Expanding the genetic and phenotypic spectrum of congenital myasthenic syndrome: new homozygous VAMP1 splicing variants in 2 novel individuals.

We report the cases of two Spanish pediatric patients with hypotonia, muscle weakness and feeding difficulties at birth. Whole-exome sequencing (WES) uncovered two new homozygous VAMP1 (Vesicle Associated Membrane Protein 1) splicing variants, NM_014231.5:c.129+5 G > A in the boy patient (P1) and c.341-24_341-16delinsAGAAAA in the girl patient (P2). This gene encodes the vesicle-associated membrane protein 1 (VAMP1) that is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. VAMP1 has a highly variable C-terminus generated by alternative splicing that gives rise to three main isoforms (A, B and D), being VAMP1A the only isoform expressed in the nervous system. In order to assess the pathogenicity of these variants, expression experiments of RNA for VAMP1 were carried out. The c.129+5 G > A and c.341-24_341-16delinsAGAAAA variants induced aberrant splicing events resulting in the deletion of exon 2 (r.5_131del; p.Ser2TrpfsTer7) in the three isoforms in the first case, and the retention of the last 14 nucleotides of the 3' of intron 4 (r.340_341ins341-14_341-1; p.Ile114AsnfsTer77) in the VAMP1A isoform in the second case. Pathogenic VAMP1 variants have been associated with autosomal dominant spastic ataxia 1 (SPAX1) and with autosomal recessive presynaptic congenital myasthenic syndrome (CMS). Our patients share the clinical manifestations of CMS patients with two important differences: they do not show the typical electrophysiological pattern that suggests pathology of pre-synaptic neuromuscular junction, and their muscular biopsies present hypertrophic fibers type 1. In conclusion, our data expand both genetic and phenotypic spectrum associated with VAMP1 variants.

Performance of the new MC-80 automated digital cell morphology analyser in detection of normal and abnormal blood cells: Comparison with the CellaVision DM9600.

INTRODUCTION: Mindray MC-80 is an automated system for digital imaging of white blood cells (WBCs) and their pre-classification. The objective of this work is to analyse its performance comparing it with the CellaVision® DM9600. METHODS: A total of 445 samples were used, 194 normal and 251 abnormal: acute leukaemia (100), myelodysplastic syndromes/myeloproliferative neoplasms (33), lymphoid neoplasms (50), plasma cell neoplasms (14), infections (49) and thrombocytopenia (5). WBC pre-classification values with the MC-80 and DM9600 were compared with (1) the microscope, (2) Mindray BC-6800Plus differentials in only normal samples, and (3) confirmed or reclassified images (post-classification). Pearson's correlation, Lin's concordance, Passing-Bablok regression, and Bland-Altman plots were used. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values for abnormal cells using the MC-80 were calculated. RESULTS: The PPV and NPV were above 98% and 99%, for normal samples. For immature granulocytes (IG), NPV and PPV were 100% and 74.2%. When comparing the WBC differentials using the MC-80, the microscope and the BC-6800Plus, no differences were found except for basophils and IG. Our results showed good agreement between the pre- and post-classification of normal WBC, including IG, quantified by high correlation and concordance values (0.91-1). Sensitivity and specificity for blasts were 0.984 and 0.640. The MC-80 detected abnormal lymphocytes in 30% of the smears from patients with lymphoid neoplasm. Plasma cell identification was better using the DM9600. The sensitivity and specificity for erythroblast detection were 1 and 0.890. CONCLUSION: We found that the MC-80 shows high performance for WBC differentials for both normal samples and patients with haematological diseases.

Kimura disease as an uncommon cause of persistent hypereosinophilia: a diagnostic challenge.

Kimura disease (KD) is an unusual inflammatory disease of unknown etiology. Despite being described many years ago, KD might cause diagnostic difficulty or be confused with other conditions. Here, we present a 33-year-old Filipino woman who was referred to our hospital for evaluation of persistent eosinophilia and intense pruritus. Blood analysis and peripheral blood smear review showed high eosinophil counts (3.8 x109/L, 40%) that did not show morphological abnormalities. Besides, high serum IgE concentration was detected (33,528 kU/L). Serological tests were positive for Toxocara canis and treatment with albendazol was initiated. Nevertheless, increased eosinophil counts were still present after several months, alongside with high serum IgE concentrations and intense pruritus. During her follow-up, an inguinal adenopathy was detected. The biopsy revealed lymphoid hyperplasia with reactive germinal centers and massive eosinophil infiltration. Proteinaceous deposits of eosinophilic material were also observed. All these findings, together with peripheral blood eosinophilia and high IgE concentrations, confirmed the diagnosis of KD. The diagnosis of KD should be considered in the differential diagnosis of long-standing unexplained eosinophilia in association with high IgE concentrations, pruritus and lymphadenopathies.

