Ultrasound targeted microbubble destruction using docetaxel and Rose Bengal loaded Microbubbles for targeted Chemo-Sonodynamic therapy treatment of prostate cancer.

Docetaxel (DTX) chemotherapy is commonly used in the treatment of patients with advanced prostate cancer demonstrating modest improvements in survival. As these patients are often elderly and the chemotherapy treatment is not targeted, it is often poorly tolerated. More targeted approaches that increase therapeutic efficacy yet reduce the amount of toxic chemotherapy administered are needed. In this manuscript, we investigate the potential of ultrasound targeted microbubble destruction (UTMD) to deliver a combination of docetaxel chemotherapy and Rose Bengal mediated sonodynamic therapy (SDT) in pre-clinical prostate cancer models. A Rose Bengal modified phospholipid was synthesized and used as a component lipid to prepare a microbubble (MB) formulation that was also loaded with DTX. The DTX-MB-RB formulation was used in the UTMD mediated treatment of androgen sensitive and androgen resistant 3D spheroid and murine models of prostate cancer. Results from the 3D spheroid experiments showed UTMD mediated DTX-MB-RB chemo-sonodynamic therapy to be significantly more effective at reducing cell viability than UTMD mediated DTX or SDT treatment alone. In an androgen sensitive murine model of prostate cancer, UTMD mediated DTX-MB-RB chemo-sonodynamic therapy was as effective as androgen deprivation therapy (ADT) at controlling tumour growth. However, when both treatments were combined, a significant improvement in tumour growth delay was observed. In an androgen resistant murine model, UTMD mediated DTX-MB-RB chemo-sonodynamic therapy was significantly more effective than standard DTX monotherapy. Indeed, the DTX dose administered using the DTX-MB-RB formulation was 91% less than standard DTX monotherapy. As a result, UTMD mediated DTX-MB-RB treatment was well tolerated while animals treated with DTX monotherapy displayed significant weight loss which was attributed to acute toxic effects. These results highlight the potential of UTMD mediated DTX-MB-RB chemo-sonodynamic therapy as a targeted, well tolerated alternative treatment for advanced prostate cancer.

Relationship Between Electroencephalography and Seizure Outcome in Typical Absence Seizures in Children.

BACKGROUND: Typical absence seizures (TAS) are seen in idiopathic generalized epilepsy. Electroencephalography (EEG) contributes to syndrome characterization and counseling in an area where genetics does not currently play a significant role. Prominent interictal EEG findings are seen in juvenile absence epilepsy (JAE) and are thus thought to be associated with less favorable outcome in any TAS case despite lack of evidence. Our study evaluates EEG findings and their association with seizure outcomes in children with TAS. METHODS: Retrospective cohort study of 123 children over 10 years with extensive EEG analysis and medical record review. Phone interviews ascertained longer-term outcomes. EEG reviewers were unaware of outcomes. RESULTS: Total cohort included 123 children with phone review completed in 98. Median follow-up was 5 years 9 months. Seizure freedom was seen in 59% off antiseizure medicines (ASMs). Interictal findings included focal discharges in 29%, fragments of spike-wave (SW) discharges in 82.1%, and generalized interictal discharges in 63.4%. Interictal SW was more likely in those who slept (100%, 18 of 18) versus those who did not (57%, 60 of 105) (P < 0.001). Outcome analysis found no associations between focal or generalized interictal findings and seizure freedom, relapse off ASM, occurrence of other seizure types, or response to first ASM. CONCLUSION: Focal and generalized interictal EEG discharges are common in children with TAS and are not associated with poorer outcomes. These interictal findings were traditionally associated with JAE rather than childhood absence epilepsy and were thus believed to be associated with potentially poorer outcome, which is probably not the case.

Autonomic instability, arrhythmia and visual impairment in a new presentation of MTFMT-related mitochondrial disease.

