Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA sequencing applied to retinitis pigmentosa

BACKGROUND:
The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa.
METHODS:
A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.
RESULTS AND CONCLUSIONS:
Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A?T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Respiratory viral infection in lower airways of asymptomatic children.

Aim: The aim of this study was to determine if asthmatic children have viruses more commonly detected in lower airways during asymptomatic periods than normal children. Methods: Fifty-five asymptomatic children attending elective surgical procedures (14 with stable asthma, 41 normal controls) underwent non-bronchoscopic bronchoalveolar lavage. Differential cell count and PCR for 13 common viruses were performed. Results: Nineteen (35%) children were positive for at least one virus, with adenovirus being most common. No differences in the proportion of viruses detected were seen between asthmatic and normal ‘control’ children. Viruses other than adenovirus were associated with higher neutrophil counts, suggesting that they caused an inflammatory response in both asthmatics and controls (median BAL neutrophil count, 6.9% for virus detected vs. 1.5% for virus not detected, p = 0.03). Conclusions: Over one-third of asymptomatic children have a detectable virus (most commonly adenovirus) in the lower airway; however, this was not more common in asthmatics. Viruses other than adenovirus were associated with elevated neutrophils suggesting that viral infection can be present during relatively asymptomatic periods in asthmatic children.

Decreased prevalence of atopic diseases in children with diabetes

Objective: To test the hypothesis that atopic diseases in early life are associated with a reduced risk (protection) for the development of type 1 diabetes in childhood.

Respiratory syncytial virus NS1 protein degrades STAT2 by using the elongin-cullin E3 ligase

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.

The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein v.,as isolated from [S-35]methionine- and [P-33]orthophosphate-labelled IRSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role or tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580. an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependant tyrosine kinase activity and that this modification influences its cellular distribution.

Inflammation markers are associated with Cardiovascular diseases risk in adolescents The Young Hearts Project 2000

Purose: The traditional approach for identifying subjects at risk from cardiovascular diseases (CVD) is to determine the extent of clustering of biological risk factors adjusted for lifestyle. Recently, markers of endothelial dysfunction and low grade inflammation, including high sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecules (sICAM), and soluble vascular adhesion molecules (sVCAM), have been included in the detection for high risk individuals. However, the relationship of these novel biomarkers with CVD risk in adolescents remains unclear. The purpose of this study, therefore, was to establish the association of hsCRP, sICAM, and sVCAM with CVD risk in an adolescent population.
Methods: Data from the Young Hearts 2000 cross-sectional cohort study, carried out in 1999-2001, were used. From a total of 2,017 male and female participants, 95 obese subjects were identified and matched according to age, sex, and cigarette smoking, with 95 overweight and 95 normal-weight adolescents. Clustered CVD risk was computed using a sum of Z-scores of biological risk factors. The relationship was described using multiple linear regression analyses.
Results: hsCRP, sICAM, and sVCAM showed significant associations with CVD risk. hsCRP and sICAM had a positive relation with CVD risk, whereas sVCAM showed an inverse relationship. In this study, lifestyle factors showed no relation with CVD risk.
Conclusion: The results fit the hypothesized role of low grade inflammation and endothelial dysfunction in CVD risk in asymptomatic adolescents. The inverse relationship of VCAM, however, is hard to explain and indicates the complex mechanisms underlying CVD. Further research is needed to draw firm conclusions on the biomarkers used.

Supplemental oxygen and hyperbaric treatment at high altitude: Cardiac and respiratory response

Introduction: The most effective treatment for high altitude sickness is prompt descent. However, rapid descent is sometimes impossible and alternative solutions are desirable. Supplemental oxygen at ambient pressure and hyperbaric oxygen in a hyperbaric tent have both been demonstrated to improve symptoms and increase arterial oxygenation (SaO(2)) in those with high altitude sickness; however, their use in combination has not previously been described in a controlled study. Methods and Results: In this feasibility study, the SaO(2) of six healthy, well-acclimatized participants rose from 76.5 to 97.5% at 4900 m and 72.5 to 96.0% at 5700 m following the administration of oxygen via a nasal demand circuit (33 ml of oxygen per pulse) inside a hyperbaric tent (107 mmHg above ambient barometric pressure) (p

Unravelling the genetic basis of renal diseases; from single gene to multifactorial disorders

Chronic kidney disease is common with up to 5% of the adult population reported to have an estimated glomerular filtration rate of