Differential gene expression analysis of placentas with increased vascular resistance and pre-eclampsia using whole-genome microarrays.

Pre-eclampsia is a pregnancy complication characterized by hypertension and proteinuria. There are several factors associated with an increased risk of developing pre-eclampsia, one of which is increased uterine artery resistance, referred to as "notching". However, some women do not progress into pre-eclampsia whereas others may have a higher risk of doing so. The placenta, central in pre-eclampsia pathology, may express genes associated with either protection or progression into pre-eclampsia. In order to search for genes associated with protection or progression, whole-genome profiling was performed. Placental tissue from 15 controls, 10 pre-eclamptic, 5 pre-eclampsia with notching, and 5 with notching only were analyzed using microarray and antibody microarrays to study some of the same gene product and functionally related ones. The microarray showed 148 genes to be significantly altered between the four groups. In the preeclamptic group compared to notch only, there was increased expression of genes related to chemotaxis and the NF-kappa B pathway and decreased expression of genes related to antigen processing and presentation, such as human leukocyte antigen B. Our results indicate that progression of pre-eclampsia from notching may involve the development of inflammation. Increased expression of antigen-presenting genes, as seen in the notch-only placenta, may prevent this inflammatory response and, thereby, protect the patient from developing pre-eclampsia.

Characterisation of insulin-resistant phenotype of cultured rat primary adipose cells.

AIMS/HYPOTHESIS: We characterised insulin resistance, metabolic defects and endocrine dysfunction in cultured adipose cells and examined the autocrine or paracrine roles of cytokines/adipokines in the progression of insulin resistance. MATERIALS AND METHODS: Rat primary adipose cells were prepared and cultured for 24 and 48 h. Insulin resistance and gene expression were examined by glucose uptake assay, cDNA microarray and real-time RT-PCR. RESULTS: After 24 h in culture, the fold increase of insulin-stimulated glucose uptake in adipose cells was markedly reduced; after 48 h the response of the cells to insulin decreased. cDNA microarray analysis showed that the expression of 514 genes was altered in adipose cells after 24 h in culture. The dysregulated genes included those involved in the citric acid cycle and in fatty acid and pyruvate metabolism. Specifically, the following genes were all downregulated: genes encoding lipolytic and lipogenic enzymes; uncoupling protein 1 and 2 genes; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha gene. This indicates that lipolytic and lipogenic activity, as well as mitochondria capacity decline in adipose cells cultured for 24 h. The mRNAs encoding 40 adipokines were also dysregulated in cultured cells. Strikingly, the dysregulated adipokines in cultured cells and in freshly isolated adipose cells from insulin-resistant Zucker fa/fa rats displayed a similar pattern with regard to protein functions. Also striking was the fact that progression of insulin resistance was promoted by the adipokines secreted from insulin-resistant adipose tissue or cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that the impairment of metabolism and endocrine dysfunction in cultured adipose cells mimics the insulin resistance occurring in vivo. Cytokines and adipokines appear to play a critical role in the progression of insulin resistance in adipose cells.

Gene expression profiling of human placentas from preeclamptic and normotensive pregnancies.

The aim of this study was to investigate patterns of gene expression in placental samples from patients with preeclampsia (PE), persistent bilateral uterine artery notching (without PE), and normal controls. This study included placental tissue from nine women with PE, seven with uncomplicated pregnancies and five with bilateral uterine artery notching in Doppler velocimetry tracings. Human cDNA microarrays with 6500 transcripts/genes were used and the results verified with real-time PCR and in-situ hybridization. Multidimensional scaling method and random permutation technique demonstrated significant differences among the three groups examined. Within the 6.5K arrays, 6198 elements were unique cDNA clones representing 5952 unique UniGenes and 5695 unique LocusLinks. Multidimensional scaling plots showed 5000 genes that met our quality criteria; among these, 366 genes were significantly different in at least one comparison. Differences in three genes of interest were confirmed with real-time PCR and in-situ hybridization; acid phosphatase 5 was shown to be overexpressed in PE samples and calmodulin 2 and v-rel reticuloendotheliosis viral oncogene homolog A (RELA) were downregulated in PE and uterine artery notch placentas. In conclusion downregulation of RELA and calmodulin 2 might represent an attempt by the placenta to compensate for elevations in intracellular calcium, possibly caused by hypoxia and/or apoptosis, in both pregnancies with uterine artery notching and preeclampsia.

