Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders.

Understanding the genetic factors underlying neurodevelopmental and neuropsychiatric disorders is a major challenge given their prevalence and potential severity for quality of life. While large-scale genomic screens have made major advances in this area, for many disorders the genetic underpinnings are complex and poorly understood. To date the field has focused predominantly on protein coding variation, but given the importance of tightly controlled gene expression for normal brain development and disorder, variation that affects non-coding regulatory regions of the genome is likely to play an important role in these phenotypes. Herein we show the importance of 3 prime untranslated region (3'UTR) non-coding regulatory variants across neurodevelopmental and neuropsychiatric disorders. We devised a pipeline for identifying and functionally validating putatively pathogenic variants from next generation sequencing (NGS) data. We applied this pipeline to a cohort of children with severe specific language impairment (SLI) and identified a functional, SLI-associated variant affecting gene regulation in cells and post-mortem human brain. This variant and the affected gene (ARHGEF39) represent new putative risk factors for SLI. Furthermore, we identified 3'UTR regulatory variants across autism, schizophrenia and bipolar disorder NGS cohorts demonstrating their impact on neurodevelopmental and neuropsychiatric disorders. Our findings show the importance of investigating non-coding regulatory variants when determining risk factors contributing to neurodevelopmental and neuropsychiatric disorders. In the future, integration of such regulatory variation with protein coding changes will be essential for uncovering the genetic causes of complex neurological disorders and the fundamental mechanisms underlying health and disease.

First wave fetal thymocytes expressing V3J gamma 1C gamma 1-V delta 1J delta 2C delta T cell receptors are not required for alpha beta T cell receptor rearrangement and expression.

We have determined the fetal expression of V gamma 3, delta and beta TcRs in mice transgenic for the V gamma 1.1J4C gamma 4 TcR chain. The first wave thymocytes appearing at day 14 and disappearing by day 17 in normal mice was absent from the transgenic mice. However, both mice had an almost identical number of gamma delta-bearing thymocytes throughout gestation. Therefore, it is most likely that the V gamma 3J gamma 1C gamma 1 chain was replaced in the transgenic mice by the V gamma 1.1J gamma 4C gamma 4 transgene. The appearance, although slightly earlier for the transgenic mice, of alpha beta-bearing thymocytes was also very similar between transgenic and control mice during gestation. These data suggest that whatever the role of the first wave thymocytes expressing V3-V delta 1 TcRs is, it most likely is not required for the rearrangement, expression and maturation of the alpha beta TcR repertoire. We are currently analyzing a series of gamma delta transgenic mice to determine whether other restricted populations of gamma delta-bearing T cells are involved in specific aspects of immune development or function.

Expression of a T cell receptor gamma-chain (V gamma 1.1J gamma 4C gamma 4) transgene in mice influences T cell receptor ontogeny and thymic architecture during development.

In this report, we have characterized T cell ontogeny in mice transgenic for the V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) TCR chain gene by analyzing the T cell surface phenotype and microarchitecture of the transgenic lymphoid organs during development. The first wave of V gamma 3J gamma 1C gamma 1 TCR+ thymocytes that appear in the thymus at day 14 of gestation were virtually absent in the TCR gamma 4 transgenic mice. However, comparable levels of total gamma delta+ thymocytes were present in the TCR gamma 4 transgenic mice, suggesting that these thymocytes expressed a transgene receptor. In addition, the appearance of significant numbers of TCR alpha beta+ T cells occurred earlier (day 17 of fetal development) in the transgenic thymus. After birth, thymuses from TCR gamma 4 transgenic mice were characterized by a consistent increase in the number of TCR gamma delta+ thymocytes and a transient increase (between 2 and 4 wk of age) in the absolute number of TCR+ thymocytes (2- to 4-fold) compared with thymuses from normal mice. Immunohistologic analysis of the thymus, spleen, and lymph nodes from 2-day and 3-wk-old transgenic mice showed significant differences to controls only in regions harboring mature, functional T cell subsets which may be the result of bidirectional signaling between lymphocyte subsets (influenced by transgene expression) and stromal elements. The results demonstrate that striking differences, leading to the earlier appearance of mature T cells, occurred in both the fetal and neonatal thymus and periphery of mice that expressed a TCR gamma 4 transgene. These findings are consistent with the hypothesis that expression of the TCR gamma 4 chain gene, early in ontogeny, has a positive influence on the development of the T cell compartment.

