Engrailed-2 Research Articles

Autism Associated Gene, ENGRAILED2, and Flanking Gene Levels Are Altered in Post-Mortem Cerebellum

BackgroundPrevious genetic studies demonstrated association between the transcription factor ENGRAILED2 (EN2) and Autism Spectrum Disorder (ASD). Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C) functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, SEROTONIN RECEPTOR5A (HTR5A), INSULIN INDUCED GENE1 (INSIG1), CANOPY1 HOMOLOG (CNPY1), RNA BINDING MOTIF PROTEIN33 (RBM33), and SONIC HEDGEHOG (SHH). These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed.MethodsqRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression.Results EN2 levels are increased in affected A-C/G-T individuals (p = .0077). Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T) and CNPY1 (G-T/G-T) levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels.Conclusions EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples

Importance of HOX genes in normal prostate gland formation, prostate cancer development and its early detection

The aims of this paper were to review the published literature on the role of HOX genes in the development of the normal prostate gland and in prostate cancer and to discuss the potential role of the HOX family member, Engrailed-2 (EN2), as a diagnostic test of PCa. Hox genes were first described in the fruit fly Drosphila melanogaster, where they specify the body plan and control the formation of body segments. They belong to a family of homeodomain-containing transcription factors that determine cell and tissue identity during normal embryonic development. They have been shown to be re-expressed by several different types of cancers. Studies have shown that different Hox genes are responsible for the development of the separate lobes of the prostate gland, the seminal vesicles and the epididymis. All HOX13 paralogues are expressed in the adult human prostate, suggesting the possibility of similarities between the function and expression of HOX genes within urological structures at similar anterior-posterior positions. The oncogenic and tumour suppressor signalling pathways associated with PCa converge on the HOX gene network, which ultimately controls gene expression, affecting tumour formation and metastatic progression. The Engrailed genes (EN1 and EN2) from the HOX gene family show a very high degree of functional conservation during embryonic development. Urinary EN2 is being investigated as a potential diagnostic marker of early PCa. It is secreted into the urine by PCa cells but not by normal prostatic tissue. A recent study has shown an association between urinary EN2 levels and cancer volume in radical prostatectomy specimens. The ability to predict tumour volume could inform the treatment decision-making process for patients with localized PCa choosing between active surveillance and radical treatment options.

Role of Engrailed-2 (EN2) as a prostate cancer detection biomarker in genetically high risk men

Controversy surrounds the use of PSA as a biomarker for prostate cancer detection, leaving an unmet need for a novel biomarker in this setting; urinary EN2 may identify individuals with clinically relevant prostate cancer. Male BRCA1 and BRCA2 mutation carriers are at increased risk of clinically significant prostate cancer and may benefit from screening. Urine samples from 413 BRCA1 and BRCA2 mutation carriers and controls were evaluated. Subjects underwent annual PSA screening with diagnostic biopsy triggered by PSA > 3.0 ng/ml; 21 men were diagnosed with prostate cancer. Urinary EN2 levels were measured by ELISA and had a sensitivity of 66.7% and specificity of 89.3% for cancer detection. There was no statistically significant difference in EN2 levels according to genetic status or Gleason score. Urinary EN2 may be useful as a non-invasive early biomarker for prostate cancer detection in genetically high-risk individuals.

Expression of Engrailed-2 (EN2) protein in bladder cancer and its potential utility as a urinary diagnostic biomarker