Mitochondrial methionyl-tRNA formyltransferase (MTFMT) is required for the initiation of translation in mitochondria. Pathogenic variants in MTFMT have been described in association with clinical presentations with Leigh syndrome, as well with as multisystem involvement (particularly cardiac and ocular involvement). There is a spectrum of severity, but many reported presentations have been milder with a better prognosis than other pathogenic variants associated with Leigh syndrome. We describe the case of a 9-year-old boy homozygous for a pathogenic MTFMT variant (c.626C > T/p.Ser209Leu) who presented with hypertensive crisis on a background of hyperphagia and visual impairment. His clinical course was complicated by supraventricular tachycardia and severe autonomic instability, requiring intensive care unit admission. He also developed seizures, neurogenic bladder and bowel and had a markedly abnormal eye examination with bilateral optic atrophy. Magnetic resonance image brain showed abnormal high T2/fluid-attenuated inversion recovery signal within the dorsal brainstem and in the right globus pallidus with some reduced diffusivity. Despite recovery from the acute neurological and cardiac manifestations, he has ongoing deficits in his gross motor skills and continues to have hyperphagia with rapid weight gain (approx. 20 kg in 2 years). Ophthalmic findings are persistent. This case expands the phenotype associated with MTFMT disease.

Room to improve: The diagnostic journey of Spinal Muscular Atrophy.

AIMS: To highlight the current diagnostic pathway for children with Spinal Muscular Atrophy (SMA) in Ireland. We look to identify points along the diagnostic pathway that may impede a timely diagnosis, and argue that newborn screening for SMA is the single best measure to remediate these delays. METHODS: Through retrospective chart review and an online questionnaire, we gathered SMA patient data outlining clinical characteristics and the route to diagnosis of the SMA cohort attending the National SMA Treatment centre at Children's Health Ireland. RESULTS: We found that 32 children were diagnosed with SMA in Ireland in the 15-years from 2007 to 2021, with twelve cases of SMA type I. Muscle weakness is the most commonly reported initial sign, and the GP is usually the first health provider to address parental concerns. Patients commonly experience delays in diagnosis due to factors such as varied SMA clinical phenotypes, and a lack of experience or awareness of SMA amongst community based health care practitioners. In spite of this, when patients do gain early access to tertiary diagnostics through prenatal or neonatal genetic testing, they then report rapid diagnosis and initiation of disease modifying therapy in the crucial pre-symptomatic window. CONCLUSION: We conclude that delays to diagnosis inherent within the current Irish system are pervasive and arise prior to engagement with tertiary services. All of these delays are remediable through the establishment of a dedicated SMA newborn screening programme.

Biomarkers to assess the risk of bladder cancer in patients presenting with haematuria are gender-specific.

Introduction: Haematuria is a common red flag symptom of urinary tract cancer. Bladder cancer (BC) is the most common cancer to present with haematuria. Women presenting with haematuria are often underdiagnosed. Currently, no gender-specific tests are utilized in clinical practice. Considerable healthcare resources are needed to investigate causes of haematuria and this study was set up to help identify markers of BC. The aim of the study was to define biomarker algorithms in haematuria patients using an expanded panel of biomarkers to diagnose BC and investigate if the algorithms are gender-specific. Materials and Methods: A total of n=675 patients with a history of haematuria were recruited from Northern Ireland hospitals. Patients were collected on a 2:1 ratio, non-BC (control) n=474: BC n=201. A detailed clinical history, urine and blood samples were collected. Biomarkers, known to be involved in the pathobiology underlying bladder carcinogenesis were investigated. Biomarkers differentially expressed between groups were investigated using Wilcoxon rank sum and linear regression. Results: Biomarkers were gender specific. Two biomarker-algorithms were identified to triage haematuria patients; male - u_NSE, s_PAI-1/tPA, u_midkine, u_NGAL, u_MMP-9/TIMP-1 and s_prolactin (u=urine; s=serum); sensitivity 71.8%, specificity 72.8%; AUROC 0.795; and female urine biomarkers - IL-12p70, IL-13, midkine and clusterin; sensitivity 83.7%, specificity 79.7%; AUROC 0.865. Addition of the clinical variable infection to both algorithms increased both AUROC to 0.822 (DeLong p=0.014) and to 0.923 (DeLong p=0.004) for males and females, respectively. Combining clinical risk factors with biomarker algorithms would enable application of the algorithms to triage haematuria patients. Conclusion: Using gender-specific biomarker algorithms in combination with clinical risks that are associated with BC would allow clinicians to better manage haematuria patients and potentially reduce underdiagnosis in females. In this study, we demonstrate, for the first time, that blood and urine biomarkers are gender-specific when assessing risk of BC in patients who present with blood in their urine. Combining biomarker data with clinical factors could improve triage when referring patients for further investigations.