A novel de novo variant in CASK causes a severe neurodevelopmental disorder that masks the phenotype of a novel de novo variant in EEF2.

We report a 9-year-old Spanish boy with severe psychomotor developmental delay, short stature, microcephaly and abnormalities of the brain morphology, including cerebellar atrophy. Whole-exome sequencing (WES) uncovered two novel de novo variants, a hemizygous variant in CASK (Calcium/Calmodulin Dependent Serine Protein Kinase) and a heterozygous variant in EEF2 (Eukaryotic Translation Elongation Factor 2). CASK gene encodes the peripheral plasma membrane protein CASK that is a scaffold protein located at the synapses in the brain. The c.2506-6 A > G CASK variant induced two alternative splicing events that account for the 80% of the total transcripts, which are likely to be degraded by NMD. Pathogenic variants in CASK have been associated with severe neurological disorders such as mental retardation with or without nystagmus also called FG syndrome 4 (FGS4), and intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH). Heterozygous variants in EEF2, which encodes the elongation factor 2 (eEF2), have been associated to Spinocerebellar ataxia 26 (SCA26) and more recently to a childhood-onset neurodevelopmental disorder with benign external hydrocephalus. The yeast model system used to investigate the functional consequences of the c.34 A > G EEF2 variant supported its pathogenicity by demonstrating it affects translational fidelity. In conclusion, the phenotype associated with the CASK variant is more severe and masks the milder phenotype of EEF2 variant.

Central Regulation of Brown Fat Thermogenesis in Response to Saturated or Unsaturated Long-Chain Fatty Acids.

Sensing of long-chain fatty acids (LCFA) in the hypothalamus modulates energy balance, and its disruption leads to obesity. To date, the effects of saturated or unsaturated LCFA on hypothalamic-brown adipose tissue (BAT) axis and the underlying mechanisms have remained largely unclear. Our aim was to characterize the main molecular pathways involved in the hypothalamic regulation of BAT thermogenesis in response to LCFA with different lengths and degrees of saturation. One-week administration of high-fat diet enriched in monounsaturated FA led to higher BAT thermogenesis compared to a saturated FA-enriched diet. Intracerebroventricular infusion of oleic and linoleic acids upregulated thermogenesis markers and temperature in brown fat of mice, and triggered neuronal activation of paraventricular (PaV), ventromedial (VMH) and arcuate (ARC) hypothalamic nuclei, which was not found with saturated FAs. The neuron-specific protein carnitine palmitoyltransferase 1-C (CPT1C) was a crucial effector of oleic acid since the FA action was blunted in CPT1C-KO mice. Moreover, changes in the AMPK/ACC/malonyl-CoA pathway and fatty acid synthase expression were evoked by oleic acid. Altogether, central infusion of unsaturated but not saturated LCFA increases BAT thermogenesis through CPT1C-mediated sensing of FA metabolism shift, which in turn drive melanocortin system activation. These findings add new insight into neuronal circuitries activated by LCFA to drive thermogenesis.

Drug-drug interactions of ritonavir-boosted SARS-CoV-2 protease inhibitors in solid organ transplant recipients: experience from the initial use of lopinavir-ritonavir.