Despite significant advances in our understanding of the molecular pathology of bladder cancer, it remains a significant health problem with high morbidity and mortality associated with muscle-invasive bladder cancer (stages T2+), and high costs associated with the surveillance of non-muscle-invasive bladder cancer (NMIBC, stages Ta/T1/Tis). Moreover, current diagnostic biomarkers are suboptimal and of poor utility for low grade disease and surveillance. In this study, we show that the Engrailed-2 (EN2) transcription factor is expressed in, and secreted by, bladder cancer cell lines and patient tumour specimens, justifying an evaluation of urinary EN2 as a diagnostic biomarker in bladder cancer using archived samples from an established biospecimen collection. In patients with NMIBC, urinary EN2 was detected in most cases with an overall sensitivity of 82% and specificity of 75%. The sensitivity for stage Ta and T1 tumours was 71% and 76%, respectively, and 94% for stage T2+ tumours. This compares favourably with existing markers. The sensitivity for tumour grades 1, 2 and 3 was 69%, 78% and 87%, respectively. Thus urinary EN2 has the potential to be a more sensitive and specific protein biomarker for NMIBC than currently available tests.

Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum

The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.

Correlation of Urinary Engrailed-2 Levels to Tumour Volume and Pathological Stage in Men Undergoing Radical Prostatectomy

The aim of this study was to assess the relationship between pre-prostatectomy urinary Engrailed-2 (EN2), a transcription factor secreted by prostate cancer cells, with tumour volume and pathological characteristics in resected prostate specimens. First pass urine samples (10 ml) without prior prostatic massage were collected and stored at –80°C. EN2 levels were measured using an enzyme-linked immunoabsorbent assay. Tumour volume in the prostatectomy specimens was determined histologically. 57 men undergoing RP in one urological cancer network were evaluated. EN2 was detected in 85% of RP patients. EN2 correlated with tumour volume (but not total prostatic volume) in a linear regression analysis, with increasing pathological T stage and margin positivity. Using three “cutoff levels” of tumour volume (0.5 ml, 1.3 ml and 2.5 ml) to define “significant disease”, men with “significant disease” had markedly higher levels of urinary EN2 (p Levels of urinary EN2 may be useful in predicting tumour volume in men with prostate cancer by potentially identifying men with small volume “insignificant” disease. This study justifies a larger multicentre evaluation of urinary EN2 levels as a biomarker of PC significance using cancer volume, pathological and PSA criteria.

Engrailed protein: A cancer-specific marker in epithelial ovarian cancer.

e15517 Background: Ovarian cancer is a common malignancy, accounting for 4% of all cancers and 5% of all cancer deaths in women in the Western world. Engrailed-2 (EN2) is a homeodomain–containing transcription factor that is essential during early development. It is dysregulated in a number of cancers and its presence in urine is diagnostic of prostate cancer. We have investigated its role as a cancer-specific marker in epithelial ovarian cancer (EOC). Methods: Ovarian tumours and normal tissue were collected from patients with epithelial ovarian cancer (EOC) and tested by semi-quantitative RT-PCR as well as by immunohistochemistry. EN2 expression relative to B-actin was calculated using the Livak comparative Ct method (2-DDCt). All slides were scored on a 0-3 scale based on intensity, by two independent assessors. Results: We examined 118 EOC specimens, of which 64 were tested by immunohistochemistry and 54 by RT-PCR. 89 (75.42%) were serous, 18 (15.25%) endometroid, 7 (5.93%) mucinous, and 4 (3.39%) clear cell tumours. Normal ovarian tissue was used as a control. 104 (88.14%) tumours expressed EN2 whilst 14 (11.86%) were negative. Of the EN2 positive tumours examined using immunohistochemistry, 28 (51.85%) strongly expressed the antigen (score of 2-3), whereas 26 (48.15%) were weakly positive (score of 1). Normal tissue did not express EN2 and this difference was statistically significant (p<0.05). There was no correlation with tumour type, grade and stage. EN2 expressing mucinous tumours showed improved overall survival (p=0.0253). There was no correlation for survival or PFS for other histological subtypes. Conclusions: EN2 is a cancer-specific marker in epithelial ovarian cancer. Further work is currently ongoing to evaluate its significance in ascites, blood and urine. We plan to further evaluate its role as a diagnostic biomarker by examining blood, urine, and ascites.

