Omega-3 and Omega-6 fatty acids and risk of psychotic outcomes in the ALSPAC birth cohort.

BACKGROUND: Long chain polyunsaturated fatty acid (PUFA) levels have been implicated in the pathology of psychotic disorders. We investigated the relationship between childhood PUFA levels and later psychotic experiences (PE's) in a large birth cohort. METHODS: Plasma levels of Ω-3 and Ω-6 fatty acids (FA's) were assayed at ages 7 and 16 years. PE's were assessed at ages 12 and 18 years using a semi-structured interview. Primary outcome was any PE's at 18 years; sensitivity analyses examined incident PE's between ages 12 and 18 years, persistent PE's (at 12 and 18) and psychotic disorder at 18 years. Genetic instruments for Ω-3 and Ω-6 were derived and used in a multivariable Mendelian Randomization analysis. RESULTS: Higher levels of Ω-6 FA's AA, OA and AdA at age 7 years were weakly associated with a reduced risk for PE's at 18 years, however, effect sizes were small and attenuated after adjusting for confounders (strongest evidence for OA; adjusted OR, 0.842; 95% CI, 0.711, 0.998; p, 0.048). Total Ω-6 levels at age 16 years were associated with an increased odds of psychotic disorder at age 18 years. However, there was no association between Ω-6/Ω-3 ratio and psychosis outcomes, nor with genetic instruments of total Ω-3 or Ω-6 levels. CONCLUSIONS: There is no strong evidence that total plasma Ω-3 FA levels or Ω-6/Ω-3 ratios in childhood and mid-adolescence are associated with increased risk for PE's or psychotic disorder, but very marginal evidence that alterations in the Ω-6 pathway at developmental time points might influence risk2.

Childhood maltreatment and transition to psychotic disorder independently predict long-term functioning in young people at ultra-high risk for psychosis.

BACKGROUND: Individuals identified as at ultra-high risk (UHR) for psychosis are at risk of poor functional outcome regardless of development of psychotic disorder. Studies examining longitudinal predictors of poor functioning have tended to be small and report only medium-term follow-up data. We sought to examine clinical predictors of functional outcome in a long-term longitudinal study. METHOD: Participants were 268 (152 females, 116 males) individuals identified as UHR 2-14 years previously. A range of clinical and sociodemographic variables were assessed at baseline. Functioning at follow-up was assessed using the Social and Occupational Functioning Assessment Scale (SOFAS). RESULTS: Baseline negative symptoms, impaired emotional functioning, disorders of thought content, low functioning, past substance use disorder and history of childhood maltreatment predicted poor functioning at follow-up in univariate analyses. Only childhood maltreatment remained significant in the multivariate analysis (p < 0.001). Transition to psychosis was also significantly associated with poor functioning at long-term follow-up [mean SOFAS score 59.12 (s.d. = 18.54) in the transitioned group compared to 70.89 (s.d. = 14.00) in the non-transitioned group, p < 0.001]. Childhood maltreatment was a significant predictor of poor functioning in both the transitioned and non-transitioned groups. CONCLUSIONS: Childhood maltreatment and transition to psychotic disorder independently predicted poor long-term functioning. This suggests that it is important to assess history of childhood maltreatment in clinical management of UHR individuals. The finding that transition to psychosis predicts poor long-term functioning strengthens the evidence that the UHR criteria detect a subgroup at risk for schizophrenia.

Sex-specific modulation of spinal nociception by alpha2-adrenoceptors: differential regulation by estrogen and testosterone.

Sex-related differences in antinociception produced by the activation of alpha(2)-adrenoceptors (alpha(2)-ARs) have been reported, however, the precise role of gonadal steroids is still unknown. Hence, we hypothesized that estrogen and testosterone modulate antinociceptive effects of clonidine (an alpha(2)-AR agonist) on N-methyl-D-aspartate- (NMDA) and heat-induced spinal nociception. We also investigated whether estrogen or testosterone alters the expression of alpha(2A)-adrenoceptors in the spinal cord. Sprague-Dawley (SD) rats were implanted with PE10 cannulae in the intrathecal space of the lumbosacral spinal cord and divided into male, proestrous and diestrous female, ovariectomized (OVX), estradiol-treated OVX (OVX+E), castrated male (GDX), testosterone (GDX+T) and estradiol-treated castrated male (GDX+E) groups. Clonidine dose-dependently inhibited NMDA-induced scratching behavior in the male and OVX groups but to a significantly lesser extent in the OVX+E group. It also increased the tail withdrawal latency in the male, OVX, diestrous and GDX+T groups but not in the OVX+E, proestrous, GDX and GDX+E groups. Levels of alpha(2A)-AR mRNA were significantly higher in the OVX, estradiol-treated OVX, GDX and GDX+E animals. In contrast, alpha(2A)-AR protein levels were higher in estradiol-treated OVX, GDX, GDX+T and GDX+E animals as compared with the male. Indeed, no correlations were observed between changes in the mRNA or protein levels of alpha(2A)-AR and behavioral observations. These results support our hypothesis that sex-related differences in alpha(2)-AR-mediated modulation of spinal nociception are gonadal hormone-dependent: estrogen attenuates antinociceptive effects in females whereas testosterone is required for the expression of antinociception in males. In addition, results also revealed that the mechanism of action of gonadal hormones may not involve a global alternation in expression of alpha(2A)-AR in the spinal cord. Estrogen-induced attenuation of alpha(2)-AR-mediated inhibition of nociception could contribute to the higher prevalence of pain syndromes in women.

Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells.

Ewing's sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of five ETS proteins. While the majority of tumors express EWS/FLI1 fusions, some Ewing's tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural differences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its C-terminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors differ greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural differences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.

Hypopigmentation of a papillary carcinoma arising in a black thyroid.

We report a case of an unpigmented papillary carcinoma arising in a black thyroid induced by minocycline. Black thyroid syndrome is an unusual pigmented change seen almost exclusively in patients on minocycline, apparently resulting from an oxidative interaction between thyroid peroxidase and the drug. Twenty-six cases have previously been reported in the English literature, nine of which described an associated thyroid neoplasm. Four of these nine neoplasms were described as pale or hypopigmented. The nature of the lesion against the background of pigmentation suggests diminished function of the thyroid peroxidase in this clonal population.

EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts.

Ewing's sarcoma is the least differentiated member of the peripheral primitive neuroectodermal (pPNET) tumor family. Chromosomal translocations involving the EWS gene and five different Ets family transcription factor genes create fusion genes encoding aberrant transcription factors and are implicated in the vast majority of Ewing's sarcoma cases. Here, NIH 3T3 fibroblasts were infected with control (tk-neo or RAS) and two different EWS/ETS-expressing retroviruses. In vitro studies of established polyclonal lines expressing the two EWS/ETS genes, either EWS/FLI1 or EWS/ETV1, showed induction of cytokeratin 15 gene expression. Both fusion genes also caused characteristic gross morphologic, histologic, and ultrastructural changes in NIH 3T3 cells when transformed cell lines were injected into CB-17-scid mice. Native NIH 3T3 cells with a spindled cell morphology were converted to polygonal cells with high nucleo-cytoplasmic ratios that continued to express abundant cytokeratin. Extracellular collagen deposition was abolished, rough endoplasmic reticulum was markedly diminished, and rudimentary cell-cell attachments appeared. Most strikingly, neurosecretory-type dense core granules like those seen in pPNET were now evident. This murine model, created in mesenchyme-derived NIH 3T3 cells, demonstrated new characteristics of both neuroectodermal and epithelial differentiation and resembled small round cell tumors microscopically.

EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction.

The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.

EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic.

EWS/FLI1, a fusion gene found in Ewing's sarcoma, encodes a transcriptional regulator and promotes cellular transformation by modulating the transcription of specific target genes. We have found that EWS/FLI1 and structurally related fusion proteins upregulate manic fringe (MFNG), a recently described member of the Fringe gene family instrumental in somatic development. MFNG is also expressed in human tumour-derived cell lines expressing EWS/FLI1. Overexpression of MFNG in NIH 3T3 cells renders them tumorigenic in mice with severe combined immunodeficiency disease (SCID). These data demonstrate that part of the oncogenic effect of EWS/FLI1 is to transcriptionally deregulate a member of a family of morphogenic genes.

EAT-2 is a novel SH2 domain containing protein that is up regulated by Ewing's sarcoma EWS/FLI1 fusion gene.

The EWS/FLI1 fusion protein is created by the translocation between chromosomes 11 and 22 that appears in most Ewing's sarcomas. This chimeric protein has been demonstrated to be an aberrant transcription factor. Genes up regulated by EWS/FLI1 but not by full-length FLI1 were identified by representational difference analysis (RDA). We have characterized a novel gene, EWS/FLI1 activated transcript 2 (EAT-2) that was cloned from a murine cDNA library using a differentially expressed RDA fragment. EAT-2 expression is seen within 4-8 h of EWS/FLI1 induction. Its expression correlates with transformation of NIH3T3 cells by chimeric proteins related to EWS/FLI1 but not by unrelated genes. EAT-2 is expressed in normal murine tissues and contains a unique but biochemically functional SH2 domain. An homologous sequence in the human genome has been identified and mapped to chromosome 1q22. Human EAT-2 transcripts were identified by reverse transcriptase-polymerase chain reaction (RT-PCR) in Ewing's sarcoma cell tumour cell lines. EAT-2's unique structure and correlation with transformation make it a candidate for playing a role in the transformation of NIH3T3 cells and the oncogenesis of Ewing's sarcoma.