A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells.

AIM: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm's canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs. METHODS: The TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments. RESULTS: SCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1,120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects. CONCLUSION: These experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

Genome-wide scan in a large complex pedigree with predominantly male schizophrenics from the island of Kosrae: evidence for linkage to chromosome 2q.

It is widely accepted that founder populations hold promise for mapping loci for complex traits. However, the outcome of these mapping efforts will most likely depend on the individual demographic characteristics and historical circumstances surrounding the founding of a given genetic isolate. The 'ideal' features of a founder population are currently unknown. The Micronesian islandic population of Kosrae, one of the four islands comprising the Federated States of Micronesia (FSM), was founded by a small number of settlers and went through a secondary genetic 'bottleneck' in the mid-19th century. The potential for reduced etiological (genetic and environmental) heterogeneity, as well as the opportunity to ascertain extended and statistically powerful pedigrees makes the Kosraen population attractive for mapping schizophrenia susceptibility genes. Our exhaustive case ascertainment from this islandic population identified 32 patients who met DSM-IV criteria for schizophrenia or schizoaffective disorder. Three of these were siblings in one nuclear family, and 27 were from a single large and complex schizophrenia kindred that includes a total of 251 individuals. One of the most startling findings in our ascertained sample was the great difference in male and female disease rates. A genome-wide scan provided initial suggestive evidence for linkage to markers on chromosomes 1, 2, 3, 7, 13, 15, 19, and X. Follow-up multipoint analyses gave additional support for a region on 2q37 that includes a schizophrenia locus previously identified in another small genetic isolate, with a well-established recent genealogical history and a small number of founders, located on the eastern border of Finland. In addition to providing further support for a schizophrenia susceptibility locus at 2q37, our results highlight the analytic challenges associated with extremely large and complex pedigrees, as well as the limitations associated with genetic studies of complex traits in small islandic populations.

Probe generation directly from small numbers of cells for DNA microarray studies.

Recently, we described a technique that allows us to prepare probes for expression profiling from 0.5-1 microgram RNA without template or signal amplification. However, we were unable to use this method to study cells harvested by needle biopsy, cell sorting, or laser capture microdissection. Here we give a new protocol for amplifying RNA with multiple reaction cycles and preparing fluorescent probes from approximately 10 cells. We use random 9-mers with a T3 RNA polymerase recognition sequence on the 5' end for every round of cDNA synthesis except the first. The latter is primed with oligo(dT) with a T7 RNA polymerase recognition sequence on the 5' end. Results were highly reproducible and reliable, and the products generated using our method seemed comparable to those produced using the RiboAmp RNA kit when both were used to do two cycles of amplification. To test our method's utility, we lysed cells directly into reverse transcription buffer containing RNase inhibitor and performed three rounds of RNA amplification. The expression profiles of mouse C2 and NIH 3T3 cells obtained with 11,232-element arrays using amplified RNAs were similar to those seen when probes were prepared from unamplified templates.

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues.

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.

Neurologic and psychiatric manifestations in a family with a mutation in exon 2 of the guanosine triphosphate-cyclohydrolase gene.

OBJECTIVE: To investigate the range of clinical features to correlate genotypic and phenotypic manifestations in hereditary progressive and/or levodopa-responsive dystonia due to a defect in the guanosine triphosphate-cyclohydrolase (GCH1) gene. DESIGN AND SETTING: A large family from Texas was studied in an ambulatory setting by clinicians in genetics, neurology, and psychiatry using structured interviews and examinations. PATIENTS: The family was selected after neurometabolic investigations of a young boy (proband) with foot dystonia and fatigue and his father, who had a long history of anxiety and depression. Results of metabolic studies showed decreased levels of metabolites of biopterin and biogenic amines in cerebrospinal fluid. Subsequently, a novel mutation (37-base pair deletion) in exon 2 of the GCH1 gene was demonstrated in 11 family members. There was no observed female sex bias, but there was a wide variability of motor dysfunctions in family members. Approximately 50% had clinical deafness and a similar number had significant psychiatric dysfunction, including depression and anxiety. CONCLUSION: Study of additional families with hereditary progressive and/or levodopa-responsive dystonia using modern molecular methods will be necessary to confirm the neuropsychiatric spectrum of this disorder, in which important clinical features may be unrecognized and thus inappropriately managed.

