Abstract
Beside the well-known polyglutamine expansions involved in several neurodegenerative disorders, convergent recent findings pointed to the expansion of polyalanine stretches as a disease mechanism in congenital malformations, skeletal dysplasia and nervous system anomalies. Polyalanine stretches have been predicted in roughly 500 human proteins among which nine have been ascribed to disease phenotype by expansion of polyalanines. The function of polyalanine stretches is largely unknown. This paper aims to review the rapidly growing evidences for a disease-causing mechanism common to expansion of homopolymeric tracts whatever the amino acid involved is.
The identification of a new type of mutation, by expansion of CAG trinucleotide repeats coding for polyglutamines in neurodegenerative disorders in human, inspired much enthusiasm in the 1990s. Polymerase slippage has long been assumed to be the disease-causing mechanism. Therefore, imperfect trinucleotide repeats have been discounted as candidate loci for such mutations (reviewed in 1). Some years later, however, imperfect nucleotide repeat expansions encoding alanines were shown to lead to diseases in human according to a different mechanism, namely unequal allelic homologous recombination during meiosis and/or mitosis. The scope of this paper is to review a rapidly growing area concerning this new type of mutation.
It has been known for long that homopolymeric tracts of amino acids are extremely abundant in eukaryotic proteins (2). In humans 20% of proteins contain at least one (3). In decreasing order, the most frequent is glutamic acid, proline, alanine, serine, leucine, glycine and glutamine (4). Alanine (A) is a hydrophobic, non-polar amino acid. Polyalanines have been found in 494 human proteins (5). Under physiological conditions in vitro, alanine stretches form b sheets that are extremely resistant to chemical denaturation and enzymatic degradation (6). Above a threshold of 19 alanines, the polypeptide aggregates and forms intracellular inclusions, leading to cell death (7,8). Interestingly, alanine stretches do not exceed 20 alanines in human and are relatively short homopolymeric repeats when compared with polyglutamine (poly Q).
Alanine tracts are coded by imperfect trinucleotide repeats (GCN), among which GCG is significantly over-represented in the polyalanine coding sequence (5). It is stable during both meiosis and mitosis (7). However, polymorphisms in length are frequent involving more than 30% of tracts longer than seven alanines and correlating with the length of both the overall tract and the number of a single codon in a row (5). Thanks to the growing genome sequence data available in several species, recent in silico studies concerning homopolymeric tracts have provided valuable information. First of all, polyalanines are frequent in eukaryotic cells, and preferentially found in transcription factors (TFs; 36% of proteins with polyalanine stretches in humans) (3, 5) . Comparative proteic sequence data showed that alanine tracts are: (i) longer in eukaryotic cells than in prokaryotic cells, where no tracts longer than nine alanines are found; (ii) poorly conserved among vertebrates and longer in mammals than in other vertebrates; and (iii) of recent and independent appearance in paralogous genes. This last finding is a strong argument for the hypothesis of convergent molecular evolution (5,8) although the functional role(s) of polyalanine stretches remains unknown as well as the nature of the negative selective pressure involved. Indeed, alanine tracts lie outside of

The identification of a new type of mutation, by expansion of CAG trinucleotide repeats coding for polyglutamines in neurodegenerative disorders in human, inspired much enthusiasm in the 1990s. Polymerase slippage has long been assumed to be the disease-causing mechanism. Therefore, imperfect trinucleotide repeats have been discounted as candidate loci for such mutations (reviewed in 1). Some years later, however, imperfect nucleotide repeat expansions encoding alanines were shown to lead to diseases in human according to a different mechanism, namely unequal allelic homologous recombination during meiosis and/or mitosis. The scope of this paper is to review a rapidly growing area concerning this new type of mutation.
HOMOPOLYMERIC STRETCHES OF ALANINE
It has been known for long that homopolymeric tracts of amino acids are extremely abundant in eukaryotic proteins (2) . In humans 20% of proteins contain at least one (3) . In decreasing order, the most frequent is glutamic acid, proline, alanine, serine, leucine, glycine and glutamine (4) . Alanine (A) is a hydrophobic, non-polar amino acid. Polyalanines have been found in 494 human proteins (5) . Under physiological conditions in vitro, alanine stretches form b sheets that are extremely resistant to chemical denaturation and enzymatic degradation (6) . Above a threshold of 19 alanines, the polypeptide aggregates and forms intracellular inclusions, leading to cell death (7, 8) . Interestingly, alanine stretches do not exceed 20 alanines in human and are relatively short homopolymeric repeats when compared with polyglutamine (poly Q).
