Alport syndrome: our knowledge update

Alport syndrome is a progressive multisystem disease associated with variants in genes COL4A3, COL4A4, COL4A5.The syndrome is an important genetic cause of kidney failure, including women with X-linked disease. Given the unfavorable natural history of Alport nephropathy and benefit from early treatment with angiotensin-converting enzyme inhibition, it is necessary to change our diagnostic approach in patients with persistent glomerular hematuria and management of patients with Alport syndrome. This review presents the ethiology, pathogenesis, genotype and phenotype heterogeneity of the syndrome and expert clinical practice recommendations to enhancing early diagnosis and achieving optimal outcomes in Alport syndrome.

Simultaneous Homozygous Mutations in SLC12A3 and CLCNKB in an Inbred Chinese Pedigree

Gitelman syndrome (GS) and Bartter syndrome (BS) type III are both rare, recessively inherited salt-losing tubulopathies caused by SLC12A3 and CLCNKB mutations, respectively. We described a 48-year-old male patient with fatigue, carpopedal spasm, arthralgia, hypokalemic alkalosis, mild renal dysfunction, hypomagnesemia, hypocalciuria, hyperuricemia, normotension, hyperreninemia and chondrocalcinosis in knees and Achilles tendons. His parents are first cousin. Genetic analysis revealed simultaneous homozygous mutations in SLC12A3 gene with c.248G>A, p.Arg83Gln and CLCNKB gene with c.1171T>C, p.Trp391Arg. The second younger brother of the proband harbored the same simultaneous mutations in SLC12A3 and CLCNKB and exhibited similar clinical features except normomagnesemia and bilateral kidney stones. The first younger brother of the proband harbored the same homozygous mutations in CLCNKB and exhibited clinical features of hypokalemia, normomagnesemia, hypercalciuria and hyperuricemia. Potassium chloride, spironolactone and potassium magnesium aspartate were prescribed to the proband to correct electrolytic disturbances. Benzbromarone and febuxostat were prescribed to correct hyperuricemia. The dose of potassium magnesium aspartate was subsequently increased to alleviate arthralgia resulting from calcium pyrophosphate deposition disease (CPPD). To the best of our knowledge, we are the first to report an exceptionally rare case in an inbred Chinese pedigree with simultaneous homozygous mutations in SLC12A3 and CLCNKB. GS and BS type III have significant intrafamilial phenotype heterogeneity. When arthralgia is developed in patients with GS and BS, gout and CPPD should both be considered.

A Family Based Whole Exome Sequence Study to Indentify Modifier Genes for Phenotype Heterogeneity Between Severe and Non-Severe Thalassemia Patients

* Corresponding author: Prof. Anupam Basu, Department of Zoology, The University of Burdwan, PurboBarddhaman, West Bengal- 713104, India, Email- [email protected] & b are equal contribution Whole Expanded Exome Sequencing Study of two families father, mother and index cases (trio) was undertaken for two E/ beta thalassemia subjects with same HBB genotype. Approximately 200ng of DNA was taken from each individual and shared into 300-400bp fragment. Then the shared fragments are end repair. Klenow exonuclease was used to add an adapter. After adapter ligation 10 cycle PCR amplification was done for each sample. The targeted Exome was captured by the Agilent Sure select XT Human all Exome V6+ UTR kit as per the manufacturer’s protocol. Captured library was then amplified 10 cycles with 8 bp index sequence for each sample. Then the indexed capture library was pooled together. Pair end sequencing of the pooled library was performed in Illumina HiSeq2500 using Illumina HiSeq SBS kit. Finally, both the genes, inherited and denovo, from both the subjects were separately functionally annotated by DAVID online tools 6.8. Functionally annotation result shows that in case of subject-1, 6 KEGG pathway were involved. These are Adherent junction, Protein digestion and absorption, Inflamatory Bowel Disease, Amoebiasis, PPAR signaling pathway and glycolysis or gluconeogenesis. Interestingly in case of subject-2, only 2 KEGG pathway were found, Thyroid hormone synthesis and carbon metabolism.

Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems

Background The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins. Results In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy. Conclusions To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Triple-A Syndrome (TAS): An In-Depth Overview on Genetic and Phenotype Heterogeneity

Triple-A Syndrome (TAS) is a rare autosomal recessive disorder characterized by three cardinal symptoms: alacrimia, achalasia and adrenal insufficiency due to ACTH insensitivity. Various progressive neurological abnormalities and skin changes have been described in association with the syndrome. The disease is caused by mutation in the AAAS gene on chromosome 12q13. Mutations in AAAS were identified in more than 90% of individuals and families with TAS. The protein encoded by AAAS was termed ALADIN and is part of the WD repeat family of proteins, that have been found to be involved in many different functions such as protein-protein interaction, RNA processing, cytoskeleton assembly, control of cell division, signal transduction and apoptosis. Immunohistochemical analysis showed that mutated or truncated ALADIN localizes to the cytoplasm rather than to the nuclear pore complex. The exact function of ALADIN and the mechanisms that lead to the ACTH-resistant adrenal phenotype remains largely unknown. Nonetheless, recent studies provided some insights on the role of ALADIN as a member of the Nuclear Pore Complex not only implicated in the import of proteins involved in DNA repair and oxidative stress homeostasis but also in the strengthening of the mitotic spindle assembly. Early identification of the syndrome is challenging, given the rarity of the condition and high phenotypic heterogeneity even among members of the same family. In this review, we aim to summarize the current knowledge of clinical and molecular profile of patients with TAS and recommendations for the diagnosis, management, and follow-up of patients.

Clinical characteristics and disease progression of retinitis pigmentosa associated with PDE6B mutations in Korean patients

Due to the genotype–phenotype heterogeneity in retinitis pigmentosa (RP), molecular diagnoses and prediction of disease progression is difficult. This study aimed to report ocular and genetic data from Korean patients with PDE6B-associated RP (PDE6B-RP), and establish genotype–phenotype correlations to predict the clinical course. We retrospectively reviewed targeted next-generation sequencing or whole exome sequencing data for 305 patients with RP, and identified PDE6B-RP in 15 patients (median age, 40.0 years). Amongst these patients, ten previously reported PDE6B variants (c.1280G > A, c.1488del, c.1547T > C, c.1604T > A, c.1669C > T, c.1712C > T, c.2395C > T, c.2492C > T, c.592G > A, and c.815G > A) and one novel variant (c.712del) were identified. Thirteen patients (86.7%) experienced night blindness as the first symptom at a median age of 10.0 years. Median age at diagnosis was 21.0 years and median visual acuity (VA) was 0.20 LogMAR at the time of genetic analysis. Nonlinear mixed models were developed and analysis revealed that VA exponentially decreased over time, while optical coherence tomography parameters linearly decreased, and this was related with visual field constriction. A high proportion of patients with the c.1669C > T variant (7/9, 77.8%) had cystoid macular edema; despite this, patients with this variant did not show a higher rate of functional or structural progression. This study will help clinicians predict functional and structural progression in patients with PDE6B-RP.

Evolution of Mutational Landscape and Tumor Immune-Microenvironment in Liver Oligo-Metastatic Colorectal Cancer

Genetic dynamics underlying cancer progression are largely unknown and several genes involved in highly prevalent illnesses (e.g., hypertension, obesity, and diabetes) strongly concur to cancer phenotype heterogeneity. To study genotype-phenotype relationships contributing to the mutational evolution of colorectal cancer (CRC) with a focus on liver metastases, we performed genome profiling on tumor tissues of CRC patients with liver metastatic disease and no co-morbidities. We studied 523 cancer-related genes and tumor-immune microenvironment characteristics in primary and matched metastatic tissues. We observed a loss of KRAS and SMAD4 alterations and a high granzyme-B+ T-cell infiltration when the disease did not progress. Conversely, gain in KRAS, PIK3CA and SMAD4 alterations and scarce granzyme-B+ T-cells infiltration were observed when the tumor evolved towards a poly-metastatic spread. These findings provide novel insights into the identification of tumor oligo-metastatic status, indicating that some genes are on a boundary line between these two clinical settings (oligo- vs. poly-metastatic CRC). We speculate that the identification of these genes and modification of their evolution could be a new approach for anti-cancer therapeutic strategies.