YAC and cosmid contigs spanning the Batten disease (CLN3) region at 16p12.1-p11.2.

A yeast artificial chromosome (YAC) contig has been constructed in 16p12.1-p11.2 that encompasses three loci (D16S288, D16S299, and D16S298) closely linked to the gene causing Batten disease or juvenile-onset neuronal ceroid lipofuscinosis (CLN3). The physical map has been ordered using 42 sequence tagged sites. Four genes, interleukin-4 receptor (IL4R), phenol-preferring phenol sulfotransferase (STP), monoamine-preferring phenol sulfotransferase (STM), and sialophorin (SPN), have been mapped to the YAC contig. A partial genomic restriction map has been constructed to confirm the order and distances between D16S298, predicted to be the locus closest to CLN3. The overlapping genomic clones are a valuable resource for cloning the Batten gene (CLN3) and other genes in the region.

Refined localization of the Batten disease gene (CLN3) by haplotype and linkage disequilibrium mapping to D16S288-D16S383 and exclusion from this region of a variant form of Batten disease with granular osmiophilic deposits.

Haplotype analysis in a collaborative collection of 143 families with juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten (Spielmeyer-Vogt-Sjögren) disease has permitted refined localization of the disease gene, CLN3, which was assigned to chromosome 16 in 1989. Recombination events in four maternal meioses delimit new flanking genetic markers for CLN3 which localize the gene to the chromosome interval 16p12.1-11.2 between microsatellite markers D16S288 and D16S383. This narrows the position of CLN3 to a region of 2.1 cM, a significant reduction from the previous best interval. Using haplotypes, analysis of the strong linkage disequilibrium that exists between genetic markers within the D16S288-D16S383 interval and CLN3 shows that CLN3 is in closest proximity to loci D16S299 and D16S298. Analysis of markers across the D16S288-D16S383 region in four families with a variant form of JNCL characterized histologically by cytosomal granular osmiophilic deposits (GROD) has excluded linkage of the gene locus to the CLN3 region of chromosome 16, suggesting that JNCL with GROD is not an allelic form of JNCL.

Chromosome 16 microdeletion in a patient with juvenile neuronal ceroid lipofuscinosis (Batten disease).

The gene that is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease--CLN3--has been localized to 16p12, and the mutation shows a strong association with alleles of microsatellite markers D16S298, D16S299, and D16S288. Recently, haplotype analysis of a Batten patient from a consanguineous relationship indicated homozygosity for a D16S298 null allele. PCR analysis with different primers on DNA from the patient and his family suggests the presence of a cytogenetically undetectable deletion, which was confirmed by Southern blot analysis. The microdeletion is embedded in a region containing chromosome 16-specific repeated sequences. However, putative candidates for CLN3, members of the highly homologous sulfotransferase gene family, which are also present in this region in several copies, were not deleted in the patient. If the microdeletion in this patient is responsible for Batten disease, then we conclude that the sulfotransferase genes are probably not involved in JNCL. By use of markers and probes flanking D16S298, the maximum size of the microdeletion was determined to be approximately 29 kb. The microdeletion may affect the CLN3 gene, which is expected to be in close proximity to D16S298.

RAG-1 and RAG-2 gene expression and V(D)J recombinase activity are enhanced by protein phosphatase 1 and 2A inhibition in lymphocyte cell lines.

Expression of the recombination activating genes, RAG-1 and RAG-2, in lymphocytes, has been shown to depend on second messenger systems. An increase in intracellular cAMP upon stimulation with caffeine increases RAG expression while activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) results in decreased RAG expression. The stringent regulation of recombination appears to be partially dependent on protein kinase activities which, alone, are not likely to be sufficient to regulate recombinase activity. We provide evidence implicating a role for serine/threonine phosphatases in the signal transduction pathway which regulates RAG gene expression and consequently the recombination process in lymphocytes. The cell permeable tumor promoter, calyculin-A (CLA), which is a potent inhibitor of the type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A, respectively), was shown to upregulate the expression of RAG-1 and RAG-2 in pre-B as well as mature B- and T-lymphocyte cell lines. Although agents such as caffeine known to increase intracellular cAMP levels induce RAG expression, synergy between CLA and caffeine was not detected in pre-B cells. An in vivo assessment of recombination activity after transfection of pre-B cells with an extrachromosomal recombination vector revealed a moderate increase in recombinase activity which paralleled RAG expression after CLA stimulation. Although increased cAMP levels in pre-B cells has been associated with upregulation of RAG expression we found no such upregulation in a surface immunoglobulin M positive (sIgM+) cell line, WEHI-231, and a T cell receptor positive (TCR+) murine cell line, EL-4. Moreover, in these mature lymphocyte cell lines there was no evidence of synergy in the regulation of RAG-1 and RAG-2 mRNA upon stimulation with CLA and caffeine. These results suggest novel intracellular mechanisms for the upregulation of RAG gene expression and confirm a role for type 1 and 2A phosphatases in the control of RAG gene expression and recombinase activity in lymphocyte cell lines.

