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Are there CAG repeat expansion-related disorders outside the central nervous system?
Brain Research Bulletin, Vol. 56, Nos. 3/4, pp. 259 –264, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00663-3
Are there CAG repeat expansion-related disorders outside the central nervous system? Paola Ferro, Raffaella dell’Eva and Ulrich Pfeffer* Laboratory of Molecular Biology, National Cancer Research Institute, Genova, Italy ABSTRACT: Expansions of poly-glutamine tracts in proteins that are expressed in the central nervous system cause neurodegenerative diseases. The altered proteins accumulate over long periods of time, forming nuclear inclusions, and lead to neuronal cell death. A similar mechanism could also be operant in non-dividing cells outside the central nervous system because nuclear inclusions are not limited to neurons. In addition, variations of the repeat length within the normal range may affect cellular function as it has been shown for the androgen receptor that is involved in neoplastic degeneration of several tissues. We have identified a poly-glutamine/poly-proline repeat in the homeobox gene DLX6. DLX genes are expressed in non-proliferative cells of the apical ectodermal ridge of developing limbs. Ablation of these cells leads to limb malformation. We propose that CAG triplet expansions in this gene could lead to cell death in the apical ectodermal ridge causing limb malformations. Indeed, autosomal dominant limb malformations with increasing severity in successive generations have been linked to the chromosomal region that contains DLX6. The analysis of a limited number of patients affected by split hand/ foot malformation so far revealed only a slight modifier effect of repeat length within the normal range and no expansions have been detected. © 2001 Elsevier Science Inc.
salvage [29,53,59]. Recent findings from cell culture and from transgenic mice link poly-glutamine dependent neurodegeneration to apoptosis of cells expressing proteins with expanded repeats [6,23,30,36,52]. TISSUE SPECIFIC CELL DEATH Although the pathogenic CAG repeat containing proteins are expressed in many cell types, cellular degeneration is observed only in the central nervous system. This could be explained by tissue specific factors that mediate cytotoxicity [37,42,66]. In a recent report, however, Kennedy and Shelbourne show a particular somatic instability of an expanded huntington gene repeat in transgenic mice in the striatum [28]. This work establishes a correlation between the phase of disease during which neuronal damage in the various brain tissues becomes manifest and the degree of somatic instability of the repeat. Similar to what has been observed for the androgen receptor (AR) [63], somatic instability of the CAG repeat could depend on the expression level of the huntington gene in the striatum. Cytotoxicity of the expanded Machado Joseph’s disease gene CAG repeat is higher in nondividing cells [73] suggesting that the expansion dependent cell death might occur preferentially during the G0 phase of the cell cycle. Cytotoxic effects may also be much less apparent in tissues with a high regeneration potential where dying cells are easily substituted. Short living cells might never reach the toxicity threshold of proteins containing the expanded poly-glutamine repeat. Moreover, CAG repeat expansion dependent apoptosis follows specific pathways involving caspases [31,50,67,70]. Differential expression of components of the apoptotic pathway could determine a differential susceptibility of specific tissues. However, the link between CAG repeat toxicity and apoptosis awaits to be confirmed in patients. Aggregates of proteins containing expanded poly-glutamine tracts sequester heat shock proteins and proteasome components and aggregation is suppressed by over-expression of molecular chaperones that provide cellular protection [8,25,27,30,33,40,61,68]. Again, the variable cellular array of those chaperones may influence poly-glutamine toxicity.
KEY WORDS: Neurodegeneration, Anticipation, Apical ectodermal ridge, DLX, Homeobox gene, Limb morphogenesis, Polyglutamine, Triplet repeat expansion, Striatum.
