Ion Channels in Gliomas-From Molecular Basis to Treatment.

Ion channels provide the basis for the nervous system's intrinsic electrical activity. Neuronal excitability is a characteristic property of neurons and is critical for all functions of the nervous system. Glia cells fulfill essential supportive roles, but unlike neurons, they also retain the ability to divide. This can lead to uncontrolled growth and the formation of gliomas. Ion channels are involved in the unique biology of gliomas pertaining to peritumoral pathology and seizures, diffuse invasion, and treatment resistance. The emerging picture shows ion channels in the brain at the crossroads of neurophysiology and fundamental pathophysiological processes of specific cancer behaviors as reflected by uncontrolled proliferation, infiltration, resistance to apoptosis, metabolism, and angiogenesis. Ion channels are highly druggable, making them an enticing therapeutic target. Targeting ion channels in difficult-to-treat brain tumors such as gliomas requires an understanding of their extremely heterogenous tumor microenvironment and highly diverse molecular profiles, both representing major causes of recurrence and treatment resistance. In this review, we survey the current knowledge on ion channels with oncogenic behavior within the heterogeneous group of gliomas, review ion channel gene expression as genomic biomarkers for glioma prognosis and provide an update on therapeutic perspectives for repurposed and novel ion channel inhibitors and electrotherapy.

Ensuring equity: Pharmacogenetic implementation in rural and tribal communities.

Implementation strategies for pharmacogenetic testing have been largely limited to major academic medical centers and large health systems, threatening to exacerbate healthcare disparities for rural and tribal populations. There exists a need in Montana (United States)-a state where two-thirds of the population live in rural areas and with a large proportion of tribal residents-to develop novel strategies to make pharmacogenetic testing more broadly available. We established partnerships between University of Montana (UM) and three early adopter sites providing patient-centered care to historically neglected populations. We conducted 45 semi-structured interviews with key stakeholders at each site and solicited participant feedback on the utility of a centralized pharmacogenetic service at UM offering consultations to patients and providers statewide via telehealth. For settings serving rural patients-tribal and non-tribal-participants described healthcare facilities without adequate infrastructure, personnel, and funding to implement pharmacogenetic services. Participants serving tribal communities stressed the need for ethical practices for collecting biospecimens and returning genetic results to patients, largely due to historical and contemporary traumas experienced by tribal populations with regard to genetic research. Participants expressed that pharmacogenetic testing could benefit patients by achieving therapeutic benefit sooner, reducing the risk of side effects, and improving adherence outcomes for patients with limited access to follow-up services in remote areas. Others expressed concern that financial barriers to pharmacogenetic testing for patients of lower socioeconomic status would further exacerbate inequities in care. Participants valued the role of telehealth to deliver pharmacogenetic consults from a centralized service at UM, describing the ability to connect providers and patients to resources and expertise as imperative to driving successful pharmacogenetic implementation. Our results support strategies to improve access to pharmacogenetic testing for neglected patient populations and create opportunities to reduce existing healthcare inequities. By exploring critical challenges for pharmacogenetic implementation focused on serving underserved communities, this work can help guide equitable frameworks to serve as a model for other resource-limited settings looking to initiate pharmacogenetic testing.

N-glycome analysis detects dysglycosylation missed by conventional methods in SLC39A8 deficiency.

Congenital disorders of glycosylation (CDG) are a growing group of inborn metabolic disorders with multiorgan presentation. SLC39A8-CDG is a severe subtype caused by biallelic mutations in the manganese transporter SLC39A8, reducing levels of this essential cofactor for many enzymes including glycosyltransferases. The current diagnostic standard for disorders of N-glycosylation is the analysis of serum transferrin. Exome and Sanger sequencing were performed in two patients with severe neurodevelopmental phenotypes suggestive of CDG. Transferrin glycosylation was analyzed by high-performance liquid chromatography (HPLC) and isoelectric focusing in addition to comprehensive N-glycome analysis using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). Atomic absorption spectroscopy was used to quantify whole blood manganese levels. Both patients presented with a severe, multisystem disorder, and a complex neurological phenotype. Magnetic resonance imaging (MRI) revealed a Leigh-like syndrome with bilateral T2 hyperintensities of the basal ganglia. In patient 1, exome sequencing identified the previously undescribed homozygous variant c.608T>C [p.F203S] in SLC39A8. Patient 2 was found to be homozygous for c.112G>C [p.G38R]. Both individuals showed a reduction of whole blood manganese, though transferrin glycosylation was normal. N-glycome using MALDI-TOF MS identified an increase of the asialo-agalactosylated precursor N-glycan A2G1S1 and a decrease in bisected structures. In addition, analysis of heterozygous CDG-allele carriers identified similar but less severe glycosylation changes. Despite its reliance as a clinical gold standard, analysis of transferrin glycosylation cannot be categorically used to rule out SLC39A8-CDG. These results emphasize that SLC39A8-CDG presents as a spectrum of dysregulated glycosylation, and MS is an important tool for identifying deficiencies not detected by conventional methods.