Assessment of face validity of a disease model of nonsense mutation Duchenne muscular dystrophy: a multi-national Delphi panel study.

OBJECTIVE: The objective of this study was to assess the face validity of a disease model evaluating the cost-effectiveness of ataluren for the treatment of nonsense mutation Duchenne muscular dystrophy (nmDMD). METHODS: This was a Delphi panel study comprising of physicians with first-hand experience of ataluren for the treatment of nmDMD. Consensus was sought for previously unvalidated model data, including patient health status and quality of life measured using the Health Utility Index (HUI), mortality, informal caregiving, and the expected benefit of early ataluren treatment across four states: (1) ambulatory, (2) non-ambulatory, not yet requiring ventilation support, (3) non-ambulatory, night-time ventilation support, and (4) non-ambulatory, full-time ventilation support. RESULTS: Nine experts from five countries participated in the Delphi panel. Consensus was obtained for all questions after three panel rounds (except for two HUI-questions concerning hand function [dexterity]). Consensus HUI-derived utilities for state (1) were 1.0000 for ataluren on top of best supportive care (BSC) and 0.7337 for BSC alone. Corresponding estimates for state (2) were 0.3179 and 0.2672, for state (3) 0.1643 and 0.0913, and for state (4) -0.0732 and -0.1163. Consensus mortality rates for states (1), (2), and (3) were 4%, 13%, and 33%, and life expectancy in state (4) was agreed to be 3 years. Panelists further agreed that two informal caregivers typically provide day-to-day care/support to patients with nmDMD, and that starting treatment with ataluren at 2 versus 5 years of age would be expected to delay loss of ambulation by an additional 2 years, and initiation of night-time and full-time ventilation support by an additional 3 years, respectively. LIMITATIONS: The main limitation concerns the size of the Delphi panel, govern primarily by the rarity of the disease. CONCLUSION: This study confirms the face validity of key clinical parameters and assumptions underlying the ataluren cost-effectiveness model.

Clinical features and predictive biomarkers for bladder cancer in patients with type 2 diabetes presenting with haematuria.

AIMS: To identify clinical features and protein biomarkers associated with bladder cancer (BC) in individuals with type 2 diabetes mellitus presenting with haematuria. MATERIALS AND METHODS: Data collected from the Haematuria Biomarker (HaBio) study was used in this analysis. A matched sub-cohort of patients with type 2 diabetes and patients without diabetes was created based on age, sex, and BC diagnosis, using approximately a 1:2 fixed ratio. Randox Biochip Array Technology and ELISA were applied for measurement of 66 candidate serum and urine protein biomarkers. Hazard ratios and 95% confidence intervals were estimated by chi-squared and Wilcoxon rank sum test for clinical features and candidate protein biomarkers. Diagnostic protein biomarker models were identified using Lasso-based binominal regression analysis. RESULTS: There was no difference in BC grade, stage, and severity between individuals with type 2 diabetes and matched controls. Incidence of chronic kidney disease (CKD) was significantly higher in patients with type 2 diabetes (p = 0.008), and CKD was significantly associated with BC in patients with type 2 diabetes (p = 0.032). A biomarker model, incorporating two serum (monocyte chemoattractant protein 1 and vascular endothelial growth factor) and three urine (interleukin 6, cytokeratin 18, and cytokeratin 8) proteins, predicted incidence of BC with an Area Under the Curve (AUC) of 0.84 in individuals with type 2 diabetes. In people without diabetes, the AUC was 0.66. CONCLUSIONS: We demonstrate the potential clinical utility of a biomarker panel, which includes proteins related to BC pathogenesis and type 2 diabetes, for monitoring risk of BC in patients with type 2 diabetes. Earlier urology referral of patients with type 2 diabetes will improve outcomes for these patients. TRIAL REGISTRATION: http://www.isrctn.com/ISRCTN25823942.