INTRODUCTION There are many genes in the human genome containing CAG triplet repeats within the coding region. When this has been analyzed, the lengths of these repeats are polymorphic in the normal human population, with some exceptions. Polymorphic repeat length is probably a function of reduced stability of such repeats which increases dramatically above a threshold of about 35 to 40 repeats, giving rise to different neurodegenerative diseases. It is not clear whether all CAG repeats are polymorphic or they all undergo occasional expansions nor whether such expansions would lead to diseases (for recent reviews see [14,44]) and other articles in this special issue). De novo repeat expansions have also been described [32,41,57,69,72] but the mutation rate is not known. CAG repeat diseases show dominant inheritance. A cytotoxic gain-of-function, apparently independent from the gene context [43], appears to be the main pathogenetic mechanism. Proteins carrying expanded glutamine stretches form aggregates in nuclear inclusions. It is not clear whether these aggregates are themselves pathogenic or whether they are the results of an attempted cellular
EFFECTS OF CAG REPEAT EXPANSIONS IN NON-NEURONAL TISSUES If the expression level of the disease protein, cell proliferation rate, regeneration potential, expression profile of mediators of apoptosis and of molecular chaperones co-operate to determine the cytotoxic effect of an expanded poly-glutamine repeat, tissue spe-
* Address for correspondence: Ulrich Pfeffer, Ph.D. Laboratory of Molecular Biology, National Cancer Research Institute, L. R. Benzi 10, 16132 Genova, Italy. Fax: ⫹39-010-5737-231; E-mail: [email protected]
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cific cell death could be explained without implicating tissue specific cellular factors. Nuclear inclusions have been found in non-neuronal tissues including scrotal skin, dermis, kidney, heart, and testis of spinobulbar muscular atrophy (SBMA) patients [35]. These inclusion were morphologically indistinguishable from those seen in neurons, reacted with the same subset of epitope specific anti-AR antibodies and were similarly ubiquinated. These observations suggest that the formation of nuclear inclusions follows the same pathway in neurons and non-neuronal cells. Nuclear inclusions were also detected in skeletal muscle, heart, liver, adrenal glands, pancreas, kidney, stomach, and duodenum of transgenic mice carrying the amino-terminal portion of the Huntington’s disease (HD) protein with an expanded repeat [51]. In these tissues progressive organ atrophy, though to variable extents, is observed, indicating that expanded CAG repeat containing proteins can lead to cellular death in non-neuronal tissues. This finding is also underpinned by the observation that the same transgenic mice develop diabetes [21] and that the islets of Langerhans contain nuclear inclusions [51]. Diabetes is also more frequent in HD patients as compared to the normal population [15]. HD patients also show hepatic symptoms in correlation to a reduced survival of hepatocytes [4].
other CAG repeat disease genes and are not expected to assume the particular conformation that is believed to be pivotal to the disease process [45]. However, cytoplasmic, non-ubiquitinated aggregates have been observed in SCA-6 patients and cells transfected with the SCA-6 containing an expanded CAG repeat undergo apoptotic cell death [24]. Pathogenicity of the short SCA-6 repeat expansions could therefore be analogous to other CAG repeat expansion diseases. Alternatively, the limited expansions might lead to ataxia via the functional inactivation of the ion channel. Inactivating splice site mutations of the same gene lead to a similar, though episodic, form of ataxia [77]. Thus at least in these two cases CAG repeat length polymorphisms within the range that is considered normal for most polyglutamine proteins can have functional consequences. Similar effects might show up also for other poly-glutamine proteins once their function will be unraveled. This appears particularly interesting for the wild-type HD protein that has been reported to protect neuronal cells from apoptotic stimuli [48]. If this activity is influenced by the repeat length within the normal range these polymorphisms might modify many aspects of neuronal function and pathology including neuronal aging.
FUNCTIONAL ASPECTS OF REPEAT LENGTH VARIATION WITHIN THE NORMAL RANGE
ARE THERE DEVELOPMENTAL CAG REPEAT EXPANSION EFFECTS?
CAG repeat lengths are highly polymorphic in the normal population and they are translated into polyglutamine stretches located in most cases in the amino-terminal part of the proteins. Above the threshold of about 35 to 40 glutamine residues the stretch assumes a particular structure which is believed to be responsible for polyglutamine pathogenicity [45]. Little is known about the function of polyglutamine regions within the normal range because information on the function of the proteins involved in trinucleotide expansion diseases is only beginning to emerge. The AR is a ligand-dependent transcription factor [65]. The human AR contains a poly-glutamine repeat in exon 1 whose expansions to over 40 CAGs cause Kennedy’s disease or SBMA. The AR CAG repeat is polymorphic in the normal human population (CAG8 –35) [34]. The transactivation potential of the AR is inversely correlated to repeat length either as a direct consequence of altered receptor function [9,38] or indirectly due to reduced AR messenger RNA and protein levels in cells carrying alleles with longer CAG repeats [10]. This is also reflected by the fact that patients suffering from Kennedy’s disease often show mild androgen insensitivity [2,56]. The receptor is involved in the control of cellular proliferation and differentiation in androgen target tissues, where the hormonal signal determines either a mitogenic response, as e.g., in the prostate [12], or an anti-mitogenic response, as, e.g., in the breast [3]. The interpretation of the androgenic signal is thus dependent on cell type-specific receptor functions. In keeping with this, men carrying AR alleles containing shorter CAG repeats face a higher risk of prostate cancer [18,19]. Women who carry a mutated allele of the breast and ovarian cancer susceptibility gene, BRCA-1, show an increased risk to develop the disease and a lower mean age at diagnosis, when they also carry AR alleles with longer CAG repeats [47]. We have reported recently that in 10% of colon cancers the AR CAG repeat undergoes somatic reductions that probably contribute to tumor progression [17]. Spinocerebrellar ataxia type 6 (SCA-6) is determined by CAG repeat expansions in the gene encoding the (1A-subunit of the voltage dependent calcium channel (CACNAIA) that is highly expressed in the cerebellum [75,77]. Expansions from the normally 4 –18 to 21–30 repeats correlate with progressive cerebrellar ataxia. These expansions are within the range that is normal for