Alanine tracts are coded by imperfect trinucleotide repeats (GCN), among which GCG is significantly over-represented in the polyalanine coding sequence (5) . It is stable during both meiosis and mitosis (7) . However, polymorphisms in length are frequent involving more than 30% of tracts longer than seven alanines and correlating with the length of both the overall tract and the number of a single codon in a row (5) . Thanks to the growing genome sequence data available in several species, recent in silico studies concerning homopolymeric tracts have provided valuable information. First of all, polyalanines are frequent in eukaryotic cells, and preferentially found in transcription factors (TFs; 36% of proteins with polyalanine stretches in humans) (3, 5) . Comparative proteic sequence data showed that alanine tracts are: (i) longer in eukaryotic cells than in prokaryotic cells, where no tracts longer than nine alanines are found; (ii) poorly conserved among vertebrates and longer in mammals than in other vertebrates; and (iii) of recent and independent appearance in paralogous genes. This last finding is a strong argument for the hypothesis of convergent molecular evolution (5, 8) although the functional role(s) of polyalanine stretches remains unknown as well as the nature of the negative selective pressure involved. Indeed, alanine tracts lie outside of Human Molecular Genetics, Vol. 13, Review Issue 2 # Oxford University Press 2004; all rights reserved other known functional domains in their proteins. However, alanine repeats (as L, P and Q amino acid repeats) do locate preferentially at the N-terminal (4). It is unlikely that they are simply tolerated, non-essential insertions considering their phylogenic evolution and the clear-cut threshold observed in human diseases resulting from expansions of poly A. They have been regarded as flexible spacer elements located between functional domains of the protein and therefore essential to protein conformation, protein -protein interactions and/or DNA binding (3, 9) . However, although preferentially located in the N-terminal end of proteins when present, their position can be highly variable even within a family of proteins such as the Hox TFs, for example (5) .
EXPANSION OF ALANINE STRETCHES AND HUMAN DISEASE
An alanine expansion was first identified as the diseasecausing mechanism in synpolydactyly syndrome (SPD) (10) . Since then, similar mutations have been described in eight additional autosomal dominant or X-linked disorders responsible, in most of the cases, for congenital malformations and/or mental retardation (Table 1) . Skeletal malformations (SPD, HFGS, CCD) and abnormalities of the nervous system (HPE, XLAG, XLMR þ GH, CCHS) are indeed over-represented (7/9). Recent general or focused reviews detail the clinical presentations of each disease (49 -52) . All but one gene involved encodes a TF (Fig. 1) . The exception concerns the oculo-pharyngeal myotonic dystrophy syndrome (OPMD), an atypical case on several accounts. Indeed, OPMD is a late-onset, progressive disease resulting from, and only from, in-frame duplications leading to polyalanine expansions ranging from þ2 to þ7 alanines in the PABPN1 protein (involved in mRNA polyadenylation) (7) . The size of the alanine expansion leading to a disease phenotype is the smallest ever described (Table 1) . Moreover, the þ1 alanine expansion, found in 2% of the French Canadian population (7, 39) may act either as a trans modifier allele of a larger expansion in severe OPMD cases, or as a disease-causing allele responsible for a milder form of the disease in homozygous patients (7) . Although a causal relationship remains to be proven, it may be worth mentioning that an expansion of 10 alanines in the 15 alanines tract of the TBX1 protein has been identified in a patient with conotroncal cardiac defect (53) .
GENOTYPE/PHENOTYPE CORRELATIONS IN HUMAN DISEASES RESULTING FROM POLYALANINE EXPANSIONS
Does the severity of the phenotype vary according to the length of the alanine tract? Does it vary between patients harboring a polyalanine expansion when compared with other types of mutations within the same gene? Positive correlation between the size of the expansion and the severity of the phenotype is convincing for the HOXD13 expansions in SPD (9) . Intrafamilial variation in SPD phenotype is most marked in families with the smallest expansions (9) . Moreover, SPD has a semi-dominant mode of inheritance since patients harboring a homozygous duplication (leading to a þ7 or a þ9 alanines expansion of the HOXD13 protein) have a more severe phenotype when compared with heterozygous patients for the same mutation (10, 11, 54) suggesting a dose effect of the mutant protein. Interestingly, hypospadias is observed only in the SPD patients harboring the longest polyalanine expansion [þ14 alanines (9) ], and incomplete penetrance has been observed in carriers of heterozygous in-frame duplications leading to alanine expansions in HOXD13 [þ7 and þ10 alanines (10) ] and in HOXA13 [þ 6 alanines (22)].