Troponin-T type 1 (TNNT1)-related nemaline myopathy: unique respiratory phenotype and muscle pathology findings.

Biallelic pathogenic variants in the troponin T type 1 (TNNT1) gene cause a severe form of congenital nemaline myopathy. Typical features include severe motor delay, proximal contractures and weakness, pectus carinatum, chest wall rigidity and tremor. If left untreated, respiratory failure leads to early death at a median age of 18 months. Here we report on three non-Amish, unrelated patients harbouring novel TNNT1 variants. The peculiar combination of respiratory muscle weakness and chest wall stiffness caused early severe hypoventilation warranting the use of high pressures on BiPAP ventilator, with subsequent rapid escalation of pressures delivered with limited efficacy secondary to the extreme rib cage stiffness. Severe respiratory impairment occurred despite a relatively milder motor involvement in one patient. Muscle biopsies from two individuals showed predominant involvement of type 1 fibres, abundant nemaline bodies, marked fibrosis and loss of TNNT1 protein. We aim to increase the awareness of the challenges of managing respiratory support in patients with this unique respiratory phenotype.

THUMPD1 bi-allelic variants cause loss of tRNA acetylation and a syndromic neurodevelopmental disorder.

Covalent tRNA modifications play multi-faceted roles in tRNA stability, folding, and recognition, as well as the rate and fidelity of translation, and other cellular processes such as growth, development, and stress responses. Mutations in genes that are known to regulate tRNA modifications lead to a wide array of phenotypes and diseases including numerous cognitive and neurodevelopmental disorders, highlighting the critical role of tRNA modification in human disease. One such gene, THUMPD1, is involved in regulating tRNA N4-acetylcytidine modification (ac4C), and recently was proposed as a candidate gene for autosomal-recessive intellectual disability. Here, we present 13 individuals from 8 families who harbor rare loss-of-function variants in THUMPD1. Common phenotypic findings included global developmental delay, speech delay, moderate to severe intellectual deficiency, behavioral abnormalities such as angry outbursts, facial dysmorphism, and ophthalmological abnormalities. We demonstrate that the bi-allelic variants identified cause loss of function of THUMPD1 and that this defect results in a loss of ac4C modification in small RNAs, and of individually purified tRNA-Ser-CGA. We further corroborate this effect by showing a loss of tRNA acetylation in two CRISPR-Cas9-generated THUMPD1 KO cell lines. In addition, we also show the resultant amino acid substitution that occurs in a missense THUMPD1 allele identified in an individual with compound heterozygous variants results in a marked decrease in THUMPD1 stability and RNA-binding capacity. Taken together, these results suggest that the lack of tRNA acetylation due to THUMPD1 loss of function results in a syndromic form of intellectual disability associated with developmental delay, behavioral abnormalities, hearing loss, and facial dysmorphism.

Response to treatment and outcomes of infantile spasms in Down syndrome.

