Analysis of mRNA transcripts improves the success rate of molecular genetic testing in OTC deficiency

BACKGROUND: Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea metabolism that can lead to hyperammonemic crises and orotic aciduria. To date, a total of 341 causative mutations within the OTC gene have been described. However, in about 20% of the patients with enzymatically confirmed OTC deficiency no mutation can be detected when sequencing of genomic DNA analyzing exons and adjacent intronic segments of the OTC gene is performed. METHODS: Standard genomic DNA analysis of the OTC gene in five consecutive patients from five families revealed no mutation. Hence, liver tissue was obtained by needle sampling or open biopsy and RNA extracted from liver was analyzed. RESULTS: Complex rearrangements of the OTC transcript (three insertions and two deletions) were found in all five patients. CONCLUSION: In patients with a strong suspicion of OTC deficiency despite normal results of sequencing exonic regions of the OTC gene, characterization of liver OTC mRNA is highly effective in resolving the genotype. Liver tissue sampling by needle aspiration allows for both enzymatic analysis and RNA based diagnostics of OTC deficiency.

INTEGRATED ASSESSMENT OF ANTHROPOGENIC, CLIMATE, AND POLICY INDUCED CHANGES OF PHOSPHORUS EXPORT IN THE UNITED STATES LAURENTIAN GREAT LAKES WATERSHEDS

Anthropogenic activities have increased phosphorus (P) loading in tributaries to the Laurentian Great Lakes resulting in eutrophication in small bays to most notably, Lake Erie. Changes to surface water quality from P loading have resulted in billions of dollars in damage and threaten the health of the world’s largest freshwater resource. To understand the factors affecting P delivery with projected increasing urban lands and biofuels expansion, two spatially explicit models were coupled. The coupled models predict that the majority of the basin will experience a significant increase in urban area P sources while the agriculture intensity and forest sources of P will decrease. Changes in P loading across the basin will be highly variable spatially. Additionally, the impacts of climate change on high precipitation events across the Great Lakes were examined. Using historical regression relationships on phosphorus concentrations, key Great Lakes tributaries were found to have future changes including decreasing total loads and increases to high-flow loading events. The urbanized Cuyahoga watersheds exhibits the most vulnerability to these climate-induced changes with increases in total loading and storm loading , while the forested Au Sable watershed exhibits greater resilience. Finally, the monitoring network currently in place for sampling the amount of phosphorus entering the U.S. Great Lakes was examined with a focus on the challenges to monitoring. Based on these interviews, the research identified three issues that policy makers interested in maintaining an effective phosphorus monitoring network in the Great Lakes should consider: first, that the policy objectives driving different monitoring programs vary, which results in different patterns of sampling design and frequency; second, that these differences complicate efforts to encourage collaboration; and third, that methods of funding sampling programs vary from agency to agency, further complicating efforts to generate sufficient long-term data to improve our understanding of phosphorus into the Great Lakes. The dissertation combines these three areas of research to present the potential future impacts of P loading in the Great Lakes as anthropogenic activities, climate and monitoring changes. These manuscripts report new experimental data for future sources, loading and climate impacts on phosphorus.

Localization of DJ-1 mRNA in the mouse brain

DJ-1 is mutated in autosomal recessive, early onset Parkinson's disease but the exact localization of the DJ-1 gene product in the mammalian brain is largely unknown. We aimed to evaluate the DJ-1 mRNA expression pattern in the mouse brain. Serial coronal sections of brains of five male and five female adult mice were investigated by using in situ hybridization with a DJ-1 specific 35S-labeled oligonucleotide probe. Hybridized sections were analyzed after exposure to autoradiography films and after coating with a photographic emulsion. DJ-1 was heterogeneously expressed throughout the mouse central nervous system. A high expression of DJ-1 mRNA was detected in neuronal and non-neuronal populations of several structures of the motor system such as the substantia nigra, the red nucleus, the caudate putamen, the globus pallidus, and the deep nuclei of the cerebellum. Furthermore, DJ-1 mRNA was also highly expressed in non-motor structures including the hippocampus, the olfactory bulb, the reticular nucleus of the thalamus, and the piriform cortex. The high expression of DJ-1 mRNA in brain regions involved in motor control is compatible with the occurrence of parkinsonian symptoms after DJ-1 mutations. However, expression in other regions indicates that a dysfunction of DJ-1 may contribute to additional clinical features in patients with a DJ-1 mutation.

Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

Expression of atrophy mRNA relates to tendon tear size in supraspinatus muscle

Skeletal muscle atrophy and fatty infiltration develop after tendon tearing. The extent of atrophy serves as one prognostic factor for the outcome of surgical repair of rotator cuff tendon tears. We asked whether mRNA of genes involved in regulation of degradative processes leading to muscle atrophy, ie, FOXOs, MSTN, calpains, cathepsins, and transcripts of the ubiquitin-proteasome pathway, are overexpressed in the supraspinatus muscle in patients with and without rotator cuff tears. We evaluated biopsy specimens collected during surgery of 53 consecutive patients with different sizes of rotator cuff tendon tears and six without tears. The levels of corresponding gene transcripts in total RNA extracts were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Supraspinatus muscle atrophy was assessed by MRI. The area of muscle tissue (or atrophy), decreased (increased) with increasing tendon tear size. The transcripts of CAPN1, UBE2B, and UBE3A were upregulated more than twofold in massive rotator cuff tears as opposed to smaller tears or patients without tears. These atrophy gene products may be involved in cellular processes that impair functional recovery of affected muscles after surgical rotator cuff repair. However, the damaging effects of gene products in their respective proteolytic processes on muscle structures and proteins remains to be investigated.

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.