RICERCA ITALIANA     

Ataxias with oculomotor apraxia: clinical and genetic study

Università degli Studi di Roma "La Sapienza"

Abstract

Hereditary autosomal recessive cerebellar ataxias (ARCAs) are rare diseases. The most common is Friedreich's ataxia (FA) that accounts for 30-40% of the cases in Caucasian populations. Other aetiologies, including ataxia telangiectasia (A-T), autosomal recessive spastic ataxia of Charlevoix-Saguenay and ataxia with vitamin E deficiency, are less frequent. A subgroup of ARCA associated withoculomotor apraxia but different from A-T has been identified (Aicardi et al., 1988; Barbot et al., 2001). Based on linkage studies, ataxia with oculomotor apraxia recently was found to encompass at least two types, type 1 (AOA1) (Moreira et al., 2001a) and type 2 (AOA2) (Bomont et al., 2000; Nemeth et al., 2000). AOA1, located to 9p13, is characterized by the association of CA with cerebellar atrophy on MRI, frequent choreic movements at onset which regress with the course of the disease, oculomotor apraxia, severe peripheral neuropathy, occasional mild mental retardation, hypercholesterolaemia and hypoalbuminaemia (Bomont et al., 2000; Tachi et al., 2000; Moreira et al., 2001a; Shimazaki et al., 2002; Le Ber et al., 2003; Tranchant et al., 2003). The defective gene, aprataxin gene (APTX), mapped to 9p13, was found to code for a new histidine-triad protein, named aprataxin, possibly involved in DNA single strand break repair (Date et al., 2001; Moreira et al., 2001b). In 2000, Nemeth and colleagues, and Bomont and colleagues independently reported linkage to 9q34 of autosomal recessive ataxia resembling A-T but with a later age at onset, without the extra-neurological features of A-T (Watanabe et al., 1998; Nemeth et al., 2000). The subsequent identification of families with both oculomotor apraxia and elevated serum AFP level suggested that this represents a single new entity, named AOA2, and allowed the critical region to be narrowed to a 4 cM interval (Moreira et al., 2002). Recently Moreira and colleagues have identified causative mutations in 15 families, which allows them to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein. The mutated gene in AOA2 encodes a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination. Moreira et al., 2004. The predicted protein encoded by the gene mutated in AOA2 is 2,677 amino acids long and contains at its C terminus a classical seven-motif domain found in the superfamily 1 of helicases. In particular, it shares extensive homologies with the fungal Sen1p proteins and so it was named senataxin (SETX). A recent clinical study by Le Ber and colleagues (Le Ber et al., 2004) showed that AOA2 can be found in Europe, North Africa and the West Indies, and its relative frequency represents ~8% of non-Friedreich ARCA, which is more frequent than ataxia telangiectasia and ataxia with oculomotor apraxia type 1 (AOA1), in our series of adult patients. In adults, AOA2 may be, therefore, the most frequent cause of ARCA identified so far, after Friedreich's ataxia.
Our study is aimed at collecting a large series of patients with ARCA of pediatric (UO2 and UO4) and adult age (UO1 and UO3), characterizing the clinical (UO1, UO3 e UO4), oculographic (UO3), neuroradiologic (UO1,UO3 e UO4) phenotype, performing molecular analyses of the APTX (UO2) and SENTX (UO1) gene in order of identify patients with AOA1 and AO2 respectively, and funcional studies about the molecular mechanisms responsible of these diseases (UO2). <<<