CAG repeat expansion related cell death apparently occurs prevalently in non-dividing cells. Specialized developmental organizers often show a strictly determined number of cells that upon differentiation do not proliferate. If CAG repeat expansion related cell death depends on the amount of toxic protein in the cell, the toxic threshold of the expanded protein will be reached during the limited life of developmental organizer cells only when it is expressed at very high levels. Clarke et al.[11] analyzed the kinetics of metabolic decline in the caudate nuclei of Huntington’s disease patients and concluded that only mathematical models that assume a constant risk of death for the single neuron fit the data collected. According to this hypothesis, accumulation of expanded CAG repeat region containing proteins would determine a higher probability for the neuronal cell to die, perhaps by apoptosis, to a yet unknown insult. An analogous situation has been observed in Drosophila mutants carrying the rdgC mutation in a protein phosphatase involved in phototransduction: the mutation alone is not sufficient to determine retinal degeneration that occurs only in the presence of light [60]. Just like in these mutants also in HD the hypothetic trigger must be unavoidable or even constitutive because the repeat expansion is a cause of the disease and not a mere risk factor. Developmental organizer tissues formed by non-proliferative cells are committed to apoptosis after they have fulfilled their organizing task. If expanded glutamine repeat containing proteins are expressed at a high level in such cells, sensitivity to the apoptotic stimulus could drastically rise leading to untimely cell death and disturbed developmental organization. The apical ectodermal ridge (AER), a thickened epithelium on the limb bud, is an example for such a tissue. It is formed by small number of cells that control limb-bud outgrowth during development [64]. Factors that induce formation and, later on, re-absorption of the AER are only partially known. Removal of the AER from chick limb buds leads to malformation of the limbs. Removal early during development leads to proximal truncation while removal at later stages leads to more distal defects indicating that the AER controls the complete period of limb outgrowth [49,54,62].
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THE DISTAL-LESS HOMOLOGUE DLX6 We have recently identified a CAG/CCG repeat in the so far unknown first exon of the homeobox gene DLX6 [46], one of seven mammalian homologues of the Drosophila distal-less gene. DLX6 is located in the same genomic region as its homologue DLX5 in a tail-to-tail position [58]. The two genes, like the other two DLX pairs, are believed to be regulated in tandem. For DLX5 strong expression in the AER of the developing limb has been shown [1,16,74]. The human DLX6 gene is located on chromosome 7q22 in the critical interval for the split hand/split foot malformation 1 [55] (SHFM1, OMIM 183600). Other three loci have also been linked to SHFM. The malformation exists in a variety of forms and occurs either sporadically or familiarly. Phenotypic alterations consist in the absence of the middle ray of hands and/or feet with a more or less pronounced syndactyly of the remaining digits and a deep cleft between the digits. In severe cases, syndactyly and ectrodactyly occur in combination [5]. According to Zlotogora [76], SHFM1 can be divided into two subtypes, with (type II) or without (type I) aplasia of long bones. These two types are also distinct by their penetrance, which is nearly complete for type I and reduced to about 66% for type II. The malformation is inherited as an autosomal dominant trait but in families where SHFM is associated to other long bone deficiencies, marked non-Mendelian transmission is observed. This includes skipping of generations (reduced penetrance), increased severity in successive generations ranging from monomelic to tetramelic forms and from simple split hand/split foot to tibial or ulnar aplasia (variable expressivity) and overtransmission from affected fathers to sons (segregation distortion) [76]. In several SHFM families the phenotypic presentation of the malformations drastically increases in successive generations. In one particular family, the first generation for which information is available showed classical split hand malformation; in the following five generations the severity of the malformation increased leading to aplasia of distal portions of the limbs to nearly complete agenesis of the entire limbs in a child born without arms and legs [20]. The longitudinal outgrowth of the limbs is regulated by the AER and removal of this control region leads to limb malformations [54,62]. CAG repeat expansion related cell death in the AER could therefore lead to limb malformations and further expansions in following generations could increase the severity of the malformation by the anticipation of cell death corresponding to early removal of the AER. Impaired AER viability has also been shown for p63 mutations [39,71] that cause the SHFM-related ectrodactyly, ectodermal displasia, cleft palate syndrome [7] and for the mouse model of dactylaplasia [13]. These malformation syndromes, however, do not map to the genomic region containing DLX6. On this background we started to search for polymorphisms in the general population. We could identify five alleles with 11 to 20 CAGs whereas no polymorphisms could be detected for the CCG repeat containing 9 triplets [46]. Two alleles with 20 and 19 CAG repeats account respectively for 28% and 69% of the tested population, 3% had shorter alleles. Unfortunately, we had no access to samples of the family with clear sign of anticipation described by Helal et al. [20] nor to those of the few families with less drastic signs of anticipation. Therefore our analysis is so far limited to 38 sporadic cases and to familiar cases with few members affected so that no linkage studies are possible. We have also analyzed possible modifier effects in two families with reduced penetrance without clear signs of increasing severity in successive genera-
FIG. 1. Allele frequencies (panel a) and genotype frequencies (panel b) of healthy controls, unaffected parents, mothers and fathers of sporadic SHFM patients and sporadic SHFM patients.