OBJECTIVES: To review the drug-drug interactions between tacrolimus and lopinavir/ritonavir in 23 patients who received solid organ transplant during the first wave of COVID-19 and to determine the efficacy as well as safety of prednisone monotherapy. METHODS: Observational study performed between March and June 2020 in solid organ transplant recipients admitted with an established diagnosis of SARS-CoV-2 infection who received lopinavir/ritonavir (≥2 doses). Once lopinavir/ritonavir therapy was initiated, calcineurin inhibitor treatment was temporarily switched to prednisone monotherapy (15-20 mg/d) to avoid drug-drug interactions and toxicity. After lopinavir/ritonavir treatment completion, immunosuppressive treatment was restarted with reduced doses of prednisone-tacrolimus (target minimum blood concentration -C0- approximately 5 ng/mL). Patients were observed for 3 months to confirm the absence of rejection. RESULTS: The median time from discontinuation of tacrolimus to initiation of lopinavir/ritonavir was 14 hours (interquartile range [IQR], 12-15) and from discontinuation of lopinavir/ritonavir to resumption of tacrolimus 58 hours (IQR, 47-81). The duration of lopinavir/ritonavir treatment was 7 days (IQR, 5-7). Nine of the 21 (42.8%) patients on tacrolimus treatment had C0 above the cutoff point after lopinavir/ritonavir initiation, despite having been substituted with prednisone before lopinavir/ritonavir initiation. Three patients had very high concentrations (>40 ng/mL) and developed toxicity. No episodes of acute rejection were diagnosed. DISCUSSION: We did not observe toxicity in patients for whom tacrolimus was discontinued 24 hours before starting lopinavir/ritonavir and reintroduced at half dose 48 to 72 hours after lopinavir/ritonavir discontinuation. Prednisone monotherapy during lopinavir/ritonavir therapy was safe with no episodes of acute rejection. Experience with lopinavir/ritonavir may be applicable to the use of nirmatrelvir/ritonavir, but larger multicentre studies are needed to confirm these findings.

Clinical usefulness of circulating tumor markers.

Tumor markers are a heterogeneous group of substances released by cancer cells into bloodstream, but also expressed by healthy tissues. Thus, very small concentrations can be present in plasma and serum from healthy subjects. Cancer patients tend to show increased levels correlating with tumor bulk, but false positive results could be present in patients with benign conditions. The correct interpretation of TM results could be challenging and many factors should be considered, from pre-analytical conditions to patient concomitant diseases. In this line, the Clinical Chemistry and Laboratory Medicine journal has made important contributions though several publications promoting the adequate use of TM and therefore improving patient safety. TM measurement offers valuable information for cancer patient management in different clinical contexts, such as helping diagnosis, estimating prognosis, facilitating early detection of relapse and monitoring therapy response. Our review analyzes the clinical usefulness of tumor markers applied in most frequent epithelial tumors, based on recent evidence and guidelines.

Local Welfare Systems and Health Inequalities: The Effects of Institutional Overlapping and Local Variations.

A growing research agenda shows the importance of local welfare systems in understanding socio-spatial inequalities in health. Welfare services provided by local governments overlap with those provided by other levels of government. Thus, differences in the provision of welfare services between municipalities could explain differences in residents' health, moderating the magnitude of health inequalities if local governments deploy actions capable of positively influencing the social determinants of health. This article attempts to analyse this idea in the Spanish case, exploring the influence of local policies according to the orientation of municipal spending on three indicators of the population's health status: self-perceived health, healthy practices and activity limitations due to health problems. A multilevel cross-sectional study was designed using information from two waves of the 2006-2007 and 2011-2012 National Health Survey for the population aged 15 years and older (N = 31,378) residing in Spanish municipalities of 20,000 inhabitants or over (N = 373). The results show that the magnitude of inequalities in self-perceived health, in the adoption of healthy practices and in daily activity limitations by social class are smaller as municipalities" spending was oriented towards policy areas considered as redistributive. Therefore, the proposed institutional overlap thesis could help understand the role of subnational governments on the magnitude of health inequalities, as well as in comparative analysis between countries with institutional systems in which local governments have a greater or lesser capacity to provide welfare services.

First splicing variant in HECW2 with an autosomal recessive pattern of inheritance and associated with NDHSAL.

We report the clinical and genetic features of a Caucasian girl who presented a severe neurodevelopmental disorder with drug-resistant epilepsy, hypotonia, severe gastro-esophageal reflux and brain magnetic resonance imaging anomalies. WES uncovered a novel variant in homozygosis (g.197092814_197092824delinsC) in HECW2 gene that encodes the E3 ubiquitin-protein ligase HECW2. This protein induces ubiquitination and is implicated in the regulation of several important pathways involved in neurodevelopment and neurogenesis. Furthermore, de novo heterozygous missense variants in this gene have been associated with neurodevelopmental disorder with hypotonia, seizures, and absent language (NDHSAL). The homozygous variant of our patient disrupts the splice donor site of intron 22 and causes the elimination of exon 22 (r.3766_3917+1del) leading to an in-frame deletion of the protein (p.Leu1256_Trp1306del). Functional studies showed a twofold increase of its RNA expression, while the protein expression level was reduced by 60%, suggesting a partial loss-of-function mechanism of pathogenesis. Thus, this is the first patient with NDHSAL caused by an autosomal recessive splicing variant in HECW2.

