A de novo YY1 missense variant expanding the Gabriele-de Vries syndrome phenotype and affecting X-chromosome inactivation.

Yin and Yang 1 gene (YY1; MIM#600,013) is recognized as a dual transcriptional activating and repressing factor, RNA-binding protein, and 3D chromatin regulator, with multi roles in neurodevelopmental and maintenance pathways. YY1 haploinsufficiency caused either by heterozygous sequence variants or deletions involving the whole gene has been recently associated with Gabriele-de Vries syndrome (GADEVS), a rare congenital autosomal dominant condition, leading to intellectual disability (ID) and multiple physical/behavioural abnormalities. Herein, we describe clinical and molecular findings from a Brazilian female harbouring a de novo missense pathogenic variant in YY1 gene (NM_003403.5:c.1106A > G; p.Asn369Ser) found by whole exome sequencing with potential implications for protein structure and function. Undescribed or uncommon clinical features in this patient included non-febrile seizures, severe scoliosis, hearing impairment, and chorioretinitis. Further bioinformatics analyses using YY1-other protein interaction networks reinforced the involvement of YY1 interactors in such phenotypes, in exception of chorioretinitis. Moreover, X-chromosome inactivation (XCI) skewing was evidenced in the patient and attributed to the haploinsufficiency of YY1, which direct and indirectly interacts with numerous XCI key regulators. Besides expanding the mutational and phenotype spectrum of GADEVS, our results highlight the role of YY1 as an essential autosomal regulator of XCI epigenetic process.

Molecular and clinical insights into complex genomic rearrangements related to MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is caused by copy number variation (CNV) spanning the MECP2 gene at Xq28 and is a major cause of intellectual disability (ID) in males. Herein, we describe two unrelated males harboring non-recurrent complex Xq28 rearrangements associated with MDS. Copy number gains were initially detected by quantitative real-time polymerase chain reaction and further delineated by high-resolution array comparative genomic hybridization, familial segregation, expression analysis and X-chromosome inactivation (XCI) evaluation in a carrier mother. SNVs within the rearrangements and/or fluorescent in situ hybridization (FISH) were used to assess the parental origin of the rearrangements. Patient 1 exhibited an intrachromosomal rearrangement, whose structure is consistent with a triplicated segment presumably embedded in an inverted orientation between two duplicated sequences (DUP-TRP/INV-DUP). The rearrangement was inherited from the carrier mother, who exhibits extreme XCI skewing and subtle psychiatric symptoms. Patient 2 presented a de novo (X;Y) unbalanced translocation resulting in duplication of Xq28 and deletion of Yp, originated in the paternal gametogenesis. Neurodevelopmental trajectory and non-neurological symptoms were consistent with previous reports, with the exception of cerebellar vermis hypoplasia in patient 2. Although both patients share the core MDS phenotype, patient 1 showed MECP2 transcript levels in blood similar to controls. Understanding the molecular mechanisms related to MDS is essential for designing targeted therapeutic strategies.

Understanding the Landscape of X-linked Variants Causing Intellectual Disability in Females Through Extreme X Chromosome Inactivation Skewing.

Intellectual disability (ID) affects 30% more males than females. This sex bias can be attributed to the enrichment of genes on the X chromosome playing essential roles in the central nervous system and their hemizygous state on males. Moreover, as a result of X chromosome inactivation (XCI), most genes on one of the X chromosomes in female somatic cells are epigenetically silenced, so that females carrying X-linked variants are not expected to be so severely affected as males. Consequently, the knowledge about X-linked ID (XLID) in females is still scarce. Herein, we used extreme XCI skewing (≥ 90%) to predict X-linked variants in females with idiopathic ID. XCI profiles from 53 probands were estimated from blood and buccal mucosa through a methylation-sensitive AR/RP2 assay. DNA samples with extreme XCI skewing were then submitted to array-comparative genomic hybridization and whole-exome sequencing. Seven females (13.2%) exhibited extreme XCI skewing, a percentage significantly higher than expected for healthy females in our population. XLID-potentially related variants were identified in five patients with extreme XCI skewing, including one pathogenic rstructural rearrangement [der(X) chromosome from a t(X;2)] and four single nucleotide variants in NLGN4X, HDAC8, TAF1, and USP9X genes, two of which affecting XCI escape genes. XCI skewing showed to be an outstanding approach for the characterization of molecular mechanisms underlying XLID in females. Beyond expanding the spectrum of variants/phenotypes associated with ID, our results pointed to compensatory biological pathways underlying XCI and uncover new insights into the involvement of escape genes on XLID, impacting genetic counseling.

