Germline DNA copy number variation in individuals with Argyrophilic grain disease reveals CTNS as a plausible candidate gene

SHORT COMMUNICATION

Germline DNA copy number variation in individuals with Argyrophilic grain disease reveals CTNS as a plausible candidate gene

Darine Villela^I; Lilian Kimura^I; David Schlesinger^II; Amanda Gonçalves^III; Peter L. Pearson^I; Claudia K. Suemoto^IV; Carlos Pasqualucci^V; Ana Cristina Krepischi^III; Lea T. Grinbergand^V; Carla Rosenberg^I

^IDepartamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil

^IIInstituto Israelita de Ensino e Pesquisa Albert Einstein, São Paulo, SP, Brazil

^IIIInstituto Nacional de Ciência e Tecnologia em Oncogenômica, Hospital AC Camargo, São Paulo, SP, Brazil

^IVDepartamento de Medicina Interna, Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil

^VDepartamento de Patologia, Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil

^{Send correspondence to}

ABSTRACT

Argyrophilic grain disease (AGD) is a progressive neurodegenerative disease of the human brain that has never been associated to a particular gene locus. In the present study, we report the results of a CNV investigation in 29 individuals whose anatomopathologic investigation of the brain showed AGD. Rare CNVs were identified in six patients (21%), in particular a 40 kb deletion at 17p13.2 encompassing the CTNS gene. Homozygote mutations in CTNS are known to cause cystinosis, a disorder characterized by the intralysosomal accumulation of cystine in all tissues. We present the first CNV results in individuals presenting AGD and a possible candidate gene implicated in the disorder.

Keywords: Argyrophilic grain disease, copy number variations, CNVs, array-CGH, CTNS.

Argyrophilic grain disease (AGD) is a neurodegenerative disorder of the aged human brain morphologically characterized by the presence of argyrophilic grains (AG) mainly in limbic areas. Several studies have shown frequent association of AGD with other tauopathies such as Alzheimer's and Pick's diseases, and its prevalence increases significantly with age, present in up to 40% of all 85 years or older individuals (Tolnay and Clavaguera, 2004). However, the cause of AGD remains unknown; the disease seems to be sporadic but genetic studies have failed to discover a link between AGD and a particular gene locus.

The contribution of DNA copy number variations (CNVs) in the phenotypes of various complex diseases has been widely demonstrated over the past years. In fact, CNVs are increasingly recognized to be a prevalent form of common genetic variation in the human population. Even though a great number of studies have demonstrated the role of CNVs in the etiolo hiatric disorders (Lee and Lupski, 2006; Cook and Scherer, 2008) there are no reports of specific CNVs related do AGD. Here, we identified rare constitutive CNVs in individuals with AGD, in particular one at 17p13.2 that points to deletions in the cystinosin, lysosomal cystine transporter gene, CTNS, as strong candidate to cause AGD.

The Brain Bank of the Brazilian Aging Brain Study Group (BBBABSG) (Grinberg et al., 2007) provided DNA from blood samples of 29 individuals with AGD. The individuals of our group ranged in age from 50 to 89 and included females and males. Table 1 presents the characterization of all cases investigated in this study. The neuropathological diagnosis of AGD was based, at least, on the presence of AG in hippocampal CA1 area (CA -Cornu Ammonis) and entorhinal region, as well as in pretangles in hippocampal CA2 area in sections immunostained with the phosphor-tau antibody (PHF1, 1:1000, gift from Peter Davies, NY) according to accepted criteria (Braak and Braak, 1987). The subject's clinical and functional statuses were assessed through a knowledgeable informant based on a validated clinical protocol. The protocol includes a series of semi-structured scales and questionnaires that cover major functional abilities and were validated for assessment with an informant (Grinberg et al., 2007). BBBABSG's procedures were approved by the Ethical Board of University of São Paulo Medical School and the next-of-kin agreed to participate and signed an informed written consent.

