Characterization of a novel founder MSH6 mutation causing Lynch syndrome in the French Canadian population.

We identified an MSH6 mutation (c.10C>T, p.Gln4*) causing Lynch syndrome (LS) in 11 French Canadian (FC) families from the Canadian province of Quebec. We aimed to investigate the molecular and clinical implications of this mutation among FC carriers and to assess its putative founder origin. We studied 11 probands and 27 family members. Additionally 6433 newborns, 187 colorectal cancer (CRC) cases, 381 endometrial cancer (EC) cases and 179 additional controls, all of them from Quebec, were used. Found in approximately 1 of 400 newborns, the mutation is one of the most common LS mutations described. We have found that this mutation confers a greater risk for EC than for CRC, both in the 11 studied families and in the unselected cases: EC [odds ratio (OR) = 7.5, p < 0.0001] and CRC (OR = 2.2, p = 0.46). Haplotype analyses showed that the mutation arose in a common ancestor, probably around 430-656 years ago, coinciding with the arrival of the first French settlers. Application of the results of this study could significantly improve the molecular testing and clinical management of LS families in Quebec.

Expanded clinical and molecular spectrum of guanidinoacetate methyltransferase (GAMT) deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine biosynthesis, characterized by excessive amounts of guanidinoacetate in body fluids, deficiency of creatine in the brain, and presence of mutations in the GAMT gene. We present here 8 new patients with GAMT deficiency along with their clinical, biochemical and molecular data. The age at diagnosis of our patients ranges from 0 to 14 years. The age of onset of seizures usually ranges from infancy to 3 years. However, one of our patients developed seizures at age 5; progressing to myoclonic epilepsy at age 8 years and another patient has not developed seizures at age 17 years. Five novel mutations were identified: c.37ins26 (p.G13PfsX38), c.403G>T (p.D135Y), c.507_521dup15 (p.C169_S173dup), c.402C>G (p.Y134X) and c.610_611delAGinsGAA (p.R204EfsX63). Six patients had the c.327G>A (last nucleotide of exon 2) splice-site mutation which suggests that this is one of the most common mutations in the GAMT gene, second only to the known Portuguese founder mutation, c.59G>C (p.W20S). Our data suggests that the clinical presentation can be variable and the diagnosis may be overlooked due to unawareness of this disorder. Therefore, GAMT deficiency should be considered in the differential diagnosis of progressive myoclonic epilepsy as well as in unexplained developmental delay or regression with dystonia, even if the patient has no history of seizures. As more patients are reported, the prevalence of GAMT deficiency will become known and guidelines for prenatal diagnosis, newborn screening, presymptomatic testing and treatment, will need to be formulated.

Phenotypic variability among patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome homozygous for the delF188 mutation in SLC25A15.

BACKGROUND: Hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (OMIM 238970) is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15. To date, 22 different mutations of the SLC25A15 gene have been described in 49 patients belonging to 31 unrelated families. OBJECTIVE: To further delineate the phenotypic spectrum of HHH syndrome from a description of a genetically homogeneous cohort of patients and identify prognostic factors based on long-term follow-up. METHODS: Sixteen French-Canadian patients were retrospectively and prospectively clinically assessed. RESULTS: Owing to a founder effect, 15 of the 16 patients were homozygous for the F188del mutation in the SLC25A15 gene. The main clinical features at presentation were liver dysfunction (6/16) and neurological disease (9/16), including chronic neurological symptoms (6/9) and acute encephalopathy (3/9). Hyperammonaemia was not constant and usually mild and uncommon after start of treatment. Long-term follow-up showed that variable intellectual impairment and lower limb spasticity often occur, together or separately, with no obvious relationship to age at diagnosis and compliance with treatment. CONCLUSION: We report the largest known cohort to date of patients with HHH syndrome. A similar range of severity occurred in the clinical course and outcome of patients homozygous for delF188 and in the 33 other reported patients compiled from the literature. The poor clinical outcome of some patients with HHH syndrome despite early treatment and repeatedly normal plasma ammonia levels emphasises the need to better understand the pathophysiology and to reconsider the therapeutic goals for HHH.

Pyruvate dehydrogenase deficiency presenting as intermittent isolated acute ataxia.