Network Profiling of Brain-Expressed X-Chromosomal MicroRNA Genes Implicates Shared Key MicroRNAs in Intellectual Disability.

MicroRNAs are endogenous non-protein-coding RNA molecules that regulate post-transcriptional gene expression. The majority of human miRNAs are brain-expressed and chromosome X is enriched in miRNA genes. We analyzed the genomic regions of 12 brain-expressed pre-miRNAs located on chromosome X coding for 18 mature miRNAs, aiming to investigate the involvement of miRNA sequence variants on X-linked intellectual disability (XLID). Genomic DNA samples from 135 unrelated Brazilian males with intellectual disability, suggestive of X-linked inheritance, were amplified through polymerase chain reaction and sequenced by Sanger sequencing. Although no sequence variations have been identified, suggesting an intense selective pressure, further computational analysis evidenced that eight mature miRNAs (miR-221-3p/222-3p, miR-223-3p, miR-361-5p, miR-362-5p, miR-504-5p.1, miR-505-3p.1, and miR-505-3p.2) act as critical regulators of X-linked and autosomal ID genes in a fully connected network. Enrichment approaches identify transcription regulation, nervous system development, and regulation of cell proliferation as the main common biological processes among the target ID genes. Besides, a clustered chromosomal coverage of the imputed miRNAs target genes and related regulators was found on X chromosome. Considering the role of miRNAs as fine-tuning regulators of gene expression, a systematic analysis of miRNAs' expression could uncover part of the genetic landscape subjacent to ID, being urgently necessary in patients with this condition, particularly XLID.

rs3851179 Polymorphism at 5' to the PICALM Gene is Associated with Alzheimer and Parkinson Diseases in Brazilian Population.

Alzheimer's (AD) and Parkinson's diseases (PD) share clinical and pathological features, suggesting that they could have common pathogenic mechanisms, as well as overlapping genetic modifiers. Here, we performed a case-control study in a Brazilian population to clarify whether the risk of AD and PD might be influenced by shared polymorphisms at PICALM (rs3851179), CR1 (rs6656401) and CLU (rs11136000) genes, which were previously identified as AD risk factors by genome-wide association studies. For this purpose, 174 late-onset AD patients, 166 PD patients and 176 matched controls were genotyped using TaqMan® assays. The results revealed that there were significant differences in genotype and allele frequencies for the SNP PICALM rs3851179 between AD/PD cases and controls, but none for CR1 rs6656401 and CLU rs11136000 intronic polymorphisms. After stratification by APOE ε4 status, the protective effect of the PICALM rs3851179 A allele in AD cases remains evident only in APOE ε4 (-) carriers, suggesting that the APOE ε4 risky allele weakens its protective effect in the APOE ε4 (+) subgroup. More genetic studies using large-sized and well-defined matched samples of AD and PD patients from mixed populations as well as functional correlation analysis are urgently needed to clarify the role of rs3851179 in the AD/PD risk. An understanding of the contribution of rs3851179 to the development of AD and PD could provide new targets for the development of novel therapies.

Finding FMR1 mosaicism in Fragile X syndrome.

OBJECTIVE: Almost all patients with Fragile X Syndrome (FXS) exhibit a CGG repeat expansion (full mutation) in the Fragile Mental Retardation 1 gene (FMR1). Here, the authors report five unrelated males with FXS harboring a somatic full mutation/deletion mosaicism. METHODS: Mutational profiles were only elucidated by using a combination of molecular approaches (CGG-based PCR, Sanger sequencing, MS-MLPA, Southern blot and mPCR). RESULTS: Four patients exhibited small deletions encompassing the CGG repeats tract and flanking regions, whereas the remaining had a larger deletion comprising at least exon 1 and part of intron 1 of FMR1 gene. The presence of a 2-3 base pairs microhomology in proximal and distal non-recurrent breakpoints without scars supports the involvement of microhomology mediated induced repair (MMBIR) mechanism in three small deletions. CONCLUSION: The authors data highlights the importance of using different research methods to elucidate atypical FXS mutational profiles, which are clinically undistinguishable and may have been underestimated.

