Funktionelle Untersuchung der Bedeutung der Mutation G832A des CLCNKA-Promotors bei einer Patientin mit renalem Salzverlust und Taubheit

Hereditary salt-losing tubulopathies are rare, but potentially life-threatening disorders. A better understanding of the genetic mechanisms underlying these diseases can lead to both earlier diagnosis of the affected patient and possibly a specific therapy, ultimately improving the care of our patients. Bartter´s syndrome with sensorineural deafness (BSND) is a particularly severe form of these tubulopathies, manifesting antenatally with polyhydramnios, subsequent premature labour and postnatally with deafness and renal salt wasting resulting in failure to thrive or even death. The molecular basis of this disorder was uncovered between 2001 and 2008. The chloride channels ClC-Ka and -Kb are expressed both in the kidney and the inner ear and are essential to renal salt reabsorption and the generation of the endochochlear electrochemical gradient. Mutations in either both ClC-channels, or their common subunit Barttin can lead to BSND. Unfortunately, for a significant number of patients with different forms hereditary salt-losing tubulopathies, the mechanism behind their disease is still unknown. This study presents one such patient with a BSND phenotype, lacking the typical genetic defects. The study shows that in this patient only one among the known genes leading to salt-losing tubulopathies, CLCKNB (encoding ClC-Kb), is affected. Since these mutations typically lead to a more benign phenotype than the one exhibited by this patient, the search for mutations was extended to the promoter of CLCNKA, the gene encoding the second chloride channel typically affected in BSND patients. Alongside several known single nucleotide polymorphisms only one previously unknown mutation, dubbed G832A, was found in CLCNKA´s promoter This mutation affects a nucleotide sequence commonly recognized by certain transcription factors. To elucidate the functional relevance of G832A, an established in vitro model for promoter-controlled gene expression was used in three different kidney cell lines. These experiments showed a reduction of up to 77.5% of the expression of genes controlled by a promoter with the G832A mutation compared to the unaffected wildtype promoter Although one should always be careful when translating in vitro findings to phenotypes in vivo, these results, combined with the knowledge that this patient´s phenotype typically results from mutations in both CLCNKA and CLCNKB, strongly hint that G832A contributes significantly to this patient´s disorder. Ultimately, this study, apart from elucidating the molecular basis of another patient´s salt-losing tubulopathy, sheds a little more light on the still insufficiently understood field of compensatory mechanisms in patients with renal salt-wasting – or deafness. The step from the rather benign phenotype of classical Bartter´s syndrome (where only CLCNKB is affected) to the much more severe antenatal from with sensorineural deafness seems to be a short one – only one mutation in CLCNKA´s promoter seems to be enough. It might be possible that this is due to an insufficient compensatory upregulation of CLCNKA, and the experiments elucidating this question should be subject of further studies.