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Recent emerging evidence indicates that changes in gene expression levels are linked to human evolution. We have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position -220C, which is the site of action of valproic acid. Reversion of this nucleotide to the ancestral A-allele has been detected in patients with degrees of deficit in higher brain cognitive functions. This mutation has since been reported in the 1000 genomes database at an approximate frequency of <0.0004 in humans (rs138452745). In the study reported here, we present update on the status of rs138452745 across evolution, based on the Ensembl and NCBI databases. The DNA pulldown assay was also used to identify the proteins binding to the C- and A-alleles, using two cell lines, SK-N-BE and HeLa. Consistent with our previous findings, the C-allele is human-specific, and the A-allele is the rule across all other species (N=38). This nucleotide resides in a block of 12-nucleotides that is strictly conserved across evolution. The DNA pulldown experiments revealed that in both SK-N-BE and HeLa cells, the transcription repressor BEN domain containing 3 (BEND3) binds to the human-specific C-allele, whereas the nuclear factor I (NFI) family members, NF1A, B, C, and X, specifically bind to the ancestral A-allele. This binding pattern is consistent with a previously reported decreased promoter activity of the C-allele vs. the A-allele. We propose that there is a link between binding of BEND3 to the CALR rs138452745 C-allele and removal of NFI binding site from this nucleotide, and the evolution of human-specific higher brain functions. To our knowledge, CALR rs138452745 is the first instance of enormous nucleotide conservation across evolution, except in the human species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Calreticulin (CALR) is a multi-functional protein that is strictly conserved across species. Two mRNA transcripts have been recognized for the CALR gene in humans, which use a common promoter sequence. We have recently reported mutations in the CALR promoter that co-occur with psychosis. One of those mutations at -220A increases gene expression in human BE(2)-C and HEK-293 cell lines. This mutation is the first instance of a functional cognition-deficit mutation reversing a human gene promoter to the primitive type. In the current study, we analyzed the effect of the most widely-used mood-stabilizing drug, valproic acid (VPA), on nucleotide -220 in two neuronal cell lines, LAN-5 and N2A. Remarkably, VPA increased gene expression in the cells with the wild-type -220C construct, whereas a dramatic decrease in gene expression was observed in the cell lines with the mutant construct (p<0.000004 and p<0.016, respectively). We also sequenced the 600-bp CALR promoter, and the highly conserved intron 1 sequence in an independent sample of patients afflicted with major psychiatric disorders and controls. A new case of major depressive disorder with psychotic features with the -220A mutation was identified. A novel 1-bp insertion was also detected in intron 1 at IVSI-310, in a case of amphetamine-induced psychosis. As for the psychosis-linked CALR promoter mutations identified to-date, the IVSI mutation was not detected in the control pool. This mutation creates a RREB-1 transcription factor binding site within the first intron. Our present findings identify the site of action of VPA in the CALR promoter, and introduce a novel mutation in a case of substance-induced psychosis in the first intron of CALR., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tissue-specific expression of the CALR gene in the brain gray matter in late-adolescence and early adulthood coincides with the expression of the psychoses phenotypes. Indeed, increased expression of the chaperone genes in the prefrontal cortex has been reported in patients affected by schizophrenia. We have previously reported cases of psychosis-associated mutations in the CALR gene promoter. One of those mutations at -48 was found to increase the expression of the gene in comparison with the wild type sequence. A recently identified mutation at -220 reverts the conserved block harboring nucleotide -220 to the ancestral type, and has an approximate prevalence of 0.7% in psychoses. In this study, we analyzed the functional implication of this mutation in the human neuroblastoma cell line BE(2)-C, and non-neural Human Embryonic Kidney 293 (HEK-293), and show that the -220A mutation results in a constitutive increase in the expression of the CALR gene (p<0.0003). We checked homology of the first 1000-bp CALR promoter sequence across species, and found that nucleotide -220C is the only human-unique nucleotide in that stretch. The -220A mutation, on the other hand, co-occurs with severe cognition deficit in humans, and is the rule across the species except humans. To our knowledge, the -220A mutation is the first reported instance of a cognition-deficit-associated mutation which reverses a human gene promoter to the primitive type. It may be speculated that, at least the basal transcription of the CALR gene, relating to the proximal promoter region, has been decreased during the process of evolution to humans., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Development-dependent, tissue-specific expression of the calreticulin (CALR) gene in the gray matter coincides with the expression of psychoses phenotypes. We have recently reported instances of mutations within the core promoter sequence of the gene in schizoaffective disorder. In view of the mounting evidence on the genetic overlap in the psychiatric spectrum, we investigated this gene in a spectrum of patients afflicted with schizophrenia, schizoaffective disorder and major affective disorder. We found that a unique mutation at nucleotide -220 from the transcription start site, located at a conserved genomic block in the promoter region of the gene, co-occurs with the spectrum of psychoses (p<0.005). This mutation reverts the human promoter sequence to the ancestral type observed in chimpanzee, mouse, and several other species, implying that the genomic block harboring nucleotide -220 may be involved in the evolution of human-specific higher-order functions of the brain (e.g. language, conceptual thinking, and judgment), that are ubiquitously impaired in psychoses. We propose that CALR is not only a promising candidate in the spectrum of psychoses, but also, a gene that may be important in the human-unique brain processes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We have recently reported the first case of mutation in the core promoter sequence of the human calreticulin gene in a family case of schizoaffective disorder. Remarkably, this gene coincides with a region of suggested linkage at 19p13.2, identified in a whole genome scan [Hamshere et al. (2005); Arch Gen Psychiatry 62;1081-1088]. The identified mutation was located at the conserved position -48 from the transcription start site, and was shown to be of functional effect, resulting in the aberrant expression of the gene. Following screening of the gene in 60 independent cases of schizoaffective disorder, we report novel germ-line mutations at positions -205 C > T and the conserved exon 5 (c: 682 C > T, pro228ser) in two unrelated cases of schizoaffective disorder. These mutations were disease-specific, and as for the -48 G > C mutation, neither was detected in a control population of 370 individuals, indicating a contribution of 3.17% in this sample series. To our knowledge, this is the first instance of disease-specific mutations in schizoaffective disorder, which warrants systematic screening of the regulatory and coding regions of the calreticulin gene in this disorder., ((c) 2009 Wiley-Liss, Inc.)

