Metabolic Brain Disease https://doi.org/10.1007/s11011-020-00640-0 ORIGINAL ARTICLE Fabry disease: GLA deletion alters a canonical splice site in a family with neuropsychiatric manifestations Patrícia Varela 1 & Gerson Carvalho 2 & Renan Paulo Martin 1,3 & João Bosco Pesquero 1 Received: 12 August 2020 / Accepted: 30 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020 Abstract Fabry disease (FD) is a rare X-linked glycosphingolipidosis caused by mutations in GLA, a gene responsible for encoding αgalactosidase A, an enzyme required for degradation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in all cells of the body. FD patients present a broad spectrum of clinical phenotype and many symptoms are shared with other diseases, making diagnosis challenging. Here we describe a novel GLA variant located in the 5′ splice site of the intron 3, in four members of a family with neuropsychiatric symptoms. Analysis of the RNA showed the variant promotes alteration of the wild type donor site, affecting splicing and producing two aberrant transcripts. The functional characterization showed absence of enzymatic activity in cells expressing both transcripts, confirming their pathogenicity. The family presents mild signs of FD, as angiokeratoma, cornea verticillata, acroparesthesia, tinnitus, vertigo, as well as accumulation of plasma lyso-Gb3 and urinary Gb3. Interestingly, the man and two women present psychiatric symptoms, as depression or schizophrenia. Although psychiatric illnesses, especially depression, are frequently reported in patients with FD and studies have shown that the hippocampus is an affected brain structure in these patients, it is not clear whether the Gb3 accumulation in the brain is responsible for these symptoms or they are secondary. Therefore, new studies are needed to understand whether the accumulation of Gb3 could produce neuronal alterations leading to psychiatric symptoms. Keywords Fabry disease . Splice site mutation . α-Galactosidase A . Psychiatric symptoms, GLA gene . Neurological manifestations . Depression . Schizophrenia Introduction Fabry Disease (FD, OMIM # 301500) is a rare and progressive X-linked glycosphingolipidosis. The origin of the disease is attributed to pathogenic mutations in the GLA, a gene responsible for encoding the α-galactosidase A enzyme (α-Gal A; EC 3.2.1.22). The deficiency or absence of the α-Gal A results in progressive accumulation of globotriaosylceramide Patrícia Varela and Gerson Carvalho contributed equally to this work. * João Bosco Pesquero jbpesquero@gmail.com 1 Center for Research and Molecular Diagnostic of Genetic Diseases – Department of Biophysics, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 9o andar, São Paulo 04039-032, Brazil 2 Medical Genetics Unit, Hospital de Apoio de Brasília, Brasília, DF, Brazil 3 McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA (Gb3) and other sphingolipids in the lysosomes, mainly in kidney, skin, eyes, heart, and brain (Oliveira and Ferreira 2019). The GLA gene spans 12 kb of genomic DNA and is divided into seven coding exons; most pathogenic mutations are located within the exons; however, many variants are also described in introns and splice site regions. Variants located in splice site regions could cause diseases due to change the RNA processing, producing an RNA decay (Sterne-Weiler and Sanford 2014) or a nonfunctional protein, leading to FD (Abramowicz and Gos 2018). FD presents a broad spectrum of clinical phenotype, classified as classical and non-classical or atypical (Arends et al. 2017). In the classical form, the most common symptoms are acroparasthesia, angiokeratomas, abdominal pain, gastrointestinal, cardiac, renal, and cerebrovascular involvement. The non-classical phenotype usually promotes late onset symptoms and affects only one organ (Oliveira and Ferreira 2019). Central and peripheral nervous system manifestation, as neuropathic pain, hearing loss, recurrent headache, and as the disease progresses, cerebrovascular symptoms, are Metab Brain Dis described in FD patients. Stroke is a common manifestation in FD patients, and several mechanisms could be involved in brain vasculopathy, as Gb3 accumulation in the blood vessels endothelium (Moore et al. 2007), changes in blood flow, prothrombotic state (Hilz et al. 2004) and increased reactive oxygen species (Moore et al. 2004). In addition to cerebrovascular symptoms, neuropsychiatric symptoms are often described in FD, with depression being the most reported disorder, affecting 15 to 62% of patients (Wang et al. 2007; Crosbie et al. 