The serotonin transporter gene as a QTL for ADHD.

Molecular studies of attention deficit hyperactivity disorder (ADHD) have identified susceptibility genes for the categorically diagnosed disorder using operational diagnostic criteria. Here, we take a QTL approach to mapping genes for ADHD using a composite continuous index of ADHD behavior in a large epidemiological sample. Previous studies of clinical ADHD suggest that two functional polymorphisms in the serotonin transporter gene (SLC6A4), one in the 5'-regulatory region of the gene (5-HTTLPR) and the other a VNTR (5-HTTVNTR) in the second intron, as well as a single nucleotide polymorphism in the 3'-untranslated region (3'-UTR SNP), may be associated with the disorder. Here, we investigate these polymorphisms as well as an additional ten SNPs spread across the gene. We found significant association with the long (L) allele of the 5-HTTLPR; P = 0.019, but neither the 5-HTTVNTR nor the 3'-UTR SNP were significantly associated. Significant associations (P < 0.05) were found for a further 5 the 10 other markers tested. We found evidence for two haplotype blocks spanning the region. We found strong evidence for association with the first haplotype block (comprised of four markers), with the significance of a combined primary and secondary test of association reaching an empirical P value = 0.0054 for the global test and an empirical P value = 0.00081 for the largest local test. Thus, we show here that SLC6A4, which has a major influence on brain serotonin availability, may be a QTL for ADHD.     

No association between CHRNA7 microsatellite markers and attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable, common psychiatric disorder of childhood that probably involves several genes. There are several lines of evidence suggesting that the nicotinic system may be functionally significant in ADHD. First, nicotine promotes the release of dopamine and has been shown to improve attention in adults with ADHD, smokers, and nonsmokers. Second, ADHD is a significant risk factor for early initiation of cigarette smoking in children and maternal cigarette smoking appears to be a risk factor for ADHD. Finally, animal studies in rats and monkeys also suggest that nicotine may be involved in attentional systems and locomotor activity. The nicotinic system has previously been studied in schizophrenia where the neuronal nicotinic acetylcholine receptor alpha 7 subunit gene (CHRNA7) has been implicated in decreased P50 inhibition and attentional disturbances in patients with schizophrenia and in many of their nonschizophrenic relatives. Three known microsatellite markers (D15S165, D15S1043, and D15S1360) near the nicotinic acetylcholine alpha 7 receptor gene, CHRNA7, were studied in 206 ADHD parent-proband trios of children aged 5-16 with ADHD according to DSM-IV criteria. Children with known major medical or psychiatric conditions or mental retardation (IQ < 70) were excluded from the study. Markers D15S165 and D15S1360 were in linkage disequilibrium. The extended Transmission Disequilibrium Test analyses demonstrated no evidence that variation at the microsatellite markers D15S1360, D15S1043, and D15S165 influences susceptibility to ADHD. However, it remains possible that the CHRNA7 gene and other nicotinic system genes may be involved in conferring susceptibility to ADHD.     

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

BACKGROUND: The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES: Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN: The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION: Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.     

Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires

A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g. 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.     

Reovirus-like agent (rotavirus) from lambs.

Rotavirus particles were demonstrated by electron microscopy in the feces of lambs with diarrhea. Rotavirus antigen was synthesized in cell cultures infected with filtrates of the diarrheic feces, but the virus was not adapted to grow serially in cell cultures. An antigenic relationship between rotaviruses from lambs, pigs, and calves was demonstrated by immunofluorescence. Colostrum-deprived lambs were infected with the lamb rotavirus, and the virus was passaged in lambs. Viral replication occurred in the villous epithelial cells of the small intestine, and the virus was excreted in the feces up to 78 h postinfection. Diarrhea was not observed in the experimentally infected lambs.     

Accuracy of presumptive criteria for culture diagnosis of Neisseria gonorrhoeae in low-prevalence populations of women.

The accuracy of presumptive criteria for identification of Neisseria gonorrhoeae was assessed in two separate populations of women with a low prevalence of gonorrhea. Of the presumptively positive cervical isolates available for confirmation, 98.5% were identified as N. gonorrhoeae. Of 25 isolates that could not be confirmed, 20 failed to grow on subculture, and of the remaining 5, only 2 (0.6% of all viable recoveries) were identified as nongonococcal isolates (N. meningitidis). Our study confirms earlier findings that meningococcal isolates are rarely found in endocervical specimens. The benefits from early treatment and counselling of women for gonorrhea on the basis of presumptive criteria outweight the risks occasioned by the rarely encountered nongonococcal isolate.     

Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.

The authors have used morphometric, immunocytochemical, and electron optical techniques to study fibrin deposits associated with villi from 14 normal term placentas, and have examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Morphometric analysis of 3477 villous profiles showed that 5.5% of villi examined had fibrin deposition at sites of syncytial denudation and that fibrin deposition was highly associated with villous epithelial denudation, as evidenced by loss of cytokeratin staining. The perivillous fibrin deposits were strongly immunoreactive for the B beta chain of fibrin II, consistent with local thrombolytic cleavage of fibrinogen to fibrin. Deposits were frequently surfaced by a discontinuous layer of cytokeratin-positive trophoblastic cells that showed type IV basement membrane collagen immunoreactivity at the interface between trophoblast and fibrin. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and the trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and a new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, showed morphologic differentiation into a trophoblast layer like that on term villi. The authors suggest that epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions can modulate trophoblastic differentiation.