Circulating Endocannabinoids and the Polymorphism 385C>A in Fatty Acid Amide Hydrolase (FAAH) Gene May Identify the Obesity Phenotype Related to Cardiometabolic Risk: A Study Conducted in a Brazilian Population of Complex Interethnic Admixture.

The dysregulation of the endocannabinoid system is associated with cardiometabolic complications of obesity. Allelic variants in coding genes for this system components may contribute to differences in the susceptibility to obesity and related health hazards. These data have mostly been shown in Caucasian populations and in severely obese individuals. We investigated a multiethnic Brazilian population to study the relationships among the polymorphism 385C>A in an endocannabinoid degrading enzyme gene (FAAH), endocannabinoid levels and markers of cardiometabolic risk. Fasting plasma levels of endocannabinoids and congeners (anandamide, 2-arachidonoylglycerol, N-oleoylethanolamide and N-palmitoylethanolamide) were measured by liquid chromatography-mass spectrometry in 200 apparently healthy individuals of both genders with body mass indices from 22.5 ± 1.8 to 35.9 ± 5.5 kg/m2 (mean ± 1 SD) and ages between 18 and 60 years. All were evaluated for anthropometric parameters, blood pressure, metabolic variables, homeostatic model assessment of insulin resistance (HOMA-IR), adiponectin, leptin, C-reactive protein, and genotyping. The endocannabinoid levels increased as a function of obesity and insulin resistance. The homozygous genotype AA was associated with higher levels of anandamide and lower levels of adiponectin versus wild homozygous CC and heterozygotes combined. The levels of anandamide were independent and positively associated with the genotype AA position 385 of FAAH, C-reactive protein levels and body mass index. Our findings provide evidence for an endocannabinoid-related phenotype that may be identified by the combination of circulating anandamide levels with genotyping of the FAAH 385C>A; this phenotype is not exclusive to mono-ethnoracial populations nor to individuals with severe obesity.

Novel microduplications at Xp11.22 including HUWE1: clinical and molecular insights into these genomic rearrangements associated with intellectual disability.

Recently, we defined a minimal overlapping region for causal Xp11.22 copy number gains in males with intellectual disability (ID), and identified HECT, UBA and WWE domain-containing protein-1 (HUWE1) as the primary dosage-sensitive gene, whose overexpression leads to ID. In the present study, we used this minimal interval to search for HUWE1 copy number variations by quantitative polymerase chain reaction in a large cohort of Brazilian males with idiopathic ID. We detected two unrelated sporadic individuals with syndromic ID carrying unique overlapping duplications encompassing HUWE1. Breakpoint junction analysis showed a simple tandem duplication in the first patient, which has probably arisen by microhomology-mediated break-induced repair mechanism. In the second patient, the rearrangement is complex having an insertion of an intrachromosomal sequence at its junction. This kind of rearrangement has not been reported in Xp11.22 duplications and might have emerged by a replication- or recombination-based mechanism. Furthermore, the presence of infantile seizures in the second family suggests a potential role of increased KDM5C expression on epilepsy. Our findings highlight the importance of microduplications at Xp11.22 to ID, even in sporadic cases, and reveal new clinical and molecular insight into HUWE1 copy number gains.

Associação entre a homocisteína e os polimorfismos do gene da MTHFR em mulheres brasileiras obesas / Association between homocysteine and polymorphisms in MTHFR in Brazilian obese women

