Comprehensive strategy improves the genetic diagnosis of different polycystic kidney diseases.

Polycystic kidney disease (PKD) is known to occur in three main forms, namely autosomal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD) and syndromic PKD (SPKD), based on the clinical manifestations and genetic causes, which are diagnosable from the embryo stage to the later stages of life. Selection of the genetic test for the individuals with diagnostic imaging reports of cystic kidneys without a family history of the disease continues to be a challenge in clinical practice. With the objective of maintaining a limit on the time and medical cost of the procedure, a practical strategy for genotyping and targeted validation to resolve cystogene variations was developed in our clinical laboratory, which combined the techniques of whole-exome sequencing (WES), Long-range PCR (LR-PCR), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to work in a stepwise approach. In this context, twenty-six families with renal polycystic disorders were enrolled in the present study. Thirty-two variants involving four ciliary genes (PKD1, PKHD1, TMEM67 and TMEM107) were identified and verified in 23 families (88.5%, 23/26), which expanded the variant spectrum by 16 novel variants. Pathogenic variations in five foetuses of six families diagnosed with PKD were identified using prenatal ultrasound imaging. Constitutional biallelic and digenic variations constituted the pathogenic patterns in these foetuses. The preliminary clinical data highlighted that the WES + LR PCR-based workflow followed in the present study is efficient in detecting divergent variations in PKD. The biallelic and digenic mutations were revealed as the main pathogenic patterns in the foetuses with PKD.

Loss of ciliary transition zone protein TMEM107 leads to heterotaxy in mice.

Cilia in most vertebrate left-right organizers are involved in the original break in left-right (L-R) symmetry, however, less is known about their roles in subsequent steps of the cascade - relaying the signaling and maintaining the established asymmetry. Here we describe the L-R patterning cascades in two mutants of a ciliary transition zone protein TMEM107, revealing that near-complete loss of cilia in Tmem107null leads to left pulmonary isomerism due to the failure of the midline barrier. Contrary, partially retained cilia in the node and the midline of a hypomorphic Tmem107schlei mutant appear sufficient for the formation of the midline barrier and establishment and maintenance of the L-R asymmetry. Despite misregulation of Shh signaling in both mutants, the presence of normal Lefty1 expression and midline barrier formation in Tmem107schlei mutants, suggests a requirement for cilia, but not necessarily Shh signaling for Lefty1 expression and midline barrier formation.

TMEM107 Is a Critical Regulator of Ciliary Protein Composition and Is Mutated in Orofaciodigital Syndrome.

The proximate causes of multiple human genetic syndromes (ciliopathies) are disruptions in the formation or function of the cilium, an organelle required for a multitude of developmental processes. We previously identified Tmem107 as a critical regulator of cilia formation and embryonic organ development in the mouse. Here, we describe a patient with a mutation in TMEM107 that developed atypical Orofaciodigital syndrome (OFD), and show that the OFD patient shares several morphological features with the Tmem107 mutant mouse including polydactyly and reduced numbers of ciliated cells. We show that TMEM107 appears to function within cilia to regulate protein content, as key ciliary proteins do not localize normally in cilia derived from the Tmem107 mouse mutant and the human patient. These data indicate that TMEM107 plays a key, conserved role in regulating ciliary protein composition, and is a novel candidate for ciliopathies of unknown etiology.

TMEM107 inhibits EMT and invasion of NSCLC through regulating the Hedgehog pathway.

BACKGROUND: Transmembrane protein 107 (TMEM107) is a key regulator of the cilium composition and Hedgehog signaling. Lower TMEM107 gene copies are correlated with poor prognosis in non-small cell lung carcinoma (NSCLC). However, TMEM107 protein expression, localization, and function in NSCLC remain unclear. METHODS: We first evaluated TMEM107 expression in 12 newly diagnosed cases of NSCLC and paired adjacent healthy tissues by western blotting. We then used an immunohistochemical method to detect TMEM107 expression in 106 paraffin-embedded NSCLC and corresponding normal samples and analyzed its relationship with clinicopathological parameters. Moreover, we determined the impact of TMEM107 upregulation and downregulation on invasion, EMT and Hedgehog pathway in NSCLC cells. RESULTS: Our results showed that TMEM107 is localized in the cytoplasm and that its expression was lower in NSCLC. TMEM107 expression was positively correlated with cell differentiation and negatively correlated with lymph node metastasis. In A549 and HCC460 cells, downregulation of TMEM107 facilitated cell invasion and upregulated the expression of the Hedgehog pathway target protein Gli1, invasion-associated proteins N-cadherin, vimentin, MMP2, and MMP9, and epithelial-mesenchymal transition (EMT), and inhibited the expression of E-cadherin. Treatment with the Hedgehog pathway inhibitor GANT61 attenuated TMEM107-knockdown-induced EMT and invasiveness. CONCLUSIONS: These results indicate that TMEM107 inhibits EMT and invasion by negatively regulating Hedgehog signaling and that it is downregulated in NSCLC. KEY POINTS: TMEM107 expression is lower in NSCLC tissues and correlates with poor prognosis TMEM107 inhibits invasion of NSCLC cells TMEM107 inhibits EMT of NSCLC cells Downregulation of TMEM107 activates the Hedgehog signaling pathway Downregulation of TMEM107 promotes EMT and migration in NSCLC by activating the Hedgehog signaling pathway.

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and ß gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation. SIGNIFICANCE: Protein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible.