Genotype-phenotype correlation in contactin-associated protein-like 2 (CNTNAP-2) developmental disorder.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Leu226Trp CACNA1A variant associated with juvenile myoclonic epilepsy with and without intellectual disability.

OBJECTIVE: Epilepsy is a disease of Central Nervous System (CNS) characterized by abnormal brain activity and recurrent seizures and is considered a clinically and genetically heterogeneous disease. Here, we investigated pathogenic genetic alteration and described the clinical characteristics of three Iranian family members affected by Idiopathic Generalized Epilepsy (IGE) with and without intellectual disability. METHODS: A non-consanguineous Iranian family with juvenile myoclonic epilepsy was enrolled in the study. The comprehensive neurological evaluation included motor and sensory skills, vision, hearing, speech, coordination, and mood. Whole-exome Sequencing (WES) was performed on the proband to detect probable pathogenic variant, and after the filtering process, probable variants were evaluated with familial segregation analysis using Sanger sequencing. RESULTS: Using WES, we identified a heterozygous missense substitution (NM_023035.3:c.T677G:p.Leu226Trp) in CACNA1A gene in the studied family with juvenile myoclonic epilepsy with and without intellectual disability and psychiatric phenotype. Considering the patients' clinical synopsis, familial segregation analysis, and literature review, we postulated this variant to be causative of the disease. Indeed, the resulting missense mutation of Leu226Trp affects a highly conserved residue supporting our hypothesis that this mutation is potentially pathogenic. CONCLUSION: To the best of our knowledge, this is the first report of juvenile myoclonic epilepsy related to CACNA1A gene. Our results provide evidence for expanding the clinical and molecular findings related to the CACNA1A gene.

Transcription factor mesenchyme homeobox protein 2 (MEOX2) modulates nociceptor function.

Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. We have previously identified dysregulation of MEOX2 in fibroblasts from Congenital Insensitivity to Pain patients, and confirmed that btn, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, we used a Meox2 heterozygous (Meox2+/- ) mouse model to characterise its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localises in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioural, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarisation. Mechanistically, we noted decreased expression of Scn9a and Scn11a genes encoding Nav 1.7 and Nav 1.9 voltage-gated sodium channels respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2+/- DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, we propose a novel role of MEOX2 in primary afferent nociceptor neurons for the maintenance of a transcriptional programme required for proper perception of acute and inflammatory noxious stimuli.

FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder.

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

AXIN-1 protein expression and localization in glioblastoma.

The etiology and pathogenesis of tumors of the central nervous system are still inadequately explained. In the present study the expression patterns of a critical molecular component of wnt signaling pathway - axin I was investigated in 42 patients with glioblastoma, the most aggressive form of glial tumors. Immunostaining and image analysis revealed the quantity and localization of the protein. Downregulation of this tumor suppressor expression was observed in 31% of tumors when compared to the levels of axin in healthy brain tissues. Axin was observed in the cytoplasm in 69% of glioblastoma samples, in 21.4% in both the cytoplasm and nucleus and 9.5% had expression solely in the nucleus. Mean values of relative axin's expression obtained by image analysis showed that the highest relative quantity of axin was measured when the protein was in the nucleus and the lowest relative quantity of axin when the protein was localized in the cytoplasm. Investigation on axin's existence at the subcellular level in glioblastomas suggests that axin's expression and spatial regulation is a dynamic process. Despite increasing knowledge on glioma biology and genetics, the prognostic tools for glioblastoma still need improvement. Our findings on expression of axin 1 may contribute to better understanding of glioblastoma molecular profile.

Network analysis reveals rare disease signatures across multiple levels of biological organization.

Rare genetic diseases are typically caused by a single gene defect. Despite this clear causal relationship between genotype and phenotype, identifying the pathobiological mechanisms at various levels of biological organization remains a practical and conceptual challenge. Here, we introduce a network approach for evaluating the impact of rare gene defects across biological scales. We construct a multiplex network consisting of over 20 million gene relationships that are organized into 46 network layers spanning six major biological scales between genotype and phenotype. A comprehensive analysis of 3,771 rare diseases reveals distinct phenotypic modules within individual layers. These modules can be exploited to mechanistically dissect the impact of gene defects and accurately predict rare disease gene candidates. Our results show that the disease module formalism can be applied to rare diseases and generalized beyond physical interaction networks. These findings open up new venues to apply network-based tools for cross-scale data integration.

Expanding the known phenotype of Mullegama-Klein-Martinez syndrome in male patients.