Genetic mapping of the Batten disease locus (CLN3) to the interval D16S288-D16S383 by analysis of haplotypes and allelic association.

CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease, has been localized by genetic linkage analysis to chromosome 16p between loci D16S297 and D16S57. We have now further refined the localization of CLN3 by haplotype analysis using two new microsatellite markers from loci D16S383 and SPN in the D16S297-D16S57 interval on a larger collaborative family resource consisting of 142 JNCL pedigrees. Crossover events in 3 maternal meioses define new flanking markers for CLN3 and localize the gene to the interval at 16p12.1-p11.2 between D16S288 and D16S383, which corresponds to a genetic distance of 2.1 cM. Within this interval 4 microsatellite loci are in strong linkage disequilibrium with CLN3, and extended haplotype analysis of the associated alleles indicates that CLN3 is in closest proximity to loci D16S299 and D16S298.

Incidence and origin of "null" alleles in the (AC)n microsatellite markers.

Twenty-three (AC)n repeat markers from chromosome 16 were typed in the parents of the 40 CEPH (Centre d'Etude du Polymorphisme Humain) families. Where parents were informative, the entire families were then typed. There were seven markers in which null alleles were demonstrated, as recognized by the apparent noninheritance, by a sib, of a parental allele. Four of these markers showed a null allele in a single sibship, while in the other three at least 30% of the CEPH sibships were shown to have a null allele segregating. One null allele was sequenced and shown to be the result of an 8-bp deletion occurring within the priming sequence for PCR amplification of the (AC)n repeats. In gene mapping or in application to diagnosis, the presence of a segregating null allele will not corrupt the linkage data but could result in loss of information. In isolated instances a segregating null allele may be interpreted as nonpaternity. The presence of a null allele may generate misleading data when individuals are haplotyped to determine the presence of linkage disequilibrium with a disease gene.

Fine genetic mapping of the Batten disease locus (CLN3) by haplotype analysis and demonstration of allelic association with chromosome 16p microsatellite loci.

Batten disease, juvenile onset neuronal ceroid lipofuscinosis, is an autosomal recessive neurodegenerative disorder characterized by accumulation of autofluorescent lipopigment in neurons and other cell types. The disease locus (CLN3) has previously been assigned to chromosome 16p. The genetic localization of CLN3 has been refined by analyzing 70 families using a high-resolution map of 15 marker loci encompassing the CLN3 region on 16p. Crossovers in three maternal meioses allowed localization of CLN3 to the interval between D16S297 and D16S57. Within that interval alleles at three highly polymorphic dinucleotide repeat loci (D16S288, D16S298, D16S299) were found to be in strong linkage disequilibrium with CLN3. Analysis of haplotypes suggests that a majority of CLN3 chromosomes have arisen from a single founder mutation.

A microsatellite marker within the duplicated D16S79 locus has a null allele; significance for linkage mapping.

The dinucleotide repeat 16AC66f3 was characterised from D16S79A within a duplicated section of 16p13.11, which is duplicated on all normal chromosome 16's. This marker has a common null allele caused by polymorphism within one of the primer sites. A redesigned primer overcame this problem; however, this allowed amplification of two dinucleotide repeats, at D16S79A and D16S79B, with an overlapping and uninterpretable distribution of alleles. Thus, the 16AC66F3 marker with a null allele is potentially useful for linkage mapping, as it avoids the ambiguity associated with the genotyping of homologous AC repeats at this duplicated locus. The distribution of additional D16S79 RFLPs flanking FRA16A is clarified.

High-resolution cytogenetic-based physical map of human chromosome 16.

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.

Isolation and characterisation of (AC)n microsatellite genetic markers from human chromosome 16.

A cosmid library of human chromosome 16 has been subcloned, and (AC)n microsatellite positive clones have been identified and sequenced. Oligonucleotide primers flanking the repeat were designed and synthesized for (AC)n microsatellites with n greater than 16. These microsatellite loci were then mapped by PCR using a somatic cell hybrid panel of human chromosome 16, and their heterozygosities and allele frequencies determined. Fourteen (AC)n microsatellites were mapped to discrete physical intervals of human chromosome 16 defined by a mouse/human hybrid panel. Nine of these have expected heterozygosities ranging between 0.60 and 0.79, four have expected heterozygosities between 0.02 and 0.49, and one detected three loci where the alleles could not be resolved.