not exceeding 20 residues.1,2 These proteins are particularly enriched for transcription regulators and in particular homeobox containing proteins, which account for the largest single functional group of proteins containing polyalanine tracts. Eight out of nine polyalanine tract expansion disorders are associated with transcription factors, four of which are homeobox genes (Table 1). The disease causing expanded polyalanine tracts are stably transmitted across multiple generations1 and generally lead to congenital disorders arising from a loss of function of transcription factors during development.

Table 1. Diseases caused by expanded polyalanine tract mutations.

Table 1

Diseases caused by expanded polyalanine tract mutations.

Expansions of polyglutamine tracts also form the basis of several human diseases.3 Unlike the congenital disorders of the expanded polyalanine tracts, these diseases are characterised by expansion of unstable tri-nucleotide repeats4 and result in neuronal dysfunction from mid-life progressing to severe neurodegeneration.5 The large expansions of polyglutamine tracts are thought to contribute to the generation of mis-folded protein intermediates that eventually lead to aggregates in susceptible neuronal sub-types, a hallmark of the associated disease states.6,7 In contrast to polyglutamine disorders the human data on in vivo aggregation of expanded polyalanine tract mutant proteins are not yet available. The only exception is PABPN1, a protein involved in mRNA polyadenylation, in which expanded polyalanine tract mutations cause later onset oculo-pharyngeal myotonic dystrophy (OPMD). OPMD is clinically associated with the presence of pathological filamentous inclusions in muscle fibre nuclei.8,9 The challenge remains to identify the pathogenic mechanism(s) underlying expanded polyalanine tracts that contribute to associated disease states, knowledge required for the design of potential therapeutic strategies.

CONGENITAL DISORDERS CAUSED BY MUTATIONS IN GENES WITH EXPANDED POLYALANINE TRACTS

Of the nine hereditary diseases caused by expansions of polyalanine tracts, eight are congenital disorders (Table 1).1 The diseases range from disturbances to the body plan, incorrect development of reproductive structures including the ovaries, limbs, bones and the autonomous ventilation system as well as the incorrect development and function of the brain including the hypothalamic—pituitary axis (Table 1). Two of the six transcription factors are located on the X-chromosome and as such the hemizygous males are affected and heterozygote female carriers are normally asymptomatic. The remaining six of the eight transcription factors and the remaining member, a poly(A) binding protein PABPN1, mutated in these diseases are located on autosomes. The mode of transmission for these is generally autosomal dominant (with varying degrees of penetrance) meaning heterozygotes carrying the mutant allele present clinically with the disease.

Complete penetrance of autosomal dominant diseases occurs due to mutations in HOXA13, ZIC2 and PABPN1. Expansion mutations have been identified in each of the three large polyalanine tracts (14, 12 and 18 residues) of HOXA13, with all mutations leading to hand-foot-genital syndrome.10-14 The same clinical outcome is also observed due to mutations deleting or truncating HOXA13. Similarly, heterozygous loss of function mutations due to frameshift and truncation mutations indicate haploinsufficiency of ZIC2 contributes to the aetiology of Holoprosencephaly (HPE).15 The same clinical phenotype also manifests in patients with a loss or partial loss-of-function of the transcription factor due to expanded polyalanine tract mutations interfering with DNA binding and transcriptional activation.16 The only adult onset disease caused by expanded polyalanine tract disorders is OPMD due to mutations in PABPN1 (Table 1). Moreover, unlike all the other genes with disease causing expanded polyalanine tracts, PABPN1 is not a transcription factor but rather a protein involved in polyadenylation of mRNA precursors.17 This protein contains a single polyalanine tract of 10 residues that when expanded cause OPMD18 (Table 1). Inheritance of OPMD is generally in an autosomal dominant fashion with heterozygous mutation carriers displaying two to seven additional alanines in this tract. A polymorphism of one additional alanine residue is noted in two percent of the population. In the homozygous state this polymorphism results in autosomal recessive OPMD, while as a compound heterozygote with a pathogenic mutation of two additional alanines, this polymorphism results in a more severe phenotype.18 The only other type of disease causing mutation in this gene also results in an expanded polyalanine tract, although by a 35G-C transversion.19 This mutation alters the glycine residue immediately 3-prime of the alanine tract to an alanine. As this residue is followed by a further two alanine residues in the normal protein, this mutation essentially results in an uninterrupted tract of 13 alanines.

