Hans H. Seydewitz

Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens

Abstract Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%–2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the “5T” allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for ΔF508 and none was compound heterozygous for ΔF508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for ΔF508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the “5T allele” was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G→A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

Cholinergic ion secretion in human colon requires coactivation by cAMP

Cl-secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca2+concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current ( I sc) [change (Δ) in I sc = -79.4 ± 7.5 μA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl- exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (Δ I sc = -42.4 ± 7.2 μA/cm2) that was inhibited by basolateral trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Δ I sc = 167.7 ± 32.7 μA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive I sc was probably due to activation of apical K+channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I sc. Under these conditions, CCH induced a large further increase in lumen-negative I sc(Δ I sc = -100.0 ± 21.0 μA/cm2). We conclude that CCH acting via [Ca2+]ican induce Cl- secretion only in the presence of cAMP, i.e., when luminal Cl- channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.

Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia.

Human airway epithelia express Ca2+-activated Cl− channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl− secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl− secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl− secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl− secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

Glycogen storage disease type I: diagnosis and phenotype/genotype correlation

Abstract. Glycogen storage disease type Ia (GSD Ia) is caused by mutations in the G6PC gene encoding the phosphatase of the microsomal glucose-6-phosphatase system. GSD Ia is characterized by hepatomegaly, hypoglycemia, lactic acidemia, hyperuricemia, hyperlipidemia and short stature. Other forms of GSD I (GSD I non-a) are characterized by the additional symptom of frequent infections caused by neutropenia and neutrophil dysfunction. GSD I non-a is caused by mutations in a gene encoding glucose-6-phosphatase translocase (G6PT1). We report on the molecular genetic analyses of G6PC and G6PT1 in 130 GSD Ia patients and 15 GSD I non-a patients, respectively, and provide an overview of the current literature pertaining to the molecular genetics of GSD I. Among the GSD Ia patients, 34 different mutations were identified, two of which have not been described before (A65P; F177C). Seventeen different mutations were detected in the GSD I non-a patients. True common mutations were identified neither in GSD Ia nor in GSD I non-a patients. Conclusion: Glycogen storage disease type Ia and and type I non-a are genetically heterogenous disorders. For the diagnosis of the various forms of glycogen storage disease type I, molecular genetic analyses are reliable and convenient alternatives to the enzyme assays in liver biopsy specimens. Some genotype-phenotype correlations exist, for example, homozygosity for one G6PC mutation, G188R, seems to be associated with a glycogen storage disease type I non-a phenotype and homozygosity for the 727G>T mutation may be associated with a milder phenotype but an increased risk for hepatocellular carcinoma.

Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes

The flavonoid genistein has been shown to activate a Cl− conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and ΔF508‐CFTR were activated by genistein while both cyclic AMP (KvLQT1) and Ca2+ (SK4) activated K+ channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non‐CF) and CF patients and in the presence of amiloride (10 μmol l−1; mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non‐CF airway epithelia, genistein (50 μmol l−1; mucosal side) increased lumen negative Isc but had no additional effects on tissues pre‐stimulated with IBMX and forskolin (100 μmol l−1 and 1 μmol l−1; both sides). In non‐CF rectal biopsies, in the presence of amiloride (10 μmol l−1; mucosal side) and indomethacin (10 μmol l−1; basolateral side), genistein increased lumen negative Isc and enabled cholinergic (carbachol; CCH, 100 μmol l−1; basolateral side) stimulation of Cl− secretion indicating activation of luminal CFTR Cl− channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated Isc. In CF airway and intestinal tissues genistein failed to induce Cl− secretion. Thus, genistein is able to activate luminal CFTR Cl− conductance in non‐CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K+ conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