tions, one with a translocation involving the locus containing DLX6 and one in linkage with another locus. We did not encounter DLX6 CAG repeat expansions in the patients analyzed. We were, however, able to show an additional allele containing 21 CAG repeats, only present in one sporadic patient and absent from 90 healthy subjects (67 from the general population, 23 parents of sporadic patients). Because samples from the parents of this patient were not available for analysis we cannot decide whether the unusual allele is a rare allele or a de novo expansion. From the analysis of allele frequencies (Fig. 1a) a very slight trend towards longer alleles is observed in sporadic patients where 32.9% had alleles with 20 or 21 repeats (27.6% in controls). The healthy mothers of sporadic patients had similarly low numbers of longer alleles (25.0%), while the allele was definitely underrepresented in the fathers of patients (5.6%). The genotype frequency (Fig. 1b) shows a complementary trend with the mothers
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showing 14.3% homozygosis for the long CAG20 allele while 6.3% were to be expected from the allele frequency. In the families of sporadic cases analyzed, the patients inherited a long allele from their mothers and, with one exception, not from their fathers. These observations so far constitute a trend but do not reach statistical significance except for the genotype frequencies control versus fathers (odds ratio⫽7.76 confidence interval 0.93–354.7; p ⫽ 0.037, Fisher test); More patients must be analyzed in order to establish a role for longer DLX6 alleles in SHFM. However, this trend is interesting because overtransmission of SHFM from affected fathers to affected sons has been described [26]. The modifier effect could be much stronger than suggested by our study if one takes into account that four loci have been linked to SHFM but the gene has been identified only in one case [7,22]. Moreover, genetic origin of the malformation is not certain for sporadic cases. In order to increase the statistical power of our analysis more patients together with their parents must be analyzed and the modifier effect should be analyzed for sporadic cases in correlation to the only known causative mutation in the p63 gene. CONCLUSIONS Poly-glutamine repeat proteins are expressed in many tissues. The cytotoxic effect apparently depends on several factors among which the proliferation status and the regeneration potential of the tissue that expresses the disease protein play a prominent role. In the adult, non-regenerating tissues, such as islets of Langerhans in the pancreas, could be targeted by poly-glutamine induced susceptibility to apoptotic insults. Our hypothesis that the same could occur during development in non-proliferating organizer cells is based on the one-hit model of neurodegeneration proposed by Clarke et al. [11]. We have tried to test this hypothesis for the DLX6 gene that contains a poly-glutamine/poly-proline tract. DLX6 appeared to be a promising candidate because it is expressed in a non-proliferative tissue with a crucial function for limb morphogenesis, it maps to a chromosomal region that has been linked to a limb malformation and the syndrome shows variable expressivity and increasing severity in successive generations. We consider these features as the developmental equivalent of anticipation. Our hypothesis could have been directly confirmed or dismissed if we would have had access to the most informative samples but the family denied collaboration. The analysis of sporadic cases and their parents indicates a potential modifier effect of longer repeat alleles and further analyses are needed to reach statistically significant conclusions. Modifier effects that depend on the repeat length have also been described for the AR [17–19,47] and might be common to many CAG repeat containing genes. DLX6 is also expressed in the brain, at least during development [58], and therefore this gene is also a candidate for “classic” CAG repeat expansion related neurodegenerative diseases. Further studies on poly-glutamine proteins outside the central nervous system should consider that CAG repeat expansions in genes that are highly expressed in non-proliferative tissues with special developmental functions may cause malformations of the corresponding structures. Databank screens reveal many developmental genes that contain such repeats. Furthermore, CAG repeat polymorphisms within the normal range may influence gene function, especially in the case of transcription factors and their coactivators, thereby modifying related cellular functions and their pathological degeneration.
ACKNOWLEDGEMENTS
We thank Fiorella Gurrieri, Giovanni Camera, and Marco Seri for providing patient samples. We also thank Douglas Noonan for critically reading the manuscript and Adriana Albini for continuous support.