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Isolation and characterization of the human homeobox gene HOX D1.

Homeobox genes, first identified in Drosophila, encode transcription factors that regulate embryonic development along the anteroposterior axis of an organism. Vertebrate homeobox genes are described on the basis of their homology to the genes found within the Drosophila Antennapedia and Bithorax homeotic gene complexes. Mammals possess four paralogous homeobox (HOX) gene clusters, HOX A, HOX B, HOX C and HOX D, each located on different chromosomes, consisting of 9 to 11 genes arranged in tandem. We report the characterization of the human HOX D1 gene. This gene consists of two exons, encoding a 328 amino acid protein, separated by an intron of 354 bp. The human HOX D1 protein is one amino acid longer (328 amino acids) than the mouse protein (327 amino acids) and is 82% identical to the mouse HOX D1 homolog. The DNA binding homeodomain region of the human protein exhibits a 97% and 80% identity between mouse Hoxd1 and Drosophila labial homeodomains, respectively. The exon/intron and intron/exon splice junctions are conserved in position between human and mouse genes. Determination of the human HOX D1 gene structure permits the use of PCR based analysis of this gene for the assessment of mutations, for diseases that link to the HOXD cluster (such as Duanes Retraction Syndrome (DRS)), or polymorphisms associated with human variation. Molecular characterization of the HOXD1 gene may also permit analysis of the functional role of this gene in human neurogenisis.

Molecular cloning and functional characterization of a vasotocin receptor subtype that is expressed in the shell gland and brain of the domestic chicken.

In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin congruent with vasopressin > oxytocin congruent with mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.

Localization of a gene for Duane retraction syndrome to chromosome 2q31.

Duane retraction syndrome (DRS) is a congenital eye-movement disorder characterized by a failure of cranial nerve VI (the abducens nerve) to develop normally, resulting in restriction or absence of abduction, restricted adduction, and narrowing of the palpebral fissure and retraction of the globe on attempted adduction. DRS has a prevalence of approximately 0.1% in the general population and accounts for 5% of all strabismus cases. Undiagnosed DRS in children can lead to amblyopia, a permanent uncorrectable loss of vision. A large family with autosomal dominant DRS was examined and tested for genetic linkage. After exclusion of candidate regions previously associated with DRS, a genomewide search with highly polymorphic microsatellite markers was performed, and significant evidence for linkage was obtained at chromosome 2q31 (D2S2314 maximum LOD score 11.73 at maximum recombination fraction. 0). Haplotype analysis places the affected gene in a 17.8-cM region between the markers D2S2330 and D2S364. No recombinants were seen with markers between these two loci. The linked region contains the homeobox D gene cluster. Three of the genes within this cluster, known to participate in hindbrain development, were sequenced in affected and control individuals. Coding sequences for these genes were normal or had genetic alterations unlikely to be responsible for the DRS phenotype. Identifying the gene responsible for DRS may lead to an improved understanding of early cranial-nerve development.

Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene.

Cataracts are a significant public health problem. Here, we describe the genetic alteration responsible for a progressive form of cataract, segregating as an autosomal dominant trait in a three-generation pedigree. Unlike most autosomal dominant cataracts, these are not clinically apparent at birth but are initially observed in the first year or two of life. The opacification evolves relatively slowly, generally necessitating removal of the lens in childhood or early adolescence. A genome-wide search in our kindred revealed linkage at 2q33-35 where the gamma-crystallin gene cluster resides. A single base alteration resulting in an Arg- 14 --> Cys (R14C) substitution in gammaD-crystallin was subsequently identified. Protein modeling suggests that the effect of this mutation is a subtle one, affecting the surface properties of the crystallin molecule rather than its tertiary structure, consistent with the fact that the patients' lenses are normal at birth. This is the first gene defect shown to be responsible for a noncongenital progressive cataract, and studying the defective protein should teach us more about the mechanisms underlying cataract formation.

Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease.

A missense mutation in the human alpha synuclein gene was recently identified in some cases of familial Parkinson's disease (FPD). We have developed an antibody that recognizes the C-terminal 12 amino acids of the human alpha synuclein protein and have demonstrated that alpha synuclein is an abundant component of the Lewy bodies found within the degenerating neurons of patients with Parkinson's disease (PD). The presence of alpha synuclein in Lewy bodies of sporadic PD patients suggests a central role for alpha synuclein in the pathogenesis of PD.

Mapping a gene involved in regulating dietary cholesterol absorption. The sitosterolemia locus is found at chromosome 2p21.

The molecular mechanisms regulating the amount of dietary cholesterol retained in the body as well as the body's ability to selectively exclude other dietary sterols are poorly understood. Studies of the rare autosomal recessively inherited disease sitosterolemia (OMIM 210250) may shed some light on these processes. Patients suffering from this disease appear to hyperabsorb both cholesterol and plant sterols from the intestine. Additionally, there is failure of the liver's ability to preferentially and rapidly excrete these non-cholesterol sterols into bile. Consequently, people who suffer from this disease have very elevated plasma plant sterol levels and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. Identification of this gene defect may therefore throw light on regulation of net dietary cholesterol absorption and lead to an advancement in the management of this important cardiovascular risk factor. By studying 10 well-characterized families with this disorder, we have localized the genetic defect to chromosome 2p21, between microsatellite markers D2S1788 and D2S1352 (maximum lodscore 4.49, theta = 0.0).

PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).

Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.

Methods for precise sizing, automated binning of alleles, and reduction of error rates in large-scale genotyping using fluorescently labeled dinucleotide markers. FUSION (Finland-U.S. Investigation of NIDDM Genetics) Study Group.

Large-scale genotyping is required to generate dense identity-by-descent maps to map genes for human complex disease. In some studies the number of genotypes needed can approach or even exceed 1 million. Generally, linkage and linkage disequilibrium analyses depend on clear allele identification and subsequent allele frequency estimation. Accurate grouping or categorization of each allele in the sample (allele calling or binning) is therefore an absolute requirement. Hence, a genotyping system that can reliably achieve this is necessary. In the case of affected sib-pair analysis without parents, the need for accurate allele calling is even more critical. We describe methods that permit precise sizing of alleles across multiple gels using the fluorescence-based, Applied Biosystems (ABI) genotyping technology and discuss ways to reduce genotyping error rates. Using database utilities, we show how to minimize intergel allele size variation, to combine data effectively from different models of ABI sequencing machines, and automatically bin alleles. The final data can then be converted into a format ready for analysis by statistical genetic packages such as MENDEL.

Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions.

A subset of familial and sporadic amyotrophic lateral sclerosis (ALS-a fatal disorder characterised by progressive motor neuron degeneration) cases are due to mutations in the gene encoding Cu,Zn superoxide dismutase (SOD1). Two mutations which have been successfully used to generate transgenic mice that develop an ALS-like syndrome are glycine 85 to arginine (G85R) and glycine 93 to alanine (G93A) with the mutant SOD1 allele overexpressed in a normal mouse genetic background. No ALS-like phenotype is observed in mice overexpressing wild-type SOD1 or mice without any SOD1 activity. These dominant mutations, which do not necessarily decrease SOD1 activity, may confer a gain of function that is selectively lethal to motor neurons. The yeast interaction trap system allowed us to determine whether these mutations in SOD1 caused novel protein interactions not observed with wild-type SOD1 and which might participate in the generation of the ALS phenotype. Two proteins, lysyl-tRNA synthetase and translocon-associated protein delta, interact with mutant forms of SOD1 but not with wild-type SOD1. The specificity of the interactions was confirmed by the coimmunoprecipitation of mutant SOD1 and the expressed proteins. These proteins are expressed in ventral cord, lending support to the relevance of this interaction to motor neuron disease.