Genetic features of Huntington disease in Cuban population: Implications for phenotype, epidemiology and predictive testing

Journal of the Neurological Sciences 335 (2013) 101–104

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Genetic features of Huntington disease in Cuban population: Implications for phenotype, epidemiology and predictive testing Yaimeé Vázquez-Mojena a,⁎, Leonides Laguna-Salvia b, José M. Laffita-Mesa a, Yanetza González-Zaldívar a, Luis E. Almaguer-Mederos a, Roberto Rodríguez-Labrada a, Dennis Almaguer-Gotay a, Pedro Zayas-Feria a, Luis Velázquez-Pérez a,⁎⁎ a b

Centre for Research and Rehabilitation of Hereditary Ataxias (CIRAH), Holguín, Cuba Department of Neurology, Clinical & Surgical Hospital, Holguín, Cuba

a r t i c l e

i n f o

Article history: Received 6 June 2013 Received in revised form 22 August 2013 Accepted 26 August 2013 Available online 3 September 2013 Keywords: Huntington's disease HTT gene CAG repeats Molecular testing Intermediate alleles Epidemiology Chorea Neurodegenerative disease

a b s t r a c t Huntington disease is the most frequent polyglutamine disorder with variable worldwide prevalence. Although some Latin American populations have been studied, HD prevalence in Cuban population remains unknown. In order to characterize the disease in Cuba, the relative frequency of HD was determined by studying 130 patients with chorea and 63 unrelated healthy controls, emphasizing in the molecular epidemiology of the disease. Sixtytwo patients with chorea belonging to 16 unrelated families carried a pathological CAG expansion in the HTT gene, ranging from 39 to 67 repeats. Eighty-three percent of them come from the eastern region of the country. A significant inverse correlation between age at onset and expanded CAG repeats was seen. Intermediate alleles in affected individuals and controls represented 4.8% and 3.97% respectively, which have been a putative source of de novo mutation. This study represents the largest molecular characterization of Huntington disease in the Cuban population. These results may have significant implications for an understanding of the disease, its diagnosis and prognosis in Cuban patients, giving health professionals the tools to implement confirmatory genetic testing, pre-symptomatic testing and clinical trials in this population. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeats in the first exon of the HTT gene producing a long polyglutamine tract in the huntingtin protein [1]. The disease is characterized by motor dysfunction, involuntary choreic movements, cognitive impairment and psychiatric manifestations [2]. The size of the HTT CAG repeat contributes significantly to the onset and disease phenotype [3]. In the general population, the CAG repeat length varies from 6 to 35 trinucleotides [4]. Alleles carrying b 26 CAG repeats are considered normal, whereas those with 27–35 triplets are intermediate. Interestingly, some individuals carrying intermediate alleles have developed the HD phenotype [5,6]. Pathological expansions exceed 35 CAG repeats [7,8], alleles carrying 36–39 CAG repeats exhibit reduced penetrance while ≥40 CAGs are fully penetrant HD alleles [9].

HD is the most frequent polyglutamine disorder with a variable worldwide prevalence according to the ethnic background [10–12] and to the geographic differences in HTT haplotypes [12]. The prevalence in European descendants, including the United States and Canada [13], is about 5–10 per 100 000 inhabitants [14]. The disease reaches higher prevalence rates in specific regions, such as Maracaibo Lake (Zulia, Venezuela), Tasmania Island (Australia) and Moray Firth (Scotia) [15] whereas considerably reduced frequencies of HD have been reported in Japan, China, Finland, South African black populations and North American black populations [13,16]. Although some Latin American populations have been studied, little is known about the prevalence rate of HD in these countries, with the exception of Venezuela [13,17]. HD prevalence in the Cuban population is unknown. In order to characterize the disease in Cuban patients, we determined the relative frequency of HD in a large group of patients with chorea, emphasizing the molecular epidemiology of the disease. 2. Methods

⁎ Correspondence to: Y. Vázquez-Mojena, Carretera Central, km. 5½ vía Habana, CP 80100, Holguín, Cuba. Tel.: +53 24 424090. ⁎⁎ Correspondence to: L. Velázquez-Pérez, Reparto Edecio, Pérez, Carretera Vía Habana, CP 80100, Holguín, Cuba. Tel.: +53 24 462296; fax: +53 24 424090. E-mail addresses: [email protected] (Y. Vázquez-Mojena), [email protected] (L. Velázquez-Pérez). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.08.037

2.1. Subjects A total of 130 patients exhibiting chorea (54 m; 76 f), were tested for the HTT mutation in the Centre for Research and Rehabilitation of Hereditary Ataxias (CIRAH). A control group was recruited, composed of

