Hepatocyte nuclear factor 1beta (HNF1beta) abnormalities have been recognized to cause congenital anomalies of the kidney and urinary tract (CAKUT), predominantly affecting bilateral renal malformations. To further understand the spectrum of HNF1beta related phenotypes, we performed HNF1B gene mutation and deletion analyses in Japanese patients with renal hypodysplasia (n = 31), unilateral multicystic dysplastic kidney (MCDK; n = 14) and others (n = 5). We identified HNF1B alterations in 5 out of 50 patients (10%). De novo heterozygous complete deletions of HNF1B were found in 3 patients with unilateral MCDK. Two of the patients showed contralateral hypodysplasia, whereas the other patient showed a radiologically normal contralateral kidney with normal renal function. Copy number variation analyses showed 1.4 Mb microdeletions involving the whole HNF1B gene with breakpoints in flanking segmental duplications. We also identified 1 novel truncated mutation (1007insC) and another missense mutation (226G>T) in patients with bilateral hypodysplasia. HNF1B alterations leading to haploinsufficiency affect a diverse spectrum of CAKUT. The existence of a patient with unilateral MCDK with normal renal function might provide genetic insight into the etiology of these substantial populations of only unilateral MCDK. The recurrent microdeletions encompassing HNF1B could have a significant impact on the mechanism of HNF1B deletions.
The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.
To achieve DNA sequencing using a solid-state nanopore, it is necessary to reduce the electric noise current. The noise current can be decreased by reducing the capacitance (C) of the nanopore device. However, we found that an electric-charge difference (ΔQ) between the electrolyte in one chamber and the electrolyte in another chamber occurred. For low capacitance devices, this electric-charge imbalance can lead to unexpectedly high voltage (ΔV = ΔQ/C) which disrupted the membrane when the two electrolytes were independently poured into the chambers. We elucidated the mechanism for the generation of initial defects and established new procedures for preventing the generation of defects by connecting an electric bypass between the chambers.
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