Identification of differentially expressed microRNAs across the developing human brain.

We present a spatio-temporal assessment of microRNA (miRNA) expression throughout early human brain development. We assessed the prefrontal cortex, hippocampus and cerebellum of 18 normal human donor brains spanning infancy through adolescence by RNA-seq. We discovered differentially expressed miRNAs and broad miRNA patterns across both temporal and spatial dimensions, and between male and female prefrontal cortex. Putative target genes of the differentially expressed miRNAs were identified, which demonstrated functional enrichment for transcription regulation, synaptogenesis and other basic intracellular processes. Sex-biased miRNAs also targeted genes related to Wnt and transforming growth factor-beta pathways. The differentially expressed miRNA targets were highly enriched for gene sets related to autism, schizophrenia, bipolar disorder and depression, but not neurodegenerative diseases, epilepsy or other adult-onset psychiatric diseases. Our results suggest critical roles for the identified miRNAs in transcriptional networks of the developing human brain and neurodevelopmental disorders.

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer.

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples. METHODS: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs). RESULTS: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system. CONCLUSION: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

Genetic expression of Wilson's disease in cell culture: a diagnostic marker.

Wilson's disease fibroblasts have an elevated intracellular copper concentration as compared to cultured control cells. A decreased ratio of copper to protein was observed in cytoplasmic protein (or proteins) having a molecular weight greater than or equal to 30,000 in Wilson's disease cells. The results of this culture study indicate its potential importance in the early unequivocal diagnosis of this disorder.

Leydig cell hypoplasia due to inactivation of luteinizing hormone receptor by a novel homozygous nonsense truncation mutation in the seventh transmembrane domain.

Inactivating mutations in the LH receptor are the predominant cause for male pseudohermaphroditism in subjects with Leydig cell hypoplasia (LCH). The severity of the mutations, correlates with residual receptor activities. Here, we detail the clinical presentation of one subject with complete male pseudohermaphroditism and LCH. We identify within the proband and her similarly afflicted sibling a homozygous T to G transversion at nucleotide 1836 in exon 11 of the LH/CGR gene. This causes conversion of a tyrosine codon into a stop codon at codon 612 in the seventh transmembrane domain, resulting in a truncated receptor that lacks a cytoplasmic tail. In vitro, in contrast to cells expressing a normal LHR, cells transfected with the mutant cDNA exhibit neither surface binding of radiolabeled hCG nor cAMP generation. In vitro expression under the control of the LHR signal peptide of either a wild type or mutant LHR-GFP fusion protein shows no differences in receptor cellular localization. In conclusion, the in vitro studies suggest that residues in the seventh transmembrane domain and cytoplasmic tail are important for receptor binding and activation without playing a major role in receptor cellular trafficking.

Functional genomics of human brain development and implications for autism spectrum disorders.

Transcription of the inherited DNA sequence into copies of messenger RNA is the most fundamental process by which the genome functions to guide development. Encoded sequence information, inherited epigenetic marks and environmental influences all converge at the level of mRNA gene expression to allow for cell-type-specific, tissue-specific, spatial and temporal patterns of expression. Thus, the transcriptome represents a complex interplay between inherited genomic structure, dynamic experiential demands and external signals. This property makes transcriptome studies uniquely positioned to provide insight into complex genetic-epigenetic-environmental processes such as human brain development, and disorders with non-Mendelian genetic etiologies such as autism spectrum disorders. In this review, we describe recent studies exploring the unique functional genomics profile of the human brain during neurodevelopment. We then highlight two emerging areas of research with great potential to increase our understanding of functional neurogenomics-non-coding RNA expression and gene interaction networks. Finally, we review previous functional genomics studies of autism spectrum disorder in this context, and discuss how investigations at the level of functional genomics are beginning to identify convergent molecular mechanisms underlying this genetically heterogeneous disorder.

Elevated spermidine and spermine levels in the blood of psoriasis patients.

Samples of blood, anticoagulated with EDTA, from 11 patients with psoriasis and 11 individuals without psoriasis were analyzed for their polyamine content. The average spermidine level in patients with psoriasis was approximately twice that of the controls and the average spermine level was three times that of the controls. The level of spermidine and spermine in the skin of two patients with psoriasis were significantly depressed as compared to those of controls.

Luteinizing hormone receptor mutations in disorders of sexual development and cancer.

