Pitfalls in the molecular genetic diagnosis of Leber hereditary optic neuropathy (LHON).

Pathogenetic mutations in mtDNA are found in the majority of patients with Leber hereditary optic neuropathy (LHON), and molecular genetic techniques to detect them are important for the diagnosis. A false-positive molecular genetic error has adverse consequences for the diagnosis of this maternally inherited disease. We found a number of mtDNA polymorphisms that occur adjacent to known LHON-associated mutations and that confound their molecular genetic detection. These transition mutations occur at mtDNA nt 11779 (SfaNI site loss, 11778 mutation), nt 3459 (BsaHI site loss, 3460 mutation), nt 15258 (AccI site loss, 15257 mutation), nt 14485 (mismatch primer Sau3AI site loss, 14484 mutation), and nt 13707 (BstNI site loss, 13708 mutation). Molecular genetic detection of the most common pathogenetic mtDNA mutations in LHON, using a single restriction enzyme, may be confounded by adjacent polymorphisms that occur with a false-positive rate of 2%-7%.     

Refined mapping of the gene for thiamine-responsive megaloblastic anemia syndrome and evidence for genetic homogeneity

Thiamine-responsive megaloblastic anemia (TRMA, also known as Rogers syndrome, OMIM 249270) is a rare autosomal recessive disorder characterized by a triad of megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Patients respond, to varying degrees, to treatment with megadoses of thiamine. We have recently shown genetic linkage of the TRMA gene to a 16-centimorgan (cM) region on 1q23.2–1q23.3 based on the analysis of four large, inbred families of Alaskan, Italian, and Israeli-Arab origin. Here we narrow the TRMA interval down to 4 cM based on genetic recombination, homozygosity mapping, and linkage disequilibrium (highest LOD score of 12.5 at D1S2799, at a recombination fraction of 0). We provide further evidence that the TRMA gene is located in this region and confirm the homogeneity of the disease. In this analysis, we genotyped seven additional families of diverse ethnic origin (Pakistani, Indian, Italian, Brazilian, and Japanese), and analyzed additional markers in two previously reported families showing evidence of linkage disequilibrium in a large area of their haplotypes. The multi-system manifestations of TRMA suggest that thiamine has a pivotal role in a multiplicity of physiological processes. Mapping the TRMA gene and understanding the molecular basis of the disease might, thus, shed light on the role of thiamine in common disorders such as deafness, anemia, and diabetes. Received: 16 April 1998 / Accepted: 6 July 1998     

Electrochemical characteristics of ion secretion in malpighian tubules of the New Zealand alpine weta (Hemideina maori)

Characteristics of ion and fluid secretion were investigated in isolated Malpighian tubules of the New Zealand Alpine Weta (Hemideina maori). Fluid secretion by tubules in iso-osmotic saline (500mOsm) occurred at a rate of 15+/-3nlh(-1) and was enriched in K(+) (approx. 125mmoll(-1)) relative to the saline (10mmoll(-1)). Maximal fluid secretion (112nlh(-1)) during simultaneous exposure to hypo-osmolality and dibutyryl cAMP resulted in an 8.8x increase in the quantity of K(+) secreted, compared to only a 2.4x increase in Na(+) secretion. Measurements of intracellular ion activities and membrane potentials indicated that Na(+) and K(+) were transported against a strong electrochemical gradient across the apical surface, regardless of saline osmolality. On the basolateral surface, there was a large driving force for Na(+) entry, while K(+) was distributed near its equilibrium potential. Neither bumetanide nor ouabain in the bathing saline had a significant effect on fluid secretion, but Ba(2+) and amiloride decreased fluid secretion by 79 and 57%, respectively. The effect of Ba(2+) on fluid secretion was consistent with a high basolateral permeability to K(+), relative to Na(+) and Cl(-). These results indicate that the characteristics of fluid secretion in this primitive insect are largely conserved with characteristics reported for other insects.     

