Effects of RTTN gene mutations and the need for complementary cDNA analysis for some transcripts: case report

QJM ◽  
2021 ◽  
Vol 114 (Supplement_1) ◽  
Author(s):  
Afshin PourMirza ◽  
Azza Abd El Moneim Attia ◽  
Mohamed Issa Tawil ◽  
Dina A Amin
Keyword(s):  
Primary Cilia ◽  
Human Fibroblasts ◽  
Gene Mutations ◽  
Normal Structure ◽  
Axial Rotation ◽  
Diagnostic Tools ◽  
Growth Delay ◽  
Postnatal Life ◽  
Cellular Division ◽  
Exact Function

Abstract RTTN is a gene coding for Rotatin protein which has been localized at ciliary body and centriole of human fibroblasts. Its exact function in human is not well identified. However, it is thought to play important role in maintenance of normal structure of primary cilia and hence in left to right organ specification, axial rotation, development of notochord as well as early cellular division in mice. Overall 28 cases have been reported with homozygous pathogenic mutations of RTTN gene till 2019 to our knowledge. They presented with primary microcephaly, short stature, polymicrogyria with or without seizures (MPPS). These symptoms can be summarized in microcephaly, generalized growth delay, malformation of the developing cortex (MDC) and hence neurodevelopmental delay with or without seizures. Meanwhile all of these signs usually appear in late prenatal or even postnatal life which emphasizes the need for other diagnostic tools in case of detection of RTTN variables of unknown significance or in case of accidental detection of multiple transcripts by NGS in prenatal genetic testing. A case with prenatal diagnosis of two different RTTN gene transcripts in fetal WES analysis. One of the transcripts showed RTTN homozygous gene mutation while the other transcript was normal. This article presents the multidisciplinary approach followed in prenatal as well as postnatal period. It also highlights the importance of gene expression analysis in prenatal genetics.

scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

Pulmonary hypertension genes as major diagnostic tools

ESC CardioMed ◽  
2018 ◽  
pp. 2490-2493
Author(s):  
Mélanie Eyries ◽  
Barbara Girerd ◽  
David Montani ◽  
David-Alexandre Tregouët ◽  
Marc Humbert ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Genetic Mutations ◽  
Incomplete Penetrance ◽  
Hereditary Haemorrhagic Telangiectasia ◽  
Sporadic Cases ◽  
Pulmonary Arterial

A few genes have been shown to be major predisposing factors for pulmonary hypertension and are responsible for heritable forms of the disease. However, for nearly all genes described, not all mutation carriers develop the disease (autosomal transmission with incomplete penetrance) explaining the presence of genetic mutations in apparently sporadic cases. Beside mutations in major genes (BMPR2 for pulmonary arterial hypertension and EIF2AK4 for recessive heritable pulmonary veno-occlusive disease), other genes have been involved in a very limited number of cases (KCNK3, CAV1, and Smad8). Gene mutations are also been found as part of syndromic diseases (ACVRL1 mutations in hereditary haemorrhagic telangiectasia and TBX4 in small patella syndrome).

scholarly journals Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ramona Jühlen ◽  
Valérie Martinelli ◽  
Chiara Vinci ◽  
Jeroen Breckpot ◽  
Birthe Fahrenkrog
Keyword(s):  
Primary Cilium ◽  
Broad Spectrum ◽  
Genetic Heterogeneity ◽  
Primary Cilia ◽  
Neuromuscular Disorders ◽  
Human Fibroblasts ◽  
Post Translational Modifications ◽  
Proximity Ligation ◽  
Clinical Disorders ◽  
Foetal Movement

Abstract Ciliopathies are clinical disorders of the primary cilium with widely recognised phenotypic and genetic heterogeneity. Here, we found impaired ciliogenesis in fibroblasts derived from individuals with fetal akinesia deformation sequence (FADS), a broad spectrum of neuromuscular disorders arising from compromised foetal movement. We show that cells derived from FADS individuals have shorter and less primary cilia (PC), in association with alterations in post-translational modifications in α-tubulin. Similarly, siRNA-mediated depletion of two known FADS proteins, the scaffold protein rapsyn and the nucleoporin NUP88, resulted in defective PC formation. Consistent with a role in ciliogenesis, rapsyn and NUP88 localised to centrosomes and PC. Furthermore, proximity-ligation assays confirm the respective vicinity of rapsyn and NUP88 to γ-tubulin. Proximity-ligation assays moreover show that rapsyn and NUP88 are adjacent to each other and that the rapsyn-NUP88 interface is perturbed in the examined FADS cells. We suggest that the perturbed rapsyn-NUP88 interface leads to defects in PC formation and that defective ciliogenesis contributes to the pleiotropic defects seen in FADS.