Exposure to atypical antipsychotic drugs such as valproate increases the expression of chaperones that assist in the folding of proteins in the endoplasmic reticulum (ER) including calreticulin, GRP78/BiP, GRP94, and PD1. This neuroprotective role may be involved in the pathophysiology of neuropsychiatric disorders such as schizophrenia and bipolar disorder. The 5'-flanking region of the human calreticulin gene was screened in 100 cases of schizophrenia by PCR/SSCA between -485 and +1 basepair (bp) relative to the transcription start site. A G > C point mutation was detected at -48 in a case of paranoid schizophrenia, which was not detected in 280 unrelated control subjects (560 chromosomes). This is the first report of mutation in relation with the calreticulin gene. The -48G > C mutation creates a CpG site at the core promoter region of the gene. The role of this mutation remains to be clarified in the pathophysiology of the disease., (Copyright 2006 Wiley-Liss, Inc.)

The majority of single nucleotide polymorphism association studies in psychiatric disorders suffer from lack of reproducibility, necessitating re-sequencing genes for the identification of novel rare variants and deleterious mutations. We have recently reported three low frequency mutations in the proximal promoter of the human calreticulin (CALR) gene at positions -48C, -205T and -220A that co-occur with the spectrum of psychoses, including schizophrenia, schizoaffective disorder, and bipolar disorder type I. The frequency of those mutations was estimated at < 0.0007, and none of those mutations have been detected in the control population (p<0.005). Mutations -48C and -220A were found to increase the expression of the CALR gene. The third mutation at -205T was detected in an isolate case of schizoaffective disorder. In this study, we examined the function of this mutation in the human neuroblastoma BE(2)-C, and non-neural Human Embryonic Kidney-293) cell lines, and show that in contrast with other mutations in the promoter region, which increase expression of the gene, the -205T mutation significantly decreased gene expression in those cell lines in comparison with the wild-type C-nucleotide (p<0.0005, and p<0.017, respectively). We propose that a deviation from normalcy in the level of CALR in either direction is associated with psychosis. We also propose that the psychoses spectrum, at least in part, is a collection of functional mutations that are absent from the control population. Re-sequencing of the candidate gene promoter regions may unravel other mutations linked with psychosis. [ABSTRACT FROM AUTHOR]

Tissue-specific expression of the calreticulin (CALR) gene in the brain gray matter in late-adolescence and early adulthood coincides with the expression of the psychoses phenotypes. We report functional mutations at −48, −205 and −220 in the promoter of the human CALR gene that co-occur with the spectrum of psychoses including schizophrenia, schizoaffective disorder, and bipolar disorder type I. None of those mutations have been detected in the control population (p<0.005). Mutation −220 reverts the conserved block harboring nucleotide −220 to the ancestral type. We analyzed the functional implication of this mutation in the human neuroblastoma cell line BE(2)-C, and non-neural Human Embryonic Kidney 293 (HEK-293), and show that the −220A mutation results in a constitutive increase in the expression of the CALR gene (p<0.0003). We checked homology of the first 1000-bp CALR promoter sequence across species, and found that nucleotide −220C is the only human-unique nucleotide in that stretch. The −220A mutation, on the other hand, co-occurs with severe cognition deficit in humans, and is the rule across the species except humans. The −220A mutation is the first reported instance of a cognition-deficit-associated mutation which reverses a human gene promoter to the primitive type. It may be speculated that, at least the basal transcription of the CALR gene, relating to the proximal promoter region, has been decreased during the process of human evolution. We propose that the psychosis spectrum is, at least in part, a collection of functional mutations that are non-existent in the control population. [ABSTRACT FROM AUTHOR]

The chromosomal region, 19p 13.2, has been suggested to be linked with schizoaffective disorder (Hamshere et al. 2005). This region harbors the calreticulin gene, protein encoded by which is an endoplasmic Ca+2-binding molecular chaperone. Development-dependent, tissue-specific expression of this gene in the gray matter coincides with the expression of psychoses phenotypes. We have recently reported instances of mutations within the core promoter (−48 G>C, −205 C>T) and coding sequence [exon 5 (c: 682 C>T, pro228ser)] of the gene in schizoaffective disorder. In view of the mounting evidence on the genetic overlap in the psychiatric spectrum, we investigated this gene in 386 patients afflicted with schizophrenia, schizoaffective disorder, major affective disorder and 600 controls by PCR/SSCA. We found that a unique mutation within the core promoter of the gene, located at a conserved genomic block, co-occurring with four cases of psychoses including schizophrenia, schizoaffective disorder and bipolar disorder type I. This unique mutation reverts the human promoter sequence to the ancestral types observed in chimpanzee, rhesus Macaque and mouse, implying that the genomic block harboring this nucleotide may be involved in the evolution of human-specific higher-order functions of the brain (i.e. cognition), that are ubiquitously impaired in psychoses. Our findings propose that calreticulin is not only a promising candidate in the spectrum of psychoses, but also, a gene that may be important in the process of human-unique higher-level functions of the brain. [ABSTRACT FROM AUTHOR]