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.

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.

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.

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.

Differences in gene expression profiles from asbestos-treated SPARC-null and wild-type mouse lungs.

The role of SPARC in the in vivo lung response to crocidolite asbestos was addressed by instillation of crocidolite asbestos in a series of wild-type or SPARC-null mice. Animals were sacrificed at one week, one month, and three months post-instillation to assess the impact of SPARC on multiple stages in the development of fibrosis. RNA was harvested from 10 animals/time point, pooled, and used to probe a mouse array containing approximately 10,000 probes. Gene expression data were analyzed for fold change, and for broader functional group alterations. As expected, the one-week time point displayed alterations in genes involved in immune recognition, energy utilization, and growth factor production. Later time points showed expression alterations for genes involved in protein degradation, Wnt receptor signaling, membrane protein activity, and transport. Molecules in the Wnt pathway have been implicated in bone growth, mediation of fibroblast activity, and have been directly linked to SPARC regulation.

Potential surrogate markers for gamma-hydroxybutyrate administration may extend the detection window from 12 to 48 hours.

The exogenous administration of gamma-hydroxybutyrate (GHB) as a drug of abuse, and especially in date rape sexual assaults, has recently increased. Chromatographic techniques are used to detect GHB in blood or urine, with a window of detection limited to 12 h. This brief window makes the proof of administration problematic in most rape cases. This study is aimed to extend the window of detection through surrogate markers of GHB administration. Microarray technology is used in a DBA/2J mouse model to detect gene expression changes in peripheral blood after GHB exposure at times as long as 96 h post exposure. This study focuses on two of the most significantly altered transcripts, epiregulin and phosphoprotein enriched in astrocytes 15 (Pea-15). Both genes have increased the ribonucleic acid expression (8.5- and 4.6-fold upregulation at 96 h, respectively) in GHB-dosed mice (1 g/kg) as compared with the control. To confirm these results at the protein level, an intracellular flow cytometric assay is developed to detect protein level changes in the peripheral blood of both these potential biomarkers after GHB exposure. These results suggest that after further development, epiregulin and Pea-15 may prove to be significant surrogate markers in the indirect detection of GHB administration.

Suppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposure.

The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1alpha, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma-type cancers.

Gene expression changes after exposure to six-mix in a mouse model.

The exposure of Libby MT residents to amphibole-contaminated vermiculite is well known. To explore the gene-environment interactions in the development of asbestos-related diseases (ARD), a mouse model of asbestos exposure using Six-mix (a combination of amphibole fibers gathered from six sites at the Libby vermiculite mine), crocidolite asbestos, or saline as a negative control was used to determine both gene expression responses by using mouse 10,000 oligonucleotide array and to visualize these changes histologically. Mice were sacrificed and whole lungs harvested for histology and microarray analysis six months following exposure via intratracheal instillation. Using an arbitrary cutoff of 1.25-fold change, genes whose RNA expression levels were specifically altered in response to the different amphibole exposures were grouped into categories by a gene ontology analysis program, GoMiner. Our hypothesis was that assessment of asbestos-responsive genes would provide a better understanding of response mechanisms. These experiments have provided new candidates for genes involved in the asbestos response pathways.

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice.

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.