Individuals with expanded polyalanine tract mutations often display milder phenotypic consequences compared to individuals with complete loss of function of a given gene. However, the polyalanine tract mutation phenotype is often coupled with broader clinical features. In some cases these mild features may be difficult to recognise on the more severe background of a complete loss of function. However, in some instances these findings imply that in addition to the loss (or partial loss) of function of the mutant allele, these expanded polyalanine tracts may in some cases confer a gain of abnormal function on the protein. One such example is a deletion of the RUNX2 locus which cause the severe skeletal abnormality Cleidocranial Dysplasia (CCD).20,21 RUNX2 has a tract of 23 uninterrupted glutamine residues followed by 17 uninterrupted alanine residues on the N-terminal side of the functional Runt domain.20 When an alanine tract is expanded by recurrent 30 bp duplication resulting in 10 additional alanine residues, the affected individuals have a phenotype of minor craniofacial features of CCD, with the additional features of brachydactyly of hands and feet.20 In the case of FOXL2, 30% of all mutations in this gene are expansions of the single 14-residue polyalanine tract to 19 or 24 residues.22 Although phenotypic variability is noted in patients with these mutations, the predominate disease outcome is Blepharophimosis ptosis epicantus inversus syndrome (BPES-II). This is a less severe form than BPES-I more commonly caused by mutations leading to PTC and or loss of function of the forkhead domain of FOXL2.23 Essentially, BPES-I is the likely outcome of a null allele of FOXL2 where BPES-II is likely due to a hypomorphic allele.24 Further to this, despite BPES being an autosomal dominant disorder, a recent report observed autosomal recessive inheritance in homozygotes with the Ala19 allele.25

The penetrance of the disease and severity of the clinical phenotype increases with the length of the expanded polyalanine tract in several genes including HOXD13 and PHOX2B.26,27 HOXD13 has three polyalanine tracts, two less than seven residues and one with 15 residues. Expansions to the longest tract were first identified in families with Synpolydactyly (SPD).28,29 Subsequent analysis identifying a range of mutations expanding the longest polyalanine tract with variable clinical phenotypes and truncation and amino acid substitution mutations in patients with SPD1 (Table 1).26,30-32 In patients with the smallest expansion mutations, phenotypic variation is not only seen within families (intrafamilial) but also within individuals, such that one hand or foot can be affected while the other is completely normal.26 In the case of PHOX2B, frameshift mutations and a variety of mutations expanding the longest of two C-terminal polyalanine tracts (Table 2) are associated with Congenital Central Hypoventilation syndrome (CCHS).27,33-35 The severity of the respiratory phenotype, related symptoms and age of onset, all correlate with increasing length of expanded polyalanine tract in PHOX2B.27

Table 2. Proposed mechanisms of polyalanine tract mutations.

Table 2

Proposed mechanisms of polyalanine tract mutations.

The remaining two transcription factors, ARX and SOX3 are located on the X-chromosome. With only one X chromosome active in any given cell, X-linked inheritance is more complex than simply grouping as either dominant or recessive.36 In males, all cells have the same copy of the maternal X-chromosome, while in females dosage compensation occurs as a result of random inactivation of one of the two X-chromosomes in every cell. Disease phenotypes due to polyalanine tract expansion mutations in both these X chromosome genes are predominantly seen in males while the heterozygous female carriers are generally not affected or have very mild clinical presentations.37-39