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Are there CAG repeat expansion-related disorders outside the central nervous system? Paola Ferro, Raffaella dell’Eva and Ulrich Pfeffer* Laboratory of Molecular Biology, National Cancer Research Institute, Genova, Italy ABSTRACT: Expansions of poly-glutamine tracts in proteins that are expressed in the central nervous system cause neurodegenerative diseases. The altered proteins accumulate over long periods of time, forming nuclear inclusions, and lead to neuronal cell death. A similar mechanism could also be operant in non-dividing cells outside the central nervous system because nuclear inclusions are not limited to neurons. In addition, variations of the repeat length within the normal range may affect cellular function as it has been shown for the androgen receptor that is involved in neoplastic degeneration of several tissues. We have identified a poly-glutamine/poly-proline repeat in the homeobox gene DLX6. DLX genes are expressed in non-proliferative cells of the apical ectodermal ridge of developing limbs. Ablation of these cells leads to limb malformation. We propose that CAG triplet expansions in this gene could lead to cell death in the apical ectodermal ridge causing limb malformations. Indeed, autosomal dominant limb malformations with increasing severity in successive generations have been linked to the chromosomal region that contains DLX6. The analysis of a limited number of patients affected by split hand/ foot malformation so far revealed only a slight modifier effect of repeat length within the normal range and no expansions have been detected. © 2001 Elsevier Science Inc.
salvage [29,53,59]. Recent findings from cell culture and from transgenic mice link poly-glutamine dependent neurodegeneration to apoptosis of cells expressing proteins with expanded repeats [6,23,30,36,52]. TISSUE SPECIFIC CELL DEATH Although the pathogenic CAG repeat containing proteins are expressed in many cell types, cellular degeneration is observed only in the central nervous system. This could be explained by tissue specific factors that mediate cytotoxicity [37,42,66]. In a recent report, however, Kennedy and Shelbourne show a particular somatic instability of an expanded huntington gene repeat in transgenic mice in the striatum [28]. This work establishes a correlation between the phase of disease during which neuronal damage in the various brain tissues becomes manifest and the degree of somatic instability of the repeat. Similar to what has been observed for the androgen receptor (AR) [63], somatic instability of the CAG repeat could depend on the expression level of the huntington gene in the striatum. Cytotoxicity of the expanded Machado Joseph’s disease gene CAG repeat is higher in nondividing cells [73] suggesting that the expansion dependent cell death might occur preferentially during the G0 phase of the cell cycle. Cytotoxic effects may also be much less apparent in tissues with a high regeneration potential where dying cells are easily substituted. Short living cells might never reach the toxicity threshold of proteins containing the expanded poly-glutamine repeat. Moreover, CAG repeat expansion dependent apoptosis follows specific pathways involving caspases [31,50,67,70]. Differential expression of components of the apoptotic pathway could determine a differential susceptibility of specific tissues. However, the link between CAG repeat toxicity and apoptosis awaits to be confirmed in patients. Aggregates of proteins containing expanded poly-glutamine tracts sequester heat shock proteins and proteasome components and aggregation is suppressed by over-expression of molecular chaperones that provide cellular protection [8,25,27,30,33,40,61,68]. Again, the variable cellular array of those chaperones may influence poly-glutamine toxicity.
KEY WORDS: Neurodegeneration, Anticipation, Apical ectodermal ridge, DLX, Homeobox gene, Limb morphogenesis, Polyglutamine, Triplet repeat expansion, Striatum.