Urinary engrailed‐2 (EN2) levels predict tumour volume in men undergoing radical prostatectomy for prostate cancer

What's known on the subject? and What does the study add? There are a lot of potential prostate cancer biomarkers being evaluated. All aim to improve on the sensitivity and specificity of PSA. EN2 was recently shown by our group to have better sensitivity and specificity than PSA. EN2 is a simple ELISA test and is not dependent on other parameters, even PSA, unlike all the other current biomarkers under evaluation. To date, no marker correlates with the amount of cancer present - the present study shows this positive correlation with EN2 in men undergoing prostatectomy. The potential utility of this work is that by knowing that the level of EN2 corresponds to the amount of cancer present, irrelevant of tumour grade and number of cancer foci, we can define an EN2 level corresponding to small cancers, which can then undergo surveillance. We are conducting a further study that is aimed at determining whether the levels of EN2 in urine can indicate 'significant' vs 'non-significant cancer' using the threshold of 0.5 mL cancer (after Epstein's work). To evaluate the relationship between levels of a recently described prostate cancer biomarker engrailed-2 (EN2) in urine and cancer volume in men who had undergone radical prostatectomy (RP) for prostate cancer. To date, prostate-specific antigen (PSA) levels have not reliably predicted prostate cancer volume. Reliable volume indicator biomarker(s) may aid management decisions, e.g. active treatment vs active surveillance. Archived patient samples from the Aarhus Prostate Cancer Project, Denmark, were assessed. Pre-treatment mid-stream urines, without preceding prostatic massage, were collected and stored at -80 °C. Urinary EN2 levels were measured by a recently published enzyme-linked immunosorbent assay. In all, 88 of the whole cohort of 125 men (70%) were positive for EN2 in their urine (>42.5 µg/L); 38/58 (65%) men where cancer volume data was available. There was no statistical relationship between urinary EN2 levels and serum PSA levels. PSA levels did not correlate with tumour stage, combined Gleason grade, total prostatic weight or cancer volume. There was a strong statistical relationship between urinary EN2 and prostate cancer volume by linear regression (P = 0.006). Higher EN2 levels correlated with tumour stage T1 vs T2 (P = 0.027). Pre-surgical urinary EN2 levels were associated with increasing tumour stage and closely reflected the volume of cancer in RP specimens. Given the ease of collection (no prostatic massage required) and the simplicity, low cost and robustness of the assay, EN2 may become a useful biomarker in not only identifying which patients have prostate cancer but may also facilitate risk stratification by indicating the burden of tumour volume.

Profile of William C. Skarnes

Profile of William C. Skarnes

Abstract 1297: Engrailed-2 (EN2) represses the transcription of the CST6 tumour suppressor gene in prostate cancer

Abstract Engrailed-2 (EN2) is a homeodomain-containing transcription factor that is expressed in prostate and breast tumours but not in normal adult tissue. Although EN2 protein is known to be secreted by prostate tumours and is a potential urine based biomarker of this disease, its function in prostate tumours is unknown. In this study we have used siRNA-mediated knock down of EN2 as well as forced over expression in prostate cancer cells. Although neither treatment has a significant effect on cell survival, we identify two key transcriptional targets of EN2, the tumour suppressor gene CST6 and the Death Receptor ligand TRAIL, the transcription of which are repressed and activated by EN2, respectively. EN2 synergises with epigenetic changes to silence CST6, and may function to block CST6 expression very early in the development of malignancy. Further, we show that EN2 protein can regulate gene transcription in non-malignant prostate cells when supplied externally, indicating that EN2 secretion by prostate tumour cells may be able to modify its microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1297. doi:1538-7445.AM2012-1297

Abstract B41: Urinary EN2 levels predict tumor volume in men undergoing radical prostatectomy for prostate cancer