2009; Schermuly et al. 2011; Laney et al. 2013; Bolsover et al. 2014; Löhle et al. 2015; Körver et al. 2020). Other disorders, such as psychoses, although rare, are also reported in patients with FD (Liston et al. 1973; Shen et al. 2007; Segal et al. 2010; Schermuly et al. 2011; Sawada et al. 2015). Studies have shown that in the brain, the accumulation of Gb3 is not restricted to vessels, but also occurs in brain regions as the dorsal motor nucleus of the vagus, substantia nigra, neocortex, hippocampus (Okeda and Nisihara 2008), brainstem, amygdala, hypothalamus and entorhinal cortex (deVeber et al. 1992). Furthermore, a study with (include), α-Gal A deficient mice showed a neurophatological and neurodegenerative phenotype associated with the disruption of the autophagy-lysosome pathway, in cerebellum, pons, hippocampus and cortex (Nelson et al. 2014). Some of these areas are known to be affected in neurodegenerative and neuropsychiatric diseases (Wise et al. 2017; Kalmady et al. 2017; Santos et al. 2018; Barch et al. 2019; Chen et al. 2019), indicating that neuropsychiatric symptoms found in patients with FD may be due to the accumulation of Gb3 in specific regions of the brain. However, the pathophysiological basis of FD in the central nervous system are poorly understood and it is a matter of debate whether psychiatric symptoms are caused by the disease or if are a causality. In this study, we report the molecular and functional analysis of a novel GLA mutation found in two-generation family present neuropsychiatric manifestations, such as depression and schizophrenia. Sequencing DNA and RNA were analyzed by Sanger sequencing. To the DNA isolation, peripheral blood samples were collected and genomic DNA was extracted using QIAamp DNA Blood Mini (Qiagen, Hilden, Germany), according to the manufacturer’s conditions. The seven exons of GLA gene were amplified by polymerase chain reaction (PCR) and each amplicon was purified and sequenced. Sequences were compared with the DNA reference (NG_007119), confirmed by reverse strand sequencing and analyzed as described by Turaça et al. (2012). Total RNA was isolated from peripheral blood sample. The extracted RNA was subjected to reverse transcription, and the cDNA was amplified by PCR using specific sense and antisense primers. The fragments were purified and sequencing as described by Varela et al. (2019). In vitro characterization of the novel mutation The in vitro characterization was conducted as described by Varela et al. (2020a). Briefly, the human cervical cancer cell line (HeLa, ATCC® CCL-2™) were used to the transfection of full-length cDNA sequence encoding human α-Gal A (NM_000169) and the mutants. The mutant plasmids were constructed using specific primers (Exxtend Biotechnology, Paulínia, Brazil) for the human GLA gene (Transcript 1 - forward 5′ TGG GGA GTA GAT CTG CTA AAA TTT GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AGG A 3′ and reverse 5’ TCC TAT TCA GGG CCA AGG ACA TGT GCT TAT AAC CAT CAA ATT TTA GCA GAT CTA CTC CCC A 3′ / Transcript 2 - forward 5’ CGA TTG ATG CCC AGA CCT TTG CTG ACT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AG 3′ and reverse 5’CTA TTC AGG GCC AAG GAC ATG TGC TTA TAA CCA GTC AGC AAA GGT CTG GGC ATC AAT GTC G 3′). The transfection confirmation was performed by GLA expression using quantitative real-time PCR, as described previously by Varela et al. (2020a). Methods Patients α-Galactosidase A activity measurement We analyzed four members of a two-generation family, three women (22, 51, 49 years old) and a young man (18 years old) with suspicion of FD after the identification of cornea verticilatta in the index case (Fig. 1a). The Medical Research and Ethical Committee of Universidade Federal de São Paulo approved the research protocols and consent forms used in this study (0585/07 and 0354/18). Written information consent was obtained from all individuals included in this study. The protein extraction and enzyme measurement were performed as described by Lukas et al. (2013). The released 4methylumbelliferone was determined by fluorescence measurement using a fluorometer SpectraMax M2 (Molecular Devices), fluorescence readings were corrected by blanks, and the results were compared to a 4-methylumbelliferone calibration curve (Muller et al. 2010). Enzymatic activities were expressed as micromoles of hydrolyzed substrate per liter of blood per hour (μmol/L blood/h). Metab Brain Dis Fig. 1 Pedigree and molecular analysis of the RNA. a Pedigree of a family with the novel variant c.547 + 1_547 + 6GTAATG. b cDNA sequencing from a wild-type