Human male sexual development is regulated by chorionic gonadotropin (CG) and luteinizing hormone (LH). Aberrant sexual development caused by both activating and inactivating mutations of the human luteinizing hormone receptor (LHR) have been described. All known activating mutations of the LHR are missense mutations caused by single base substitution. The most common activating mutation is the replacement of Asp-578 by Gly due to the substitution of A by G at nucleotide position 1733. All activating mutations are present in exon 11 which encodes the transmembrane domain of the receptor. Constitutive activity of the LHR causes LH releasing hormone-independent precocious puberty in boys and the autosomal dominant disorder familial male-limited precocious puberty (FMPP). Both germline and somatic activating mutations of the LHR have been found in patients with testicular tumors. Activating mutations have no effect on females. The molecular genetics of the inactivating mutations of the LHR are more variable and include single base substitution, partial gene deletion, and insertion. These mutations are not localized and are present in both the extracellular and transmembrane domain of the receptor. Inactivation of the LHR gives rise to the autosomal recessive disorder Leydig cell hypoplasia (LCH) and male hypogonadism or male pseudohermaphroditism. Severity of the clinical phenotype in LCH patients correlates with the amount of residual activity of the mutated receptor. Females are less affected by inactivating mutation of the LHR. Symptoms caused by homozygous inactivating mutation of the LHR include polycystic ovaries and primary amenorrhea.

Complex de novo rearrangement involving four chromosomes and ten break points with interstitial deletions and duplication.

We describe an unusual de novo case of two interstitial deletions (5q22----5q31; 9q13----9p22) and one duplication (9q22----9p34) resulting from a 10-breakpoint, complex chromosome rearrangement of chromosomes 1, 5, 8, and 9 in a profoundly retarded woman.

Atypical Menkes steely hair disease.

Menkes steely hair disease (MSHD) is a rare disorder which typically results in severe mental retardation and death in early childhood. A 21-month-old boy with an atypical milder form was presented by Procopis et al. [1981]. A second child with the atypical form is presented here who has survived to age 9 years and is doing well clinically.

Description of a large kindred with autosomal dominant inheritance of branchial arch anomalies, hearing loss, and ear pits, and exclusion of the branchio-oto-renal (BOR) syndrome gene locus (chromosome 8q13.3).

It has been suggested that branchio-oculo-facial (BOF) syndrome, deafness with ear pits, and associated conditions [MIM nos. 125100, 120502], and branchio-oto-renal (BOR) [MIM no. 113650] or Melnick-Fraser syndrome represent phenotypic variants of the BOR syndrome, which is inherited in an autosomal dominant (AD) manner and has variable clinical expression. Recently, the BOR gene was mapped to chromosome region 8q13.3 and its sequence was identified as the human homolog of the Drosophila eyes absent (EYA1) gene. We studied an extended family with AD inheritance of branchial arch anomalies (BAA), hearing loss, and ear pits, whose phenotype differed from that of patients with BOR in that none of the affected members had renal abnormalities or lacrimal duct stenosis. Fifteen affected members were studied; ear pits were present in all of them, whereas hearing loss and other BAA were present in 40 and 20%, respectively. Blood was collected from 31 patients; DNA was extracted by standard methods and amplified using primers from microsatellite sequences flanking the BOR locus on chromosome 8q13.3 (D8S1807, D8S530, and D8S543). Linkage analysis was performed under two models of AD inheritance with different penetrance: 100% and 80%. In both cases, the logarithm of odds (LOD) scores produced were significantly less than -2; exclusion of the 8q13.3 locus was also confirmed by multipoint LOD score analysis. We conclude that, in one large family with AD inheritance of BAA, hearing loss and ear pits, the BOR locus was excluded. This represents the first documentation of heterogeneity in branchio-oto anomalies, syndromes with phenotypes similar to BOR syndrome.

Testicular seminoma in a patient with a constitutively activating mutation of the luteinizing hormone/chorionic gonadotropin receptor.

A white man who had been diagnosed, 35 years previously at the age of 27 months, to have precocious puberty, was later determined to have familial male-limited precocious puberty (FMPP), on the basis of his family history, increased serum testosterone, prepubertal concentrations of follicle stimulating hormone and luteinizing hormone, and Leydig cell hyperplasia. Recently, this diagnosis was confirmed by molecular genetic analysis that demonstrated the presence of a heterozygous constitutive activating mutation of the luteinizing hormone/chorionic gonadotropin receptor. This dominant gain-of-function Asp578Gly mutation has been shown constitutively to activate the receptor in the absence of the agonist, leading to enhanced synthesis of cAMP and, in turn, to increased, sustained production of testosterone. In 1994, this patient was found to have a testicular seminoma. He represents the first case of a testicular germ cell tumor described in an FMPP patient, raising the possibility of a potentially harmful effect of prolonged increased concentrations of sex hormones, with onset early in life, upon the cellular components of the testes.

Cloning of a human acid sphingomyelinase cDNA with a new mutation that renders the enzyme inactive.

