Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Combined heart and liver transplantation in a patient supported by left ventricular assist device (LVAD) with propionic acidemia.

Propionic acidemia (PA) is a rare inherited metabolic disease due to inborn errors of metabolism. PA results in the accumulation of abnormal organic acid metabolites in multiple systems, mainly the central nervous system and the heart. Cardiac complications include dilated cardiomyopathy (DCM) and carry a 40-50% increased mortality risk. Liver transplantation (LT) is required in PA patients when medical treatment fails and may prevent or slow down the cardiomyopathy progression. However, severe heart disease may be a serious contraindication to LT. We present a complicated case of a PA patient, supported with a Left Ventricular Assist Device, who underwent a heart and Liver transplant. PA patients are at increased risk for metabolic acidosis during surgery, with increased anion gap and hyperammonemia. A strict multi-disciplinary approach is needed to prevent and treat metabolic decompensation. The patient had a successful heart and liver transplant after a strict treatment protocol in the pre, intra, and post-operative periods. His case highlights the complexity of PA patients and the increased risk for metabolic decompensation during surgery and provides an insight into how to manage such complicated patients.

Seudoartrosis después de una osteotomía en Chevron: incidencia, tratamiento, seguimiento y evolución / Nonunion after Chevron osteotomy. Incidence, treatment, follow-up, and outcomes

Introducción: El hallux valgus es el trastorno más común del primer dedo del pie. Provoca dolor, discapacidad funcional y altera los patrones de la marcha. Las deformidades leves o moderadas se han corregido con osteotomías distales del primer metatarsiano, como la osteotomía en chevron, un procedimiento seguro, pero no exento de complicaciones. Los objetivos de este estudio fueron determinar la incidencia de seudoartrosis por dicha osteotomía y comunicar nuestro método terapéutico, el seguimiento y la evolución. materiales y métodos: Se realizó un estudio multicéntrico, retrospectivo que incluyó a pacientes operados entre 2009 y 2018. Se evaluaron 1156 osteotomías en chevron como tratamiento del hallux valgus leve o moderado en 1017 pacientes (rango etario 16-83 años; promedio 57.5) realizadas por 4 cirujanos experimentados. El criterio de inclusión fue que el paciente contara con estudios por imágenes compatibles con seudoartrosis a los 6 meses de la cirugía. Resultados: Se evaluó a 5 pacientes con diagnóstico de seudoartrosis después de una osteotomía en chevron para tratar el hallux valgus. Los puntajes promedio de la AOFAS fueron 51 antes del tratamiento del hallux valgus y 87,8 después del tratamiento de la seudoartrosis. Conclusiones: La incidencia de seudoartrosis fue del 0,4% en el posoperatorio alejado. Nuestro abordaje y el tratamiento de la seudoartrosis lograron una excelente mejoría clínica y funcional en todos los pacientes operados. Nivel de Evidencia: III

Introduction: Hallux valgus is the most common disorder of the first toe. It causes pain, functional impairment, and alters gait patterns. Mild to moderate deformities are tipically corrected with distal osteotomies of the first metatarsal, such as the chevron osteotomy, a safe procedure, but not without complications. The objectives of this study were to determine the incidence of pseudarthrosis following this osteotomy and report our therapeutic method, follow-up, and outcomes. Materials and methods: A retrospective multicenter study was carried out, which included patients operated on between 2009 and 2018. A total of 1156 chevron osteotomies were evaluated as a treatment for mild to moderate hallux valgus in 1017 patients (age range 16 -83 years; average 57.5 years) performed by 4 experienced surgeons. The inclusion criterion was that the patient had imaging studies compatible with pseudarthrosis six months after surgery. Results: We evaluated five patients who met our criterion. The average AOFAS (American Orthopedic Foot and Ankle Society) scores were 51 before hallux valgus treatment and 87.8 after pseudarthrosis treatment. Conclusion: The incidence of pseudarthrosis was 0.4% in the distant postoperative period. Our approach and treatment of pseudarthrosis achieved excellent clinical and functional improvements in all operated patients. Level of Evidence: III

Accelerated artificial aging and color stability in resin-based cements / Envejecimiento artificial acelerado y estabilidad de color de medios de fijación a base de resinas