GLA transcript and two aberrant transcripts found in the individuals with the mutation found in the intron 3, which produces mRNAs with deletions of part of exon 3. The figure shown, in the transcript 1 were deleted 36pb, corresponding to amino acids 172 to 183, promoting the shortening of the protein without generate frame-shift (p.Cys172_Gly183del). In the transcript 2, 62 pb were deleted from the exon 3, the figure shows alteration of the open reading frame, promoting formation of a premature stop codon (p.Trp162_Gly163insLeufs*) Statistical analysis enzyme activity of 0.06 μmol/L/h (reference >2.2 μmol/L/h). The Fig. 1a show the family pedigree. The index case’s mother presents acroparesthesia and major depression since adolescence. Her sister, the index case’s aunt, was diagnosed with schizophrenia at the second decade of life; she also presents acroparesthesia; her son was 18 years old at the diagnosis and presents angiokeratoma, acroparaesthesia, and depressed mood. Renal dysfunction and nephrotic proteinuria were not detected. Brain and heart magnetic resonance imaging (MRI) were performed in all individuals and no changes were detected. The family’s clinic and laboratorial data are shown in the Table 1. All experiments were performed in four separate duplicates in order to confirm the consistency of the results. Statistical evaluation utilized was one-way analyses of variance (ANOVA) with Turkey as post-hoc, performed using IBM SPSS® software (version 18). The level of significance was set at p < 0.05. Results Clinical features and genetic analysis The index case was a 22-year-old female patient referred to a geneticist for presenting cornea verticillata. At the clinical evaluation, the presence of mild FD signs as acroparesthesia, tinnitus and vertigo were detected. Laboratory analysis of substrate accumulation was performed and showed accumulation of lyso-Gb3 (6.1 ng/mL – reference <1.8 ng/mL) and urinary Gb3 (60.6 μg/mg – reference <15 μg Gb3/mg creatinine). Clinical and laboratorial data are shown in Table 1. After clinical and laboratory results, the molecular investigation showed a novel GLA deletion in the intron 3 (c.547 + 1_547 + 6GTAATG); this mutation deleted the canonical donor 5’ss of the intron 3. This variant was not described in the literature and is absent in the consulted population genetic database (gnomAD and ABraOM) (Karczewski et al. 2020; Naslavsky et al. 2017). Therefore, the in-silico analysis used to identify splicing motif alteration predicted this variant as probably affecting the splicing (Desmet et al. 2009). After finding the mutation in the index patient, the family was also analyzed by DNA sequencing, lyso-Gb3, urinary Gb3, and enzymatic assays (for male individual). The mutation, as well as accumulation of lyso-Gb3 and urinary Gb3, was found in the patient’s mother, an aunt and a male cousin. The affected male showed α-Gal A deficiency, presenting an RNA analysis In order to confirm the impact of the novel mutation c.547 + 1_547 + 6delGTAATG in the mRNA processing, the cDNA from the male affected patient was sequenced. The sequencing showed two transcripts produced from the mutated GLA. The transcript 1 presented a deletion of 36 bp (p.Cys172_Gly183del), this deletion promotes shortening of twelve amino acids in the α-Gal A protein, without formation of a frame-shift and a premature stop codon (Fig. 1b). In the second transcript (transcript 2) 62 pb were deleted between the amino acids 163 and 183 (p.Trp162_Gly163insLeu*). In this transcript, the glycine 163 was exchanged for a leucine and after that, a premature stop codon was formed (Fig. 1b). Functional characterization It is possible to observe that the absence of the canonical 5’ss of intron 3 promoted the formation of two new splice donor sites into exon 3 (Fig. 2a). Even though the shortening of transcript 1 does not change the reading frame, in both transcripts the Cys172 was deleted; this amino acid forms a disulfide bond with Cys142 and both amino acids are part of the αGal A active site (Fig. 2b). Therefore, the deletion of the Metab Brain Dis Table 1 Clinical and laboratory characteristics of individuals with novel GLA variant General Information / Symptoms Neurological Symptoms Patient Gender Age at diagnosis Lyso-Gb3 Urinary Gb3* Enzyme activity (μmol/h/L) ** Index Case Female 22 y 6.1 ng/mL# 60.6 μg/mg NA Mother Female 51 y 4.1 ng/mL# 49.3 μg/mg NA Aunt Female 49 y 3.6 ng/mL# 33.7 μg/mg NA Cousin Male 18 y 101 ng/mL## 522.8 μg/mg 0.06 Angiokeratoma Cornea verticillata Tinnitus Vertigo Acroparaesthesia Depression