INTRODUCTION There are many genes in the human genome containing CAG triplet repeats within the coding region. When this has been analyzed, the lengths of these repeats are polymorphic in the normal human population, with some exceptions. Polymorphic repeat length is probably a function of reduced stability of such repeats which increases dramatically above a threshold of about 35 to 40 repeats, giving rise to different neurodegenerative diseases. It is not clear whether all CAG repeats are polymorphic or they all undergo occasional expansions nor whether such expansions would lead to diseases (for recent reviews see [14,44]) and other articles in this special issue). De novo repeat expansions have also been described [32,41,57,69,72] but the mutation rate is not known. CAG repeat diseases show dominant inheritance. A cytotoxic gain-of-function, apparently independent from the gene context [43], appears to be the main pathogenetic mechanism. Proteins carrying expanded glutamine stretches form aggregates in nuclear inclusions. It is not clear whether these aggregates are themselves pathogenic or whether they are the results of an attempted cellular
EFFECTS OF CAG REPEAT EXPANSIONS IN NON-NEURONAL TISSUES If the expression level of the disease protein, cell proliferation rate, regeneration potential, expression profile of mediators of apoptosis and of molecular chaperones co-operate to determine the cytotoxic effect of an expanded poly-glutamine repeat, tissue spe-
* Address for correspondence: Ulrich Pfeffer, Ph.D. Laboratory of Molecular Biology, National Cancer Research Institute, L. R. Benzi 10, 16132 Genova, Italy. Fax: ⫹39-010-5737-231; E-mail: [email protected]
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cific cell death could be explained without implicating tissue specific cellular factors. Nuclear inclusions have been found in non-neuronal tissues including scrotal skin, dermis, kidney, heart, and testis of spinobulbar muscular atrophy (SBMA) patients [35]. These inclusion were morphologically indistinguishable from those seen in neurons, reacted with the same subset of epitope specific anti-AR antibodies and were similarly ubiquinated. These observations suggest that the formation of nuclear inclusions follows the same pathway in neurons and non-neuronal cells. Nuclear inclusions were also detected in skeletal muscle, heart, liver, adrenal glands, pancreas, kidney, stomach, and duodenum of transgenic mice carrying the amino-terminal portion of the Huntington’s disease (HD) protein with an expanded repeat [51]. In these tissues progressive organ atrophy, though to variable extents, is observed, indicating that expanded CAG repeat containing proteins can lead to cellular death in non-neuronal tissues. This finding is also underpinned by the observation that the same transgenic mice develop diabetes [21] and that the islets of Langerhans contain nuclear inclusions [51]. Diabetes is also more frequent in HD patients as compared to the normal population [15]. HD patients also show hepatic symptoms in correlation to a reduced survival of hepatocytes [4].
other CAG repeat disease genes and are not expected to assume the particular conformation that is believed to be pivotal to the disease process [45]. However, cytoplasmic, non-ubiquitinated aggregates have been observed in SCA-6 patients and cells transfected with the SCA-6 containing an expanded CAG repeat undergo apoptotic cell death [24]. Pathogenicity of the short SCA-6 repeat expansions could therefore be analogous to other CAG repeat expansion diseases. Alternatively, the limited expansions might lead to ataxia via the functional inactivation of the ion channel. Inactivating splice site mutations of the same gene lead to a similar, though episodic, form of ataxia [77]. Thus at least in these two cases CAG repeat length polymorphisms within the range that is considered normal for most polyglutamine proteins can have functional consequences. Similar effects might show up also for other poly-glutamine proteins once their function will be unraveled. This appears particularly interesting for the wild-type HD protein that has been reported to protect neuronal cells from apoptotic stimuli [48]. If this activity is influenced by the repeat length within the normal range these polymorphisms might modify many aspects of neuronal function and pathology including neuronal aging.
FUNCTIONAL ASPECTS OF REPEAT LENGTH VARIATION WITHIN THE NORMAL RANGE
ARE THERE DEVELOPMENTAL CAG REPEAT EXPANSION EFFECTS?
CAG repeat lengths are highly polymorphic in the normal population and they are translated into polyglutamine stretches located in most cases in the amino-terminal part of the proteins. Above the threshold of about 35 to 40 glutamine residues the stretch assumes a particular structure which is believed to be responsible for polyglutamine pathogenicity [45]. Little is known about the function of polyglutamine regions within the normal range because information on the function of the proteins involved in trinucleotide expansion diseases is only beginning to emerge. The AR is a ligand-dependent transcription factor [65]. The human AR contains a poly-glutamine repeat in exon 1 whose expansions to over 40 CAGs cause Kennedy’s disease or SBMA. The AR CAG repeat is polymorphic in the normal human population (CAG8 –35) [34]. The transactivation potential of the AR is inversely correlated to repeat length either as a direct consequence of altered receptor function [9,38] or indirectly due to reduced AR messenger RNA and protein levels in cells carrying alleles with longer CAG repeats [10]. This is also reflected by the fact that patients suffering from Kennedy’s disease often show mild androgen insensitivity [2,56]. The receptor is involved in the control of cellular proliferation and differentiation in androgen target tissues, where the hormonal signal determines either a mitogenic response, as e.g., in the prostate [12], or an anti-mitogenic response, as, e.g., in the breast [3]. The interpretation of the androgenic signal is thus dependent on cell type-specific receptor functions. In keeping with this, men carrying AR alleles containing shorter CAG repeats face a higher risk of prostate cancer [18,19]. Women who carry a mutated allele of the breast and ovarian cancer susceptibility gene, BRCA-1, show an increased risk to develop the disease and a lower mean age at diagnosis, when they also carry AR alleles with longer CAG repeats [47]. We have reported recently that in 10% of colon cancers the AR CAG repeat undergoes somatic reductions that probably contribute to tumor progression [17]. Spinocerebrellar ataxia type 6 (SCA-6) is determined by CAG repeat expansions in the gene encoding the (1A-subunit of the voltage dependent calcium channel (CACNAIA) that is highly expressed in the cerebellum [75,77]. Expansions from the normally 4 –18 to 21–30 repeats correlate with progressive cerebrellar ataxia. These expansions are within the range that is normal for