Principal Investigator

Carlo CASALI Universita' degli Studi di ROMA

Research Objectives

This project is aimed at collecting and characterizing genetically a large series of patients with Autosomal Recessive Cerebellar Ataxia (ARCA) after the recent identification of two new disease entities AOA1 and AOA2 and the mutated genes, APTX and SETX, respectively (Le Ber et al., 2003; Le Ber et al., 2004). The clinical presentation includes cerebellar ataxia and oculomotor apraxia (Ataxia with ocumototor apraxia, AOA). The actual prevalence is still unknown. While AOA1 is only relatively frequent among childhood onset patients (Le Ber et al., 2003), AOA2 is probably the most frequent form of ARCA in adults after Friedreich's ataxia (10% of ARCA) (Le Ber et al., 2004). Available data show that AOA2 is present in Europe (Moreira et al., 2004). For example an initial attempt at identifying patients suitable for genetic analysis of SETX gene in the existing series at the UO1, 2 and 3, gave encouraging results. Many individuals have been identified with ocular apraxia and/or elevated alpha fetoprotein, a laboratory marker of AOA2. The APTX gene has been shown to be causative of AOA1 in 2002 (Moreira et al., 2002), while SETX mutations have only been reported in march 2004 in AOA2 patients (Moreira et al., 2004). As a consequence little is known about the prevalence of mutated genes in the Italian poplulation of ataxic patients and about the different allele variants associated with the disease. Our study is aimed at adressing some of these questions.
For that purpose the resources of the different UOs will be of advantage as follows:
1. UO1 (Casali; La Sapienza Roma I Fac): availability of a large series (more 50), in whom other genetic causes have been previously excluded. Technical expertise of performing genetic analyses of the SETX gene (AOA2).
2. UO2 (Chessa, LaSapienza Roma II Fac): access to a large series of ataxic children with ataxia and oculomotor apraxia, registered in the Italian Register of Ataxia-Telangectasia (A-T) (oculomotor apraxia is a feature of A-T). In all patients mutations of ATM and hMRE11 genes, responsible of classic and variant A-T, have been previously excluded. Technical expertise of performing genetic analyses of the APTX gene (AOA1).
3. UO3 (Palmeri, Siena): availability of a large series of ataxic patients, mainly adults, suitable for APTX and SETX gene screening; availability of speciphic softwares developed for quantitative analysis of ocular movements (ASTIDET) and post-processing analysis of brain MRI imaging (SIENAX) for a quantitative analysis of cerebellar atrophy.
4. UO4 (Sorge, Catania): availability of a large series of ataxic children and shared expertise with the UO3 of the SIENAX software.
We believe this project represents the first systematic effort at identifying patients with AOA1 and AOA2 in Italy, describing causative mutations in APTX ans SETX, respectively, and attempting genotype-phenotype correlations. <<<

First Results

Recruitment of a large series of patients with AOA1 (15-20 individuals) and AOA2 (20-30 individuals)
Detailed information about the clinical presentation, disease course, ecc.
Acquisition of electrophysiologic and neuroimaging data
Oculographic study
Bank of DNA from patients and their relatived
Bank of lymphoblastoid linesResults of the mutational analyses of APTX and SETX genes in the Italian population
Possible identification of novel mutations and founder effect in restricted poulations
Genetic counseling and molecular diagnosis for subjects at risk, healthy carriers and prenatal diagnosis
Epidemiologic data (prevalence; frequency of the mutated genes at the heterozygote state)
Reappraisal a posteriori of the clinical features of AOA1 and 2 in genetically confirmed patients; validation of clinical criteria for diagnosis
Assessment of inter and intrafamilia variability
Qualitative and quantitative evaluation of oculomotor apraxia
Qualitative and quantitative description of cerebellar and extracerebellar atrophy also for diagnostic purposes
Phenotype-genotype correlation for different mutations
Result of funcional studies in lymphoblastoid cell lines <<<