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63 healthy subjects (25 m; 38 f. who had no family history or clinical signs suggestive of HD or other neurological disorders. The clinical diagnosis of patients was made by the referring neurologists. Informed consent was obtained from all subjects. The study was approved by the Institutional Ethics Committee of the Center. 2.2. Molecular genetics measures Genomic DNA was extracted from peripheral blood leucocytes using standard procedures [18]. The CAG size was estimated through PCR amplification, using primers that flank the CAG repeat tract [19,20], and polyacrylamide gel electrophoresis. 2.3. Statistical analysis Statistical analysis was conducted using the commercially available Statistica software package (StatSoft, Inc., 2003 STATISTICA data analysis software system, version 6. www.statsoft.com). The normality of all variables was assessed through the Kolgomorov–Smirnov test. Mean comparisons were performed using the Mann–Whitney U test. Statistical significance was considered when p b 0.05. Correlation was assessed by the Spearman test.

Fig. 2. Age of onset and CAG correlation: Significant inverse correlation between age of onset and expanded CAG repeats r = −0.82 for Spearman's correlation coefficient p = 0.000001.

3. Results Sixty-two out of 130 (47.7%) patients with chorea belonging to 16 unrelated families carried a pathological CAG repeat expansion in the HTT gene. Among them, 83% came from the eastern region of the country, specifically from the Municipality of Banes. The HD patients' mean age of onset was 38 ± 12.56 years (range 9–65 years). All HD patients were heterozygotes for a pathological mutation. Sixteen different alleles were identified, in the range of expanded alleles from 39 to 67 trinucleotides (mean 45.3) (Fig. 1). The distribution of expanded alleles was asymmetrical (skewness: 2.48 ± 0.38) and did not fit a normal distribution (D = 0.24; p b 0.05). Chromosomes carrying 45 repeats were the most frequent in HD patients (16.7%). Fullypenetrant alleles (N40 CAG) were observed in 98.3% of the HD mutation carriers. The longest expanded chromosomes carried 55, 67 and 59 units, corresponding to cases of infantile and juvenile HD respectively. There was a significant inverse correlation between age at onset and expanded CAG repeats (r = −0.82; p = 0.000001) (Fig. 2).

Thirteen different normal alleles were identified in HD patients, ranging from 13 to 29 units (mean 18.4). In this series, 17.7% of the chromosomes had ≥21 CAG units and intermediate alleles represented 4.8% of the total. No correlation was demonstrated between the normal CAG repeats and the age of onset in HD patients (r = 0.0012; p =0.9955). No statistically significant differences were detected between the expanded allele CAG size in patients with paternal versus maternal inheritance (p = 0.53, with the median CAG repeat length of 46.2 ± 5.89 vs. 45.3 ± 3.41 respectively) (Fig. 3). CAG repeat instability in HD chromosomes was analyzed in seven sib-ship transmissions, four maternally and three paternally inherited. CAG size was stable in four transmissions; the remaining showed an increase in 6, 13 and 20 CAG units respectively; the last two corresponding to paternal transmission. In order to gain insights into the CAG allele frequency at the HTT locus for the Cuban population, a series of 126 chromosomes from unaffected individuals was analyzed. All controls had unexpanded CAG repeats. We found 17 different alleles ranging from 12 to 31 CAG repeats

Fig. 1. Distribution of (CAG)n size of the Huntington disease gene in a Cuban population.

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Fig. 3. Parental inheritance and CAG repeats: Paternal and maternal inheritance of CAG repeat expansions.

(mean: 18.3; SD: 3.54). The alleles carrying 16 repeats were the most frequent (23.0%), followed by alleles with 17 units (19.8%). Intermediate alleles were observed in 5 subjects, accounting for 3.97%.