ABSTRACT The aim of this study was to determine color change after accelerated artificial ageing (AAA) of different composite cements that are used with veneers. Five cylindrical test specimens, 15 mm in diameter and 2 mm thick, were made from a single layer of each of the following: RelyX Veneer 3M ESPE (RX), Paracore White Coltene (PC), Solocem White Opaque Coltene (SO), Resin Duo Cement Densell (DC), Panavia V5 Paste Kuraray Noritake (PA) and Panavia F2.0 Kuraray Noritake (PF) (30 specimens altogether). The specimens were light cured following manufacturers' instructions using a Coltolux LED (Coltene) unit. Initial color was determined using an Easyshade - Vita Zahnfabrik Spectrophotometer. Then, the specimens were subjected to AAA for two weeks (336 hours) with cycles of 4 hours of UV light at 60 °C and 4 hours of vapor condensation at 50 °C, successively, after which color was recorded again. Color change was determined for each specimen according to the difference in shade on the Vita scale before and after AAA. Results were analyzed using Kruskal Wallis test. Mean and standard deviation for each group were: RX 8.40 (1.52); PC 8.60 (3.13); SO 6.40 (3.51); DC 10.00 (0.00); PA 7.60 (3.29); PF 2.00 (0.00). The Kruskal Wallis test showed significant difference for material (p<0.05), and comparison of means showed difference between Panavia F2.0 and the other materials. A table providing equivalence between the Vita Classic and CIELAB scales was used to transfer the recorded colors to the CIELAB scale, and the color difference ΔE was calculated for each group, where ΔL, Δa and Δb are the differences in the L, a and b values before and after the AAA. The mean and standard deviation were analyzed statistically by the ANOVA test and Tukey's test. Mean and standard deviation for each group were: RX 14.94 (2.02); PC 14.51 (4.02); SO 12.08 (4.53); DC 16.31 (0.00); PA 10.9 (3.38); PF 7.24 (0.00). The ANOVA test showed significant difference for material (p<0.05). Tukey's test showed two groups (PF-DC, RX, PA). Under the experimental conditions of this study, it can be concluded that accelerated ageing significantly affects the color stability of the resin based cements tested.

RESUMEN El objetivo de este trabajo fue determinar el cambio de coloración de distintos medios de fijación a base de resinas con indicación para carillas estéticas luego de someterse al envejecimiento artificial acelerado (AAA). Se confeccionaron 30 probetas cilíndricas de 15 mm de diámetro y 2 mm de espesor con RelyX Veneer 3M ESPE (RX), Paracore White Coltene (PC), Solocem White Opaque Coltene (SO), Resin Duo Cement Densell (DC), Panavia V5 Paste Kuraray Noritake (PA) y Panavia F2.0 Kuraray Noritake (PF) de una sola capa de material. Se polimerizaron según las indicaciones del fabricante con unidad de curado Coltolux LED (Coltene). Cada grupo quedó conformado con 5 probetas de cada material. Se tomó el color con el espectrofotómetro Easyshade de Vita Zahnfabrik. A continuación, se sometieron a AAA por dos semanas (336 horas) con ciclos de 4 horas de radiaciones ultravioletas a 60°C Y 4 horas de condensación de vapor de agua a 50°C sucesivamente. Una vez terminado este proceso se volvió a registrar el color. El cambio de color se evaluó dentro de la escala de color, ordenada en función del valor. El dato registrado fue la diferencia en la posición inicial y final en esta escala. Los resultados obtenidos para cada grupo fueron analizados por medio de la prueba de Kruskal Wallis. Los valores de media y desvío estándar de cada grupo fueron: RX 8,40 (1,52); PC 8,60 (3,13); SO 6,40 (3,51); DC 10,00 (0,00); PA 7,60 (3,29); PF 2,00 (0,00). El análisis con la prueba de Kruskal Wallis mostró diferencia significativa para el factor material (p<0.05) y la comparación de medias mostró diferencia entre PF y el resto de los materiales. Por otra parte, con una tabla de equivalencia entre los colores en escala de Vita Classic y CIE Lab, se hicieron el pasaje de los valores registrados a la escala de CIE Lab y se calculó la diferencia de color ΔE de cada grupo antes y después del AAA. La media y el desvío estándar fueron analizados estadísticamente por el test de ANOVA y prueba de Tukey. Los valores de media y desvío estándar de cada grupo fueron: RX 14,94 (2,02); PC 14,51 (4,02); SO 12,08 (4,53); DC 16,31 (0,00); PA 10,9 (3,38); PF 7,24 (0,00). En la evaluación de los resultados con ANOVA se encontró diferencia significativa entre los materiales evaluados (p<0,05). La comparación de medias con prueba de Tukey mostró la presencia de dos grupos (PF-DC, RX, PA). En las condiciones experimentales de este trabajo puede concluirse que el envejecimiento acelerado afecta significativamente la estabilidad de color de los cementos utilizados en este trabajo.

Intertwined leukocyte balances in tumours and peripheral blood as robust predictors of right and left colorectal cancer survival.