Psychiatric Symptoms + + + + + – – + – – – + + Major Depression + – – – + – Schizophrenia + – – – + + Depressed mood Legend: y = years; (+) presence of symptom; (−) absence of symptom; NA = Not applicable; Lyso-Gb3 analysis were performed in two different laboratories, for # the reference value is <1.8 ng/mL and for ## the reference value is <5.0 ng/mL; * reference value <15 μg Gb3 / mg creatinine; ** reference value: >2.2 μmol/L/h Cys172 could impact the enzymatic function. To confirm the impact on the enzyme, the functional characterization was performed. Both transcripts generated from the mutated mRNA resulting from the splicing error caused by variant c.547 + 1_547 + 6delGTAATG were produced in vitro by a site directed mutagenesis protocol. GLA mRNA levels were measured in the transfected HeLa cells (WT α-Gal A, transcript 1 and transcript 2) and shown to be increased at least tenfold, when compared to the endogenous GLA expression in HeLa cells. We did not observe significant difference between the expression of the GLA WT mRNA when compared with the first transcript. However, the second transcript showed higher GLA mRNA expression when compared to transcript 1 and WT (Fig. 3a). The α-Gal A activity was also evaluated in the HeLa transfected cells (WT, transcript 1 and transcript 2), as well as in cells without transfection. The overexpression of WT GLA mRNA led to a sevenfold increase in the α-Gal A enzymatic activity compared to endogenous α-Gal A. No difference in αGal A activity was observed in the cells transfected with the transcripts 1 and 2, when compared with endogenous α-Gal A in HeLa cells. The α-Gal A activity resulting from transcripts 1 and 2 was normalized to the activity of the wild-type α-Gal A and did not present any residual activity (Fig. 3b). Discussion Pathogenic mutations in GLA gene cause α-Gal A deficiency and consequently, Gb3 accumulation leading to FD, a rare and multisystemic disorder. To date, the Human Gene Mutation Database described over a thousand variants in the GLA of patients with presumed FD. Several GLA variants occur only in one family (Turaça et al. 2012) and even in mutations found in more than one family the pathogenicity of some are still controversial (Varela et al. 2020b). Thus, functional characterization of novel variants is essential to confirm their pathogenicity. In this study, we present a novel splice site mutation found in four members of a family. The mRNA sequencing showed that the deletion of six bases in the 5’ss of the intron 3 created two aberrant transcripts with partial deletion of exon 3. The functional characterization was used to confirm the pathogenicity. The mRNA expression of the transfected cells showed that all plasmids either containing WT GLA sequence or both mutated transcripts were super expressed in HeLa cells. However, although transcript 2 had a significantly increased expression in relation to the transcript 1 and WT, the in vitro test showed increase of the α-Gal A activity only in the WT transfected cells and absence of residual activity in the cells transfected with the two mutated transcripts, confirming alternative transcripts pathogenicity. The disruption of the canonical donor site due to the aforementioned intronic mutation might lead the transcription machinery to use preferentially weak donor splice sites, both located 5′ to the canonical one, leading to an exon shrinkage and the formation of these two novel alternative transcripts. Transcript 1 presents a deletion of 36 pb comprising part of exon 3 and two bases of exon 4 (residues 172 to 184). This deletion does not generate a frameshift and premature stop codon, however, the cysteine 172 form a disulfide bond with cysteine 142, the disruption of this bond could lead to an improper formation of the catalytic site, due to their spatial proximity along with the missing of additional residues deleted with the alternative transcript. Variants affecting cysteines that form disulfide bonds are associated Metab Brain Dis Fig. 2 The deletion of 5’ss promotes shortening of the exon 3, through the formation of new splicing donor sites. a The new variant c.547 + 1_ 547 + 6delGTAATG deletes a canonical splicing site; the interruption of the canonical donor site leads the transcription machinery to use preferably weak donor splice sites, located within exon 3, promoting the shortening of the two transcripts. b The figure highlights the active site of α-Gal A enzyme. It is possible note that the amino acid Cys172, deleted in the two transcripts, is part of the active site of the enzyme and