When looking at the X-linked genes ARX and SOX3 we can see an emerging genotype-phenotype correlation between the length of the polyalanine tract expansion and the severity of the disorder. Of the four polyalanine tracts in ARX, the two N-terminal tracts are expanded contributing to ~60% of all mutations reported in this gene.40 Expansions of the first polyalanine tract from 16 to 17, 19, 23 and 27 alanines result in increasingly severe phenotypes from nonsyndromic intellectual disability, to X-linked infantile spasms and Ohtahara Syndrome.40 The second polyalanine tract is also expanded by mutations increasing the 12-residue tract to 20 and 27 residues.40-42 Expansion of the second polyalanine tract by a 24 bp duplication is the most frequent mutation reported for ARX and leads to an unusual breadth of variation in the clinical presentations, both within and between families.37 Interestingly, a duplication of 33 bp in the same tract gives rise to a less severe phenotype than would otherwise be expected.43 While the overall number of alanine residues increases from 12 to 22, the tract is interrupted by a glycine after the tenth alanine residue. This glycine interruption likely ameliorates the severity of this polyalanine tract expansion by a yet to be defined mechanism. It is difficult to determine whether the expanded polyalanine tract mutations in ARX cause disease due to a dominant gain of function or due to a loss of function. A complete loss of function is unlikely given the comparison with loss of ARX function mutations that result in severe brain malformation phenotypes, including lissencephaly, hydranencephaly and agensis of the corpus callosum.44,45 Although partial loss of function is implied by the formation of aggregates in the nucleus and cytoplasm,46,47 a loss of transcriptional activity due to the two most frequent expanded polyalanine tracts in ARX is not supported by cell based assays.48

In the case of SOX3, over-dosage due to a duplication of the gene, as well as expansion of the first of four polyalanine tracts lead to X-linked hypopituitarism (XH), with variable pituitary deficiency and incompletely penetrant mental retardation.49,50 When the 15-residue tract is expanded by seven alanines, the patients have XH with normal cognitive function. Expansion by 11 residues leads to XH with isolated growth hormone deficiency and X-linked intellectual disability. Experimental evidence indicates that both expansions of the polyalanine tract in SOX3 are associated with decreased activity of the transcription factor.50,51 Hence, disturbance to the activity levels of SOX3, either by partial loss of function of the protein due to expanded polyalanine tract mutations or duplication of the whole gene both contribute to similar disease phenotypes.

DNA MUTATION MECHANISM(S) UNDERLYING POLYALANINE TRACT EXPANSION

The size of disease-causing polyalanine tract expansions in the nine genes reported to date varies across these genes (Table 2). The fine balance between the normal and disease-associated variation in function might be demonstrated by expansion of just one extra alanine of the 16-residue polyalanine tract of ARX, which causes nonsyndromic X-linked intellectual disability.40,52 The largest expansions of 14 residues occur in both HOXA13 and HOXD13 and lead to tracts of 32 and 29 uninterrupted alanines, respectively.14,26 The longest polyalanine tract reported to date is a 33-residue tract in PHOX2B in patients with congenital central hypoventilation syndrome.53

Both replication slippage and nonhomologous recombination (or unequal cross over of mis-paired alleles) have been proposed to explain the increase in tract length. Understanding the mechanism responsible for causing the expansion of these tracts has often been confounded by the sequence of these tracts themselves. In all nine genes, the polyalanine tracts themselves are coded, to at least some degree, by imperfect tri-nucleotide repeats. The majority of the mutations leading to disease causing expansions have been identified as partial direct repeats of these imperfect tri-nucleotide repeats, often with several sites of insertion (Table 2). Mis-pairing of normal alleles followed by unequal cross-over events54 provide a rational explanation for these types of expanded tracts. Further to this, some cases of a recurrent alanine tract expansion in ZIC2 are suggested to arise due to errors in somatic recombination (i.e., mitotic rather than meiotic) in fathers of affected individuals.55