Abstract Total tumor volume has been proposed as a guide to managing prostate cancer by immediate treatment versus active surveillance. We evaluated the relationship between levels of a recently described prostate cancer biomarker EN2 (Engrailed-2) in urine and cancer volume in men who had undergone radical prostatectomy for prostate cancer. Archived patient samples from the Aarhus Prostate Cancer Project, Denmark, were assessed. Pre-treatment mid stream urines, without preceding prostatic massage, were collected and stored at −80°C. Urinary EN2 levels were measured by a recently published Enzyme-linked Immunosorbent Assay (ELISA). 88 of the whole cohort of 125 men (70%) were positive for EN2 in their urine (>42.5 μ/L); 38/58 (65%) of men where cancer volume data was available. No statistical relationship was found between urinary EN2 levels and serum PSA. PSA levels did not correlate with tumor stage, combined Gleason grade, total prostatic weight or cancer volume. We found a strong statistical relationship between urinary EN2 and prostate cancer volume by linear regression (p=0.0058). Furthermore, higher EN2 levels correlated with tumor stage T1 versus T2 (p=0.0272). Pre-surgical urinary EN2 levels were associated with increasing tumor stage and closely reflected the volume of cancer in prostatectomy specimens. Given the ease of collection (no prostatic massage required) and the simplicity, low cost and robustness of the assay, EN2 may become a useful biomarker in not only identifying which patients have prostate cancer but may also facilitate risk stratification by indicating the burden of tumor volume. Citation Format: Hardev Pandha, Karina Sorensen, Torben Falck Orntoft, Stephen Langley, Soren Hoyer, Michael Borre, Richard Morgan. Urinary EN2 levels predict tumor volume in men undergoing radical prostatectomy for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B41.

Cut-like homeobox 1 and nuclear factor I/B mediate ENGRAILED2 autism spectrum disorder-associated haplotype function

Both common and rare variants contribute to autism spectrum disorder (ASD) risk, but few variants have been established as functional. Previously we demonstrated that an intronic haplotype (rs1861972-rs1861973 A-C) in the homeobox transcription factor ENGRAILED2 (EN2) is significantly associated with ASD. Positive association has also been reported in six additional data sets, suggesting EN2 is an ASD susceptibility gene. Additional support for this possibility requires identification of functional variants that affect EN2 regulation or activity. In this study, we demonstrate that the A-C haplotype is a transcriptional activator. Luciferase (luc) assays in mouse neuronal cultures determined that the A-C haplotype increases expression levels (50%, P < 0.01, 24 h; 250%, P < 0.0001, 72 h). Mutational analysis indicates that the A-C haplotype activator function requires both associated A and C alleles. A minimal 202-bp element is sufficient for function and also specifically binds a protein complex. Mass spectrometry identified these proteins as the transcription factors, Cut-like homeobox 1 (Cux1) and nuclear factor I/B (Nfib). Subsequent antibody supershifts and chromatin immunoprecipitations demonstrated that human CUX1 and NFIB bind the A-C haplotype. Co-transfection and knock-down experiments determined that both CUX1 and NFIB are required for the A-C haplotype activator function. These data demonstrate that the ASD-associated A-C haplotype is a transcriptional activator, and both CUX1 and NFIB mediate this activity. These results provide biochemical evidence that the ASD-associated A-C haplotype is functional, further supporting EN2 as an ASD susceptibility gene.