BACKGROUND: Colorectal cancer (CRC) accounts for 9.4% of overall cancer deaths, ranking second after lung cancer. Despite the large number of factors tested to predict their outcome, most patients with similar variables show big differences in survival. Moreover, right-sided CRC (RCRC) and left-sided CRC (LCRC) patients exhibit large differences in outcome after surgical intervention as assessed by preoperative blood leukocyte status. We hypothesised that stronger indexes than circulating (blood) leukocyte ratios to predict RCRC and LCRC patient outcomes will result from combining both circulating and infiltrated (tumour/peritumour fixed tissues) concentrations of leukocytes. AIM: To seek variables involving leukocyte balances in peripheral blood and tumour tissues and to predict the outcome of CRC patients. METHODS: Sixty-five patients diagnosed with colon adenocarcinoma by the Digestive Surgery Service of the La Paz University Hospital (Madrid, Spain) were enrolled in this study: 43 with RCRC and 22 with LCRC. Patients were followed-up from January 2017 to March 2021 to record overall survival (OS) and recurrence-free survival (RFS) after surgical interventions. Leukocyte concentrations in peripheral blood were determined by routine laboratory protocols. Paraffin-fixed samples of tumour and peritumoural tissues were assessed for leukocyte concentrations by immunohistochemical detection of CD4, CD8, and CD14 marker expression. Ratios of leukocyte concentration in blood and tissues were calculated and evaluated for their predictor values for OS and RFS with Spearman correlations and Cox univariate and multivariate proportional hazards regression, followed by the calculation of the receiver-operating characteristic and area under the curve (AUC) and the determination of Youden's optimal cutoff values for those variables that significantly correlated with either RCRC or LCRC patient outcomes. RCRC patients from the cohort were randomly assigned to modelling and validation sets, and clinician-friendly nomograms were developed to predict OS and RFS from the respective significant indexes. The accuracy of the model was evaluated using calibration and validation plots. RESULTS: The relationship of leukocyte ratios in blood and peritumour resulted in six robust predictors of worse OS in RCRC: CD8+ lymphocyte content in peritumour (CD8pt, AUC = 0.585, cutoff < 8.250, P = 0.0077); total lymphocyte content in peritumour (CD4CD8pt, AUC = 0.550, cutoff < 10.160, P = 0.0188); lymphocyte-to-monocyte ratio in peritumour (LMRpt, AUC = 0.807, cutoff < 3.185, P = 0.0028); CD8+ LMR in peritumour (CD8MRpt, AUC = 0.757, cutoff < 1.650, P = 0.0007); the ratio of blood LMR to LMR in peritumour (LMRb/LMRpt, AUC = 0.672, cutoff > 0.985, P = 0.0244); and the ratio of blood LMR to CD8+ LMR in peritumour (LMRb/CD8MRpt, AUC = 0.601, cutoff > 1.485, P = 0.0101). In addition, three robust predictors of worse RFS in RCRC were found: LMRpt (AUC = 0.737, cutoff < 3.185, P = 0.0046); LMRb/LMRpt (AUC = 0.678, cutoff > 0.985, P = 0.0155) and LMRb/CD8MRpt (AUC = 0.615, cutoff > 1.485, P = 0.0141). Furthermore, the ratio of blood LMR to CD4+ LMR in peritumour (LMRb/CD4MRpt, AUC = 0.786, cutoff > 10.570, P = 0.0416) was found to robustly predict poorer OS in LCRC patients. The nomograms showed moderate accuracy in predicting OS and RFS in RCRC patients, with concordance index of 0.600 and 0.605, respectively. CONCLUSION: Easily obtainable variables at preoperative consultation, defining the status of leukocyte balances between peripheral blood and peritumoural tissues, are robust predictors for OS and RFS of both RCRC and LCRC patients.

Soluble SIGLEC5: A New Prognosis Marker in Colorectal Cancer Patients.

Colorectal cancer (CRC) is the second most deadly and third most commonly diagnosed cancer worldwide. There is significant heterogeneity among patients with CRC, which hinders the search for a standard approach for the detection of this disease. Therefore, the identification of robust prognostic markers for patients with CRC represents an urgent clinical need. In search of such biomarkers, a total of 114 patients with colorectal cancer and 67 healthy participants were studied. Soluble SIGLEC5 (sSIGLEC5) levels were higher in plasma from patients with CRC compared with healthy volunteers. Additionally, sSIGLEC5 levels were higher in exitus than in survivors, and the receiver operating characteristic curve analysis revealed sSIGLEC5 to be an exitus predictor (area under the curve 0.853; cut-off > 412.6 ng/mL) in these patients. A Kaplan-Meier analysis showed that patients with high levels of sSIGLEC5 had significantly shorter overall survival (hazard ratio 15.68; 95% CI 4.571-53.81; p ≤ 0.0001) than those with lower sSIGLEC5 levels. Our study suggests that sSIGLEC5 is a soluble prognosis marker and exitus predictor in CRC.

Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation.

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Effect of flowable composite or glass ionomer liners on shrinkage stress of a composite resin / Efecto de recubrimientos con resinas de baja viscosidad o ionómero vitreo sobre la tensión por contracción de una resina compuesta

ABSTRACT The aim of this study was to evaluate the effect of flowable composite or glass ionomer liners on the shrinkage stress of a restorative composite resin. Fifteen previously sandblasted metal boxes were attached to a universal mechanical testing machine (INSTRON 1011, Instron Corporation). Five of these boxes were filled with Filtek Z350 XT (FXT) Universal Restorative A2 (3M ESPE) (Group 1 or Control). Two further groups of 5 boxes were prepared by interposing a layer of Vitrebond Light Cure Glass Ionomer 3M ESPE (VGI) (Group 2 or G.I.) or Filtek Z350 XT Flowable Restorative A2 3M ESPE (FFR) (Group 3 or Flowable) between the box and the composite resin, completing with the same volume of composite as in Group 1. Upon activating lightcuring, the filled boxes mounted on the testing machine were videoed for 60 seconds (40 s photoactivation and 20 s postcuring), timed with a digital chronometer. Force values were recorded in newtons and converted into stress according to contact surface. Stress values were recorded every 10 s. Results were analyzed using repeated measures ANOVA. Mean and standard deviation in kPa (stress) recorded for each group were: Control group: 126.2 (30.8); G.I.: 48.4 (18); Flowable: 27.9 (19.5). Statistical analysis showed significant differences between the control group and the rest (p<0.01), with no significant difference between groups with glass ionomer liners and flowable resin liners (G.I. and Flowable). Under the experimental conditions of this study, it can be concluded that polymerization shrinkage stress can be reduced by the presence of a liner between the preparation and the restorative material.

RESUMEN El objetivo de este trabajo fue evaluar el efecto de la colocación de una capa de Composite flow o Ionómero vítreo sobre la tensión de contracción de un composite para restauración. Se utilizaron 15 cajas metálicas previamente arenadas y conectadas a la máquina universal para ensayos mecánicos (INSTRON 1011, Instron Corporation). Cinco de estas cajas (G1) se rellenaron con Filtek Z350 XT (FXT) Universal Restorative A2 3M ESPE. Al iniciar la activación de la unidad de curado se comenzaba a registrar con una cámara de video y un cronómetro digital desde el comienzo de la activación de la lámpara hasta 60 s después, registrando los valores post curado durante 20 s. Los valores de fuerza generados por la polimerización fueron registrados en newton de cada 10 s para los 15 ensayos. Los valores fueron convertidos en tensión de contracción según la superficie de contacto. Se realizaron además dos grupos de cajas (5 en cada una) en los cuales se colocaron una capa inicial de Vitrebond Light Cure Glass Ionomer 3M ESPE (VGI) (G2 o IV) y Filtek Z350 XT Flowable Restorative A2 3M ESPE (FFR) (G3 o Flow) y se completó con el mismo volumen de composite de las del GI. Los resultados obtenidos fueron analizados por medio de ANOVA para mediciones repetidas. La media y la desviación estándar en kPa (tensión o estrés de contracción) registrado para cada grupo fueron: Grupo control: 126.2 (30.8); IV: 48.4(18); Flow: 27.9(19.5). El análisis estadístico mostró diferencias estadística mente significativas entre el grupo control y el resto (p=0.00), pero no hubo diferencias significativas entre la presencia de Ionómero vítreo o Composite Flow (IV y Flow). En las condiciones experimentales de este trabajo puede concluirse que la tensión de contracción generada durante la polimerización puede ser disminuida por la presencia de algún material interpuesto entre la preparación y el composite restaurador.

De novo mutations in the GTP/GDP-binding region of RALA, a RAS-like small GTPase, cause intellectual disability and developmental delay.

Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.

Refining the phenotype associated with GNB1 mutations: Clinical data on 18 newly identified patients and review of the literature.

De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Volumetric arc therapy for total scalp irradiation: case report for a recurrent basal cell carcinoma of the scalp.