Activity of certain enzymes and histomorphological changes in subacute intoxication of rats with selected organophosphates.

The reported investigations were carried out on female Wistar rats which were given at 72-hour interval during 117 days (40 doses) dichlorphos subcutaneously, intragastrically and percutaneously, and chlorphenvinphos, bromphenvinphos, methylbromphenvinphos and phospholine subcutaneously in doses amounting to 5% of LD50. After the 40th dose the animals were weighed and killed, blood and organs were taken for investigations. The activity of AChE was determined in the erythrocytes, brain, tibialis muscle and liver. In the serum the levels of transaminases (AspAT, AlAT), alkaline phosphatase, cholesterol and phospholipids were determined. Histomorphological and histochemical investigations included the liver, kidneys, myocardium, femoral muscle, diaphragm, hypothalamus, gastric wall and abdominal sympathetic ganglia. A statistically significant fall of body weight was observed in all intoxicated animals as compared with controls. The mean AChE activity was about 50% of the activity in the control group. A decrease was found also in the cholesterol-phospholipid ratio in the intoxicated animals. Degenerative changes in the parenchymal organs developed independently on the route of poison administration and they included particularly the liver (atrophy of parenchyma) and myocardium (hyperaemia, extravasations, early necrotic changes of muscle fibres, nuclear pyknosis). Necrosis and oedema were observed also in the neurons of sympathetic ganglia. The greatest intensity of the lesions was found after intoxication with the agents which were most potent AChE inhibitors (phospholine and chlorphenvinphos).

[Effects of trimethadione on the therapeutic efficiency of atropine and obidoxime in mice poisoned with various phosphoorganic compounds]. / Wplyw trimetadionu na skutecznosc lecznicza atropiny i obidoksymu w zatruciach myszy niektórymi zwiazkami fosforoorganicznymi.

This paper was aimed at the observation of trimethadione therapeutic effects while this preparation was used as an agent supplementing the standard therapeutic mixture (cholinolytic and reactivator) for mice poisoning with phospholine--a compound of AO 217 and DEP. The studies covered 216 female mice of Balb C strain, body weight 18-24 g. After the determination of toxic doses of phosphoroorganic preparations administered subcutaneously, therapeutic effects of a mixture of atropine sulphate (10 mg/kg i.p.) and obidoxime (40 mg/kg i.p.) administered immediately after poisoning, were observed. Then trimethadione 300 mg/kg i.p. was of in a dose of 300 mg/kg i.p was added and the effects of that agent on the efficiency of basic medicines were observed. The studies were performed during 2 and 24 hr of observation. The observations prove that the efficiency of the basic therapeutic mixture during 24 hrs of observation of mice poisonings with AO 217 and DFP was respectively: 2.31 x and 2.64 x. In phospholine poisonings no increase in therapeutic effects were found.

Acetylcholinesterase activity in several rat liver cell fractions after repeated poisoning with some organophosphates.

The reported investigations were carried out on female Wistar rats aged about 6 weeks, weighing 150 g. During 28 days the fats were given daily organophosphates (DFP, DDVP, Chlorfenvinphos, IPO-62, IPO-63, Phospholine) in a dose of 1/10 LD50 subcutaneously. The observations were conducted on groups of 8 rats. The body weight of the animals was noted on the 10th, 20th and 28th day of the experiment. On the 28th day the rats were killed and the activity of acetylcholinesterase (AChE) was determined by the biochemical method of Hestrin in liver homogenate and in the microsomal, mitochondrial and soluble fractions of liver cells. After 28 days of the experiment the body weight of the rats poisoned with these substances amounted to from 80.8% (DFP) to 90.7% (IPO-63) of that in control animals. The AChE activity was also reduced in relation to the control group ranging in the liver homogenate from 49.7% (DFP) to 75.6% (IPO-63), in the microsomal fraction from 33.0% (DFP) to 63.8% (IPO-63), in the mitochondrial fraction from 45.5% (DFP) to 72.9% (IPO-63), and in the soluble fraction from 52.8% (DFP) to 80.5% (DDVP).

