Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Associations of common genetic risk variants of the muscarinic acetylcholine receptor M2 with cardiac autonomic dysfunction in patients with schizophrenia.

OBJECTIVES: Decreased vagal modulation, which has consistently been observed in schizophrenic patients, might contribute to increased cardiac mortality in schizophrenia. Previously, associations between CHRM2 (Cholinergic Receptor Muscarinic 2) and cardiac autonomic features have been reported. Here, we tested for possible associations between these polymorphisms and heart rate variability in patients with schizophrenia. METHODS: A total of three single nucleotide polymorphisms (SNPs) in CHRM2 (rs73158705 A>G, rs8191992 T>A and rs2350782 T>C) that achieved significance (p < 5 * 10-8) in genome-wide association studies for cardiac autonomic features were genotyped in 88 drug-naïve patients, 61 patients receiving antipsychotic medication and 144 healthy controls. Genotypes were analysed for associations with parameters of heart rate variability and complexity, in each diagnostic group. RESULTS: We observed a significantly altered heart rate variability in unmedicated patients with identified genetic risk status in rs73158705 A>G, rs8191992 T>A and rs2350782 T>C as compared to genotype non-risk status. In patients receiving antipsychotic medication and healthy controls, these associations were not observed. DISCUSSION: We report novel candidate genetic associations with cardiac autonomic dysfunction in schizophrenia, but larger cohorts are required for replication.

Analysis of CACNA1C and KCNH2 Risk Variants on Cardiac Autonomic Function in Patients with Schizophrenia.

BACKGROUND: Cardiac autonomic dysfunction (CADF) is a major contributor to increased cardiac mortality in schizophrenia patients. The aberrant function of voltage-gated ion channels, which are widely distributed in the brain and heart, may link schizophrenia and CADF. In search of channel-encoding genes that are associated with both CADF and schizophrenia, CACNA1C and KCNH2 are promising candidates. In this study, we tested for associations between genetic findings in both genes and CADF parameters in schizophrenia patients whose heart functions were not influenced by psychopharmaceuticals. METHODS: First, we searched the literature for single-nucleotide polymorphisms (SNPs) in CACNA1C and KCNH2 that showed genome-wide significant association with schizophrenia. Subsequently, we looked for such robust associations with CADF traits at these loci. A total of 5 CACNA1C SNPs and 9 KCNH2 SNPs were found and genotyped in 77 unmedicated schizophrenia patients and 144 healthy controls. Genotype-related impacts on heart rate (HR) dynamics and QT variability indices (QTvi) were analyzed separately in patients and healthy controls. RESULTS: We observed significantly increased QTvi in unmedicated patients with CADF-associated risk in CACNA1C rs2283274 C and schizophrenia-associated risk in rs2239061 G compared to the non-risk allele in these patients. Moreover, unmedicated patients with previously identified schizophrenia risk alleles in KCNH2 rs11763131 A, rs3807373 A, rs3800779 C, rs748693 G, and 1036145 T showed increased mean HR and QTvi as compared to non-risk alleles. CONCLUSIONS: We propose a potential pleiotropic role for common variation in CACNA1C and KCNH2 associated with CADF in schizophrenia patients, independent of antipsychotic medication, that predisposes them to cardiac arrhythmias and premature death.

Histone demethylase KDM4C is a functional dependency in JAK2-mutated neoplasms.

Mutations of the JAK2 gene are frequent aberrations in the aging hematopoietic system and in myeloid neoplasms. While JAK-inhibitors efficiently reduce hyperinflammation induced by the constitutively active mutated JAK2 kinase, the malignant clone and abundance of mutated cells remains rather unaffected. Here, we sought to assess for genetic vulnerabilities of JAK2-mutated clones. We identified lysine-specific demethylase KDM4C as a selective genetic dependency that persists upon JAK-inhibitor treatment. Genetic inactivation of KDM4C in human and murine JAK2-mutated cells resulted in loss of cell competition and reduced proliferation. These findings led to reduced disease penetrance and improved survival in xenograft models of human JAK2-mutated cells. KDM4C deleted cells showed alterations in target histone residue methylation and target gene expression, resulting in induction of cellular senescence. In summary, these data establish KDM4C as a specific dependency and therapeutic target in JAK2-mutated cells that is essential for oncogenic signaling and prevents induction of senescence.

The androgen receptor-lncRNASAT1-AKT-p15 axis mediates androgen-induced cellular senescence in prostate cancer cells.

The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.

RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis.

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3-regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes-platinum resistance and angiogenesis.

Disruption of KCC2 in Parvalbumin-Positive Interneurons Is Associated With a Decreased Seizure Threshold and a Progressive Loss of Parvalbumin-Positive Interneurons.

GABAA receptors are ligand-gated ion channels, which are predominantly permeable for chloride. The neuronal K-Cl cotransporter KCC2 lowers the intraneuronal chloride concentration and thus plays an important role for GABA signaling. KCC2 loss-of-function is associated with seizures and epilepsy. Here, we show that KCC2 is expressed in the majority of parvalbumin-positive interneurons (PV-INs) of the mouse brain. PV-INs receive excitatory input from principle cells and in turn control principle cell activity by perisomatic inhibition and inhibitory input from other interneurons. Upon Cre-mediated disruption of KCC2 in mice, the polarity of the GABA response of PV-INs changed from hyperpolarization to depolarization for the majority of PV-INs. Reduced excitatory postsynaptic potential-spike (E-S) coupling and increased spontaneous inhibitory postsynaptic current (sIPSC) frequencies further suggest that PV-INs are disinhibited upon disruption of KCC2. In vivo, PV-IN-specific KCC2 knockout mice display a reduced seizure threshold and develop spontaneous sometimes fatal seizures. We further found a time dependent loss of PV-INs, which was preceded by an up-regulation of pro-apoptotic genes upon disruption of KCC2.

Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms.

Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.

Sepsis induces long-lasting impairments in CD4+ T-cell responses despite rapid numerical recovery of T-lymphocyte populations.

Massive apoptosis of lymphocytes is a hallmark of sepsis. The resulting immunosuppression is associated with secondary infections, which are often lethal. Moreover, sepsis-survivors are burdened with increased morbidity and mortality for several years after the sepsis episode. The duration and clinical consequences of sepsis induced-immunosuppression are currently unknown. We have used the mouse model of peritoneal contamination and infection (PCI) to investigate the quantitative and qualitative recovery of T lymphocytes for 3.5 months after sepsis with or without IL-7 treatment. Thymic output and the numbers of naive and effector/memory CD4+ and CD8+ lymphocytes quickly recovered after sepsis. IL-7 treatment resulted in an accelerated recovery of CD8+ lymphocytes. Next generation sequencing revealed no significant narrowing of the T cell receptor repertoire 3.5 months after sepsis. In contrast, detailed functional analyses of T helper (Th)-cell responses towards a fungal antigen revealed a significant loss of Th cells. Whereas cytokine production was not impaired at the single cell level, the absolute number of Th cells specific for the fungal antigen was reduced. Our data indicate a clinically relevant loss of pathogen-specific T cell clones after sepsis. Given the small number of naive T lymphocytes specific for a given antigen, this decrement of T cell clones remains undetected even by sensitive methods such as deep sequencing. Taken together, our data are compatible with long lasting impairments in CD4+ T-cell responses after sepsis despite rapid recovery of T lymphocyte populations.

MiR-145 expression and rare NOTCH1 variants in bicuspid aortic valve-associated aortopathy.

MicroRNAs (miRNAs) may serve as elegant tool to improve risk stratification in bicuspid aortic valve (BAV)-associated aortopathy. However, the exact pathogenetic pathway by which miRNAs impact aortopathy progression is unknown. Herewith, we aimed to analyze the association between circulating miRNAs and rare variants of aortopathy-related genes. 63 BAV patients (mean age 47.3±11.3 years, 92% male) with a root dilatation phenotype, who underwent aortic valve+/-proximal aortic surgery at a single institution (mean post-AVR follow-up 10.3±6.9 years) were analyzed. A custom-made HaloPlex HS panel including 20 aortopathy-related genes was used for the genetic testing. miRNAs were extracted from whole blood and miRNA analysis was performed using miRNA-specific assay. Study endpoint was the association between circulating miRNAs and rare genetic variants in the aortopathy gene panel. The study cohort was divided into a subgroup with rare variants vs. a subgroup without rare variants based on the presence of rare variants in the respective genes (i.e., at least one variant present). The genetic analysis yielded n = 6 potentially and likely pathogenic rare variants within the NOTCH1 gene as being the most common finding. Univariate analysis between blood miRNAs and NOTCH1 variants revealed a significantly lower expression of miR-145 in the subgroup of patients with NOTCH1 variants vs. those without NOTCH1 variants (i.e., delta Ct 4.95±0.74 vs. delta Ct 5.57±0.78, p = 0.04). Our preliminary data demonstrate a significant association between blood miR-145 expression and the presence of rare NOTCH1 variants. This association may be indicative of a specific pathogenetic pathway in the development of genetically-triggered bicuspid aortopathy.

Posterior column ataxia with retinitis pigmentosa coexisting with sensory-autonomic neuropathy and leukemia due to the homozygous p.Pro221Ser FLVCR1 mutation.