Validation of a Microwave-Assisted Derivatization Gas Chromatography-Mass Spectrometry Method for the Quantification of 2-Hydroxybutyrate in Human Serum as an Early Marker of Diabetes Mellitus.

Circulating levels of 2-hydroxybutyrate (2HB) are highly related to glycemic status in different metabolomic studies. According to recent evidence, 2HB is an early biomarker of the future development of dysglycemia and type 2 diabetes mellitus and may be causally related to the progression of normal subjects to impaired fasting glucose or insulin resistance. In the present study, we developed and validated a simple, specific and sensitive gas chromatography-mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HB. Liquid-liquid extraction with ethyl acetate was followed by 2 min of microwave-assisted derivatization. The method presented acceptable accuracy, precision and recovery, and the limit of quantification was 5 µM. Levels of 2HB were found to be stable in serum after three freeze-thaw cycles, and at ambient temperature and at a temperature of 4 °C for up to 24 h. Extracts derivatized under microwave irradiation were stable for up to 96 h. No differences were found in 2HB concentrations measured in serum or plasma EDTA samples. In summary, the method is useful for a rapid, precise and accurate quantification of 2HB in serum samples assessed for the evaluation of dysglycemia and diabetes mellitus.

MLIP causes recessive myopathy with rhabdomyolysis, myalgia and baseline elevated serum creatine kinase.

Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the muscular lamin A/C-interacting protein, MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients' skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans and solidifies MLIP's role in normal and diseased skeletal muscle homeostasis.

New subtype of PCH1C caused by novel EXOSC8 variants in a 16-year-old Spanish patient.

We report the case of a 16-year-old Spanish boy with cerebellar and spinal muscular atrophy, spasticity, psychomotor retardation, nystagmus, ophthalmoparesis, epilepsy, and mitochondrial respiratory chain (MRC) deficiency. Whole exome sequencing (WES) uncovered three variants (two of them novel) in a compound heterozygous in EXOSC8 gene (NM_181503.3:c.[390+1delG];[628C>T;815G>C]) that encodes the exosome complex component RRP43 protein (EXOSC8). In order to assess the pathogenicity of these variants, expression experiments of RNA and protein for EXOSC8 were carried out. The c.[390+1delG] variant produces the elimination of exon 7 (r.[345_390del]; p.[Ser116LysfsTer27]) and a decrease of the RNA expression in relation to the other allele (p.[Pro210Ser;Ser272Thr]). Furthermore, total mRNA expression is reduced by 30% and the protein level by 65%. EXOSC8 is an essential protein of the exosome core, a ubiquitously expressed complex responsible for RNA processing and degradation. Recessive mutations in EXOSC8 cause pontocerebellar hypoplasia type 1C (PCH1C), and currently, only two homozygous variants in this gene have been described. However, unlike PCH1C-affected individuals with EXOSC8 variants, our patient presents a normal supratentorial cerebral tissue (neither corpus callosum hypoplasia nor hypomyelination) with a less severe phenotype and longer survival. In conclusion, our data expand both genetic and phenotypic spectrum associated with EXOSC8 variants.

Characterization of a complex phenotype (fever-dependent recurrent acute liver failure and osteogenesis imperfecta) due to NBAS and P4HB variants.

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants.

Peripheral blood morphology review and diagnostic proficiency evaluation by a new Spanish EQAS during the period 2011-2019.

INTRODUCTION: The Spanish Haematology and Haemotherapy Society organizes peripheral blood smear review scheme, focused on the evaluation of diagnostic proficiency of participants by blood cell morphology analysis. The objective was to evaluate the efficacy of this scheme as an educational tool to improve the diagnostic proficiency of the participants. METHODS: During 2011-2019, 54 peripheral blood smears, alongside with patient details such as age, sex, blood cell counts and relevant clinical information, were sent to an average of 125 ± 13 laboratories per year. A number of 44 shipments were selected to analyse whether successive surveys of the same disease may lead to an improvement in the diagnostic success rate proposed by the laboratories. Participants were asked to select the most relevant morphological abnormalities, alongside the diagnostic orientation. Agreement of participant responses with RR was evaluated. RESULTS: Spanish laboratories showed a diagnostic proficiency greater than 80% in acute myeloid leukaemia, including acute promyelocytic leukaemia, mature B-cell neoplasms (hairy cell leukaemia and splenic marginal zone lymphoma), chronic myeloid leukaemia, sickle cell disease, Bernard-Soulier syndrome and infectious mononucleosis. It was important to note the significant improvement over the time in the successive shipments of the same disease, with a 31% and 13% increase in their diagnostic orientation success rate for acute myeloid leukaemia and acute promyelocytic leukaemia cases, respectively, 15% for mantle cell lymphoma and 6% for sickle cell disease. CONCLUSIONS: The present study provides evidence that peripheral blood smear review scheme can be a valid educational tool to improve the clinical pathologist skills in blood morphology and haematological diagnosis.