CAG repeat expansion related cell death apparently occurs prevalently in non-dividing cells. Specialized developmental organizers often show a strictly determined number of cells that upon differentiation do not proliferate. If CAG repeat expansion related cell death depends on the amount of toxic protein in the cell, the toxic threshold of the expanded protein will be reached during the limited life of developmental organizer cells only when it is expressed at very high levels. Clarke et al.[11] analyzed the kinetics of metabolic decline in the caudate nuclei of Huntington’s disease patients and concluded that only mathematical models that assume a constant risk of death for the single neuron fit the data collected. According to this hypothesis, accumulation of expanded CAG repeat region containing proteins would determine a higher probability for the neuronal cell to die, perhaps by apoptosis, to a yet unknown insult. An analogous situation has been observed in Drosophila mutants carrying the rdgC mutation in a protein phosphatase involved in phototransduction: the mutation alone is not sufficient to determine retinal degeneration that occurs only in the presence of light [60]. Just like in these mutants also in HD the hypothetic trigger must be unavoidable or even constitutive because the repeat expansion is a cause of the disease and not a mere risk factor. Developmental organizer tissues formed by non-proliferative cells are committed to apoptosis after they have fulfilled their organizing task. If expanded glutamine repeat containing proteins are expressed at a high level in such cells, sensitivity to the apoptotic stimulus could drastically rise leading to untimely cell death and disturbed developmental organization. The apical ectodermal ridge (AER), a thickened epithelium on the limb bud, is an example for such a tissue. It is formed by small number of cells that control limb-bud outgrowth during development [64]. Factors that induce formation and, later on, re-absorption of the AER are only partially known. Removal of the AER from chick limb buds leads to malformation of the limbs. Removal early during development leads to proximal truncation while removal at later stages leads to more distal defects indicating that the AER controls the complete period of limb outgrowth [49,54,62].
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THE DISTAL-LESS HOMOLOGUE DLX6 We have recently identified a CAG/CCG repeat in the so far unknown first exon of the homeobox gene DLX6 [46], one of seven mammalian homologues of the Drosophila distal-less gene. DLX6 is located in the same genomic region as its homologue DLX5 in a tail-to-tail position [58]. The two genes, like the other two DLX pairs, are believed to be regulated in tandem. For DLX5 strong expression in the AER of the developing limb has been shown [1,16,74]. The human DLX6 gene is located on chromosome 7q22 in the critical interval for the split hand/split foot malformation 1 [55] (SHFM1, OMIM 183600). Other three loci have also been linked to SHFM. The malformation exists in a variety of forms and occurs either sporadically or familiarly. Phenotypic alterations consist in the absence of the middle ray of hands and/or feet with a more or less pronounced syndactyly of the remaining digits and a deep cleft between the digits. In severe cases, syndactyly and ectrodactyly occur in combination [5]. According to Zlotogora [76], SHFM1 can be divided into two subtypes, with (type II) or without (type I) aplasia of long bones. These two types are also distinct by their penetrance, which is nearly complete for type I and reduced to about 66% for type II. The malformation is inherited as an autosomal dominant trait but in families where SHFM is associated to other long bone deficiencies, marked non-Mendelian transmission is observed. This includes skipping of generations (reduced penetrance), increased severity in successive generations ranging from monomelic to tetramelic forms and from simple split hand/split foot to tibial or ulnar aplasia (variable expressivity) and overtransmission from affected fathers to sons (segregation distortion) [76]. In several SHFM families the phenotypic presentation of the malformations drastically increases in successive generations. In one particular family, the first generation for which information is available showed classical split hand malformation; in the following five generations the severity of the malformation increased leading to aplasia of distal portions of the limbs to nearly complete agenesis of the entire limbs in a child born without arms and legs [20]. The longitudinal outgrowth of the limbs is regulated by the AER and removal of this control region leads to limb malformations [54,62]. CAG repeat expansion related cell death in the AER could therefore lead to limb malformations and further expansions in following generations could increase the severity of the malformation by the anticipation of cell death corresponding to early removal of the AER. Impaired AER viability has also been shown for p63 mutations [39,71] that cause the SHFM-related ectrodactyly, ectodermal displasia, cleft palate syndrome [7] and for the mouse model of dactylaplasia [13]. These malformation syndromes, however, do not map to the genomic region containing DLX6. On this background we started to search for polymorphisms in the general population. We could identify five alleles with 11 to 20 CAGs whereas no polymorphisms could be detected for the CCG repeat containing 9 triplets [46]. Two alleles with 20 and 19 CAG repeats account respectively for 28% and 69% of the tested population, 3% had shorter alleles. Unfortunately, we had no access to samples of the family with clear sign of anticipation described by Helal et al. [20] nor to those of the few families with less drastic signs of anticipation. Therefore our analysis is so far limited to 38 sporadic cases and to familiar cases with few members affected so that no linkage studies are possible. We have also analyzed possible modifier effects in two families with reduced penetrance without clear signs of increasing severity in successive genera-
FIG. 1. Allele frequencies (panel a) and genotype frequencies (panel b) of healthy controls, unaffected parents, mothers and fathers of sporadic SHFM patients and sporadic SHFM patients.