A cDNA encoding human acid sphingomyelinase was initially obtained by screening a placental cDNA library in lambda gt11 with a synthetic oligonucleotide probe and subsequently with partial cDNA. The full-length cDNA, hPSM55, comprised 2,376 nucleotides, with a 5' untranslated sequence of 122 nucleotides, an open reading frame of 1,884 nucleotides encoding a protein of 627 amino acids, and a 3' untranslated region of 370 bases. hPSM55 was almost identical to pASM-1FL reported by Schuchman et al. (J. Biol. Chem. 266, 8531-8539, 1991) except for a 6 base pair deletion in the signal peptide, which indicated the possible removal of valine and leucine residues between positions 36 and 37, and a 463T- to C-transition, which indicated a possible substitution of 155arginine for cystine. This cDNA was expressed in both COS-7 cells and Chinese hamster ovary cells. There was no increase in acid sphingomyelinase activity in either cell line following transfection. However, the correction of a single base change, 463C to T, in hPSM55 caused increased acid sphingomyelinase activity in transfectants. These results suggest that the mutation of nucleotide 463C to T plays an important role in the catalytic activity of acid sphingomyelinase.

Correlation of plasma propylthiouracil levels with inhibition of thyroid hormone synthesis in the rat.

The inhibition of thyroid hormone synthesis was studied in relation to plasma levels of 6-n-propyl-2-thiouracil (PTU). Na 125I (5 muCi) was injected i.p. into adult male Sprague-Dawley rats. After 30 min, graded doses of PTU (0.2 mg/kg. 0.1 mg/kg, and 0.05 mg/kg) were similarly injected. Thyroid hormone synthesis was followed by the accumulation of radioactivity into thyroid glands, which were removed at specified time intervals. PTU levels were measured spectrophotometrically at the time of sacrifice. A-ditionally, PTU (35S) was used to confirm blood levels of PTU and also to follow intrathyroidal PTU levels. Plasma PTU levels in excess of 0.18 microgram/ml completely inhibited thyroid hormone synthesis. Levels between 0.14 and 0.09 microgram/ml had a partial effect, and PTU levels less than 0.09 microgram/ml had no effect on thyroid hormone synthesis.

Glucose tolerance and plasma lipid distributions in rats fed a high-sucrose, high-cholesterol, low-chromium diet.

Male Sprague-Dawley rats were fed either a low-chromium (60 to 100 micrograms per kg of diet) or chromium-supplemented (5 mg per kg of diet), high-sucrose, high-cholesterol diet from weaning until age 18 months. Rats that were pair- and meal-fed the low-chromium diet had higher one-hour postprandial plasma glucose concentrations than their supplemented partners at ages 4 and 8 months (P less than 0.05), but not at age 12 months. One-hour postgavage (250 mg glucose/100 g body wt) glucose concentrations did not differ between dietary groups at 12 months. Plasma cholesterol concentrations increased up to age 12 months, but neither they nor postprandial triglyceride concentrations differed significantly between dietary groups. Ad libitum feeding of the low-chromium and chromium-supplemented diets was initiated at age 14 months in order to determine whether there were differences due to dietary chromium content which might not be manifest on the pair-feeding regimen. Animals of both dietary groups had significant weight gain by age 16 months, but their one-hour postgavage plasma glucose concentrations did not differ significantly. Plasma cholesterol concentrations increased significantly following institution of ad libitum feedings, but neither they nor lipoprotein cholesterol or triglyceride distributions differed significantly between dietary groups. Experimental conditions, methods, and results from this study and previous studies are compared and critically examined. We suggest that other factors in addition to dietary chromium content may contribute to the differences in glucose tolerance and plasma cholesterol concentrations described in such studies and that there is a need for improved documentation of glucose intolerance and tissue chromium concentrations in this animal model.

Metal-binding studies of metallothioneins in Menkes kinky hair disease.

Two species of metallothioneins were isolated from both normal and Menkes kinky hair disease (MKHD) patient livers. Atomic absorption determination of metals indicated that the patient liver metallothioneins had lower copper and cadmium content than normals. Isotope exchange studies, carried out by incubating native metallothioneins with copper-64 or cadmium-109 demonstrated a decreased affinity for copper and an increased affinity for cadmium in both MKHD metallothioneins. An hypothesis is proposed in which metallothionein functions as an intracellular copper carrier and is responsible for the transport of copper between the cells and the surrounding. Change in the copper affinity of the metallothioneins was suggested to be the major abnormality in MKHD.

Cell culture studies of Menkes kinky hair disease.