Total scalp irradiation may be used to treat numerous conditions including squamous and basal cell carcinomas. These conditions are relatively uncommon and patients are frequently treated with palliative intent. In this report, we describe a volumetric arc therapy technique using photon beams for curative intent in an 84 years old patient with recurrent basal cell carcinoma of the scalp. Dose was 50Gy (2Gy per session) to the planning target volume (PTV) followed by a 10 Gy boost to the macroscopic disease on the forehead. A custom made 1 cm superflab bolus helmet was used. Toxicities only consisted of Grade-1 transient radiation dermatitis and alopecia. A sustained clinical response was observed at 6 months follow-up. Volumetric arc therapy (VMAT) may offer an effective alternative modality to treat patients with very extensive scalp lesions as described in this case report.

Phenotypic Heterogeneity of Neutropenia and Gastrointestinal Illness Associated with G6PC3 Founder Mutation.

Severe congenital neutropenia type IV (SCN IV) is a syndrome of severe neutropenia, cardiac and urogenital defects, prominent superficial veins, facial dysmorphism, failure to thrive (FTT), and intermittent thrombocytopenia, caused by a glucose-6-phosphatase catalytic subunit 3 (G6PC3) gene mutation. SCN IV has been linked to glycogen storage disease type 1b as both disorders involve disruption of the glucose-6-phosphatase/glucose-6-phosphate transporter complex, leading to arrested neutrophil maturation. Emerging evidence suggests that neutrophil function plays an important role in intestinal integrity, evidenced by inflammatory bowel disease in certain neutropenic patients. Here, we report 3 unrelated Hispanic males from the Dominican Republic with classic features of SCN IV found to share an identical inherited canonical splice-site mutation of the G6PC3 gene (c.218+1G>A). All 3 patients presented with severe FTT and gastrointestinal manifestations. Two of the patients had significant improvement in growth and resolution of gastrointestional symptoms with initiation of granulocyte colony-stimulating factor. We hypothesize that the gene variant described represents a founder mutation in the Dominican Republic, the first to be described in this geographical region. We discuss the potential associations between neutropenia and gastrointestinal disease with FTT and the role of granulocyte colony-stimulating factor in improving neutrophil count and intestinal integrity and growth.

Clinical outcomes of children with abnormal newborn screening results for Krabbe disease in New York State.

BACKGROUND: Early infantile Krabbe disease is rapidly fatal, but hematopoietic stem cell transplantation (HSCT) may improve outcomes if performed soon after birth. New York State began screening all newborns for Krabbe disease in 2006. METHODS: Infants with abnormal newborn screen results for Krabbe disease were referred to specialty-care centers. Newborns found to be at high risk for Krabbe disease underwent a neurodiagnostic battery to determine the need for emergent HSCT. RESULTS: Almost 2 million infants were screened. Five infants were diagnosed with early infantile Krabbe disease. Three died, two from HSCT-related complications and one from untreated disease. Two children who received HSCT have moderate to severe developmental delays. Forty-six currently asymptomatic children are considered to be at moderate or high risk for development of later-onset Krabbe disease. CONCLUSIONS: These results show significant HSCT-associated morbidity and mortality in early infantile Krabbe disease and raise questions about its efficacy when performed in newborns diagnosed through newborn screening. The unanticipated identification of "at risk" children introduces unique ethical and medicolegal issues. New York's experience raises questions about the risks, benefits, and practicality of screening newborns for Krabbe disease. It is imperative that objective assessments be made on an ongoing basis as additional states begin screening for this disorder.Genet Med 18 12, 1235-1243.

Mutagenesis of Coronavirus nsp14 Reveals Its Potential Role in Modulation of the Innate Immune Response.

UNLABELLED: Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-ß), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response. IMPORTANCE: The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.

Newborn screening for Krabbe disease in New York State: the first eight years' experience.

PURPOSE: Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006. METHODS: Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination. RESULTS: Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease. CONCLUSIONS: The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.

TMEM107 Is a Critical Regulator of Ciliary Protein Composition and Is Mutated in Orofaciodigital Syndrome.

The proximate causes of multiple human genetic syndromes (ciliopathies) are disruptions in the formation or function of the cilium, an organelle required for a multitude of developmental processes. We previously identified Tmem107 as a critical regulator of cilia formation and embryonic organ development in the mouse. Here, we describe a patient with a mutation in TMEM107 that developed atypical Orofaciodigital syndrome (OFD), and show that the OFD patient shares several morphological features with the Tmem107 mutant mouse including polydactyly and reduced numbers of ciliated cells. We show that TMEM107 appears to function within cilia to regulate protein content, as key ciliary proteins do not localize normally in cilia derived from the Tmem107 mouse mutant and the human patient. These data indicate that TMEM107 plays a key, conserved role in regulating ciliary protein composition, and is a novel candidate for ciliopathies of unknown etiology.