Conversely, such correlations are unclear for heterozygous PABPN1 expansions in OPMD (39, 40, 42) or PHOX2B in CCHS. In the latter case, it has been proposed that the longer the alanine expansion of the PHOX2B protein, the more severe the ventilatory phenotype and the broader the spectrum of autonomic dysfunction in patients (35, 36) . Indeed, patients with a central hypoventilation syndrome of late-onset (6 months to 12 years in our series of patients) harbor the smallest expansion of the PHOX2B protein if any (þ5 alanines), whereas patients with Haddad syndrome (CCHS þ Hirschsprung disease) mostly harbor expansions of at least six alanines (33 -36) . However, one cannot accurately predict the phenotype from the genotype, since the þ7 alanines mutation is the most frequent expansion among both the isolated CCHS and the associated to Hirschsprung disease groups of patients (unpublished data). Finally, the question of correlations cannot be raised when an alanine expansion has been reported only once, as is the case for the SOX3 gene, or when all expansions reported have been similar in length (ZIC2 protein, Table 1 ).
Whenever different types of mutation within the same gene are reported as disease-causing, polyalanine expansions lie at the milder end of the phenotypic spectrum. Such is the case for ARX (32), RUNX2 (22) , HOXA13 (16, 18, 20) , FOXL2 (44, 45) and PHOX2B. It is worth noting that patients harboring polyalanine expansions do not present features seen in patients with other types of mutations such as ovarian failure in BPEIS, (44, 45) brain malformations in XL-MR linked to the ARX gene (32) or neuroblastoma in CCHS (unpublished data).
DISEASE-CAUSING MECHANISM OF ALANINE EXPANSIONS
Although it may be inappropriate to consider a common disease-causing mechanism for alanine expansions, a general discussion may be proposed (Table 2 ). When studied, the predicted expanded proteins have been shown to be present and stable (7, 52) . There are several arguments against straightforward loss-of-function as the disease-causing mechanism in disorders resulting from polyalanine expansions. Indeed, at a single locus, the phenotypes observed are correlated with the size of the expansion. In addition, the disease is milder in a polyalanine type of mutation when compared with other mutations at the same locus highly likely to result in a plain loss of function (such as deletion encompassing the gene, missense or truncating mutations within the homeodomain of the protein). This holds true both in humans (14, 17, 44, 55) and mice (56, 57) . Regarding X-linked genes with polyalanine expansions (SOX3 and ARX ), the observation of unaffected female carriers suggests that a gain-of-function of alanine expansions is unlikely (30) . However, X-inactivation pattern in female carriers is not known. A dominant negative effect on the wild-type protein and/or other proteic partners remains a possibility as shown for HOXD13 expansions in SPD (reviewed in 52).
MUTATIONAL MECHANISM LEADING TO ALANINE EXPANSIONS
Warren first suggested that an unequal allelic homologous recombination was the most likely mechanism for alanine expansions in the HOXD13 protein (1) . Several lines of evidence support this hypothesis, namely: (i) almost all variant alleles appear as in-frame duplications within the nucleotidic sequence encoding the alanine stretch and can therefore be ascribed to this mechanism; (ii) expanded alleles are stable mitotically and meiotically over generations as shown in SPD, HGS, CCHS and OPMD pedigrees (the only exception being a further expansion of one trinucleotide repeat in the PABPN1 gene); and (iii) contractions (the mirror image of expansions during the recombination event) are observed as polymorphisms in several genes for which expansions lead to human diseases (PHOX2B, ARX, RUNX2 ). This mechanism is equally likely in GC-rich homopolymeric tracts encoded by imperfect codons since the majority of non-conserved homopolymeric stretches between human and rodents are the consequence of either expansions or deletions (58) .
PHOX2B is thus far the best gene to analyse how in-frame duplications leading to alanine expansions occur. Indeed, due to poor reproductive fitness, the vast majority of cases are sporadic and de novo in-frame duplications have been reported in more than 100 unrelated CCHS patients world-wide (33 -36) . As expected, a PHOX2B polyalanine expansion of a given size can be the result of several recombination events as shown in Figure 2 for the three prevalent expansions (þ5, þ6 and þ7 alanines). Intriguingly, for each of the expansion sizes some recombination events are significantly overrepresented, whereas some are not observed (Fig. 2) . It is worth noting that the most common expansion (þ7 alanines) results from an unequal allelic recombination event involving the fewer nucleotide mismatches. It is, thus, tempting to postulate a causal relationship.