Autosomal recessive polycystic kidney disease

2015 ◽  
Author(s):  
Carsten Bergmann ◽  
Klaus Zerres
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Autosomal Recessive ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Phenotypic Variability ◽  
Amino Acid Type ◽  
Polycystic Kidney ◽  
Exact Function ◽  
The Individual

Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of childhood renal- and liver-related morbidity and mortality with variable disease expression. Many patients manifest peri- or neonatally with a mortality rate of 30–50%, whereas others survive to adulthood with only minor clinical features. ARPKD is typically caused by mutations in the PKHD1 gene that encodes a 4074-amino acid type 1 single-pass transmembrane protein called fibrocystin or polyductin. Fibrocystin/polyductin is among other cystoproteins expressed in primary cilia, basal bodies, and centrosomes, but its exact function has still not been fully unravelled. Mutations were found to be scattered throughout the gene with many of them being private to single families. Correlations have been drawn for the type of mutation rather than for the site of the individual mutation. Virtually all patients carrying two truncating mutations display a severe phenotype with peri- or neonatal demise while surviving patients bear at least one hypomorphic missense mutation. However, about 20–30% of all sibships exhibit major intrafamilial phenotypic variability and it becomes increasingly obvious that ARPKD is clinically and genetically much more heterogeneous and complex than previously thought.

scholarly journals Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing

2017 ◽  
Vol 216 (7) ◽  
pp. 1949-1957 ◽  
Author(s):  
Bernadette Carroll ◽  
Glyn Nelson ◽  
Yoana Rabanal-Ruiz ◽  
Olena Kucheryavenko ◽  
Natasha A. Dunhill-Turner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Primary Cilia ◽  
Human Fibroblasts ◽  
Mammalian Target Of Rapamycin ◽  
Senescent Cell ◽  
Cell Senescence ◽  
Growth Factor Signaling ◽  
Mtorc1 Signaling ◽  
Underlying Mechanisms

Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well understood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival.

scholarly journals Multiomic identification of factors associated with progression to cystic kidney disease in mice with nephron Ift88 disruption

2021 ◽  
Author(s):  
Chunyan Hu ◽  
Katherine Beebe ◽  
Edgar J Hernandez ◽  
Jose M Lazaro-Guevara ◽  
Monica P Revelo ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Kidney Disease ◽  
Primary Cilia ◽  
Gene Mutations ◽  
Cystic Kidney Disease ◽  
Cystic Kidney ◽  
Factors Associated ◽  
Lipidomic Analysis ◽  
Renal Histology ◽  
Chain Acyl

Ift88 gene mutations cause primary cilia loss and polycystic kidney disease (PKD) in mice. Nephron Ift88 knockout (KO) at 2 months postnatal does not affect renal histology at 4 months postnatal and causes PKD only in males by 11 months postnatal. To identify factors associated with PKD development, kidneys from 4-month-old male and female control and Ift88 KO mice underwent transcriptomic, proteomic, western, metabolomic and lipidomic analysis. mRNAs involved in extracellular matrix (ECM) synthesis and degradation were selectively upregulated in male KO mice. Proteomic analysis was insufficiently sensitive to detect most ECM components, while western analysis paradoxically revealed reduced fibronectin and collagen I in male KO mice. Only male KO mice upregulated mRNAs encoding fibrinogen subunits and receptors for VEGF and PDGF; Per2, Per3 and Nrld2 clock mRNAs were selectively decreased in male KO mice. Proteomic, metabolomic and lipidomic analysis detected a relative (vs same sex control) decrease in factors involved in fatty acid ß-oxidation in female KO, while increased or unchanged levels in male KO, mice including medium chain acyl-CoA dehydrogenase, 3-hydroxybutyrate, and acylcarnitine. Three putative mRNA biomarkers of cystogenesis in male Ift88 KO mice (similar control levels between sexes and uniquely altered by KO in males) were identified, including high levels (Fga and Sdf2l1) and low levels (Banp) in male KO mice. These findings suggest that relative alterations in renal ECM metabolism, fatty acid ß-oxidation, and other pathways precede cystogenesis in Ift88 KO mice. In addition, potential novel biomarkers of cystogenesis in Ift88 KO mice have been identified.

scholarly journals Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

2021 ◽  
Vol 22 (10) ◽  
pp. 5293
Author(s):  
Marie Christine Martens ◽  
Janin Edelkamp ◽  
Christina Seebode ◽  
Mirijam Schäfer ◽  
Susanne Stählke ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Human Fibroblasts ◽  
Human Cell Line ◽  
Epidermal Differentiation ◽  
Loss Of Function ◽  
Lentiviral Transduction ◽  
Protein Receptor ◽  
Models Comparison

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

scholarly journals A Bbs5 mouse model reveals pituitary cilia contributions to developmental abnormalities