Timescale

24 months

National and international background

Hereditary autosomal recessive cerebellar ataxias (ARCAs) are rare diseases. The most common is Friedreich's ataxia (FA) that accounts for 30±40% of the cases in Caucasian populations. Other aetiologies, including ataxia telangiectasia (A-T), autosomal recessive spastic ataxia of Charlevoix-Saguenay and ataxia with vitamin E deficiency, are less frequent. A subgroup of ARCA associated withoculomotor apraxia but different from A-T has been identified (Aicardi et al., 1988; Barbot et al., 2001). Oculomotor apraxia was described initially in children affected by congenital oculomotor apraxia as the inability to generate volitional horizontal saccades with a characteristic compensatory headthrusting and synkinetic blinking (Cogan, 1953). It is better described as intermittent saccade failure rather than a true apraxia (Harris et al., 1996; Shawkat et al., 1996). A-T is characterized by an early age at onset, the association of cerebellar ataxia (CA), oculomotor apraxia, telangiectasias, recurrent infections, cancers and markedly elevated a-foetoprotein (AFP) level. The patients usually become wheelchair-bound in their early teens (Woods et al.,1992; Stankovic et al., 1998). The ATM gene codes for a protein involved in DNA double strand break repair (Stavitsky et al., 1995; Laake et al., 2000; Campbell et al.,2003). Approximately 10% of A-T patients have mutations causing a milder phenotype (`A-T variants') that differs from the typical phenotype by a later age at onset, a longer disease duration or the absence of one characteristic feature of the disease (Gilad et al., 1998). In addition, two families presenting an A-T-like phenotype (`A-T-like syndrome') have mutations in the hMRE11 gene which maps proximal to ataxia-telangiectasia mutated gene (ATM) (Stewart et al.,1999). Based on linkage studies, ataxia with oculomotor apraxia recently was found to encompass at least two types, type 1 (AOA1) (Moreira et al., 2001a) and type 2 (AOA2) (Bomont et al., 2000; Nemeth et al., 2000). AOA1, located to 9p13, is characterized by the association of CA with cerebellar atrophy on MRI, frequent choreic movements at onset which regress with the course of the disease, oculomotor apraxia, severe peripheral neuropathy, occasional mild mental retardation, hypercholesterolaemia and hypoalbuminaemia (Bomont et al., 2000; Tachi et al., 2000; Moreira et al., 2001a; Shimazaki et al., 2002; Le Ber et al., 2003; Tranchant et al., 2003). The defective gene, aprataxin gene (APTX), mapped to 9p13, was found to code for a new histidine-triad protein, named aprataxin, possibly involved in DNA single strand break repair (Date et al., 2001; Moreira et al., 2001b). All mutations identified so far are located in exons 5, 6 and 7 (Date et al., 2001; Moreira et al., 2001b). In 2000, Nemeth and colleagues, and Bomont and colleagues independently reported linkage to 9q34 of autosomal recessive ataxia resembling A-T but with a later age at onset, without the extra-neurological features of A-T (Watanabe et al., 1998; Nemeth et al., 2000). These results were based on the study of a large Pakistani family with oculomotor apraxia but normal serum AFP level (Nemeth et al., 2000) and a large family with elevated AFP level but no oculomotor apraxia (Watanabe et al., 1998; Bomont et al.,2000). The subsequent identification of families with both oculomotor apraxia and elevated serum AFP level suggested that this represents a single new entity, named AOA2, and allowed the critical region to be narrowed to a 4 cM interval (Moreira et al., 2002). Recently Moreira and colleagues have identified causative mutations in 15 families, which allows them to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein. Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination. Moreira et al., 2004. The predicted protein encoded by the gene mutated in AOA2 is 2,677 amino acids long and contains at its C terminus a classical seven-motif domain found in the superfamily 1 of helicases. In particular, it shares extensive homologies with the fungal Sen1p proteins and so it was named senataxin (SETX). Saccharomyces cerevisiae Sen1p is involved in splicing and termination of tRNA, small nuclear RNA and small nucleolar RNA and has RNA helicase activity encoded by its C-terminal domain (Ursic at al., 1997; Rasmussen and Culbertson, 1998; Kim et al., 1999). Schizosaccharomyces pombe has two Sen1 genes. The first reported S. pombe Sen1p (Sen1p1, encoded on chromosome I) has both RNA and DNA helicase activities (Kim et al., 1999). Of all the fungal Sen1p proteins, however, the second S. pombe Sen1p (Sen1p2, encoded on chromosome II) has the highest homology with senataxin over the N-terminal domain (20% identity over 466 residues). The C-terminal domain of senataxin and the Sen1p proteins shares significant similarity with two other members of the DExxQ-box family of helicases: RENT1/Upf1, involved in nonsense mediated RNA decay (Wang et al., 2001), and IGHMBP2, defective in spinal muscular atrophy with respiratory distress (Grohmann et al., 2001) (OMIM 604320), a human disorder of motor neurons, and in mouse neuromuscular degeneration (Cox et al., 1998). Upf1 proteins have RNA helicase activity, but IGHMBP2 was initially identified as a DNA binding protein with transcriptional transactivating properties (Mizuta et al., 1993). It is therefore possible that, like S. pombe Sen1p1, senataxin has both RNA and DNA helicase activities and that senataxin acts in a DNA repair pathway, like several other proteins defective in autosomal recessive cerebellar ataxias, as in ataxia-telangiectasia (Shiloh, 2003), AOA1 (Moreira et al., 2001), ataxia-telangiectasia-like disorder (Stewart et al., 1999) and spinocerebellar ataxia with peripheral neuropathy 1 (Takashima et al., 2002). Alternatively, the results also suggest that senataxin might be a nuclear RNA helicase with a role in the splicing machinery and that the molecular pathology of AOA2 may share features with spinal muscular atrophy and spinal muscular atrophy with respiratory distress. These results add to the increasing evidence to suggest that both DNA repair and RNA splicing are key factors in several neurodegenerative disorders, including the newly identified AOA2, and further work may elucidate the role of these mechanisms in neuronal integrity and neurodegeneration. <<<