GATA6 mutations: Characterization of two novel patients and a comprehensive overview of the GATA6 genotypic and phenotypic spectrum

The first human mutations in GATA6 were described in a cohort of patients with persistent truncus arteriosus, and the phenotypic spectrum has expanded since then. This study underscores the broad phenotypic spectrum by presenting two patients with de novo GATA6 mutations, both exhibiting complex cardiac defects, pancreatic, and other abnormalities. Furthermore, we provided a detailed overview of all published human genetic variation in/near GATA6 published to date and the associated phenotypes (n = 78). We conclude that the most common phenotypes associated with a mutation in GATA6 were structural cardiac and pancreatic abnormalities, with a penetrance of 87 and 60%, respectively. Other common malformations were gallbladder agenesis, congenital diaphragmatic hernia, and neurocognitive abnormalities, mostly developmental delay. Fifty‐eight percent of the mutations were de novo, and these patients more often had an anomaly of intracardiac connections, an anomaly of the great arteries, and hypothyroidism, compared with those with inherited mutations. Functional studies mostly support loss‐of‐function as the pathophysiological mechanism. In conclusion, GATA6 mutations give a wide range of phenotypic defects, most frequently malformations of the heart and pancreas. This highlights the importance of detailed clinical evaluation of identified carriers to evaluate their full phenotypic spectrum.     

Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation

LLC-PK₁/PKE₂₀ cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833–837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797–6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pH_i from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 M), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 M). Addition of either vasopressin (2 M) or calcitonin (0.3 M) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively. Separate hormone additions to either the apical or basolateral cell surface led to effects similar to those produced by simultaneous hormone additions onto both cell surfaces, although the relative response of Na/H exchangers to either agonist is variable. In summary, these results suggest that in LLC-PK ₁/PKE₂₀ cells, vasopressin and calcitonin can act via receptor systems coupled either to adenylate cyclase or to phospholipase C. Activation of these receptor systems can lead to inhibition of Na/H-2 and stimulation of Na/H-1.     

Epinephrine activates outward rectifying K channel in Madin-Darby canine kidney cells

Patch-clamp recordings were used to study the epinephrine dependent activation of ion channels in the cell membrane of cultured subconfluent renal epithelial (MDCK) cells. The patch-current was dominated by two populations of K channels. The spontaneously active population of K channels shows an inward rectifying behavior. Addition of epinephrine to the cell exterior, after the patchpipette had been sealed to the cell membrane, increased the open probability of the inward rectifying K channel and shifted the membrane potential in the hyperpolarizing direction. The epinephrine induced hyperpolarization occurs in the range of seconds and is caused by activation of outward-rectifying K channels. The outward-rectifying K channel could not be observed under control conditions. Epinephrine activated channels always appeared in clusters of four to nine channels. Both populations of K channels are modulated in their open probability by cytoplasmic free calcium and voltage.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.     

Strain dependency of the transfer of experimental immune insulitis in mice.

Multiple treatment with low doses of streptozotocin induces hyperglycaemia with concomitant lymphocytic infiltrations into pancreatic islets (insulitis) in several mouse strains. The transfer of cellular immune reactions against islet cells by means of spleen cells was tested in two congeneic and five allogeneic strain combinations. Donor mice were treated on 5 consecutive days with 40 mg streptozotocin per kg body weight. Three weeks later, 5 x 10(7) live spleen cells were transferred into thymusless recipient mice. Insulitis which had developed in about 70% of the donors was only transferable from C57B1/6J to congeneic thymusless mice. In a second congeneic and in all allogeneic strain combinations, cellular immune reactions against pancreatic islets could not be transferred. In none of the recipients of spleen cells from diabetic donors was hyperglycaemia observed. As streptozotocin-induced cellular immune reactions against pancreatic islet cells were only transferable in one congeneic and in no allogeneic strain combinations, it is concluded that there is a genetic restriction both on the levels of donor and recipient mice.     