Adult Plant Slow Rusting Genes Confer High Levels of Resistance to Rusts in Bread Wheat Cultivars From Mexico.

Rust diseases continuously threaten global wheat production: stem rust, leaf rust, and yellow rust caused by Puccinia graminis f. sp. tritici, Puccinia triticina, and Puccinia striiformis f. sp. tritici, respectively. Recent studies indicated that the average losses from all these three rusts reached up to 15.04 million tons per year, which is equivalent to an annual average loss of around US $2.9 billion per year. The major focus of Mexican and worldwide breeding programs is the release of rust resistant cultivars, as this is considered the best option for controlling rust diseases. In Mexico, the emphasis has been placed on genes that confer partial resistance in the adult plant stage and against a broad spectrum of rust races since the 1970s. In this study, a set of the first-generation tall varieties developed and released in the 1940s and 1950s, the first semi-dwarfs, and other releases in Mexico, all of which showed different levels of rust resistance have been phenotyped for the three rust diseases and genotyped. Results of the molecular marker detection indicated that Lr34, Lr46, Lr67, and Lr68 alone or in different gene combinations were present among the wheat cultivars. Flag leaf tip necrosis was present in all cultivars and most were positive for brown necrosis or Pseudo Black Chaff associated with the Sr2 stem rust resistance complex. The phenotypic responses to the different rust infections indicate the presence of additional slow rusting and race-specific resistance genes. The study reveals the association of the slow rusting genes with durable resistance to the three rusts including Ug99 in cultivars bred before the green revolution such as Frontera, Supremo 211, Chapingo 48, Yaqui 50, Kentana 52, Bajio 52, Bajio 53, Yaqui 53, Chapingo 53, Yaktana Tardio 54, and Mayo 54 and their descendants after intercrossing and recombination. These slow rusting genes are the backbone of the resistance in the current Mexican germplasm.

MAST1 variant causes mega-corpus-callosum syndrome with cortical malformations but without cerebellar hypoplasia.

We report the case of a Caucasian Spanish origin female who showed severe psychomotor developmental delay, hypotonia, strabismus, epilepsy, short stature, and poor verbal language development. Brain magnetic resonance imaging scans showed thickened corpus callosum, cortical malformations, and dilated and abnormal configuration of the lateral ventricles without hydrocephalus. Whole-exome sequence uncovered a de novo variant in the microtubule associated serine/threonine kinase 1 gene (MAST1; NM_014975.3:c.1565G>A:p.(Gly522Glu)) that encodes for the MAST1. Only 12 patients have been identified worldwide with 10 different variants in this gene: six patients with mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations; two patients with microcephaly and cerebellar hypoplasia; two patients with autism, one patient with diplegia, and one patient with microcephaly and dysmorphism. Our patient shows a new phenotypic subtype defined by mega-corpus-callosum syndrome with cortical malformations without cerebellar hypoplasia. In conclusion, our data expand the phenotypic spectrum associated to MAST1 gene variants.

A novel de novo mutation in the PURA gene associated with a new clinical finding: large brainstem.

We report the case of a Caucasian Spanish boy, who showed profound neonatal hypotonia, feeding difficulties, apnea, severe developmental delay, epilepsy, bilateral convergent strabismus, poor verbal language development and a large brainstem. Whole-exome sequence uncovered a novel de novo mutation in the purine-rich element binding protein A gene (PURA; NM_005859.4:c.72del:p.(-Gly25AlafsTer53)) that encodes the transcriptional activator protein Pur-alpha (PURA). Mutations in this gene have been identified in patients with PURA syndrome, a rare disorder characterized by an early hypotonia, developmental delay, severe intellectual disability with or without epilepsy, and disability in expressive language development. Although, up to 75 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with a brainstem larger than normal. In conclusion, our data expand both geneticand phenotypic spectrum associated with PURA gene mutations.