Pathogenic variants in SQOR encoding sulfide:quinone oxidoreductase are a potentially treatable cause of Leigh disease.

Hydrogen sulfide, a signaling molecule formed mainly from cysteine, is catabolized by sulfide:quinone oxidoreductase (gene SQOR). Toxic hydrogen sulfide exposure inhibits complex IV. We describe children of two families with pathogenic variants in SQOR. Exome sequencing identified variants; SQOR enzyme activity was measured spectrophotometrically, protein levels evaluated by western blotting, and mitochondrial function was assayed. In family A, following a brief illness, a 4-year-old girl presented comatose with lactic acidosis and multiorgan failure. After stabilization, she remained comatose, hypotonic, had neurostorming episodes, elevated lactate, and Leigh-like lesions on brain imaging. She died shortly after. Her 8-year-old sister presented with a rapidly fatal episode of coma with lactic acidosis, and lesions in the basal ganglia and left cortex. Muscle and liver tissue had isolated decreased complex IV activity, but normal complex IV protein levels and complex formation. Both patients were homozygous for c.637G > A, which we identified as a founder mutation in the Lehrerleut Hutterite with a carrier frequency of 1 in 13. The resulting p.Glu213Lys change disrupts hydrogen bonding with neighboring residues, resulting in severely reduced SQOR protein and enzyme activity, whereas sulfide generating enzyme levels were unchanged. In family B, a boy had episodes of encephalopathy and basal ganglia lesions. He was homozygous for c.446delT and had severely reduced fibroblast SQOR enzyme activity and protein levels. SQOR dysfunction can result in hydrogen sulfide accumulation, which, consistent with its known toxicity, inhibits complex IV resulting in energy failure. In conclusion, SQOR deficiency represents a new, potentially treatable, cause of Leigh disease.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

Clinical, neuroradiological, and biochemical features of SLC35A2-CDG patients.

SLC35A2-CDG is caused by mutations in the X-linked SLC35A2 gene encoding the UDP-galactose transporter. SLC35A2 mutations lead to hypogalactosylation of N-glycans. SLC35A2-CDG is characterized by severe neurological symptoms and, in many patients, early-onset epileptic encephalopathy. In view of the diagnostic challenges, we studied the clinical, neuroradiological, and biochemical features of 15 patients (11 females and 4 males) with SLC35A2-CDG from various centers. We describe nine novel pathogenic variations in SLC35A2. All affected individuals presented with a global developmental delay, and hypotonia, while 70% were nonambulatory. Epilepsy was present in 80% of the patients, and in EEG hypsarrhythmia and findings consistent with epileptic encephalopathy were frequently seen. The most common brain MRI abnormality was cerebral atrophy with delayed myelination and multifocal inhomogeneous abnormal patchy white matter hyperintensities, which seemed to be nonprogressive. Thin corpus callosum was also common, and all the patients had a corpus callosum shorter than normal for their age. Variable dysmorphic features and growth deficiency were noted. Biochemically, normal mucin type O-glycosylation and lipid glycosylation were found, while transferrin mass spectrometry was found to be more specific in the identification of SLC35A2-CDG, as compared to routine screening tests. Although normal glycosylation studies together with clinical variability and genetic results complicate the diagnosis of SLC35A2-CDG, our data indicate that the combination of these three elements can support the pathogenicity of mutations in SLC35A2.

Overrepresentation of pregnancies conceived by artificial reproductive technology in prenatally identified fetuses with Beckwith-Wiedemann syndrome.

OBJECTIVES: In vitro fertilization (IVF) has been linked to an increased risk for imprinting disorders in offspring. The data so far have predominantly been retrospective, comparing the rate of IVF conceptions in affected patients with controls. We describe a series of fetuses with omphalocele that were tested for Beckwith-Wiedemann syndrome (BWS) and subsequently ascertained as to whether pregnancies were conceived by assisted reproductive technologies (ART). METHODS: Fetuses were tested for BWS by Southern blot, PCR based methods, and methylation analysis to identify the imprinting status at primarily the IC2 locus, KCNQ1OT1, as well as IC1, H19/IGF-2. Some fetuses were also tested for uniparental disomy of chromosome 11p. RESULTS: We tested 301 fetuses with omphalocele for BWS. Forty samples were positive. Sixteen were from IVF pregnancies, for an overall rate of 40%. Such as high proportion of IVF pregnancies in a series of BWS-positive fetuses has not been described previously. Possible factors such as twinning and ascertainment bias are discussed. CONCLUSION: We found about a 20-fold overrepresentation of IVF cases in fetuses with BWS/omphalocele when compared with the rate of ART pregnancies in the USA (p < .0001). Our series provides support for an association of IVF and BWS. Patients should be counseled about these risks and made aware of the availability of prenatal diagnosis for detection.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK-neurexin interaction.