2326 ENGRAILED-2 (EN2): A URINARY BIOMARKER FOR THE DIAGNOSIS OF PROSTATE CANCER WITHOUT DRE

You have accessJournal of UrologyProstate Cancer: Markers1 Apr 20112326 ENGRAILED-2 (EN2): A URINARY BIOMARKER FOR THE DIAGNOSIS OF PROSTATE CANCER WITHOUT DRE Hardev Pandha, Mohammed Ismail, Angie Boxall, Aagna Bhatt, Stephen Langley, Richard Hindley, and Richard Morgan Hardev PandhaHardev Pandha Guildford, United Kingdom More articles by this author , Mohammed IsmailMohammed Ismail Guildford, United Kingdom More articles by this author , Angie BoxallAngie Boxall Guildford, United Kingdom More articles by this author , Aagna BhattAagna Bhatt Guildford, United Kingdom More articles by this author , Stephen LangleyStephen Langley Guildford, United Kingdom More articles by this author , Richard HindleyRichard Hindley Basingstoke, United Kingdom More articles by this author , and Richard MorganRichard Morgan Guildford, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2573AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PC) is the second most common cause of cancer related death in men. A number of key limitations with prostate specific antigen (PSA), currently the standard detection test, has justified evaluation of new biomarkers. Recently described biomarkers are complex, expensive, often identify ‘high risk' but are not immediately diagnostic and not easily adopted into clinical practice. We have assessed the diagnostic potential of EN2 protein, a homeodomain-containing transcription factor expressed in PC cell lines and secreted into the urine by PC in men. Detection of EN2 was simple and inexpensive. METHODS EN2 expression in PC cell lines and prostate cancer tissue was determined by semi-quantative RT-PCR and immunohistochemistry. First pass urine (without prior digital rectal examination (DRE)) was collected from men presenting with urinary symptoms (referred to exclude/confirm the presence of prostate cancer) and from 3 relevant control groups. EN2 protein was measured by ELISA in urine from men with PC (n=82) and controls (combined n=102). RESULTS EN2 expression was expressed and secreted by PC cell lines and PC tissue but not by normal prostate tissue or stroma. The presence of EN2 in urine was highly predictive of PC, with a sensitivity of 66% and a specificity of 88.2%, without requirement for DRE, with an overall AUC of 0.81. There was no correlation with PSA levels. These results we confirmed independently by a second academic centre in the United Kingdom. CONCLUSIONS Urinary EN2 is a highly specific and sensitive candidate biomarker of prostate cancer. To date, the limitations of false positive PSA levels due to BPH have not been observed. No DRE was required unlike all other recently described markers, making it attractive as a screening tool in the future. No correlation with PSA was evident. A larger multicentre study to further evaluate the diagnostic potential of EN2 is justified. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e932-e933 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hardev Pandha Guildford, United Kingdom More articles by this author Mohammed Ismail Guildford, United Kingdom More articles by this author Angie Boxall Guildford, United Kingdom More articles by this author Aagna Bhatt Guildford, United Kingdom More articles by this author Stephen Langley Guildford, United Kingdom More articles by this author Richard Hindley Basingstoke, United Kingdom More articles by this author Richard Morgan Guildford, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

123 ENGRAILED-2 (EN2): A HIGHLY SPECIFIC URINARY BIOMARKER FOR THE EARLY DIAGNOSIS OF PROSTATE CANCER

Purpose: Prostate cancer (PC) is the second most common cause of cancer related death in men. A number of key limitations with prostate specific antigen (PSA), currently the standard detection test, has justified evaluation of new biomarkers. We have assessed the diagnostic potential of Engrailed-2 (EN2) protein, a homeodomain-containing transcription factor expressed in PC cell lines and secreted into the urine by PC in men. Experimental Design: EN2 expression in PC cell lines and prostate cancer tissue was determined by semi-quantative RT-PCR and immunohistochemistry. First pass urine [without prior digital rectal examination (DRE)]was collected from menpresenting withurinary symptoms (referredto exclude/confirm the presence of prostate cancer) and from controls. EN2 protein was measured by ELISA in urine from men with PC (n ¼ 82) and controls (n ¼ 102). Results: EN2 was expressed and secreted by PC cell lines and PC tissue but not by normal prostate tissue or stroma. The presence of EN2 in urine was highly predictive of PC, with a sensitivity of 66% and a specificity of 88.2%, without requirement for DRE. There was no correlation with PSA levels. These results were confirmed independently by a second academic center. Conclusions: Urinary EN2 is a highly specific and sensitive candidate biomarker of prostate cancer. A larger multicenter study to further evaluate the diagnostic potential of EN2 is justified. Clin Cancer Res; 17(5); 1090–8. � 2011 AACR.