4. Discussion This study represents the largest molecular characterization of Huntington disease in a Cuban population. The analysis of the CAG repeats in the HTT gene in Cuban families confirmed the presence of expansions in ~48% of the patients with a choreic phenotype, most of them carrying fully-penetrant expansions. This finding ranks HD as the second more frequent polyglutamine disease in Cuba, only surpassed by the Spinocerebellar Ataxia type 2 (SCA2), the latter resulting from a founder effect [21] and a large number of predisposing normal alleles [22]. In addition, the representative percentage of HD cases belonging to the eastern region of the island suggests the existence of a possible founder effect underlying the Cuban HD cases, but further epidemiological, historical and genetic approaches are pending to strengthen this hypothesis. The frequency of CAG repeat expansions in the HTT gene, among all individuals with a choreic phenotype in our sample, is lower than the one reported in other studies [23–26]. Chorea has many causes and may also be found as a part of the phenotype in a large number of genetic conditions [27,28]. However where chorea forms the dominant clinical feature, HD is the most common and should be considered in cases of chorea of uncertain origin, even in the absence of a family history of the disorder [29]. HD phenocopies have been considered responsible for 3% of the patients referred for molecular confirmation of a clinical diagnosis of HD with a negative test result [30,31]. Other authors highlight the possibility of an HD-like disease without CAG expansion in the HTT gene. In the last few years, several so-called HD phenocopies have been described, which are responsible for choreic syndromes [2,8]. Creighton et al. reported a series where 31.5% of patients had a CAG of b 36 units and underline the importance of documenting CAG studies in cases with presumed HD to avoid falsely reassuring the individuals with a ‘low risk’ result for HD when the mutation in that particular patient is not a CAG expansion in the HD gene [32]. In addition, SCA 17 and DRPLA that may resemble HD and several other loci (HDL1 and HDL2) have recently been identified [33]. Other disorders that may more rarely resemble HD are SCA1 and SCA3 [18,27], and even SCA2 [2,8,34]. Therefore, we consider that such mutations could be responsible for the clinical characteristics of the other

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affected subjects in our sample. Further research is needed to determine the genetic origin of the chorea phenotypes in the remaining cases. The range of the expanded alleles found in our study is similar to the one identified in other Latin-American populations [13,16,35–38]. Despite the differences in the regional ethnic background, this result could support previous observations about the common European origin of HD chromosomes in North and South America and the Caribbean [12,39] but further studies are needed to confirm this hypothesis in our population. In keeping with the previous reports, our study demonstrated an inverse correlation between the CAG length and the age of onset. The gender of the transmitting parent has been documented as an important factor influencing the likelihood of CAG repeat instability in HD [40]. We analyzed the differences in the stability of the CAG repeats when inherited either through maternal or through paternal transmission. We observed instability consisting of an increase in the CAG repeat size, which was responsible for the infantile onset cases, independently of the transmitting parent. Nevertheless, paternally transmitted HD alleles render the largest repeat expansions leading to infantile case and the emergence of de novo mutation for HD from expansion of an intermediate allele, supporting the important instability of the paternal transmission [40,41]. It has been suggested that the mean of the CAG size in the general population allows deducing HD prevalence, taking into account the instability of larger alleles and its risk to become expanded alleles [12,13,42]. The average of CAG repeats in our control group (18.3) is in accordance with the one described for the general population worldwide (17–20 repeats) [13,42]. Based on this hypothesis we would expect that, in terms of instability, the prevalence of HD in our study was somewhat higher than that reported for European descendants (17.8) [12]. In comparison to other Latin American populations, the Cuban population shows an average of CAG repeats higher than the one described for Argentina (17.82) [13] and Brazil (17.8) [16], lower than in Mexico (19.04) [17], but more similar to Venezuelan population where a cluster of HD with striking geographic prevalence is located [43]. Previous studies demonstrated that HTT gene intermediate alleles are specifically enriched by the same haplotype of HD patients [44,45] and consequently they are prone to the generation of de novo mutation [46,47]. The frequency of intermediate alleles in our control group is consistent with the range estimated for the general population (between 1 and 3.9%) [44]. Although intermediate alleles in HD families reach a higher percentage (4.8%), there were no significant differences in the frequency of these alleles between both groups, so we cannot reach a definite conclusion regarding the hypothesis of an association between intermediate allele frequency and HD frequency in our sample. Nevertheless, the observation of 4% of intermediate alleles in our control group deserves special attention and justifies further studies of predisposing haplogroups in larger cohorts to clarify this issue. In conclusion, the expanded alleles in the HTT gene are frequent in Cuban patients with a choreic phenotype. In the eastern region of the country there is a noteworthy frequency of HD disease suggesting a possible founder effect, which deserves further attention. The results generated from this study may have significant implications for the knowledge of the disease, its diagnosis and prognosis in HD Cuban patients, giving health professionals the tools to implement confirmatory genetic testing, pre-symptomatic testing and clinical trials in this population. Finally, further studies are highly warranted to confirm if a founder effect or a high frequency of intermediate alleles of the HTT locus account for HD prevalence in Cuba.

Funding source Cuban Ministry of Public Health.

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Conflict of interest No conflict of interest was declared. Acknowledgments We are grateful to all patients, their families and the control individuals for their participation. We would like to thank Professor Maritza Pupo-Antunez, Tania Cruz-Mariño, MD and Professor Patrick McLeod for the critical revision.

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