The dose response as well as kinetics of uptake and retention of copper and cadmium of normal and Menkes kinky hair disease (MKHD) cultured fibroblasts are described. In basal culture medium, intracellular copper concentration in MKHD fibroblasts was approximately 3 times that of control cultures. The intracellular copper concentration of MKHD cells was significantly higher than that of normal fibroblasts at medium copper concentrations below 20 microgram/ml. Death of MKHD cells occurred at medium copper concentrations between 15 and 20 microgram/ml with an intracellular copper level 3 times that at basal medium. Normal cells died at medium copper concentration above 30 microgram/ml with an intracellular copper concentration 19 times that at basal medium. These observations suggested the existence of a regulatory mechanism for maintenance and control of intracellular copper in normal fibroblasts which is effective at medium copper concentrations below 30 microgram/ml. This system is defective in MKHD fibroblasts. In basal medium MKHD and normal fibroblasts had similar intracellular cadmium concentrations; however, at higher medium cadmium concentrations MKHD cells had increased intracellular cadmium levels. The uptake of both 64Cu and 109Cd was significantly higher in MKHD cells than in normal cells, indicating that the uptake of 64Cu and 109Cd is not impaired in MKHD cells. A higher retention of 64Cu was observed in MKHD cells at both 37 degrees C and 4 degrees C. No obvious trend, however, was observed in the difference of retention of 109Cd between MKHD and normal cells. An impairment of egress of copper in MKHD cells is implicated by these results.

Polyamine compartmentalization in various human disease states.

Whole blood was separated into preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets and plasma. Each preparation was analyzed for the concentration of the polyamines putrescine, spermidine and spermine. This was done in 17 controls, 14 patients with psoriasis, four patients with hereditary elliptocytosis, two patients with chronic lymphocytic leukemia and one patient each with lung cancer, non-Hodgkin's lymphoma, sickle cell anemia with mild psoriasis, and progeria. In patients with elevated blood polyamine levels, absorption onto erythrocytes was relatively common, and the spermine/spermidine ratio was useful in localizing abnormalities and characterizing the nature of the polyamine alteration. Proliferative states were associated with elevated spermine/spermidine ratios relative to controls while this relationship was reversed in erythropathies such as hereditary elliptocytosis and sickle cell anemia.

Urinary polyamines in preterm infants.

Polyamines were determined in urine of 22 preterm infants (mean 30.0 weeks gestation) from birth to 22 weeks of age, and in full-term infants in the first week of life. A significant decline in urine putrescine and spermidine levels occurred with increased postnatal age in preterm infants. At expected term preterm infants had significantly higher levels of polyamines in urine than full-term infants at the same postconceptional age. No constant correlations between weight or linear growth velocity and urinary polyamine excretion could be established in this group of infants. Altered urine polyamine values were detected in two clinical situations: hyperbilirubinemia was associated with increased urine spermidine (and with increased spermidine/putrescine ratio), and liver disease was associated with increased levels of both putrescine and spermidine in urine.

Polyamine distribution in cellular compartments of blood and in aging erythrocytes.

Human blood was separated into pure preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets, and platelet free plasma. The mean concentrations of putrescine, spermidine, and spermine per 10(9) cells were found to be several orders of magnitude higher for leukocytes than erythrocytes. There was no significant difference between leukocyte types. Platelets and plasma had relatively low levels in proportion to the amounts contributed by erythrocytes and leukocytes to whole blood. Human erythrocytes were age-separated by density and the changes in polyamine concentrations in maturing erythrocytes were documented. There were highly significant statistical differences between young and old red blood cells for putrescine, spermidine and spermine. The clinical use of red blood cell polyamines as an indicator of the activity of the bone marrow in anemic states is suggested.

Isolation and characterization of a polyamine-peptide conjugate from human amniotic fluid.

Significant amounts of the diamine putrescine and the polyamines spermidine and spermine could be detected in human third-trimester amniotic fluid only after acid hydrolysis. This observation was interpreted to mean that these amines existed only in conjugated form in this biological fluid. Upon fractionation by ultrafiltration 90--10% of the putrescine was associated with the 1000--10 000 dalton fraction. Spermine was identified in this fraction and in a low-molecular weight fraction presumably representing acetylated derivatives. Spermidine was entirely associated with the 10 000--30 000 dalton fraction. The putrescine conjugate was purified to homogeneity by column chromatography on Biogels P10 and P6 followed by ion-exchange chromatography on DEAE-Sephadex A-25. Molecular weight by gel exclusion using peptide standards was estimated to be approx. 4600. The UV absorption spectrum of the putrescine conjugate conformed to that expected for a polypeptide. This putrescine conjugate contained 39 identified amino acids with a combined molecular weight of 4713. Putrescine was detectable by high pressure liquid chromatography only after acid hydrolysis of the conjugate. No other polyamines were detected in these hydrolyzates, nor were any polyamines demonstable in hydrolyzates of control peptides nor in pooled column washes. The identity of the putrescine determined by high pressure liquid chromatography was confirmed by a two-dimensional thin-layer chromatography method. These results establish the in vivo production of a putrescine--polypeptide conjugate in man. Such molecular species may constitute yet another metabolic pathway for polyamines or may reflect another mode of post-translational modification of polypeptide structure and function. The qualitative and quantitative analysis of polyamine conjugate in human aminotic fluid may prove to be useful in the detection of abnormalities in fetal development.