AIM: To estimate the prevalence, and evaluate presentation, treatment response, treatment side effects, and long-term seizure outcomes in all known cases of children with Down syndrome and infantile spasms on the island of Ireland. METHOD: This was a 10-year retrospective multicentre review of clinical records and investigations, focusing on treatment response, side effects, and long-term outcomes. RESULTS: The prevalence of infantile spasms in Down syndrome was 3.0% during the study period. Fifty-four infants were identified with median age of spasm onset at 201 days (interquartile range [IQR] 156-242). Spasm cessation was achieved in 88% (n=46) at a median of 110 days (IQR 5-66). The most common first-line medications were prednisolone (n=20, 37%), vigabatrin (n=18, 33.3%), and sodium valproate (n=9, 16.7%). At follow-up (median age 23.7mo; IQR 13.4-40.6), 25% had ongoing seizures and 85% had developmental concerns. Treatment within 60 days did not correlate with spasm cessation. Seventeen children (31%) experienced medication side effects, with vigabatrin accounting for 52%. INTERPRETATION: Prednisolone is an effective and well-tolerated medication for treating infantile spasms in Down syndrome. Despite the high percentage of spasm cessation, developmental concerns and ongoing seizures were common.

Infantile onset ascending hereditary spastic paralysis.

A 3½-year-old girl, presented with delayed motor development and increased tone in lower limbs along with tight tendoachilles, toe walking and bilateral clonus. There were normal antenatal and perinatal period, however, after birth there was twitching of her lower limbs. Examination showed lower limb spasticity.Brain and spinal MRI along with EEG were normal. Serum amino acids revealed hyperprolinemia type 1. Hereditary spastic paraplegia gene panel confirmed a homozygous pathogenic variant in ALS2 gene, confirming a diagnosis of infantile onset ascending hereditary spastic paraparesis. She was fitted with ankle-foot orthotics, uses a Kaye walker and is on baclofen and diazepam as she can experience spasticity and painful muscle cramps. She is being managed by a multidisciplinary team involving paediatrician, paediatric neurologist, physiotherapist, occupational therapist, speech and language therapist, dietitian and social worker. Infantile onset ascending hereditary spastic paraplegia represents a rare cause of early onset spasticity with a progressive prognosis.

Neurological involvement in children with hemolytic uremic syndrome.

Our objective was to establish the rate of neurological involvement in Shiga toxin-producing Escherichia coli-hemolytic uremic syndrome (STEC-HUS) and describe the clinical presentation, management and outcome. A retrospective chart review of children aged ≤ 16 years with STEC-HUS in Children's Health Ireland from 2005 to 2018 was conducted. Laboratory confirmation of STEC infection was required for inclusion. Neurological involvement was defined as encephalopathy, focal neurological deficit, and/or seizure activity. Data on clinical presentation, management, and outcome were collected. We identified 240 children with HUS; 202 had confirmed STEC infection. Neurological involvement occurred in 22 (11%). The most common presentation was seizures (73%). In the neurological group, 19 (86%) were treated with plasma exchange and/or eculizumab. Of the 21 surviving children with neurological involvement, 19 (91%) achieved a complete neurological recovery. A higher proportion of children in the neurological group had renal sequelae (27% vs. 12%, P = .031). One patient died from multi-organ failure.Conclusion: We have identified the rate of neurological involvement in a large cohort of children with STEC-HUS as 11%. Neurological involvement in STEC-HUS is associated with good long-term outcome (complete neurological recovery in 91%) and a low case-fatality rate (4.5%) in our cohort. What is Known: • HUS is associated with neurological involvement in up to 30% of cases. • Neurological involvement has been reported as predictor of poor outcome, with associated increased morbidity and mortality. What is New: • The incidence of neurological involvement in STEC-HUS is 11%. • Neurological involvement is associated with predominantly good long-term outcome (90%) and a reduced case-fatality rate (4.5%) compared to older reports.

Prediagnosis pathway benchmarking audit in patients with Duchenne muscular dystrophy.