Localization of the Gene for Thiamine-Responsive Megaloblastic Anemia Syndrome, on the Long Arm of Chromosome 1, by Homozygosity Mapping

Thiamine-responsive megaloblastic anemia, also known as "TRMA" or "Rogers syndrome," is an early-onset autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, and sensorineural deafness, responding in varying degrees to thiamine treatment. On the basis of a linkage analysis of affected families of Alaskan and of Italian origin, we found, using homozygosity mapping, that the TRMA-syndrome gene maps to a region on chromosome 1q23.2-23.3 (maximum LOD score of 3.7 for D1S1679). By use of additional consanguineous kindreds of Israeli-Arab origin, the putative disease-gene interval also has been confirmed and narrowed, suggesting genetic homogeneity. Linkage analysis generated the highest combined LOD-score value, 8.1 at a recombination fraction of 0, with marker D1S2799. Haplotype analysis and recombination events narrowed the TRMA locus to a 16-cM region between markers D1S194 and D1S2786. Several heterozygote parents had diabetes mellitus, deafness, or megaloblastic anemia, which raised the possibility that mutations at this locus predispose carriers in general to these manifestations. Characterization of the metabolic defect of TRMA may shed light on the role of thiamine deficiency in such common diseases.     

Induction of aquaporin-1 mRNA following cardiopulmonary bypass and reperfusion.

BACKGROUND: Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are important components of congenital cardiac surgery. Ischemia/reperfusion injury and inflammatory cascade activation result in endothelial damage and vascular leak which are clinically manifested as pulmonary edema and low cardiac output postoperatively. Newborns are particularly susceptible. Subtraction cloning is a useful method of isolating induced genes and can be applied to CPB/HCA. MATERIALS AND METHODS: We used a newborn lamb model replicating infant CPB with HCA to obtain tissues during various periods of reperfusion. We utilized subtraction cloning to identify mRNA induced in lung following CPB/HCA and reperfusion. Ribonuclease protection was used to quantify mRNA levels. RESULTS: We isolated a cDNA encoding ovine aquaporin-1 in a subtracted cDNA screen comparing control lung with lung exposed to CPB/HCA and reperfusion. Aquaporin-1 mRNA levels increased 3-fold in lung (p = .006) exposed to CPB/HCA and 6 hr of reperfusion. No induction was observed immediately following bypass or after 3 hr of reperfusion. We found no significant induction of aquaporin-1 mRNA following bypass, arrest, and reperfusion in other tissues surveyed, including ventricle, atrium, skeletal muscle, kidney, brain, and liver. CONCLUSIONS: Our finding that aquaporin-1 mRNA is reproducibly induced in lung following CPB/HCA with 6 hr of reperfusion suggests an important role for the water channel in the setting of pulmonary edema. Induction of Aquaporin-1 is late compared with other inflammatory mediators (ICAM-1, E-selectin, IL-8). Further studies are needed to determine if aquaporin-1 contributes to the disease process or if it is part of the recovery phase.     

Structure and development of ‘witches' broom’ galls in reproductive organs of Byrsonima sericea (Malpighiaceae) and their effects on host plants

Galls are anomalies in plant development of parasitic origin that affect the cellular differentiation or growth and represent a remarkable plant–parasite interaction. Byrsonima sericea DC. (Malpighiaceae) is a super host of several different types of gall in both vegetative and reproductive organs. The existence of galls in reproductive organs and their effects on the host plant are seldom described in the literature. In this paper, we present a novel study of galls in plants of the Neotropical region: the ‘witches' broom’ galls developed in floral structures of B. sericea. The unaffected inflorescences are characterised by a single indeterminate main axis with spirally arranged flower buds. The flower buds developed five unaffected brownish hairy sepals and five pairs of elliptical yellow elaiophores, five yellow fringed petals, 10 stamens and a pistil with superior tricarpellar and trilocular ovary. The affected inflorescences showed changes in architecture, with branches arising from the main axis and flower buds. The flower buds exhibited several morphological and anatomical changes. The sepals, petals and carpels converted into leaf‐like structures after differentiation. Stamens exhibited degeneration of the sporogenous tissue and structures containing hyphae and spores. The gynoecium did not develop, forming a central meristematic region, from which emerges the new inflorescence. In this work, we discuss the several changes in development of reproductive structures caused by witches' broom galls and their effects on reproductive success of the host plants.