Polymorphisms in the length of the alanine tract have been described in four genes whose polyalanine expansions mutation results in human diseases namely the PHOX2B, RUNX2, ARX and PABPN1 genes ( Table 1) . This raises the question of whether nucleotidic variation would predispose to polyalanine expansion mutation. At least for the PHOX2B protein, nucleotidic sequence encoding polyalanine contractions are not prone to expansions during transmission. Indeed, their frequency is not greater in unaffected parents of CCHS cases than in a control population (one contracted allele/240 parental alleles versus two contracted alleles/250 control alleles, respectively, unpublished data). Interestingly, nucleotidic variations of the third nucleotide of codons encoding alanine stretches have been reported only for the SPD, synpolydactyly syndrome; HFGS, hand -foot-genital syndrome; CCD, cleidocranial dysplasia; HPE, holoprosencephaly; XLMR þ GHD, X-linked mental retardation and growth hormone deficit; XLAG, X-linked mental retardation and abnormal genitalia; CCHS, congenital central hypoventilation syndrome; OPMD, oculopharyngeal muscular dystrophy; BPEIS, Blepharophimosis-ptosis-epicanthus inversus syndrome. Del, deletion; FS, frameshift mutation, MS, misense mutation; NS, nonsense mutation; PE, position effect.
HOXD13 (A.G in the 12th trinucleotide repeat) (9) and PHOX2B genes (A.C in the 14th trinucleotide repeat).
One can speculate that selection may occur against heterozygotes if prone to allelic unequal crossing-over during meiosis (species limited selective pressure). Interestingly, complex duplication events have been described in FOXL2, HOXD13 and PHOX2B genes (16,32) (Fig. 2) as if heterozygosity would favor hairpins formations in the genomic sequence. Finally, it is worth noting that somatic mosaicism for an alanine expansion in ZIC2 and PHOX2B has been observed in several unaffected parents of probands (27, 35, 36 and unpublished data), illustrating presumably rare mitotic recombination events.
WHAT CAN A POLYALANINE TRACT DO?
Lanz et al. showed that an artificial alanine tract (replacing a glutamine tract by an out of frame mutation) in the rat glucocorticoid receptor (GR) can inactivate transcription. Such an effect could be observed whatever the polyalanine tract position is in the protein, despite a length increasing threshold from the N-Ter to the C-Ter locations (59) . Interestingly, transcriptional activation is tightly correlated to the number of repeats of homopolymeric tract (both proline and glutamine tracts) (60) . Above the physiological range, transcriptional activation drops, whereas the modified protein remains stable.
Filamentous nuclear inclusions in muscle cells are the hallmark of OPMD. Their cytotoxicity, leading to cell death, may be the consequence of poly(A)RNA sequestration (61, 62) . Interestingly, when searched for, cytoplasmic aggregates have been observed in several polyalanine diseases such as HFGS, SPD, XL-MR (SOX3 gene) and CCD (62) . This finding implicates a change in localization of the mutant protein. Concerning Hoxd13, the rate of cytoplasmic aggregation correlates with the length of the repeat (63) . Moreover, as demonstrated in polyglutamine diseases (reviewed in 64), reducing polyalanine aggregation alleviates toxicity. These data cope well with the observation of alanine tracts of more than 19 alanines alone being sufficient to form aggregates (both time-dependent and repeat-length-dependent) in COS-7 cells and cause cell death (65) . Accordingly, the longest alanine tract observed in mammals is of 20 amino acids Table 1 . HD, homeodomain; ZNF, zinc-finger; PL-HD, paired-like homeodomain; HMG, high mobility group; ARS, aristaless; RNA-REC, RNA recognition; FKD, forkload.
(PHOX2B) (5) . Moreover, cell toxicity due to long alanine tracts could extend beyond the scope of trinucleotide repeats encoding alanines. Indeed, GCA repeats may be translated in alanine (GCA), glutamine (CAG) or serine (AGC) codons according to the three possible reading frames. At least in Machado -Joseph disease, large CAG repeats at the MJD-1 locus are prone to frameshifts that could be transcriptional, translational or both. Those frameshifts result in hybrid proteins containing polyglutamine/polyalanine tract that aggregate to form intranuclear inclusions both in vitro and in vivo (66) . Along these lines, it is worth mentioning that the expanded CAG/CTG repeats in Huntington disease-like 2 is located in an alternatively spliced exon of the JPH3 gene. One alternate transcript identified in normal brain encodes an alanine tract according to the open reading frame, and affected individuals present intranuclear inclusions of variable extent in the brain (67 -69) . Therefore, both polyglutamine and polyalanine expansions could lead to protein aggregation due to an alanine stretch rising above a threshold as a slow process in the former case (expected low frequency of frameshifts), and a faster one in the later case (already in-frame alanine coding sequence) and an intermediate one in OPMD where the shortest disease causing alanine tract is of 12 repeats.