To identify CNVs we performed oligonucleotide comparative genomic hybridization based on microarrays (array-CGH) using a whole-genome platform containing ~180.000 oligonucleotides (180k platform) (Oxford Gene Technologies, UK). Briefly, samples were labeled with Cy3-and Cy5-deoxycytidine triphosphates by random priming. Purification, hybridization and washing were carried out as previously reported (Krepischi et al., 2010). Scanned images of the arrays were processed using Feature Extraction software and data were analyzed with the Genomic Workbench software, both from Agilent Technologies. To distinguish CNVs, we used the Aberration Detection Method 2 statistical algorithm (ADM2) with a sensitivity threshold of 6.7. A genomic segment was considered duplicated or deleted when the log₂ ratio of the Test/Reference fluorescent intensities of a given region encompassing at least three probes was above 0.3 or below -0.3, respectively. Detected CNVs were compared to CNV data from oligoarray studies documented in the Database of Genomic Variants (DGV). The relevant CNVs were validated by dye-swap hybridizations, and only a mirror result would be confirmatory of the CNVs presence.

The array-CGH analysis revealed rare CNVs (rare defined as frequency < 0.1% of population, based on DGV) in six individuals among the 29 with AGD, none of them recurrent. To exclude that these rare CNVs represent com-genomic imbalances. Any of the rare CNVs detected are mon variants in the Brazilian population, we compiled potential candidates for the investigated phenotype; how-CNV data obtained with the same 180K array-CGH plat-ever, we highlighted the genomic 40 kb microdeletion of at form from more than 400 independent samples studied in 17p13.2 that includes the CTNS gene as especially interest-our laboratory for reasons other than dementia. None of the ing due to its relevant gene content and the lack of reports on rare CNVs documented in this study were detected in these copy number changes affecting this gene. Figure 1 shows the subjects. Table 2 summarizes the rare CNVs identified in validation of this CNV made by reverse labeling hybridizaour group and shows the genes encompassed by these tion, where it is possible to see that the alteration is mirrored.

The CTNS gene encodes a seven-transmembrane domain protein that functions to transport cystine out of lysosomes. Its activity is driven by the H⁺ electrochemical gradient across the lysosomal membrane. Mutations in this gene cause cystinosis, a rare autosomal recessive disorder characterized by the intralysosomal accumulation of cystine in all tissues (Kalatzis and Antignac, 2002). The most common mutation associated with this rare disease is a deletion of 65 kb presented in homozygosity that also includes the SHPK gene. The protein encoded by this latter gene has weak homology to several carbohydrate kinases, a class of proteins involved in the phosphorylation of sugars as they enter the cell, inhibiting return across the cell membrane (Wamelink et al., 2008). Our results show a heterozygous deletion in the CTNS gene encompassing the region of this common mutation associated with cystinosis. Literature data demonstrate that the brain is one of the last organs to be affected by the progressive cystine accumulation (Cochat et al., 1986). Cognitive impairments have been documented in some cystinosis patients, which presented deficit in visual-spatial memory (Trauner et al., 1988; Scar-vie et al., 1996). Additionally, an investigation showed that Ctns -/-knockout mice present a severe age-related memory deficit (Maurice et al., 2009). Therefore, this evidence makes CTNS a good candidate gene for susceptibility to AGD.

In conclusion, this is the first study to identify a rare CNV at 17p13.2 with AGD and links this disease with a particular gene locus, the CTNS.

Acknowledgments

This work was supported by a FAPESP grant CR (2009/00898-1), and DV was supported by a FAPESP PhD fellowship (2010/15503-0). We thank the Brazilian Aging Brain Study Group for providing DNA samples.

Received: May 16, 2013

Accepted: October 8, 2013.

Associate Editor: Carlos F.M. Menck

License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Publication Dates

• Publication in this collection
  09 Dec 2013
• Date of issue
  2013

History

• Received
  16 May 2013
• Accepted
  08 Oct 2013