On the other hand, a portion of polyalanine tracts are encoded by repeats of a single codon, often (GCG). When disease-causing mutations lead to in-frame duplication, the underlying mechanism is more difficult to attribute. For example, the first polyalanine tract of ARX is made up of a (GCG)(GCA)(GCG)10(GCA)(GCG)(GCG)2 coding for a 16 polyalanine tract.41 This tract is expanded due to an in frame insertion of seven (GCG) codons resulting in the tract of 23 alanines.56 Replication slippage, although possible, is an unlikely mechanism in this type of case. The mutation is stable across multiple generations in affected families, implying this expanded tract is stable during both meiosis and mitosis. Furthermore, the sequence this insertion arises from is much shorter than the 34-38 uninterrupted repeats that would be expected to cause replication slippage.57 A recent mutation in this polyalanine tract of ARX identified the inclusion of two imperfect tri-nucleotide repeats as well as nine of the 10 GCG repeats ((GCG)(GCA)(GCG)9) leading to a 27 polyalanine tract.58 This type of mutation is consistent with unequal cross over between mis-paired normal alleles and suggests the insertion of seven (GCG) codons could just as likely arise due to this type of mechanism.

A similar scenario is observed for the expansion of the only polyalanine tract in PABPN1. The 10 alanines within this tract are encoded by (GCG)6(GCA)4 with the first mutations identified as expansion of the (GCG) codon by as little as two and up to eight leading to (GCG)8-13 range of expansion mutations.18 Again, replication slippage has been touted as a possible mechanism underlying the mutations in this gene. Diagnostically, mutations in this gene have often been analyzed as an increased size of a specific PCR product containing the expanded region. However, closer scrutiny of these expansion mutations in a cohort of OPMD patients by direct sequencing has revealed that a third of these mutations were interspersed with a GCA codon19 and not just expansions of the GCG repeat previously reported.18 In agreement with the case for some mutations in ARX, the expansion of PABPN1 is likely due to rare, but stable events of unequal recombination of mis-aligned normal alleles for both GCG alone and GCA containing expansions.

Several polyalanine tract expansion mutations, however, can not be adequately explained by the process of unequal cross-over.26,59-61 For example, in 86 OPMD patients with expansion mutations in PABPN1 there were 13 different types of expansions, seven of which were uninterrupted GCG repeats and six had GCA interspersed in the repeat. Using a theoretical model of unequal cross over of mis-paired normal alleles based on the seven mutations with an interspersed GCA, 12 of the 13 different types of expansion mutations could be accounted for.60 The (GCG)13 allele comprises an addition of seven (GCG) codons to the (GCG)6(GCA)4 sequence and as such is not readily explained by unequal cross over. This mutation has been suggested presumably to arise due to slippage or a combination of unequal cross over and slippage.60 Similarly, there are several mutations for which recombination events cannot be deduced from the observed sequence in patients with CCHS due to expanded polyalanine tracts in PHOX2B.53 These particular mutations and the presence of somatic mosaicism in some cases support a mutational mechanism that may involve misalignment (either template or nascent strand) within a slippage model, resulting in either contraction or expansion due to replication if the unpaired sequence is not repaired.61

Fork stalling and template switching (FosTeS)62 has been suggested as an alternative mechanism to account for expansion mutations that do not fit with recombination events. Essentially, FosTeS utilizes microhomologies of just a few base pairs that act as bridges for the DNA replication fork to skip (forward or backward) along the chromosome when encountering complex genomic architecture or DNA lesions.63 This FosTeS mechanism has been suggested from the sequence of a polyalanine expansion identified in a mouse with a spontaneous mutation in the Hoxd13 gene.64 Spdh mice modeling the most common mutation in human SPD have a 21 bp in frame duplication in the polyalanine stretch of exon-1.65-67 This expansion is a straightforward reduplication of a short segment of the imperfect tri-nucleotide repeat. In contrast, the expansion mutation in Dyc mice appears to have resulted from two smaller, more complex duplications.64 When applied to the expansion in Hoxd13 in Dyc mice, FosTeS could potentially explain the sequence of the expansion. Moreover, polyalanine expansions not explained by unequal cross-over in other genes could, with only one exception, be accounted for by this complex mechanism64 (Table 2). The one exception not explained by any one of the mechanisms discussed so far is the expansion of the 15 polyalanine tract in HOXD13 by an additional 9 residues. As suggested when this mutation was first identified this particular expansion could only arise if a point mutation in one of the alanine codons has occurred in addition to either unequal crossing over or FosTeS to give rise to the duplication.26,64