FLVCR1 encodes for a ubiquitous heme exporter, whose recessive mutations cause posterior column ataxia with retinitis pigmentosa (PCARP). Recently, FLVCR1 recessive mutations were also found in two sporadic children with hereditary sensory-autonomic neuropathy (HSAN). We report the unique case of a 33-year-old Italian woman with a combination of typical PCARP, sensory-autonomic neuropathy with sensory loss to all modalities and multiple autonomic dysfuctions, and acute lymphocytic leukemia. Molecular analysis demonstrated homozygosity for the previously identified FLVCR1 p.Pro221Ser variation. The same variation, in combination with a frameshift mutation, was previously identified in an Italian child with HSAN. Functional studies carried out on patient-derived lymphoblastoid cell lines showed decreased FLVCR1a transcript, increased reactive oxygen species, excessive intracellular heme accumulation, and increased number of Annexin V positive cells. This indicates that the homozygous p.Pro221Ser FLVCR1 variation compromises the ability of FLVCR1a to export heme leading to enhanced susceptibility to programmed cell death. Our study demonstrates the existence of a phenotypic continuum among the discrete disorders previously linked to FLVCR1 mutations, and suggests that the related alteration of heme metabolism may lead to the degeneration of specific neuronal cell populations.

Genetic abnormalities in bicuspid aortic valve root phenotype: preliminary results.

OBJECTIVES: Genetic defects associated with bicuspid aortopathy have been infrequently analysed. Our goal was to examine the prevalence of rare genetic variants in patients with a bicuspid aortic valve (BAV) with a root phenotype using next-generation sequencing technology. METHODS: We investigated a total of 124 patients with BAV with a root dilatation phenotype who underwent aortic valve ± proximal aortic surgery at a single institution (BAV database, n = 812) during a 20-year period (1995-2015). Cross-sectional follow-up revealed 63 (51%) patients who were still alive and willing to participate. Systematic follow-up visits were scheduled from March to December 2015 and included aortic imaging as well as peripheral blood sampling for genetic testing. Next-generation sequencing libraries were prepared using a custom-made HaloPlex HS gene panel and included 20 candidate genes known to be associated with aortopathy and BAV. The primary end-point was the prevalence of genetic defects in our study cohort. RESULTS: A total of 63 patients (mean age 46 ± 10 years, 92% men) with BAV root phenotype and mean post-aortic valve replacement follow-up of 10.3 ± 4.9 years were included. Our genetic analysis yielded a wide spectrum of rare, potentially or likely pathogenic variants in 19 (30%) patients, with NOTCH1 variants being the most common ( n = 6). Moreover, deleterious variants were revealed in AXIN1 ( n = 3), NOS3 ( n = 3), ELN ( n = 2), FBN1 ( n = 2) , FN1 ( n = 2) and rarely in other candidate genes. CONCLUSIONS: Our preliminary study demonstrates a high prevalence and a wide spectrum of rare genetic variants in patients with the BAV root phenotype, indicative of the potentially congenital origin of associated aortopathy in this specific BAV cohort.

Stroke-like onset of brain stem degeneration presents with unique MRI sign and heterozygous NMNAT2 variant: a case report.

BACKGROUND: Acute-onset neurodegenerative diseases in older patients are rare clinical cases, especially when the degeneration only affects specific regions of the nervous system. Several neurological disorders have been described in which the degeneration of brain parenchyma originates from and/or primarily affects the brain stem. Clinical diagnosis in these patients, however, is often complicated due to a poor understanding of these diseases and their underlying mechanisms. CASE PRESENTATION: In this manuscript we report on a 73-year-old female who had experienced a sudden onset of complex neurological symptoms that progressively worsened over a period of 2 years. Original evaluation had suggested a MRI-negative stroke as underlying pathogenesis. The combination of patient's medical history, clinical examination and exceptional pattern of brain stem degeneration presenting as "kissing swan sign" in MR imaging was strongly suggestive of acute onset of Alexander's disease. This leukoencephalopathy is caused by GFAP (glial fibrilary acidic protein) gene mutations and may present with brain stem atrophy and stroke-like onset of symptoms in elderly individuals. However, a pathognomonic GFAP gene mutation could not be identified by Sanger sequencing. CONCLUSIONS: After an extended differential diagnosis and exclusion of other diseases, a definite diagnosis of the patient's condition presently remains elusive. However, whole-exome sequencing performed from patient's blood revealed 12 potentially disease-causative heterozygous variants, amongst which several have been associated with neurological disorders in vitro and in vivo - in particular the axon degeneration-related NMNAT2 gene.

Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.