The pelvic epithelium of the rat kidney: A scanning and transmission electron microscopic study

The renal pelvis of the rat is characterized by extensions called specialized fornices that penetrate into the outer zone of the outer medulla (a type II as classified by Pfeiffer, 1968, 1970). The renal pelvic epithelium, therefore, covers areas of the kidney from the inner medulla, the inner and outer stripe of the outer medulla, and the cortex. The renal pelves of seven rats were studied by transmission and scanning electron microscopy. The transitional epithelium on the nonparenchymal surface of the pelvis was three to four cell layers thick (zone 0–1). This epithelium became thinner where it covered the renal cortex (zone 1–2) or the outer medulla. Although the apical cells of the epithelium retained the asymmetric luminal unit-membrane plaques, the number of cytoplasmic fusiform vesicles decreased as one studied the epithelium progressing over the zones from cortex toward papilla. Scanning electron microscopy demonstrated a small number of surface cells of a different morphology that were characterized by apical microvilli. The number of these microvillous lining cells increased as the epithelium covering the outer (zone 2–3) and inner (zone 3–4) stripe regions of the outer medulla was viewed, until the inner medulla was entirely covered by this cell type. In a reciprocal manner, the cells with the asymmetric apical plaques decreased in numbers and in their morphologic specialization in each successive region. The epithelium surrounding the inner medulla (zone 6–7) was completely devoid of this transitional cell type. Judging from the morphologic characteristics of the epithelia, one could surmise that little exchange of urea, water, and salts would occur with the extrarenal connective tissue or the cortical parenchyma. Recycling of urea might become more important physiologically with the outer stripe parenchyma, and even more so with the increased surfaces of the inner stripe parenchyma that lined the secondary pyramid, as well as with the epithelium lining the inner medulla.     

No Association between SNP rs498055 on Chromosome 10 and Late-Onset Alzheimer Disease in Multiple Datasets

SNP rs498055 in the predicted gene LOC439999 on chromosome 10 was recently identified as being strongly associated with late-onset Alzheimer disease (LOAD). This SNP falls within a chromosomal region that has engendered continued interest generated from both preliminary genetic linkage and candidate gene studies. To independently evaluate this interesting candidate SNP we examined four independent datasets, three family-based and one case-control. All the cases were late-onset AD Caucasian patients with minimum age at onset ≥ 60 years. None of the three family samples or the combined family-based dataset showed association in either allelic or genotypic family-based association tests at p < 0.05. Both original and OSA two-point LOD scores were calculated. However, there was no evidence indicating linkage no matter what covariates were applied (the highest LOD score was 0.82). The case-control dataset did not demonstrate any association between this SNP and AD (all p-values > 0.52). Our results do not confirm the previous association, but are consistent with a more recent negative association result that used family-based association tests to examine the effect of this SNP in two family datasets. Thus we conclude that rs498055 is not associated with an increased risk of LOAD.     

Decreased expression of adhesion molecules on monocytes in recent onset IDDM.

The expression of adhesion molecules in monocytes of patients with recent onset type I diabetes was analysed. Monocytes were identified as CD14-positive cells by flow cytometry. The percentage of monocytes expression LFA-1 alpha, ICAM-1 and HLA-DR was slightly lower in recent onset type I diabetes (n = 13) compared to normal subjects (n = 15) and was significantly decreased after activation of cells with lipopolysaccharide and interferon-gamma for 5-24 hr. Receptor densities on adhesion molecule-positive monocytes and the expression of LFA-1 beta were normal. These data indicate that monocyte trafficking is abnormal in recent onset type 1 diabetes.     

Hepatitis B virus transmission in Brazilian hemodialysis units: serological and molecular follow-up

A serological and molecular study of hepatitis B virus (HBV) infection was carried out in dialysis units in Central Brazil. Between 1995 and 1999, serum samples from all HBsAg-positive hemodialysis patients (n = 43) were tested for HBeAg/anti-HBe and subtyping by monoclonal ELISA. HBV DNA was detected by PCR and positive samples were genotyped by restriction fragment polymorphism pattern (RFLP) methodology. TheHBsAg prevalence declined in this population during the survey period (12-5.8%). HBeAg and anti-HBe were detected in 23 (53.5%) and 18 (41.9%) sera, respectively. Thirty-six samples could be HBsAg subtyped: 21 were subtype ayw(3), 14 belonged to adw(2) and one was identified as adw(4). HBV DNA was present in 30 serum samples. Of these, 20 (66.7%) were genotype D, 9 (30%) genotype A, and 1 (3.3%) genotype F. In addition, the RFLP pattern could be determined in samples from 18/20 genotype D patients: D3 (10 strains), D7 (7 strains) and D4 (1 strain); from 8/9 genotype A patients: A1 (6 strains) and A3 (2 strains); and from the patient infected with genotype F: F1. Patterns D3 and D7 were associated closely with HBV infection in the two largest hemodialysis units studied. These findings confirm the value of the RFLP method as an effective molecular epidemiological tool for elucidating HBV transmission in hemodialysis units.