PATHOGENIC MECHANISMS OF POLYALANINE TRACT EXPANSIONS

The predicted mechanism of protein dysfunction for these autosomal dominant and X-linked disorders arising from expanded polyalanine tract mutations ranges from complete or partial loss of function, dominant negative effect or a gain of function.68 In the case of autosomal genes, dominant transmission of disease traits due to expanded polyalanine tracts means that heterozygotes with one wild-type allele and one mutant allele will be affected. Hence, cells specifically expressing the genes in question will essentially have both wild-type and mutant protein present in the same cell. The potential exists for expanded polyalanine tracts in the mutant protein to sequester the wild-type protein and/or other factors. This interference with either the localisation or function of these normal proteins is likely to confound the pathogenic effect of the mutation and contribute to the disease phenotype due to a dominant negative effect. This scenario is observed with longest expansion of PHOX2B, which exerts a partial dominant negative effect over the wild-type protein in addition to functional haploinsufficiency.69 A dominant negative mechanism of these expanded polyalanine tract mutations may extend to include sequestration of other proteins required for cell function. Expanded polyalanine tract mutations in HOXD13 are suggested to have a dominant negative effect not only over wild-type HOXD13, but over other wild-type HOX proteins as well.66 In support of this suggestion, mice with inactivation of the Hoxd13 alleles have a milder phenotype than encountered in human patients heterozygous for expanded polyalanine tract mutations. However, mice with inactivation of multiple Hoxd genes together (Hoxd11, Hoxd12 and Hoxd13) have SPD-like malformations in keeping with the associated clinical outcomes in patients with expanded polyalanine tract mutations in HOXD13.70

An emerging theme over the last few years has held that expansion of polyalanine tracts above a certain threshold results in degradation of the mutant protein. Depending upon the efficiency of this process, the length of the expansion in the mutant protein and overall expression levels, aggregation of the protein may occur. The threshold at which these events occur differ between proteins, but are a common finding in over-expression studies in routine and explant cell culture for FOXL2,71 HOXD13, SOX3, RUNX2, HOXA13,72 PHOX2B69 and ARX.46,47 Although these types of studies indicate aggregation is a key functional consequence of polyalanine expansion mutations, the contribution of these aggregates to the pathogenesis of disease remains to be demonstrated.

As more and more disease-causing expansions to polyalanine tracts have been identified many have begun to investigate the impact of tract length on localisation, aggregation and transcriptional activity of the mutant proteins. Using a range of different length polyalanine tracts generated as fusion constructs with GFP, Moumne and colleagues73 examined a range of constructs with normal and expanded tracts of FOXL2 identified in patients, as well as tracts expanded well above the number seen even in other disease states. The wild-type tract of 14 residues in FOXL2 is expanded to 19 and 24 in human disease (Table 2). The data showed the longer the tract, the higher the propensity of the mutant protein to aggregate and mis-localise to the cytoplasm. When the longest tract of 37 alanines in total was tested, all cells transfected had abnormal cytoplasmic sub-cellular localisation of the mutant protein.73 Functional analysis of a range of expanded polyalanine tracts in FOXL2 revealed a clear correlation between transcriptional activity of FOXL2 and the resulting clinical phenotype.74 Variants that cause the more severe type 1 BPES had significantly reduced transcriptional activity in Luciferase reporter assays using two specific reporter systems. In contrast, mutations leading to the less severe type II BPES had activity levels not different from wild-type levels. The mutation that is known to give rise to both types of BPES syndromes interestingly displayed intermediate effects on the transcriptional activity in this analysis.74 In addition to this functional haploinsufficiency, co-aggregation of wild-type protein is also noted for expanded polyalanine tract mutations in FOXL2,71 indicating a partial dominant negative effect may contribute to the dominant inheritance of BPES.