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

Truncating de novo mutations in the Krüppel-type zinc-finger gene ZNF148 in patients with corpus callosum defects, developmental delay, short stature, and dysmorphisms.

BACKGROUND: Krüppel-type zinc finger genes (ZNF) constitute a large yet relatively poorly characterized gene family. ZNF genes encode proteins that recognize specific DNA motifs in gene promotors. They act as transcriptional co-activators or -repressors via interaction with chromatin remodeling proteins and other transcription factors. Only few ZNF genes are currently linked to human disorders and identification of ZNF gene-associated human diseases may help understand their function. Here we provide genetic, statistical, and clinical evidence to support association of ZNF148 with a new intellectual disability (ID) syndrome disorder. METHODS: Routine diagnostic exome sequencing data were obtained from 2172 patients with ID and/or multiple congenital anomalies. RESULTS: In a cohort of 2172 patient-parent trios referred for routine diagnostic whole exome sequencing for ID and/or multiple congenital anomalies (MCA) in the period 2012-2016, four patients were identified who carried de novo heterozygous nonsense or frameshift mutations in the ZNF148 gene. This was the only ZNF gene with recurrent truncating de novo mutations in this cohort. All mutations resulted in premature termination codons in the last exon of ZNF148. The number of the de novo truncating mutations in the ZNF148 gene was significantly enriched (p = 5.42 × 10-3). The newly described ZNF148-associated syndrome is characterized by underdevelopment of the corpus callosum, mild to moderate developmental delay and ID, variable microcephaly or mild macrocephaly, short stature, feeding problems, facial dysmorphisms, and cardiac and renal malformations. CONCLUSIONS: We propose ZNF148 as a gene involved in a newly described ID syndrome with a recurrent phenotype and postulate that the ZNF148 is a hitherto unrecognized but crucial transcription factor in the development of the corpus callosum. Our study illustrates the advantage of whole exome sequencing in a large cohort using a parent-offspring trio approach for identifying novel genes involved in rare human diseases.

Phenotypic spectrum of GABRA1: From generalized epilepsies to severe epileptic encephalopathies.

OBJECTIVE: To delineate phenotypic heterogeneity, we describe the clinical features of a cohort of patients with GABRA1 gene mutations. METHODS: Patients with GABRA1 mutations were ascertained through an international collaboration. Clinical, EEG, and genetic data were collected. Functional analysis of 4 selected mutations was performed using the Xenopus laevis oocyte expression system. RESULTS: The study included 16 novel probands and 3 additional family members with a disease-causing mutation in the GABRA1 gene. The phenotypic spectrum varied from unspecified epilepsy (1), juvenile myoclonic epilepsy (2), photosensitive idiopathic generalized epilepsy (1), and generalized epilepsy with febrile seizures plus (1) to severe epileptic encephalopathies (11). In the epileptic encephalopathy group, the patients had seizures beginning between the first day of life and 15 months, with a mean of 7 months. Predominant seizure types in all patients were tonic-clonic in 9 participants (56%) and myoclonic seizures in 5 (31%). EEG showed a generalized photoparoxysmal response in 6 patients (37%). Four selected mutations studied functionally revealed a loss of function, without a clear genotype-phenotype correlation. CONCLUSIONS: GABRA1 mutations make a significant contribution to the genetic etiology of both benign and severe epilepsy syndromes. Myoclonic and tonic-clonic seizures with pathologic response to photic stimulation are common and shared features in both mild and severe phenotypes.

A Quality Improvement Collaborative to Improve Pediatric Primary Care Genetic Services.

OBJECTIVE: To investigate if a national pediatric primary care quality improvement collaborative (QIC) could improve and sustain adherence with process measures related to diagnosis and management of children with genetic disorders. METHODS: Thirteen practices in 11 states from the American Academy of Pediatrics' Quality Improvement Innovation Networks participated in a 6-month QIC that included regular educational opportunities, access to genetic professionals, and performance feedback. The QIC identified 11 aims related to improving diagnosis and management of children with genetic disorders. The practices evaluated adherence by reviewing patient records at baseline, monthly for 6 months (active improvement period), and then once 6 months after the QIC's conclusion to check for sustainability. Random intercept binomial regression models with practice level random intercepts were used to compare adherence over time for each aim. RESULTS: During the active improvement period, statistically significant improvements in adherence were observed for 4 of the 7 aims achieving minimal data submission levels. For example, adherence improved for family histories created/maintained at health supervision visits documenting all components of the family history (6% vs 60%, P < .001), and for patients with specific genetic disorders who received recommended care (58% vs 85%, P < .001). All 4 of these aims also demonstrated statistically significant improvements during the sustainability period. CONCLUSIONS: A national QIC reveals promise in improving and sustaining adherence with process measures related to the diagnosis and management of genetic disorders. Future research should focus on patient outcome measures and the optimal number of aims to pursue in QICs.