Fundamentos: Pesquisas nacionais brasileiras indicam aumento da obesidade e doenças cardiovasculares em mulheres. Objetivo: Determinar a frequência dos polimorfismos 677C>T e 1298A>C do gene da metilenotetra-hidrofolato redutase (MTHFR) em mulheres brasileiras obesas e avaliar sua associação com as concentrações séricas de homocisteína (Hcy),folato e cobalamina, no período após a fortificação das farinhas de trigo e milho com ácido fólico no Brasil. Métodos: Estudo transversal realizado no período de 2008 a 2009, com 133 mulheres obesas. Kits comerciais foramutilizados para realizar análises laboratoriais, incluindo mensuração de lipídeos e glicose por métodos enzimáticos; Hcy total e o folato plasmático, utilizando um imunoensaio competitivo; e cobalamina baseado em quimiluminescência. A genotipagem foi realizada por PCR, seguido por fragmento de restrição enzimática. Resultados: A média de idade dos participantes foi 39,0±4,4 anos e o índice de massa corporal, 32,5±2,1 kg/m². Distribuições dos genótipos encontradas: CC (47%), CT (44%) e TT (9%) para a posição 677 da MTHFR e AA (60%), AC (35%), e CC (5%) para a posição 1298. As concentrações de Hcy correlacionaram-se negativamente com a concentração de folato plasmático no grupo exibindo os genótipos 677CT, 1298AC ou 1298CC (r=0,554, p<0,01). Conclusão: Mulheres brasileiras obesas com genótipos 677TT estudadas apresentaram maiores concentrações de Hcy do que aquelas que apresentaram os genótipos 677CT e 677CC. Além disso, genótipos 1298CC mostraram associação com concentrações de Hcy maiores do que os genótipos 1298AC e 1298AA.

Background: Brazilian national surveys have indicated a rise in obesity and cardiovascular disease in women.Objective: To determine the frequency of 677C>T and 1298A>C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene in obese Brazilian women and to assess the potential association of these polymorphisms with serum concentrations of homocysteine (Hcy), folate and cobalamin after fortification of wheat and corn flour with folic acid in Brazil. Methods: A cross-sectional study was conducted from 2008 to 2009 with 133 obese women. Commercial kits were employed to perform laboratory analyses including measurement of lipids and glucose using enzymatic methods, total Hcy and serum folate using a competitive immunoassay and cobalamin based on chemiluminescence. Genotyping was performed by PCR, followed by restriction fragment lengthpolymorphism analysis. Results: The average age of participants was 39.0±4.4 years and mean body mass index was 32.5±2.1kg/m². The distributions of the genotypeswere CC (47%), CT (44%), and TT (9%) for the position MTHFR 677 and AA (60%), AC (35%), and CC (5%) for the position 1298. Hcy levels correlated negatively with serum folate in the group displaying the 677CT, 1298AC, or 1298CC genotypes (r=-0.554, p<0.01). Conclusion: Our findings suggest that obese Brazilian women with genotypes 677TT have higher Hcy concentrations than those carrying the genotypes 677CT and 677CC. Additionally, genotypes 1298CC are associated with higher Hcy concentrations than genotypes 1298AC and 1298AA.

KDM5C mutational screening among males with intellectual disability suggestive of X-Linked inheritance and review of the literature.

An increasing number of neurodevelopmental diseases have been associated with disruption of chromatin remodeling in eukaryotes. Lysine(K)-specific demethylase 5C (KDM5C) is a versatile epigenetic regulator that removes di- and tri-methyl groups of lysine 4 on histone H3 (H3K4) from transcriptional targets and is essential for neuronal survival and dendritic growth. Mutations in KDM5C gene, located at Xp11.22, have been reported as an important cause of both syndromic and non-syndromic X-linked intellectual disability (XLID) in males. The aim of this study was to evaluate the prevalence and spectrum of KDM5C mutations among Brazilian patients with XLID. To access the impact of KDM5C variants on XLID, a cohort of 143 males with a family history of intellectual disability (ID) suggestive of X-linked inheritance were enrolled. Common genetic causes of XLID were previously excluded and the entire coding and flanking intronic sequences of KDM5C gene were screened by direct Sanger sequencing. Seven nucleotide changes were observed: one pathogenic mutation (c.2172C>A, p.Cys724*), one novel variant with unknown value (c.633G>C, p.Arg211Arg) and five apparently benign sequence changes. In silico analysis of the variants revealed a putative creation of an Exonic Splicing Enhancer sequence by the silent c.633G>C mutation, which co-segregates with the ID phenotype. Our results point out to a KDM5C pathogenic mutational frequency of 0.7% among males with probable XLID. This is the first KDM5C screening among ID males from a country in Latin America and provides new clues about the significance of KDM5C mutations for genetic counseling.

A novel in-frame deletion affecting the BAR domain of OPHN1 in a family with intellectual disability and hippocampal alterations.