forming a disulfide bond with Cys142 with the classic FD phenotype promoting undetectable or very low residual activity (Ploos van Amstel et al. 1994; Okumiya et al. 1995; Schafer et al. 2005; Shabbeer et al. 2005; Filoni et al. 2010). Transcript 2 (p.Trp162_Gly163insLeu *) deletes 62 bp, referring to amino acids 163 to 183. This deletion promotes frame-shift and the formation of a premature stop codon at position 164. Despite the functional characterization showed that the transcripts formed by the mutation c.547 + 1_547 + 6delGTAATG do not produce α-Gal A residual activity, the main signs found in this family were acroparesthesia, tinnitus, cornea verticillata, and psychiatric symptoms; symptoms as renal, heart, or cerebrovascular were not present. In women, mild symptoms could be explained by the random inactivation of the X chromosome that creates a mosaic of cells expressing both the normal or mutant alleles in variable proportions. The more non-mutated cells are prevalent, the milder and delayed the symptoms will be presented; on the other hand, severe symptoms are more frequent when more mutated cells are prevalent (Juchniewicz et al. 2018). This new variant leads to the absence of enzyme activity and, consequently, to the accumulation of Gb3, therefore men with this mutation will show classic symptoms of FD. Classical phenotype was not observed in this family because the only hemizygote affected is an 18-year-old, and in men the most symptoms as renal and cardiac occur between the second and third decades of life, therefore, he is being monitored periodically. Interestingly, three members of this family present neuropsychiatric symptom as follows: mother presents depression; aunt presents schizophrenia; and cousin presents depressed mood. It is known that the accumulation of Gb3 found in patients with FD leads to lysosomal dysfunction. According Metab Brain Dis Fig. 3 Functional characterization of the transcripts formed from the variant c.547 + 1_547 + 6delGTAATG. a GLA mRNA expression in HeLa cells sixty hours after transfection. GAPDH gene was used to normalize the expression of GLA mRNA. Data are expressed as mean ± S.E.M. of 2-ΔCt, which represents the relative expression between GLA and GAPDH. The figure shows the comparison of the GLA expression in Hela cells (endogenous expression) and the expression in GLA WT and GLA mutant (Transcript 1 and 2). *P < 0.0001 WT (12.1 ± 0.5, N = 4) vs. HeLa (1 ± 0, N = 4); Transcript 1 (10.5 ± 0.5, N = 4) vs. HeLa; Transcript 2 (15.8 ± 0.8, N = 4) vs. HeLa; #P = 0.0014 Transcript 2 vs. Transcript 1; a. u. = arbitrary unit. b Enzymatic activity of the α-Gal A endogenous of HeLa cells compared to the enzymatic activity of WT, transcript 1 and transcript 2 overexpressed HeLa cells. Data are expressed as mean ± S.E.M. of the hydrolysis of the fluorescent substrate 4-MU-alphaD-galactopyranoside; *P < 0.0001 WT (703.2 ± 18.5, N = 4) vs. HeLa (120.3 ± 9.6, N = 4), WT vs. Transcript 1 (73.9 ± 15.9, N = 4) and WT vs. Transcript 2 (116.5 ± 16.4, N = 4). There was no difference between HeLa, Transcript 1 and Transcript 2; u.a.f. = arbitrary unit of fluorescence to Mckenna et al. (2019), lysosomal dysfunction in the brain could change the neurotransmitter homeostasis or promote neurons damage, implicating in the pathophysiology of neurological disease. In fact, neuropsychiatric symptoms, as depression (Wang et al. 2007; Crosbie et al. 2009; Schermuly et al. 2011; Laney et al. 2013; Bolsover et al. 2014; Löhle et al. 2015; Körver et al. 2020), psychosis, hallucinations, delusions, mental confusion, dementia, schizophrenia, behavioral difficulties, and psychomotor retardation are reported in patients with FD (Liston et al. 1973; Shen et al. 2007; Okeda and Nisihara 2008; Segal et al. 2010; Gairing et al. 2011; Schermuly et al. 2011; Sawada et al. 2015). As reported by to the World Health Organization, depression is the most common psychiatric disorder, affecting 322 million people worldwide, which is equivalent to 4.4% of the world population. In contrast, the incidence of schizophrenia is lower, affecting approximately 15 people per 100,000 (McGrath et al. 2004). In FD, depression affects 15 to 62% of patients (Bolsover et al. 2014; Crosbie et al. 2009; Laney et al. 2013; Löhle et al. 2015; Schermuly et al. 2011; Wang et al. 2007); however, the incidence of other psychiatric disorders has not yet been studied. Although frequent, depression has not been a recognized symptom in primary care in at least half of FD patients (Cole et al. 2007). As depression is associated with other medical