Engrailed-2 (EN2): A Tumor Specific Urinary Biomarker for the Early Diagnosis of Prostate Cancer

Prostate cancer (PC) is the second most common cause of cancer related death in men. A number of key limitations with prostate specific antigen (PSA), currently the standard detection test, has justified evaluation of new biomarkers. We have assessed the diagnostic potential of Engrailed-2 (EN2) protein, a homeodomain-containing transcription factor expressed in PC cell lines and secreted into the urine by PC in men. EN2 expression in PC cell lines and prostate cancer tissue was determined by semi-quantative RT-PCR and immunohistochemistry. First pass urine [without prior digital rectal examination (DRE)] was collected from men presenting with urinary symptoms (referred to exclude/confirm the presence of prostate cancer) and from controls. EN2 protein was measured by ELISA in urine from men with PC (n = 82) and controls (n = 102). EN2 was expressed and secreted by PC cell lines and PC tissue but not by normal prostate tissue or stroma. The presence of EN2 in urine was highly predictive of PC, with a sensitivity of 66% and a specificity of 88.2%, without requirement for DRE. There was no correlation with PSA levels. These results were confirmed independently by a second academic center. Urinary EN2 is a highly specific and sensitive candidate biomarker of prostate cancer. A larger multicenter study to further evaluate the diagnostic potential of EN2 is justified.

Family‐based studies indicate association of Engrailed 2 gene with autism in an Indian population

Engrailed 2 (EN2) is a homeobox transcription factor involved in the patterning of cerebellum during brain development. Linkage analysis and studies on knockout mice support EN2, located on chromosome 7q36.3, as a potential risk locus for autism. Candidate gene approach also suggested association of EN2 with autism spectrum disorder (ASD) in various populations. Here, we have investigated the association of five markers [rs3735653 (C/T) in exon 1; rs34808376 (GC/-) and rs6150410 (CGCATCCCC/-) in promoter region; rs1861972 (A/G) and rs1861973 (C/T) in the intron] of the gene with autism and ASD in Indian population using family-based approach. Probands have been recruited using Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) diagnostic criteria. Genotypic distributions conform to Hardy-Weinberg equilibrium. Genotyping analysis showed that the intronic single nucleotide polymorphisms (SNPs) are in complete linkage disequilibrium showing A-C and corresponding G-T allelic association. We observed significant preferential transmission of C allele of rs1861973 from the parents to affected offspring [transmission disequilibrium test (TDT): narrow diagnosis likelihood ratio statistics (LRS) = 6.63, P = 0.006; broad diagnosis LRS = 4.47, P = 0.05]. Interestingly, gender-based investigations showed significant transmission of C allele to the affected females [TDT: LRS = 7.36, P = 0.0025; haplotype-based haplotype relative risk (HHRR): LRS = 7.16, P = 0.02]. A maternal overtransmission for these alleles was also noted (TDT: LRS = 3.65, P = 0.036; HHRR: LRS = 2.81, P = 0.036). Bioinformatic analysis using TFSearch showed generation of Sp1 binding site in the presence of C allele. While Del-T haplotype formed from rs34808376-rs1861973 markers showed increased non-transmission, the Ins-C showed significant transmission suggesting protective effect and risk, respectively, conferred by these haplotypes in autism etiology. These results suggest positive genetic correlation of EN2 with autism in the Indian population.

