Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons

Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.</p

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained &gt;95% target region coverage at a read depth of 20 and &gt;50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p

Variable loss of functional activities of androgen receptor mutants in patients with androgen insensitivity syndrome

Androgen receptor (AR) mutations in androgen insensitivity syndrome (AIS) are associated with a variety of clinical phenotypes. The aim of the present study was to compare the molecular properties and potential pathogenic nature of 8 novel and 3 recurrent AR variants with a broad variety of functional assays. Eleven AR variants (p.Cys177Gly, p.Arg609Met, p.Asp691del, p.Leu701Phe, p.Leu723Phe, p.Ser741Tyr, p.Ala766Ser, p.Arg775Leu, p.Phe814Cys, p.Lys913X, p.Ile915Thr) were analyzed for hormone binding, transcriptional activation, cofactor binding, translocation to the nucleus, nuclear dynamics, and structural conformation. Ligand-binding domain variants with low to intermediate transcriptional activation displayed aberrant Kd values for hormone binding and decreased nuclear translocation. Transcriptional activation data, FxxFF-like peptide binding and DNA binding correlated well for all variants, except for p.Arg609Met, p.Leu723Phe and p.Arg775Leu, which displayed a relatively higher peptide binding activity. Variants p.Cys177Gly, p.Asp691del, p.Ala766Ser, p.Phe814Cys, and p.Ile915Thr had intermediate or wild type values in all assays and showed a predominantly nuclear localization in living cells. All transcriptionally inactive variants (p.Arg609Met, p.Leu701Phe, p.Ser741Tyr, p.Arg775Leu, p.Lys913X) were unable to bind to DNA and were associated with complete AIS. Three variants (p.Asp691del, p.Arg775Leu, p.Ile915Thr) still displayed significant functional activities in in vitro assays, although the clinical phenotype was associated with complete AIS. The data show that molecular phenotyping based on 5 different functional assays matched in most (70%) but not all cases. Copyrigh

The role of organic nutrients in structuring freshwater phytoplankton communities in a rapidly changing world

Carbon, nitrogen, and phosphorus are critical macroelements in freshwater systems. Historically, researchers and managers have focused on inorganic forms, based on the premise that the organic pool was not available for direct uptake by phytoplankton. We now know that phytoplankton can tap the organic nutrient pool through a number of mechanisms including direct uptake, enzymatic hydrolysis, mixotrophy, and through symbiotic relationships with microbial communities. In this review, we explore these mechanisms considering current and projected future anthropogenically-driven changes to freshwater systems. In particular, we focus on how naturally- and anthropogenically- derived organic nutrients can influence phytoplankton community structure. We also synthesize knowledge gaps regarding phytoplankton physiology and the potential challenges of nutrient management in an organically dynamic and anthropogenically modified world. Our review provides a basis for exploring these topics and suggests several avenues for future work on the relation between organic nutrients and eutrophication and their ecological implications in freshwater systems

High-yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing

Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.</p

Identification of a large rearrangement in CYLD as a cause of familial cylindromatosis

Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. To date, only technologies which are able to identify small point mutations in CYLD, such as sequence and WAVE analysis, were used. Here we describe the identification of a larger rearrangement identified by Quantitative PCR analysis of CYLD, indicating that a combination of these technologies is necessary when searching for pathogenic mutations in CYLD