Quality assurance checklist and additional considerations for canine clinical genetic testing laboratories: a follow-up to the published standards and guidelines.

There is currently no oversight for canine clinical genetic testing laboratories. We published an initial set of standards and guidelines with the goal of providing a basis for which canine testing laboratories could evaluate their quality assurance programs. To further those standards and guidelines, we have developed a checklist that can be used as a self-evaluation to identify gaps in their programs for continual quality improvement over time. Because there is currently no organization willing to oversee an external proficiency program, the checklist provides the first step toward an internal, self-assessment that can be used periodically to monitor improvements. In addition, we attempt to address concerns from the canine community regarding rare or private mutations, genetic screening using array-based technologies, non-peer reviewed tests that are being offered, and the clinical validity of certain mutations in particular breeds. Through coordination, conversation and hard work, the canine genetic testing community can strive to organize to improve testing and to provide more transparency to consumers and better outcomes for dogs.

Standards and guidelines for canine clinical genetic testing laboratories.

This publication represents a proposed approach to quality standards and guidelines for canine clinical genetic testing laboratories. Currently, there are no guidelines for laboratories performing clinical testing on dogs. Thus, there is no consensus set of protocols that set the minimal standards of quality among these laboratories, potentially causing variable results between laboratories, inconsistencies in reporting, and the inability to share information that could impact testing among organizations. A minimal standard for quality in testing is needed as breeders use the information from genetic testing to make breeding choices and irreversible decisions regarding spay, neuter or euthanasia. Incorrect results can have significant impact on the health of the dogs being tested and on their subsequent progeny. Because of the potentially serious consequences of an incorrect result or incorrect interpretation, results should be reviewed by and reported by individuals who meet a minimum standard of qualifications. Quality guidelines for canine genetic testing laboratories should include not only the analytical phase, but also the preanalytical and postanalytical phases, as this document attempts to address.

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae.

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE-containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.

The effect of translationally controlled tumour protein (TCTP) on programmed cell death in plants.

BACKGROUND: Translationally controlled tumour protein (TCTP), a well known protein of the animal kingdom, was shown to be a Ca(2+)-binding protein with important functions in many different cellular processes (e.g. protection against stress and apoptosis, cell growth, cell cycle progression, and microtubule organization). However, only little is known about TCTP in plants. Transcript and protein levels of plant TCTPs were shown to be altered by various stress conditions (e.g. cold, salt, draught, aluminium, and pathogen infection), and Arabidopsis thaliana TCTP (AtTCTP) was described as an important regulator of growth. The aim of this study was to further characterize plant TCTP relating to one of its major functions in animals: the protection against cell death. RESULTS: We used two different activators of programmed cell death (PCD) in plants: the mammalian pro-apoptotic protein BAX and tunicamycin, an inhibitor of glycosylation and trigger of unfolded protein response (UPR). Over-expression of AtTCTP significantly decreased cell death in tobacco leaf discs in both studies. A (45)Ca overlay assay showed AtTCTP to be a Ca(2+)-binding protein and localization experiments revealed cytosolic distribution of AtTCTP-GFP in Arabidopsis seedlings. CONCLUSIONS: Our study showed cytoprotective effects of plant TCTP for the first time. Furthermore, we showed the ability of AtTCTP to bind to Ca(2+) and its cytosolic distribution within the cell. If these results are combined, two putative modes of action can be assumed: 1) AtTCTP acts as Ca(2+) sequester, preventing PCD by reducing cytosolic Ca(2+) levels as described for animals. 2) AtTCTP could directly or indirectly interact with other cytosolic or membrane-bound proteins of the cell death machinery, thereby inhibiting cell death progression. As no homologous proteins of the anti-apoptotic machinery of animals were found in plants, and functional homologues still remain to be elucidated, future work will provide more insight.

Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae.

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4 kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.

vWDI is inherited in an autosomal dominant manner with incomplete penetrance, in the Kromfohrländer breed.

BACKGROUND: Von Willebrand disorder type I (vWDI) is known as an inherited bleeding disorder in different dog breeds following an autosomal recessive inheritance. The Kromfohrländer is a rare dog breed with an increased incidence of unclear bleeding episodes and prolonged coagulation time during/after surgery or injuries, indicating a defect in one or more critical proteins of the coagulation cascade. OBJECTIVE: The objective of this study was to determine whether the c.7437G > A mutation in the VWF gene previously shown to cause von Willebrand disorder type I in Doberman Pinscher is also linked to this disease in the Kromfohrländer breed and to serum concentrations of vWF. Furthermore, establish a possible link between bleeding phenotype, vWF serum concentrations and VWF mutation status. RESULTS: Eighty-seven Kromfohrländer were genotyped for the G > A von Willebrand type I mutation. For detection of the associated mutation we used an endpoint genotyping method. We identified the G > A von Willebrand type I mutation in 80.5% of our study population. 65.5% were heterozygous (WT/MUT) and 15.0% were homozygous for the mutation (MUT/MUT). 21% of the overall study population exhibited bleeding symptoms. 45.5% of all homozygous dogs (MUT/MUT) showed bleeding symptoms. In contrast, wild-type homozygotes exhibited no bleeding symptoms, whereas 23.2% of the heterozygotes did. VWF serum concentrations varied from 28 to 137% in wild-type dogs while in heterozygous and homozygous dogs the concentration ranged from 3 to 77% and 1 to 23%, respectively (p < 0.05). CONCLUSION: Based on our data, we found the G > A mutation in the VWF gene in the Kromfohrländer breed and the subsequent vWDI as the underlying cause for the bleeding episodes and delayed coagulation in heterozygous and homozygous dogs. Since both, heterozygotes and homozygotes show reduced vWF serum concentrations and exhibit to a certain percentage the vWD syndrome phenotype, we postulate that, in contrast to most other vWDI affected breeds, inheritance follows an autosomal dominant mode with incomplete penetrance.