The Shodair Medical Genetics Department--recent past and future developments.

Philip Pallister and John Opitz laid the ground work for a unique genetic service model in Montana that continues to flourish through ongoing support by the Montana Legislature, the Montana Department of Public Health and Human Services and the Shodair Foundation. At the heart of the model are clinical and laboratory genetic specialists based at Shodair Children's Hospital in Helena providing genetic care for patients through outreach clinics. Clinical services are supported by a state-of-the-art cytogenetics and molecular genetic laboratory as well a fetal genetic pathology program. Over the years, the reach of regular genetics clinics expanded to include large geographic areas including northwest (Kalispell), west central (Missoula), southwest (Bozeman, Butte), north central (Great Falls), and south central Montana (Billings). Building on the foundation of its world-renowned pioneers, the next generation of medical geneticists at Shodair carries the responsibility of integrating genomic medicine in the diagnosis and care of their patients, reducing inequality of services within Montana and partnering with colleagues across specialties to develop a more personalized practice of medicine.

Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm.

Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-ß signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-ß signaling, including either subunit of the TGF-ß receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-ß2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-ß signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2(+/-) mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-ß signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1(C1039G/+)) and Tgfb2 haploinsufficiency show increased TGF-ß signaling and phenotypic worsening in association with normalization of TGF-ß2 expression and high expression of TGF-ß1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-ß-mediated vasculopathies.

Neonatal cholestasis as initial manifestation of type 2 Gaucher disease: a continuum in the spectrum of early onset Gaucher disease.

Gaucher disease type 2 [OMIM #230800] is a rare lysosomal storage disorder with usual onset between 3 and 6 months of age leading to progressive neurodegeneration and death within the first 2 years of life. Rarely it may lack the characteristic symptom-free period and initially manifest prenatally or in the neonatal period. The early course of neonatal onset classic type 2 variants is not well known, and reports of early histological changes in the liver of type 2 Gaucher disease patients are scarce. We describe a patient who presented in the immediate postnatal period with cholestasis without hepatomegaly associated with hepatocellular giant-cell transformation on liver biopsy, thrombocytopenia, and failure to thrive. This was initially thought to represent neonatal giant-cell hepatitis and the correct diagnosis was not made until the age of 6 months. Hepatocellular giant transformation has not been described in the classic acute neuronopathic form of GD. However, it has been reported in congenital GD with nonimmune hydrops and neonatal hepatitis, an example of perinatal lethal Gaucher disease (PLGD), which sometimes is regarded as an entity separate from GD type 2. Our case illustrates that neonatal cholestasis may be part of a spectrum of manifestations which spans a continuum between the PLGD and classic type 2 GD. Giant cells are a nonspecific finding but may reflect the presence of a systemic inflammatory process that recently has been implicated in the brain stem degeneration associated with acute neuronopathic GD.

Tuberculosis and profound hypovitaminosis D in an infant.

A young infant presented with a febrile respiratory illness, irritability, and failure to thrive. Clinical evaluation identified that he had profound hypovitaminosis D with severe hypocalcemia and active pulmonary tuberculosis. We review the literature and discuss the current state of knowledge of the interrelationship between Mycobacterium infection and vitamin D status, and its implication to pediatricians.

Community-based intervention to manage an outbreak of MRSA skin infections in a county jail.

This article describes a community-based intervention to manage an outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin infections in a midwestern county jail. A systematic investigation conducted by a family medicine residency program identified 64 total cases and 19 MRSA cases between January 1 and December 31, 2007. Factors contributing to MRSA transmission included inadequate surveillance, lack of antibacterial soap, and a defective laundry process. All 19 isolates were CA-MRSA and all seven tested by pulsed-field gel electrophoresis (PFGE) were USA300. Four of the seven isolates showed variation of their PFGE patterns. A primary care approach using community-based resources effectively reduced the number of cases in this heterogeneous outbreak of CA-MRSA, with the last MRSA being isolated in October 2007.

Typhoid fever in a young immigrant child: a case report and review of the literature.

Typhoid fever is a systemic disease not endemic to South Dakota. We report a case of a young child, living in South Dakota since immigrating with his family as a refugee, who developed typhoid fever. Contact investigation failed to identify a source, suggesting that the infection was acquired abroad. The clinical presentation of typhoid is discussed, and the literature relevant to diagnosis, treatment and prevention is reviewed.