OBJECTIVE: To describe age and time at key stages in the Duchenne muscular dystrophy (DMD) prediagnosis pathway at selected centres to identify opportunities for service improvement. DESIGN: A multicentre retrospective national audit. SETTING: Nine tertiary neuromuscular centres across the UK and Ireland. A prior single-centre UK audit of 20 patients with no DMD family history provided benchmark criteria. PATIENTS: Patients with a definitive diagnosis of DMD documented within 3 years prior to December 2018 (n=122). MAIN OUTCOME MEASURES: Mean age (months) at four key stages in the DMD diagnostic pathway and mean time (months) of presentational and diagnostic delay, and time from first reported symptoms to definitive diagnosis. Type of symptoms was also recorded. RESULTS: Overall, mean age at definitive diagnosis, age at first engagement with healthcare professional (HCP) and age at first reported symptoms were 53.9±29.7, 49.9±28.9 and 36.4±26.8 months, respectively. The presentational delay and time to diagnosis were 21.1 (±21.1) and 4.6 (±7.9) months, respectively. The mean time from first reported symptoms to definitive diagnosis was 24.2±20.9. The percentages of patients with motor and/or non-motor symptoms recorded were 88% (n=106/121) and 47% (n=57/121), respectively. CONCLUSIONS: Majority of data mirrored the benchmark audit. However, while the time to diagnosis was shorter, a presentational delay was observed. Failure to recognise early symptoms of DMD could be a contributing factor and represents an unmet need in the diagnosis pathway. Methods determining how to improve this need to be explored.

A Randomized, Double-Blind, Placebo-Controlled, Global Phase 3 Study of Edasalonexent in Pediatric Patients with Duchenne Muscular Dystrophy: Results of the PolarisDMD Trial.

BACKGROUND: Edasalonexent (CAT-1004) is an orally-administered novel small molecule drug designed to inhibit NF-κB and potentially reduce inflammation and fibrosis to improve muscle function and thereby slow disease progression and muscle decline in Duchenne muscular dystrophy (DMD). OBJECTIVE: This international, randomized 2 : 1, placebo-controlled, phase 3 study in patients ≥4 - < 8 years old with DMD due to any dystrophin mutation examined the effect of edasalonexent (100 mg/kg/day) compared to placebo over 52 weeks. METHODS: Endpoints were changes in the North Star Ambulatory Assessment (NSAA; primary) and timed function tests (TFTs; secondary). Assessment of health-related function used the Pediatric Outcomes Data Collection tool (PODCI). RESULTS: One hundred thirty one patients received edasalonexent (n = 88) and placebo (n = 43). At week 52, differences between edasalonexent and placebo for NSAA total score and TFTs were not statistically significant, although there were consistently less functional declines in the edasalonexent group. A pre-specified analysis by age demonstrated that younger patients (≤6.0 years) showed more robust and statistically significant differences between edasalonexent and placebo for some assessments. Treatment was well-tolerated and the majority of adverse events were mild, and most commonly involved the gastrointestinal system (primarily diarrhea). CONCLUSIONS: Edasalonexent was generally well-tolerated with a manageable safety profile at the dose of 100 mg/kg/day. Although edasalonexent did not achieve statistical significance for improvement in primary and secondary functional endpoints for assessment of DMD, subgroup analysis suggested that edasalonexent may slow disease progression if initiated before 6 years of age. (NCT03703882).

Benign intracranial hypertension associated with inhaled corticosteroids in a child with asthma.

A 13-year-old male asthmatic presented to the general paediatric clinic with papilloedema identified following a check-up with his optician due to blurred vision. His asthma was well controlled on a moderate dose of inhaled corticosteroid and there had been no recent increase or decrease in the dose. A diagnosis of benign intracranial hypertension (BIH) was made based on a raised cerebrospinal fluid opening pressure, papilloedema, a normal neurological examination and normal neuroimaging. The only associated risk factor was his inhaled corticosteroids. He was commenced on acetazolamide and the inhaled corticosteroid dose was reduced, resulting in resolution of his papilloedema. This case serves to highlight that steroid side effects including BIH may occur at moderate doses of inhaled corticosteroids and that inhaled corticosteroid dose should be regularly reviewed and decreased to the lowest dose that maintains asthma control.

Exploiting a Rose Bengal-bearing, oxygen-producing nanoparticle for SDT and associated immune-mediated therapeutic effects in the treatment of pancreatic cancer.

Sonodynamic therapy (SDT) is an emerging stimulus-responsive approach for the targeted treatment of solid tumours. However, its ability to generate stimulus-responsive cytotoxic reactive oxygen species (ROS), is compromised by tumour hypoxia. Here we describe a robust means of preparing a pH-sensitive polymethacrylate-coated CaO2 nanoparticle that is capable of transiently alleviating tumour hypoxia. Systemic administration of particles to animals bearing human xenograft BxPC3 pancreatic tumours increases oxygen partial pressures (PO2) to 20-50 mmHg for over 40 min. RT-qPCR analysis of expression of selected tumour marker genes in treated animals suggests that the transient production of oxygen is sufficient to elicit effects at a molecular genetic level. Using particles labelled with the near infra-red (nIR) fluorescent dye, indocyanine green, selective uptake of particles by tumours was observed. Systemic administration of particles containing Rose Bengal (RB) at concentrations of 0.1 mg/mg of particles are capable of eliciting nanoparticle-induced, SDT-mediated antitumour effects using the BxPC3 human pancreatic tumour model in immuno-compromised mice. Additionally, a potent abscopal effect was observed in off-target tumours in a syngeneic murine bilateral tumour model for pancreatic cancer and an increase in tumour cytotoxic T cells (CD8+) and a decrease in immunosuppressive tumour regulatory T cells [Treg (CD4+, FoxP3+)] was observed in both target and off-target tumours in SDT treated animals. We suggest that this approach offers significant potential in the treatment of both focal and disseminated (metastatic) pancreatic cancer.

X-linked infantile spinal muscular atrophy (SMAX2) caused by novel c.1681G>A substitution in the UBA1 gene, expanding the phenotype.

X-linked infantile spinal muscular atrophy (SMAX2), OMIM 301830, is a rare, severe form of spinal muscular atrophy, caused by variants in the Ubiquitin like modifier-activating enzyme 1 (UBA1) gene. Clinical features reported to date include marked hypotonia, areflexia, arthrogryposis, contractures, myopathic facies and tongue fibrillations. Previous reports have included a history of contractures. We report a male patient presenting following a normal pregnancy with typical symptoms of X-linked infantile spinal muscular atrophy including hypotonia, weakness, areflexia and respiratory insufficiency, however contractures were absent. There was a significant family history of neuromuscular disease on the maternal side, with several male relatives all dying before the age of six months. Creatine Kinase was mildly elevated, MRI Brain was normal and neurophysiological testing revealed a diffuse motor neuronopathy. Genetic testing for SMN1 gene was normal. UBA1 sequencing revealed a maternally inherited hemizygous familial variant [c.1681G>A p. (Asp561Asn)], which has not been previously reported.

Compound heterozygosity with PRRT2: Pushing the phenotypic envelope in genetic epilepsies.

PRRT2 pathogenic variants have been described in benign familial infantile epilepsy, episodic ataxia, paroxysmal kinesigenic dyskinesia, and hemiplegic migraines. We describe a patient with compound heterozygous variants, infantile epilepsy with status epilepticus, paroxysmal dyskinesia and episodic ataxia. Testing revealed a pathogenic PRRT2 duplication (c.649dupC), and a likely pathogenic missense variant (c.916G>A). His presentation meets the severe phenotypic category with a combination of at least 3 neurological symptoms: seizures and status epilepticus, prolonged episodic ataxia, and paroxysmal dyskinesia. This further expands the clinical findings related to PRRT2, and suggests that compound heterozygous variants could confer a severe phenotype.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

PURPOSE: To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). EXPERIMENTAL DESIGN: Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. RESULTS: Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. CONCLUSIONS: Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.