Increased phosphorylation of human fibrinopeptide A under acute phase conditions

The High Performance Liquid Chromatography (HPLC) separation of the fibrinopeptides liberated by the action of thrombin from plasma fibrinogen in a new one-step procedure without prior purification of fibrinogen is described. Since the phosphorylated and non-phosphorylated form of fibrinopeptide A are clearly resolved by this method, the determination of the degree of phosphorylation of fibrinopeptide A from the peak heights of these peptides becomes possible. By this method the degree of phosphorylation of fibrinogen in healthy volunteers (n = 21) is found to be 23.6 +/- 3.6%. Under acute phase conditions where the synthesis rate of fibrinogen is known to be markedly enhanced the degree of its phosphorylation also increases considerably. This was demonstrated on 13 patients undergoing an elective hip joint replacement, the hip surgery being chosen as a model for the elicitation of an acute phase reaction. The degree of phosphorylation rises steeply up to 60% on the first day after operation thereafter declining slowly to normal values within about one week. The maximum of the degree of phosphorylation precedes that of the fibrinogen concentration by several days. The mechanism which leads to the higher phosphorylation of fibrinogen during increased synthesis is unknown at the moment.

Hereditary hypofibrinogenemia with fibrinogen storage in the liver

A family with hereditary autosomal dominant hypofibrinogenemia is described. The outstanding feature is massive deposition of fibrinogen/fibrin within hepatocytes, faintly visible in routine microscopic sections, but clearly demonstrable by immunohistologic techniques. Circulating fibrinogen shows normal electrophoretic mobility of Aα-, Bβ-, and γ-chains. We assume that the hereditary defect in this family interferes with fibrinogen release from hepatocytes. Clinically there are fluctuating slight elevations of serum transaminase levels. Hemostasis and wound healing are undisturbed.

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

Background The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K+ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl− secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. Methods We studied the effects of 1-EBIO on CFTR-mediated Cl− secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl− secretion. Results Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl− secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl− secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca2+-activated and clotrimazole-sensitive KCNN4 K+ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl− secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl−conductance. Conclusions We conclude that 1-EBIO potentiates Cl−secretion in native CF tissues expressing CFTR mutants with residual Cl− channel function by activation of basolateral KCNN4 K+ channels that increase the driving force for luminal Cl− exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

Glucose-6-phosphatase mutation G188R confers an atypical glycogen storage disease type 1b phenotype.

Glycogen storage disease type 1a (GSD 1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). A variant (GSD 1b) is caused by a defect in the transport of glucose-6-phosphate (G6P) into the microsome and is associated with chronic neutropenia and neutrophil dysfunction. Mutually exclusive mutations in the G6Pase gene and the G6P transport gene establish GSD 1a and GSD 1b as independent molecular processes and are consistent with a multicomponent translocase catalytic model. A modified translocase/catalytic unit model based on biochemical data in a G6Pase knockout mouse has also been proposed for G6Pase catalysis. This model suggests coupling of G6Pase activity and G6P transport. A 5-mo-old girl with hypoglycemia, hepatomegaly, and lactic acidemia was diagnosed with GSD 1a. She also developed neutropenia, neutrophil dysfunction, and recurrent infections characteristic of GSD 1b. Homozygous G188R mutations of the G6Pase gene were identified, but no mutations in the G6P translocase gene were found. We have subsequently identified a sibling and two unrelated patients with similar genotypic/phenotypic characteristics. The unusual association of neutrophil abnormalities in patients with homozygous G188R mutations in the G6Pase gene supports a modified translocase/catalytic unit model.

The location of a second in vivo phosphorylation site in the Aα-chain of human fibrinogen

Abstract By quantitative phosphorus determination on the single chains of human fibrinogen it is demonstrated that the covalently bound phosphorus of adult and fetal fibrinogen is exclusively located in the Aα-chain. The Aα-chain of fetal fibrinogen contains about twice as much phosphorus as the adult Aα-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the Aα-chain. By consecutive cleavage of the Aα-chains of fetal and adult fibrinogen with cyanogen bromide, trypsin, and chymotrypsin, separation of the resulting peptide mixtures and analysis for phosphorylated amino acids, this second phosphorylation site could be traced to Ser 345 of the Aα-chain. There is only one sequence homology between the two now known in vivo phosphorylation sites of human fibrinogen, namely that the second amino acid to the carboxyl side of the phosphorylated Ser is Glu. The sequence specificity of the up to now unidentified protein kinase phosphorylating fibrinogen allows it to be classified as a member of the group of type-2 casein kinases or casein kinases TS.