conditions, it can be neglected, since physicians focus on the primary disease (Hamilton 2006, Cole et al. 2007). Therefore, cases of depression and other psychiatric disorders may be underestimated in patients with FD. Furthermore, depression has not been seen as a symptom of FD, but as a predictable consequence in patients with an incurable disease, and it has been explained as a response to chronic pain (Cole et al. 2007; Bolsover et al. 2014; Körver et al. 2020) and/or impaired quality of life (Cole et al. 2007; Crosbie et al. 2009). However, some studies have established a relationship between brain involvement and psychiatric symptoms in FD. It is known that the hippocampus is a structure involved in psychiatric illnesses; for example, hippocampal volume deficit is an established finding in both depression and schizophrenia (Wise et al. 2017; Kalmady et al. 2017; Santos et al. 2018; Barch et al. 2019; Chen et al. 2019). Curiously, studies have shown that the hippocampus is an affected structure in FD. A post-mortem study showed the Gb3 accumulation was present in neurons in selective brain areas, including hippocampus (Okeda and Nisihara 2008). Another studied described a disruption of the autophagy-lysosome pathway associated to neuropathology and axonal degeneration in the brain regions, as hippocampus, of α-Gal A knockout mice (Nelson et al. 2014). In addition, hippocampal atrophy was found in patients with FD (Fellgiebel et al. 2012; Lelieveld et al. 2015). A longitudinal study showed that over a period of eight years there was a decline of approximately 11% in the hippocampal volume in FD patients (Lelieveld et al. 2015). Interestingly, the hippocampal atrophy cannot be explained by the vascular pathology of the disease, suggesting a probable manifestation of neuronal involvement (Fellgiebel et al. 2012; Lelieveld et al. 2015). In the present family, it was not possible to state that FD is the cause of the psychiatric symptoms. However, as these symptoms are described with more frequency in FD patients and studies point to changes in their hippocampus, further studies are needed to confirm whether the accumulation of Gb3 in neurons could cause neuropsychiatric symptoms. In summary, here we report a novel GLA mutation found in four members of a family. The mutation at the 5’ss produces two alternative non-functional transcripts, leading to accumulation of Lyso-Gb3 and urinary Gb3 in both women and men. The family presents mild neurological symptoms along with Metab Brain Dis neuropsychiatric symptoms, as depression and schizophrenia, which have not been confirmed as primary or secondary to FD. New in vitro or in vivo studies with neurons and other brain cells are needed to understand if the Gb3 accumulation promotes lysosomal dysfunction in the cells leading to changes in the neurotransmitter homeostasis or neurons damage, causing psychiatric symptoms. Acknowledgments This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2014/27198-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) - Finance Code 001. Authors’ contributions PhD. Patricia Varela: conceptualized and designed the study, designed the data collection experiments, drafted the initial manuscript, carried out the analyses, reviewed and revised the manuscript and approved the final manuscript as submitted. MD. Gerson Carvalho: providing the samples analyzed in this work, analyzed the clinical symptoms of patients, reviewed and revised the manuscript, and approved the final manuscript as submitted. PhD. Renan Paulo Martin: carried out the initial analyses, reviewed and revised the manuscript and approved the final manuscript as submitted. PhD. Joao Bosco Pesquero: conceptualized and designed the study, drafted the initial manuscript, carried out the initial analyses, reviewed and revised the manuscript and approved the final manuscript as submitted. Corresponding author and guarantor for the article. Funding This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2014/27198–8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Data availability All data generated or analyzed during this study are included in this published article. Compliance with ethical standards Competing interests The authors declare no conflict of interest and confirm independence from the sponsors. The sponsors have not influenced the content of the article. Ethical approval and patient consent statement The Research Ethics Committee of the Federal University of São Paulo, Brazil approved this protocol (0585/07 and 0354/18). 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