Oligophrenin-1 (OPHN1) is one of at least seven genes located on chromosome X that take part in Rho GTPase-dependent signaling pathways involved in X-linked intellectual disability (XLID). Mutations in OPHN1 were primarily described as an exclusive cause of non-syndromic XLID, but the re-evaluation of the affected individuals using brain imaging displayed fronto-temporal atrophy and cerebellar hypoplasia as neuroanatomical marks. In this study, we describe clinical, genetic and neuroimaging data of a three generation Brazilian XLID family co-segregating a novel intragenic deletion in OPHN1. This deletion results in an in-frame loss of exon 7 at transcription level (c.781_891del; r.487_597del), which is predicted to abolish 37 amino acids from the highly conserved N-terminal BAR domain of OPHN1. cDNA expression analysis demonstrated that the mutant OPHN1 transcript is stable and no abnormal splicing was observed. Features shared by the affected males of this family include neonatal hypotonia, strabismus, prominent root of the nose, deep set eyes, hyperactivity and instability/intolerance to frustration. Cranial MRI scans showed large lateral ventricles, vermis hypoplasia and cystic dilatation of the cisterna magna in all affected males. Interestingly, hippocampal alterations that have not been reported in patients with loss-of-function OPHN1 mutations were found in three affected individuals, suggesting an important function for the BAR domain in the hippocampus. This is the first description of an in-frame deletion within the BAR domain of OPHN1 and could provide new insights into the role of this domain in relation to brain and cognitive development or function.

Genetic analysis of PARK2 and PINK1 genes in Brazilian patients with early-onset Parkinson's disease.

Parkinson's disease is the second most frequent neurodegenerative disorder in the world, affecting 1-2% of individuals over the age of 65. The etiology of Parkinson's disease is complex, with the involvement of gene-environment interactions. Although it is considered a disease of late manifestation, early-onset forms of parkinsonism contribute to 5-10% of all cases. In the present study, we screened mutations in coding regions of PARK2 and PINK1 genes in 136 unrelated Brazilian patients with early-onset Parkinson's disease through automatic sequencing. We identified six missense variants in PARK2 gene: one known pathogenic mutation, two variants of uncertain role, and three nonpathogenic changes. No pathogenic mutation was identified in PINK1 gene, only benign polymorphisms. All putative pathogenic variants found in this study were in heterozygous state. Our data show that PARK2 point mutations are more common in Brazilian early-onset Parkinson's disease patients (2.9%) than PINK1 missense variants (0%), corroborating other studies worldwide.

Exon dosage variations in Brazilian patients with Parkinson's disease: analysis of SNCA, PARKIN, PINK1 and DJ-1 genes.

Parkinson's disease is one of the most common neurodegenerative disorders associated with aging, reaching ∼ 2% of individuals over 65 years. Knowledge achieved in the last decade about the genetic basis of Parkinson's disease clearly shows that genetic factors play an important role in the etiology of this disorder. Exon dosage variations account for a high proportion of Parkinson's disease mutations, mainly for PARKIN gene. In the present study, we screened genomic rearrangements in SNCA, PARKIN, PINK1 and DJ-1 genes in 102 Brazilian Parkinson's disease patients with early onset (age of onset ⩽ 50 years), using the multiplex ligation-dependent probe amplification method. Family history was reported by 24 patients, while 78 were sporadic cases. Screening of exon dosage revealed PARKIN and PINK1 copy number variations, but no dosage alteration was found in SNCA and DJ-1 genes. Most of the carriers harbor heterozygous deletions or duplications in the PARKIN gene and only one patient was found to have a deletion in PINK1 exon 1. Data about dosage changes are scarce in the Brazilian population, which stresses the importance of including exon dosage analysis in Parkinson's disease genetic studies.

A MECP2 missense mutation within the MBD domain in a Brazilian male with autistic disorder.

Point mutations and genomic rearrangements in the MECP2 gene are the major cause of Rett syndrome (RTT), a pervasive developmental disorder affecting almost exclusively females. MECP2 mutations were also identified in patients with autism without RTT. In this study, we present a mutational and gene dosage analysis of the MECP2 in a cohort of 60 Brazilian males with autistic features but not RTT. No duplication or deletion was identified. Sequencing analysis, however, revealed four MECP2 sequence variations. Three of them were previously discussed as non disease causing mutations and one mutation (p.T160S) was novel. It affects a highly conserved amino acid located within the MBD domain, a region of the protein involved in specific recognition and interaction with methylated CpG dinucleotides. The p.T160S variation was not found in the control sample. This mutation may represent a potential genetic factor for autistic phenotype and should be object of further studies.