Effect of preceding physostigmine administration on neuromuscular transmission disturbances in rats caused by dichlorphos and phospholine.

The reported experiments were carried out on male Wistar rats. Under general anaesthesia with chloral hydrate in situ physiological preparations were made consisting of sciatic nerve and anterior tibialis muscle. Physostigmine was injected subcutaneously and was followed after from 15 to 60 minutes by intravenous injection of DDVP or phospholine. Physostigmine effect on the blockade of tetanic muscular contractions was studied after administration of these organophosphate inhibitors of acetylcholinesterase. At the same time, the effect of these substances on acetylcholinesterase was determined in the skeletal muscle. It was found that physostigmine in a dose of 125 microgram/kg protected the tibialis muscle against the development of blockade of tetanic response. The protective effect was greatest when the time between physostogmine injection and the subsequent administration of these organophosphate inhibitors was 30--40 minutes. In the same observation period the activity of AChE in the tibialis muscle of rats was found to be highest after physostigmine injection before administration of DDVP or phospholine.

Antiesterase activity of chlorphenvinphos and phospholine in certain rat tissues after administration by different routes.

The reported investigations were carried out on female Wistar rats receiving chlorphenvinphos and phospholine in doses equalling 3/4 LD50 onto the skin and intragastrically. the levels of acetylcholinesterase (AChE) were determined in the erythrocytes, liver and tibialis muscle and in three brain areas: pontomedullary area, hypothalamus and cerebral cortex. The activity of the enzyme was determined 15 and 30 minutes, and 3, 24 and 72 hours after poisoning. A high degree of AChE inhibition was observed in the brain after chlorphenvinphos poisoning (activity fall to 15--30%) and a slight decrease of AChE activity after administration of phospholine independently of the route. After oral poisoning with both preparations the AChE activity level in the erythrocytes was from about 27% to 31%, in the liver from 18 to 23%, and in the skeletal muscle from 51 to 54%, while with the transcutaneous route of poisoning these values were: 18--23%, 41--52%, and 36--48% respectively. A high degree of enzyme restoration was observed during 72 hours of observation. AChE activity was then 60--80% of the initial value.

Protective effect of some pyridinium salts on acetylcholinesterase against organophosphate inhibition.

The protective effect of ten new synthetized mono- and bis-pyridinium salts on acetylcholinesterase (AChE) against DFP, Chlorphenvinphos, DDVP, IPO 62 and IPO 63 inhibition in vitro and in mice i.p. was studied. Moreover, abolition of tetanic block of the tibialis anterior muscle in rats after intoxication with DDVP by these salts was tested. An increase of the therapeutic effect of atropine after DFP poisoning in mice and a rise of LD50 values from 1.4 (PAN-W-15) to 5.1 times (PAN-W-14) was noted. The effect of PAN-W-14 and PAN-W-18 after inhibition by other organophosphates was found to be highest in human erythrocytes in vitro. In addition, prophylactic administration of PAN-W-14 prevented the tetanic block of the muscle in situ after DDVP intoxication.

Histopathology, histochemistry and acetylcholinesterase activity after repetitive administration of fluostigmine to albino rat.

Histopathological, histochemical and biochemical investigations were performed on the brain, sciatic nerve, skeletal muscle, heart, liver and kidney of rats which were given 5% of LD50 dose of DFP for 10 days. A decrease in AChE activity, degeneration of neurons and necrotic changes in the nuclei of hypothalamus, degeneration of myelin sheaths in sciatic nerve, a decrease in succinic dehydrogenase activity in the myocardium, and a minimal decrease of acid phosphatase activity (AcPh) in the liver were found. The biochemical determination of AChE level indicated about 30% AChE activity in erythrocytes and tibialis muscle, and 40% in the brain 1 hr after the last dose of the inhibitor and 80% and 50% respectively on the 7th day after poisoning in relation to normal values.