OBJECTIVE: The aim of this study is to report and emphasize unusual presentations of pyruvate dehydrogenase (PDH) deficiency (OMIM 312170). METHODS: PDH activity and PDHA1 gene were studied in two siblings presenting with intermittent ataxia in childhood. Similar presentations in reported PDH-deficient patients were searched for using the Medline database. RESULTS: Both patients had PDH deficiency caused by a new mutation (G585C) in the PDHA1 gene, which is predicted to replace a highly conserved glycine at codon 195 by alanine. Although this mutation lies within the thiamine pyrophosphate binding domain, there was no thiamine responsiveness IN VIVO. The patients presented recurrent episodes of acute isolated ataxia in infancy. Both had normal blood and CSF lactate levels. Although symptoms initially resolved between episodes during the first decade, both patients subsequently worsened and developed progressive and severe encephalopathy, leading to death in their twenties. The spectrum of intermittent presentations in PDH deficiency includes episodic ataxia, intermittent peripheral weakness, recurrent dystonia and extrapyramidal movement disorders. CONCLUSIONS: PDH deficiency should be considered in patients with unexplained intermittent and recurrent acute neurological symptoms. Long-term prognosis and outcome remain uncertain. PDH deficiency can occur even with normal CSF lactate concentration.

Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae from wetlands on the Savannah River Site, South Carolina.

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes.

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.

Receptor-mediated endocytosis in kidney proximal tubules: recent advances and hypothesis.

Preparation of kidney proximal tubules in suspension allows the study of receptor-mediated endocytosis, protein reabsorption, and traffic of endosomal vesicles. The study of tubular protein transport in vitro coupled with that of the function of endosomal preparation offers a unique opportunity to investigate a receptor-mediated endocytosis pathway under physiological and pathological conditions. We assume that receptor-mediated endocytosis of albumin in kidney proximal tubules in situ and in vitro can be regulated, on the one hand, by the components of the acidification machinery (V-type H+-ATPase, Cl(-)-channel and Na+/H+-exchanger), giving rise to formation and dissipation of a proton gradient in endosomal vesicles, and, on the other hand, by small GTPases of the ADP-ribosylation factor (Arf)-family. In this paper we thus analyze the recent advances of the studies of cellular and molecular mechanisms underlying the identification, localization, and function of the acidification machinery (V-type H+-ATPase, Cl(-)-channel) as well as Arf-family small GTPases and phospholipase D in the endocytotic pathway of kidney proximal tubules. Also, we explore the possible functional interaction between the acidification machinery and Arf-family small GTPases. Finally, we propose the hypothesis of the regulation of translocation of Arf-family small GTPases by an endosomal acidification process and its role during receptor-mediated endocytosis in kidney proximal tubules. The results of this study will not only enhance our understanding of the receptor-mediated endocytosis pathway in kidney proximal tubules under physiological conditions but will also have important implications with respect to the functional consequences under some pathological circumstances. Furthermore, it may suggest novel targets and approaches in the prevention and treatment of various diseases (cystic fibrosis, Dent's disease, diabetes and autosomal dominant polycystic kidney disease).

The role of the renin-angiotensin system in normotensive and hypertensive rats with varying renin status.

We have investigated the relationship between the acute blood pressure lowering effect of captopril and renin status. Differences in renin status were induced by unilateral artery clipping combined with unilateral or bilateral nephrectomy in rats. The blood pressure lowering effect of captopril correlated very closely with plasma or aortic renin across a very wide range of renin levels.

Antibodies to beta-bungarotoxin and its phospholipase inactive derivative.

Antisera were raised against the presynaptic neurotoxin beta-bungarotoxin and against its phospholipase-inactive derivative, modified by reaction with p-bromophenacyl bromide. The cross-reactivity of the antisera to other phospholipase A2 enzymes and polypeptide neurotoxins was examined. The antisera inhibited both the neurotoxic effects of beta-bungarotoxin at the frog motor endplate and the enzymatic activity of the toxin on model phospholipid membranes, although it is unlikely that the catalytic active centre is the locus of any major determinant.

A further study of the phospholipase-independent action of beta-bungarotoxin at frog end-plates.