2020 ◽  
Author(s):  
Melissa R. Bentley ◽  
Staci E. Engle ◽  
Courtney J. Haycraft ◽  
Reagan S. Andersen ◽  
Mandy J. Croyle ◽  
...  
Keyword(s):  
Cognitive Deficits ◽  
Primary Cilia ◽  
Protein Composition ◽  
Cell Types ◽  
Postnatal Life ◽  
Bardet Biedl Syndrome ◽  
Developmental Abnormalities ◽  
Conditional Allele ◽  
Craniofacial Defects ◽  
Ciliary Signaling

AbstractPrimary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl Syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay, and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain which are developmental in origin. A subset of BBS proteins assembles into the BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here we describe two new mouse models for BBS resulting from a congenital null and conditional allele of Bbs5. Bbs5 null mice develop a complex phenotype including craniofacial defects, skeletal shortening, ventriculomegaly, infertility, and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly, and pituitary abnormalities are only found when Bbs5 is mutated prior to P7 indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity independent of the age of Bbs5 loss. Compared to other animal models of BBS, Bbs5 mutant mice exhibit pathologies that suggest a specialized role for Bbs5 in ciliary function.

scholarly journals Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

2019 ◽  
Author(s):  
Ramona Jühlen ◽  
Valérie Martinelli ◽  
Chiara Vinci ◽  
Jeroen Breckpot ◽  
Birthe Fahrenkrog
Keyword(s):  
Primary Cilium ◽  
Broad Spectrum ◽  
Primary Cilia ◽  
Neuromuscular Disorders ◽  
Human Fibroblasts ◽  
Post Translational Modifications ◽  
Proximity Ligation ◽  
Tubulin Isoforms ◽  
Clinical Disorders ◽  
Foetal Movement

AbstractCiliopathies are clinical disorders of the primary cilium with widely recognised phenotypic and genetic heterogeneity. Here we found impaired ciliogenesis in fibroblasts derived from individuals with fetal akinesia deformation sequence (FADS), a broad spectrum of neuromuscular disorders arising from impaired foetal movement. We show that cells derived from FADS individuals have shorter and less primary cilia (PC), in association with alterations in post-translational modifications in α-tubulin. Similarly, siRNA-mediated depletion of two known FADS proteins, the scaffold protein rapsyn and the nucleoporin NUP88, resulted in defective PC formation. Consistent with a role in ciliogenesis, rapsyn and NUP88 localised to centrosomes and PC. By proximity-ligation assays, we show that rapsyn and NUP88 are adjacent and that both proteins are adjoining to all three tubulin isoforms (α, and γ rapsyn-NUP88 interface, as well as their contact to microtubules, is perturbed in the examined FADS cells. We suggest that the perturbed rapsyn-NUP88-tubulin interface leads to defects in PC formation and that defective ciliogenesis contributes to the pleiotropic defects seen in FADS.SummaryFibroblasts derived from fetal akinesia individuals are characterised by ciliary defects and rapsyn and NUP88 are required for proper formation of the primary cilium.

scholarly journals Covid-19 genomic analysis reveals clusters of emerging sublineages within the delta variant

2021 ◽  
Author(s):  
Evans K. Rono
Keyword(s):  
Genetic Diversity ◽  
United Kingdom ◽  
Control Strategies ◽  
Gene Mutations ◽  
Genomic Analysis ◽  
Genomic Data ◽  
Diagnostic Tools ◽  
Future Studies ◽  
Evolutionary Trajectories ◽  
New Mutations

AbstractThe emerging SARS-CoV-2 variants may potentially have enhanced transmissibility and virulence of the virus, and impacts on performance of diagnostic tools and efficacy of vaccines. Genomic surveillance provides an opportunity to detect and characterize new mutations early enough for effective deployment of control strategies. Here, genomic data from Germany and United Kingdom were examined for genetic diversity by assessing gene mutations and inferring phylogeny. Delta variant sublineages were grouped into seven distinct clusters of spike mutations located in N-terminal domain of S1 region (T95I, D138H, *D142G, Y145H and A222V) and S2 region (T719I and *N950D). The most predominant cluster was T95I mutation, with the highest frequencies (71.1% - 83.9%) in Wales, England and Scotland, and the least frequencies (8.9% - 12.1%) in Germany. Two mutations, *D142G and *N950D here described as *reverse mutations and T719I mutation, were largely unique to Germany. In a month, frequencies of D142G had increased from 55.6% to 67.8 % in Germany. Additionally, a cluster of D142G+T719I/T mutation went up from 27.7% to 34.1%, while a T95I+ D142G+N950D/N cluster rose from 19.2% to 26.2%. Although, two distinct clusters of T95I+D138H (2.6% - 3.8%) and T95I+Y145H+A222V (2.5% - 8.5%) mutations were present in all the countries, they were most predominant in Wales and Scotland respectively. Results suggest divergent evolutionary trajectories between the clusters of D142G mutation and those of T95I mutation. These findings provide insights into underlying dynamics of evolution of the delta variant. Future studies may evaluate the epidemiological and biological implications of these sublineages.

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