A similar disruption to transcriptional activity has been reported in several other proteins containing expanded polyalanine tracts including SOX3,51 PHOX2B69 and ZIC216 (Table 3). In the case of mutations in PHOX2B, the transcriptional activity on the regulatory regions of DHB and PHOX2A target genes was increasingly impaired as the length of the polyalanine tract expanded, causing both cytoplasmic retention and aggregate formation.69 Subsequent studies indicate upregulation of heat shock response prevents the formation and induces clearance of existing cytoplasmic aggregates of mutant protein.75 Components of the heat shock response pathway and members of the proteasomal machinery have been colocalised to aggregates of PABPN1, ARX and PHOX2B with expanded polyalanine tract mutations.9,46,47,53,76 Increased expression of HSP70 and recruitment to the nucleus has been implicated in reducing aggregation of ARX and PABPN1 expanded polyalanine mutants and alleviating the incidence of aggregate related cell death.46,76 Similarly, up-regulation of the heat-shock response by the application of geldanamycin prevented formation of aggregates, was able to clear preformed aggregates and partially restored transactivation activity mutant PHOX2b.75

Table 3. Effects on protein activity due to expanded polyalanine tract mutations.

Table 3

Effects on protein activity due to expanded polyalanine tract mutations.

A potential sequence of events has been described by the work of Albrecht et al65 outlining that although mutant protein may initially be translocated to the nucleus, prolonged exposure or high expression levels may cause the formation of small aggregates in perinuclear region. In turn these can grow to large inclusions around the nucleus as they trap mutant and in the case of some autosomal genes, wild-type proteins alike. Eventually, these events will hinder adequate levels of these transcriptional factors to reach the nuclear environment and interact with specific targets, leading to partial or complete loss-of-activity. This model easily accommodates the observation that mutations leading to larger expanded tract length often result in more severe phenotypes as well as the apparent dominant negative aspect of some autosomal diseases. Although appealing, this common pathogenic mechanism needs to be considered in the context that human data on in vivo aggregation of proteins with expanded polyalanine tracts is not available.

The only exception is the presence of pathological filamentous inclusions in muscle fibre nuclei in patients with OPMD due to expanded polyalanine tract mutations in PABPN1.8,9,18,77 Mis-folding and aggregation of mutant PABPN1 protein underlies the formation of insoluble inclusions which in turn sequester poly(A) RNA.8 A range of cellular factors including molecular chaperones, components of Ubiquitin-proteasome pathway and transcription cofactors are also caught within these intranuclear inclusions. Despite the possible contribution of this sequestration to disease outcomes, a loss of function in OPMD due to decreased availability of PABPN1 has been suggested.78 Expansions to the polyalanine tract in PABPN1 alter the protein conformation and change protein binding properties of interacting proteins.79 Hence, the likely pathogenic mechanism causing OPMD is a loss of function of PABPN1. This loss of function may be due to the expanded polyalanine tract leading to confirmation changes of the mutant protein and contributing to a combination of aggregation and altered protein-protein interactions.79

A growing number of the polyalanine tract expansion disorders are being modelled in mice, providing an avenue to address the role of aggregates or mis-localised mutant proteins and investigate mechanisms contributing to the disease phenotype. Heterozygous and homozygous mice generated to express a patient relevant expansion to the polyalanine tract of Hoxa13 showed no phenotypic differences to mice with deletion of this gene,13 supporting the observations in affected patients.10 Limb buds in mice with the expanded allele had normal mRNA expression and splicing but had reduced protein levels, suggesting the loss of function of the mutant protein is due to reduced levels of expanded protein, likely due to increased degradation of the mutant protein.13 However, the sub-cellular localisation or aggregation of the mutant protein in these animals was not reported. In the case of ARX there are currently two, independently generated mouse models of the expansion in the first polyalanine tract from 16 to 23Ala.80,81 Both of these genetic mouse models recapitulate many of the phenotypic features of affected human individuals and support a partial loss-of-function of the mutant Arx protein. Closer examination identified a selective reduction of Arx-positive GABAergic interneurons in the striatum of mutant mice. At the cellular level mutant Arx protein was mis-localised to the cytoplasm in 55% of interneurons throughout the cortex when examined in mature/adult mice,80 supporting previous in vitro findings.47 In contrast, no specific formation of intranuclear inclusions (or cytoplasmic accumulation) occurred in migratory cells positive for Arx expression in the ganglionic eminence at E12 and in the cortical cells at P0.81 Moreover, no specific aggregation of Arx protein was noted when the more frequent c.429_452dup was modeled.81 If aggregate formation is not an obvious pathogenic hallmark explaining the loss of function of expanded polyalanine tract mutations of ARX then alternative mechanisms such as partial loss of function, aberrant protein-protein interactions, or protein-degradation remain to be investigated.