tions, one with a translocation involving the locus containing DLX6 and one in linkage with another locus. We did not encounter DLX6 CAG repeat expansions in the patients analyzed. We were, however, able to show an additional allele containing 21 CAG repeats, only present in one sporadic patient and absent from 90 healthy subjects (67 from the general population, 23 parents of sporadic patients). Because samples from the parents of this patient were not available for analysis we cannot decide whether the unusual allele is a rare allele or a de novo expansion. From the analysis of allele frequencies (Fig. 1a) a very slight trend towards longer alleles is observed in sporadic patients where 32.9% had alleles with 20 or 21 repeats (27.6% in controls). The healthy mothers of sporadic patients had similarly low numbers of longer alleles (25.0%), while the allele was definitely underrepresented in the fathers of patients (5.6%). The genotype frequency (Fig. 1b) shows a complementary trend with the mothers
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showing 14.3% homozygosis for the long CAG20 allele while 6.3% were to be expected from the allele frequency. In the families of sporadic cases analyzed, the patients inherited a long allele from their mothers and, with one exception, not from their fathers. These observations so far constitute a trend but do not reach statistical significance except for the genotype frequencies control versus fathers (odds ratio⫽7.76 confidence interval 0.93–354.7; p ⫽ 0.037, Fisher test); More patients must be analyzed in order to establish a role for longer DLX6 alleles in SHFM. However, this trend is interesting because overtransmission of SHFM from affected fathers to affected sons has been described [26]. The modifier effect could be much stronger than suggested by our study if one takes into account that four loci have been linked to SHFM but the gene has been identified only in one case [7,22]. Moreover, genetic origin of the malformation is not certain for sporadic cases. In order to increase the statistical power of our analysis more patients together with their parents must be analyzed and the modifier effect should be analyzed for sporadic cases in correlation to the only known causative mutation in the p63 gene. CONCLUSIONS Poly-glutamine repeat proteins are expressed in many tissues. The cytotoxic effect apparently depends on several factors among which the proliferation status and the regeneration potential of the tissue that expresses the disease protein play a prominent role. In the adult, non-regenerating tissues, such as islets of Langerhans in the pancreas, could be targeted by poly-glutamine induced susceptibility to apoptotic insults. Our hypothesis that the same could occur during development in non-proliferating organizer cells is based on the one-hit model of neurodegeneration proposed by Clarke et al. [11]. We have tried to test this hypothesis for the DLX6 gene that contains a poly-glutamine/poly-proline tract. DLX6 appeared to be a promising candidate because it is expressed in a non-proliferative tissue with a crucial function for limb morphogenesis, it maps to a chromosomal region that has been linked to a limb malformation and the syndrome shows variable expressivity and increasing severity in successive generations. We consider these features as the developmental equivalent of anticipation. Our hypothesis could have been directly confirmed or dismissed if we would have had access to the most informative samples but the family denied collaboration. The analysis of sporadic cases and their parents indicates a potential modifier effect of longer repeat alleles and further analyses are needed to reach statistically significant conclusions. Modifier effects that depend on the repeat length have also been described for the AR [17–19,47] and might be common to many CAG repeat containing genes. DLX6 is also expressed in the brain, at least during development [58], and therefore this gene is also a candidate for “classic” CAG repeat expansion related neurodegenerative diseases. Further studies on poly-glutamine proteins outside the central nervous system should consider that CAG repeat expansions in genes that are highly expressed in non-proliferative tissues with special developmental functions may cause malformations of the corresponding structures. Databank screens reveal many developmental genes that contain such repeats. Furthermore, CAG repeat polymorphisms within the normal range may influence gene function, especially in the case of transcription factors and their coactivators, thereby modifying related cellular functions and their pathological degeneration.
ACKNOWLEDGEMENTS
We thank Fiorella Gurrieri, Giovanni Camera, and Marco Seri for providing patient samples. We also thank Douglas Noonan for critically reading the manuscript and Adriana Albini for continuous support.
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