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Background Uromodulin-associated Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD-UMOD) belongs to a group of autosomal dominant inherited diseases caused by mutations in the UMOD gene, which codes for uromodulin, a protein exclusively expressed in renal tubular cells of the ascending limb of the loop of Henle. The diagnosis is hampered by non-specific clinical, laboratory and histological findings. In this study, we evaluated serum uromodulin as diagnostic marker for ADTKD-UMOD in a family with a novel mutation in UMOD. Methods We investigated a family with five members suffering from chronic kidney disease of unknown origin (CKD) and three healthy members using whole exome sequencing. Serum uromodulin was measured by ELISA. The uromodulin concentration of each CKD family member was compared to reference CKD groups with similar eGFR and to non-CKD individuals in case of the healthy family members, respectively. Results Whole exome sequencing revealed novel missense mutation c.457T>G, p.(Cys153Gly) in UMOD. Serum uromodulin concentration was lower in all affected patients compared to all patients of the reference CKD groups, while healthy family members showed normal values comparable to those of the non-CKD reference group. Conclusions The mutation detected in our family leads to severely reduced serum uromodulin concentrations, distinguishing these patients clearly from CKD patients with comparable eGFR. Therefore, serum uromodulin could serve as a simple, new diagnostic marker to identify patients with ADTKD-UMOD.

Ahnak ist ein ubiquitäres Protein, das an einer Vielzahl biologischer Prozesse beteiligt ist. In der Herzmuskelzelle ist Ahnak überwiegend am Sarkolemma lokalisiert und bindet an Aktin und an die regulatorischen Beta2-Untereinheit des L-Typ-Kalzium-Kanals. Das Ziel dieser Arbeit war die Funktion von Ahnak im Herzen mit Hilfe eines Knock-out-Maus-Modells und in Bindungsstudien zu untersuchen. Morphologische Untersuchungen zeigten, dass das Längenwachstum adulter Kardiomyozyten bei Ahnakdefizienz signifikant reduziert war. Die Kontraktionseigenschaften adulter isolierter Ahnak-defizienter Kardio-myozyten (im Alter von 6 Monaten) waren ebenfalls verändert. Die Kontraktions- und Relaxaktionsgeschwindigkeiten waren erhöht. Eine Erhöhung des diastolischen Kalzium-Spiegels zeigten die Kardiomyozyten schon im Alter von 3 Monaten. Diese beobachteten phänotypischen Veränderungen lassen vermuten, dass die Aktivität des L-Typ-Kalzium-Kanals erhöht ist. In dieser Arbeit konnte das PXXP-Motiv, in der C-terminalen Ahnak-Domäne, als die hochaffine Beta2-Bindungsstelle (KD ~ 60 nM) identifiziert werden. Substitution von Prolin gegen Alanin verringerte zwar die Bindung zur Beta2-Untereinheit dramatisch (KD ~ 1 µM), hob sie aber nicht auf. In weiteren Bindungsstudien zeigte sich, dass die natürlich vorkommende Missensmutation I5236T die Bindung zur regulatorischen Beta2-Untereinheit verstärkte, dagegen verminderte die PKA-abhängige Phosphorylierung der beiden Proteinpartner die Bindung. Experimente am ganzen isoliert perfundierten Herzen zeigten, dass Ahnak-Knock-Out-Herzen geringer Beta-adrenerg stimulierbar waren. Ahnak scheint wie eine physiologische Bremse des kardialen Kalzium-Kanals zu wirken. Ahnak is an ubiquitous protein with in unique structure, which has been implicated in cell type specific functions. In cardiomyocytes, ahnak is predominantly localized at the sarcolemma and is associated with actin and with the regulatory beta2 subunit of the L-type calcium-channel. The aim of this work was to unravel the function of ahnak in the heart, using a knock-out-mouse model and binding studies. Morphological studies showed a significant decrease in the cell-length of ahnak deficient cardiomyocytes. The contractile parameters of isolated adult ahnak deficient cardiomyocytes (in the age of 6 month) were altered. The development of tension and relaxation were increased. An increase of diastolic calcium was already observed at the age of 3 month. In general the observed phenotypic changes suggested an increased activity of the L-type calcium-channel. In this study, a PXXP-motif, which locates in ahnaks C-terminus, was identified as the high affinity beta2 subunit binding site (KD ~ 60 nM). Substitution of both proline residues by alanine reduced, but did not abolish the binding (KD ~ 1 µM). Further binding studies revealed that the natural occurring ahnak missense mutation I5236T increases the binding affinity to the regulatory beta2 subunit. By contrast PKA dependant phosphorylation of both protein partners decreases the interaction. In studies with isolated perfused working heart preparations, the ahnak deficient hearts were less beta-adrenergic stimulated than hearts from wild type. Taken together ahnak seems to be a physiological brake of the cardiac calcium-channel.

Aim: To investigate the phenotypical expression of an identical mutation of the CBFA1/RUNX2 gene within a family with cleidocranial dysplasia. Patients and Method: A five-member family underwent clinical examination.Two members, father and son, showed dissimilar symptoms of cleidocranial dysplasia. The two affected patients were examined for syndrome-typical symptoms, and the genotype was determined by molecular-genetic analysis. Results: In both patients an identical missense mutation (G146R) in exon 2 of the CBFA1/RUNX2 gene was identified. In father and son the dental disturbances were similarly clearly expressed. However, the craniofacial skeleton of the son exhibited fewer dysostotic ossification features than that of the father. In the three clinically healthy family-members no mutation of the CBFA1/RUNX2 gene was found. Conclusion: In two patients with cleidocranial dysplasia an identical missense mutation in the CBFA1/RUNX2 gene leading to a divergent craniofacial phenotype was determined. The results indicate marked variability in the phenotypical expression of CBFA1/RUNX2 mutations.