Neural stem cell regulation, fibroblast growth factors, and the developmental origins of neuropsychiatric disorders

There is increasing appreciation for the neurodevelopmental underpinnings of many psychiatric disorders. Disorders that begin in childhood such as autism, language disorders or mental retardation as well as adult-onset mental disorders may have origins early in neurodevelopment. Neural stem cells (NSCs) can be defined as self-renewing, multipotent cells that are present in both the embryonic and adult brain. Several recent research findings demonstrate that psychiatric illness may begin with abnormal specification, growth, expansion and differentiation of embryonic NSCs. For example, candidate susceptibility genes for schizophrenia, autism and major depression include the signaling molecule Disrupted In Schizophrenia-1 (DISC-1), the homeodomain gene engrailed-2 (EN-2), and several receptor tyrosine kinases, including brain-derived growth factor and fibroblast growth factors, all of which have been shown to play important roles in NSCs or neuronal precursors. We will discuss here stem cell biology, signaling factors that affect these cells, and the potential contribution of these processes to the etiology of neuropsychiatric disorders. Hypotheses about how some of these factors relate to psychiatric disorders will be reviewed.

Autism-Associated Haplotype Affects the Regulation of the Homeobox Gene, ENGRAILED 2

Association analysis identified the homeobox transcription factor, ENGRAILED 2 (EN2), as a possible autism spectrum disorder (ASD) susceptibility gene (ASD [MIM 608636]; EN2 [MIM 131310]). The common alleles (underlined) of two intronic single nucleotide polymorphisms (SNPs), rs1861972 (A/G) and rs1861973 (C/T), are over-transmitted to affected individuals both singly and as a haplotype in three separate datasets (518 families total, haplotype p = .00000035). Further support that EN2 is a possible ASD susceptibility gene requires the identification of a risk allele, a DNA variant that is consistently associated with ASD but is also functional. To identify possible risk alleles, additional association analysis and linkage disequilibrium (LD) mapping were performed. Candidate polymorphisms were then tested for functional differences by luciferase (Luc) reporter transfections and electrophoretic mobility shift assays (EMSAs). Association analysis of additional EN2 polymorphisms and LD mapping with Hapmap SNPs identified the rs1861972-rs1861973 haplotype as the most appropriate candidate to test for functional differences. Luciferase reporters for the two common rs1861972-rs1861973 haplotypes (A-C and G-T) were then transfected into human and rat cell lines as well as primary mouse neuronal cultures. In all cases the A-C haplotype resulted in a significant increase in Luc levels (p < .005). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs. These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.

Increased susceptibility to kainic acid–induced seizures in Engrailed- 2 knockout mice

The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout ( En2 −/−) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2 −/− mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2 −/− mice. In keeping with these results, En2 −/− mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2 −/− mice, when compared to WT animals. Seizures were accompanied by a widespread c- fos and c- jun mRNA induction in the brain of En2 −/− but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2 −/− but not WT mice. These findings suggest that En2 −/− mice might be used as a novel tool to study the link between epilepsy and ASD.

Association of the ENGRAILED 2 (EN2) gene with autism in Chinese Han population

Human ENGRAILED 2 (EN2) gene is localized to 7q36, an autism susceptibility locus. En2 knockout mice display hypoplasia of cerebellum and a decrease in the number of Purkinje cell, which are similar to those reported for individuals with autism. Furthermore, deficits in social behavior were detected in En2(-/-) mice. Two recent studies have demonstrated that two intronic SNPs (rs1861972, rs1861973) in the EN2 gene are significantly associated with autism. To investigate whether this finding could be replicated in Chinese Han population, we performed the association study between eight single nucleotide polymorphisms (SNPs) of the EN2 gene and autism in 210 Chinese Han trios, using the family-based association test (FBAT). The present study demonstrated that a preferential transmission of the rs3824068 A-allele to affected offspring (A > G: Z = 2.399, P = 0.0165). After the Bonferroni correction, this statistical significance of preferential transmission did not remain. However, when haplotypes were constructed with multiple markers, a number of haplotypes including three two-marker haplotypes, nine three-marker haplotypes, one four-marker haplotype, and one six-marker haplotype, all of which contain the major allele A of rs3824068, displayed significantly associated with autism. These results were still significant after using the permutation method to obtain empirical P values. Thus, our data provide evidence that the EN2 gene may be implicated in the predisposition to autism in the Chinese Han population.