SHOULD SEQUENCES FLANKING ALANINE TRACTS BE CONSIDERED?
Interestingly, when focusing on the biochemical properties of amino acids composing homopolymeric stretches, the more hydrophobic the amino acid (Leu . Ala . Ser . Gln), the shorter the length of the stretches observed in proteins and the shorter the threshold in human disease (at least for alanine and glutamine tracts) (59, 70) . Therefore, not only the length of the homopolymeric stretch but also the biochemical properties of amino acids flanking the homopolymeric stretches might be considered. This was proposed for the rat GR where the threshold in the number of alanines necessary to inactivate the GR is lowered when the stretch is flanked by hydrophobic amino acids (59) . If such is the case, hydrophobic stretches could be lengthened by both unequal allelic homologous recombination (within the homopolymeric tract) and nucleotidic variation (outside of it). Among proteins bearing alanine expansions in human diseases, both the alanine tract and the flanking amino acids are conserved in human, mouse and rat orthologs (whenever data are available), even though the alanine stretches lie outside of known functional domains of the proteins.
CAN POLYALANINE CONTRACTIONS BE DISEASE-CAUSING?
So far, polyalanine contractions have not been involved with certainty in human diseases. This, however, has been suggested for the thyroid TF-2 (TTF-2) and the human achaete-scute homolog-1 (HASH-1) genes in thyroid dysgenesis and CCHS, respectively (71, 72) . Considering that many proteins with polyalanine stretches interact with each other (5) polyalanine contractions in one protein may well modify the transcriptional activity of a proteic complex in both space-and time-dependant ways, whereas in vitro transcriptional activity of the contract protein remains within the range of the wild-type protein (37, 71, 72) . Further functional characterization is needed before the effect of polyalanine contractions is established.
MOUSE MODELS
Yet, only one natural mouse model with an alanine expansion has been described; spdh mice mutant for which the Hoxd13 protein harbors a þ7 alanines expansion, as the shorter hitherto observed in SPD in human (57) . Only mice homozygous for the mutation show a limb phenotype very similar to the one observed in patients with SPD, whereas heterozygous More recently, transgenic mice expressing a Pabpn1 protein with an expansion of three alanines have been generated (76) . Mice show a late-onset myopathy phenotype and, accordingly, pathological studies revealed intranuclear inclusions consisting of mutant Pabpn1 protein and developing gradually with aging.
OTHER AMINO ACID REPEATS AND DISEASE IN HUMAN
Polyglutamine expansions have been reviewed elsewhere (64, 77, 78) . Aspartic acid repeats (GAT or GAC) of at least five codons are unfrequent in eukaryotic proteins (4) . Both in-frame deletion of one codon and duplication of two codons within the perfect five trinucleotidic repeats of the COMP gene encoding an aspartic acid stretch (lying in the calmodulin-like domain of the protein) have been identified in pseudoachondroplasia, whereas a duplication of one codon was found in a patient with multiple epiphyseal dysplasia (79, 80) . Both conditions have an autosomal dominant mode of inheritance. Strikingly, inclusions in the rough endoplasmic reticulum of chondrocytes and tendon cells are described in both diseases and contain COMP as well as other proteins of the extracellular matrix (80, 81) . Finally, although polyleucine expansions have not been described thus far, polyleucine are more aggregation-prone than polyglutamine in vitro (70) . Altogether, these data further support the view of a disease-causing mechanism common to homopolymeric tract expansions hitherto identified in human, namely protein aggregation.
PERSPECTIVES
Polyalanine expansion may be a rapidly growing diseasecausing type of mutation in vertebrates as polyalanine stretches are: (i) frequent among key players in development such as TFs; (ii) rapidly evolving in eukaryotes; and (iii) Figure 2 . Recombination events observed at the PHOX2B locus in CCHS patients. Recombination events are quoted by all possible crosses. The most frequent (over 60%) recombination event is in bold red, whereas recombination events in black or blue have been observed either once or two to three times, respectively. encoded by sequences prone to unequal allelic homologous recombination leading to either expansions or contractions.
It is tempting to speculate on a disease-causing mechanism common to all polyalanine expansions and, to a greater extent, to any amino acid expansions: protein aggregation that traps both mutant and wild-type proteins as well as other protein partners. This time-dependent and repeat-length-dependent mechanism, with variable threshold according to the amino acid involved, could therefore be regarded as gain-of-function with a dominant negative effect. If such is the case, one can expect that the insertion of a sequence encoding an in-frame hydrophobic polypeptide (alanines or leucines) in any gene might be a simple, inducible and tissue-specific knock-out strategy. This may well represent the model proposed by Herskowitz (82) .

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