Here, we report a novel case of a male patient with a hemizygous missense variant in STAG2 (p.Tyr159His) resulting in Mullegama-Klein-Martinez syndrome (MKMS), a rare X-linked cohesinopathy. He shares distinct clinical features with a previously reported male patient carrying the STAG2 variant p.Tyr159Cys, suggesting that this phenotype is determined by the position of the mutation. Additionally, our patient exhibits symptoms not previously associated with MKMS, expanding the known clinical phenotype of this rare disease.

PRDM12 Is Transcriptionally Active and Required for Nociceptor Function Throughout Life.

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.

PRDM12: New Opportunity in Pain Research.

PRDM12 is a newly identified causative gene for a type of congenital insensitivity to pain disorder, which is characterized by the inability to perceive pain. Here, we discuss the (patho)physiology of PRDM12 function and the opportunities and challenges those data provide for novel therapeutic approaches in various pain disorders.

Acute poisonings in Croatia: differences in epidemiology, associated comorbidities and final outcomes - a single-centre 15-year follow-up.

PURPOSE: To determine the demographic and social characteristics of acutely intoxicated patients in the emergency department (ED), intention of drug/chemical intake, main toxic agents, outcomes and time trends for all variables. METHODS: This prospective, observational study included acutely intoxicated patients treated in the ED of the University Hospital Centre Sisters of Charity, Zagreb, Croatia, during the years 2001, 2010 and 2015. The diagnosis was derived from patient's self-report, clinical observations and laboratory test results (quantitative test for serum ethanol levels and qualitative tests for drugs in urine). RESULTS: A total of 1593 patients were enrolled in the study (331 in 2001, 618 in 2010, 644 in 2015), with a predominance of men (55.9%, 65.2%, 70.7%, respectively). The median age was 28 [18-89], 39 [18-92] and 40 years [18-95], respectively. There was a decline in the number of suicide attempts during the study (46.2%, 22.2%, 17.1% of patients, respectively) with a predominance of women (64.1-68.2%), and an escalation in the number of unintentional overdoses by substances of abuse (50.2%, 72.3%, 81.7% of patients, respectively) with a predominance of men (74.1-79.1%). Ethanol was the main toxic agent (40.2%, 69.4%, 75.8% of patients, respectively), used primarily as a substance of unintentional overdose. Anxiolytics were the most frequently implicated pharmaceuticals among all patients (46.5%, 32.0%, 18.5% of patients, respectively) and the main substance used in suicide attempts. The number of patients hospitalized in the Intensive Care Unit (ICU) decreased during the study from 20.2% of all patients in 2001 to 7.9% in 2010 and 6.8% in 2015. They accounted for 7.2%, 5.8% and 5.6% of all ICU-treated patients, respectively. In-hospital mortality (ED and ICU) caused by acute intoxications was low (0.9%, 0.8%, 0.8%, respectively). CONCLUSIONS: The escalation of overdoses by ethanol was a major medical and public concern in Zagreb. Anxiolytics were the main substance used in suicide attempts, with a low death rate due to their good safety profile.

Changes of AXIN-1 and beta-catenin in neuroepithelial brain tumors.

In the present study changes of components of Wnt signaling pathway--axin (AXIN1) and beta-catenin (CTNNB1) in a sample of 72 neuroepithelial brain tumors were investigated. AXIN-1 gene was tested by PCR/loss of heterozygosity (LOH). Immunostaining and image analysis revealed the quantity and localization of relevant proteins. Polymorphic marker for AXIN-1, showed LOH in 11.1% of tumors. LOH was distributed to 6.3% of glioblastomas, one was found in neuroepithelial dysembrioplastic tumor and one in medulloblastoma. Down regulation of axin expression and up regulation of beta-catenin were detected in the analyzed tumors. Axin was observed in the cytoplasm in 68.8% of samples, in 28.1% in both the cytoplasm and nucleus and 3.1% had no expression. Beta-catenin was observed mainly in the nucleus and cytoplasm (59.4%). Expression in 34.4% of samples was in the cytoplasm and 6.3% showed no expression. Comparison of mean values of relative increase of axin and beta-catenin showed that they are significantly reversely proportional (P = 0.014). Relative quantity of beta-catenin in patients with gross deletion of AXIN1 was significantly higher in comparison to patients without LOH (P = 0.040). Our results demonstrate that changes of key components of the Wnt signaling play a role in neuroepithelial brain tumors.