There are examples supporting alternative pathogenic mechanisms to aggregation of expanded polyalanine tract containing proteins contributing to disease. Increases in the polyalanine tract of ZIC2 (expanded from 15 to 25 alanines) result in no difference in localisation of the mutant protein compared to the wild-type ZIC2 protein.16 Instead of aggregation leading to reduced levels of protein reaching the specific DNA target the reduced transcriptional activity of mutant ZIC2 is instead linked to altered binding of the protein to specific DNA targets.16 An elegant study recently identified, in mice homozygous for expanded polyalanine tract in hoxd13, the phenotypic consequences that arise from mutant Hoxd13 altering the regulation of a rate-limiting enzyme involved in the production of retinoic acid (RA). The subsequent, reduced levels of RA in the limb buds do not adequately repress chondrogenesis, leading to accelerated and uncontrolled differentiation of interdigital cells into chondrocytes and hence, fused digits.82

CONCLUSION

Despite the considerable progress made in recent years of understanding how expanded polyalanine tract mutations may arise, the contribution of these mutations to disease pathogenesis still remains poorly understood. Although there is a range of mechanisms potentially contributing to diseases associated with expanded polyalanine tract mutations, the loss of function appears to be a common cause. The mechanisms underlying this loss of function, however, remain to be demonstrated in most cases. A dominant negative effect in addition to functional haploinsufficiency is also likely to underpin the pathogenesis of several expanded polyalanine tract diseases. Expanded polyalanine tracts may cause increased degradation of the mutant protein, interference in the efficient binding to protein partners and/or binding to specific DNA targets required for normal transcription factor activity, thereby contributing to the disease pathogenesis.

Increasingly sophisticated techniques modelling expanded polyalanine tract mutations in the correct cellular context are likely to be required to reveal at least part of the pathogenic mechanisms contributing to the associated diseases. Unfortunately, this gene-specific strategy means a 'magic bullet' solution to these debilitating diseases caused by expanded polyalanine tract mutations is unlikely. Despite this rather bleak outlook, some hope can be derived from the early success of approaches in other neurocognitive disorders, often focusing on common sets of targets or pathways impacted by a range of disease states. Although not the focus of this chapter, several recent examples help to illustrate this concept. In the case of Fragile X syndrome (FXS), the loss of FMRP activity is predicted to result in unchecked mGluR-dependent protein synthesis, which in turn contributes to the pathogenesis of FXS.83 The understanding of the basic disease pathogenesis of FXS lead to a potential therapeutic avenue of chronically down-regulating Gp1 mGluR signalling to correct, at least in part, phenotypic outcomes of FXS.84 Similarly, the superfamily of histone deacetylases have also been put forward as potential targets of therapeutic intervention in diseases such as cancer and more recently disorders of the central nervous system, in particular Fragile X syndrome and polyglutamine diseases such as Huntington's disease and spinocerebellar ataxia.85 One lesson to be taken from these studies is that a physiologically integrated picture surrounding the expanded polyalanine tract mutations in each gene will be necessary for potential therapeutic approaches to be devised. A potential caveat would be that understanding the pathogenesis of each disease in turn might highlight areas of commonality that may be amenable to intervention, similar to the interrogation of the current data on molecular mechanisms to identify therapeutic strategies for the polyglutamine diseases.7

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