Mutational analysis of GIGYF2, ATP13A2 and GBA genes in Brazilian patients with early-onset Parkinson's disease.

In the last decade, several genes have been linked to Parkinson's disease (PD), including GIGYF2, ATP13A2 and GBA. To explore whether mutations in these genes contribute to development of PD in the Brazilian population, we screened 110 patients with early-onset PD. No clearly pathogenic mutations were identified in ATP13A2 and GIGYF2. In contrast, we identified a significantly higher frequency of known pathogenic mutations in GBA gene among the PD cases (6/110=5.4%) when compared to the control group (0/155) (P=0.0047). Our results strongly support an association between GBA gene mutations and an increased risk of PD. Mutations in GIGYF2 and ATP13A2 do not seem to represent a risk factor to the development of PD in the Brazilian population. Considering the scarcity of studies on GIGYF2, ATP13A2 and GBA mutation frequency in Latin American countries, we present significant data about the contribution of these genes to PD susceptibility.

High frequency of nonrecurrent MECP2 duplications among Brazilian males with mental retardation.

Structural variations that affect the copy number of the MECP2 gene were shown to cause mental retardation in males by driving the overexpression of this gene. To access the impact of these rearrangements in males with unexplained mental retardation, we have performed a quantitative real-time polymerase chain reaction assay using SYBR Green I chemistry to quantify MECP2 gene copy number in 145 Brazilian males with mental retardation of unknown cause. Three patients carrying MECP2 duplications (approximately 2%) were identified. The analysis of additional markers flanking the MECP2 region showed that the duplications observed are nonrecurrent. Expression studies in two of these patients revealed the overexpression of the MECP2 gene compared to the expression level observed in controls. These findings corroborate other recent reports in the literature and highlight that the overexpression of MECP2 caused by duplications involving this gene is a relatively frequent genetic cause of mental retardation in males, highlighting the importance of MECP2 gene dosage for diagnostic purposes in such cases.

LRRK2 p.G2019S mutation is not common among Alzheimer's disease patients in Brazil.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have emerged as a potential common cause for both sporadic and familial Parkinson's Disease (PD) in different populations. The pleomorphic features exhibited by LRRK2 mutation carriers and the central role of Lrrk2 protein in the proper functioning of central nervous system suggest that mutations in this protein might be involved in multiple cellular processes leading to other neurodegenerative disorders than PD. The location of LRRK2 gene on chromosome 12, close to a linkage peak for familial late-onset Alzheimer's Disease (AD), highlights that LRRK2 mutations might be involved in AD pathogenesis. We screened the most common LRRK2 mutation (p.G2019S) in a series of 180 consecutive patients clinically diagnosed with Alzheimer Disease (AD). We identified the p.G2019S in one AD patient with no PD signs, indicating that this mutation is not a common etiological factor for AD in our population (0.5%), corroborating recent data found in Norwegian, North American, Chinese and Italian populations. Nevertheless, these observations together with new information about the Lrrk2 critical multifunctionality do not rule out the possible influence of other variants within LRRK2 in AD, so that other screenings focusing in the whole extension of the LRRK2 using larger sized confirmed AD sample are urgently needed.

A MECP2 mutation in a highly conserved aminoacid causing mental retardation in a male.

MeCP2 is a protein that functions as a key factor in epigenetic transcriptional regulation. Mutations in MECP2 gene have been reported as being the major cause of Rett syndrome. These mutations may also cause a wide spectrum of neurological disorders in males. Here, we report the identification of the mutation p.P405L in a 19-year-old Brazilian male with mental retardation. This variant is localized in a highly conserved aminoacid from the carboxy terminal domain and may affect the protein function. Segregation analysis on the patient's mother revealed that this is a de novo mutation and it was not identified in the control sample. The programs SIFT, PolyPhen and A-GVGD considered that the p.P405L may be damaging. Despite the high frequency of non pathogenic variants that have been identified in this gene, our data lead us to consider the p.P405L a disease-causing mutation.