1. The effect of beta-bungarotoxin (beta-BuTx) at the frog neuromuscular junction has been investigated further in order to distinguish more clearly between phospholipase- independent and phospholipase-dependent actions on transmitter release. 2. Inhibition of the enzymatic activity, by substitution of strontium for calcium, allowed determination of the dose-response curve of the early rapid decrease in transmitter release caused by the toxin. In the presence of strontium ions there was, however, still about 7% residual enzymatic activity, and electrophysiological evidence of it could be seen in room-temperature experiments at high concentrations of beta-BuTx. This residual enzymatic activity could be suppressed by lowering the temperature to 5 degrees C. 3. In normal calcium-Ringer solution beta-BuTx produced the typical triphasic effect on the amplitude of end-plate potentials (e.p.p.s). Lowering the temperature markedly delayed an then diminished the secondary transient increase. There was, however, comparatively little temperature influence on the first rapid decrease in e.p.p. amplitude. Enzymatic assays confirmed the temperature dependence of the toxin's phospholipase activity on model phospholipid substrates. 4. The kinetics of the phospholipase-independent action of beta-BuTx were examined in strontium-Ringer compared to calcium-Ringer solution, as well as in calcium-Ringer at different temperatures. Both the time to onset of inhibition and the time to 50% inhibition of the e.p.p., during the first phase of toxin action, are temperature-dependent and briefer in calcium than in strontium-Ringer solution. It is suggested that calcium is more effective than strontium in promoting this phospholipase- independent interaction of beta-BuTx with the nerve terminal membrane.

Localization of kallikrein in the coagulating and submandibular glands of the guinea pig.

Antibody to pure kallikrein from the coagulating gland of the guinea pig was used to localize kallikrein in the gland by immunofluorescence techniques. This antibody also reacted with the guinea pig's submandibular gland kallikrein. The specific fluorescence in the coagulating gland was present diffusely in all secretory cells lining the crypts. In contrast to its diffuse location in the coagulating gland, kallikrein in the submandibular gland was specifically located in the luminal border of striated and some larger duct cells, whereas the acinar cells and interstitial tissue showed no significant fluorescence.

Studies on kallikrein in the duct systems of the salivary glands of the cat.

By correlating immunofluorescence light microscopy with electron microscope studies and with kallikrein concentrations under various conditions, we have made the following observations and conclusions about kallikrein in the submandibular and other salivary glands.1. In the submandibular gland, specific immunofluorescence to kallikrein was observed in the luminal region of the striated ducts particularly, but also in the outer epithelial cells of the stratified epithelial collecting ducts. Sympathetic nerve stimulation resulted in a reduction in intensity of specific fluorescence and in its increased localization towards the lumen. The nearly complete elimination of kallikrein from the gland by duct obstruction for four days resulted in complete disappearance of specific fluorescence in the gland. Prolonged parasympathetic nerve stimulation at frequencies which did not reduce the kallikrein concentration of the gland failed to alter the specific immunofluorescence despite copious secretion of saliva. Our results failed to reveal evidence of secretion of kallikrein either into or towards the interstitium of the gland. The luminal layer of stratified epithelial cells in the collecting ducts contained small secretory granules closely resembling those in the striated ducts. Our results are not conclusive, but do suggest that kallikrein is located in these granules whence it is secreted into the lumen of the duct.2. The parotid gland was found to contain much lower concentrations of kallikrein than the submandibular gland. This finding was associated with the presence of far fewer striated ducts in the parotid gland. Otherwise, specific fluorescence and the response to sympathetic nerve stimulation was like that of the submandibular gland. Small secretory granules in the striated and collecting ducts resembled those of the submandibular gland.3. The sublingual gland, like the parotid, had a low concentration of kallikrein and very few striated ducts. These ducts were unevenly distributed and were concentrated in only a few lobules of the gland. Specific immunofluorescence was seen only in sections containing striated ducts.4. The possible physiological role of kallikrein in the salivary glands is discussed.

Direct evidence for the location of kallikrein in the striated ducts of the cat's submandibular gland by the use of specific antibody.

Kallikrein was located in the apical portion of the striated duct cells of the cat's submandibular gland by an immunohistochemical technique. This portion only of these cells showed an intense band of specific fluorescence. There was no evidence of specific fluorescence in the acinar and demilune cells nor in the interstitial tissue or blood besells. In some sections the collecting ducts showed a very fine fluorescent luminal rim.