Multiorgan manifestations of COL4A1 and COL4A2 variants and proposal for a clinical management protocol.

COL4A1/2 variants are associated with highly variable multiorgan manifestations. Depicting the whole clinical spectrum of COL4A1/2-related manifestations is challenging, and there is no consensus on management and preventative strategies. Based on a systematic review of current evidence on COL4A1/2-related disease, we developed a clinical questionnaire that we administered to 43 individuals from 23 distinct families carrying pathogenic variants. In this cohort, we extended ophthalmological and cardiological examinations to asymptomatic individuals and those with only limited or mild, often nonspecific, clinical signs commonly occurring in the general population (i.e., oligosymptomatic). The most frequent clinical findings emerging from both the literature review and the questionnaire included stroke (203/685, 29.6%), seizures or epilepsy (199/685, 29.0%), intellectual disability or developmental delay (168/685, 24.5%), porencephaly/schizencephaly (168/685, 24.5%), motor impairment (162/685, 23.6%), cataract (124/685, 18.1%), hematuria (63/685, 9.2%), and retinal arterial tortuosity (58/685, 8.5%). In oligosymptomatic and asymptomatic carriers, ophthalmological investigations detected retinal vascular tortuosity (5/13, 38.5%), dysgenesis of the anterior segment (4/13, 30.8%), and cataract (2/13, 15.4%), while cardiological investigations were unremarkable except for mild ascending aortic ectasia in 1/8 (12.5%). Our multimodal approach confirms highly variable penetrance and expressivity in COL4A1/2-related conditions, even at the intrafamilial level with neurological involvement being the most frequent and severe finding in both children and adults. We propose a protocol for prevention and management based on individualized risk estimation and periodic multiorgan evaluations.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Atypical Ocular Coloboma in Tuberous Sclerosis-2: Report of Two Novel Cases.

ABSTRACT: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystemic disorder caused by mutations in either TSC1 or TSC2 genes and is characterized by hamartomas in multiple organs. The most frequent and best-known ocular manifestation in TSC is the retinal hamartoma. Less frequent ocular manifestations include punched out areas of retinal depigmentation, eyelid angiofibromas, uveal colobomas, papilledema, and sector iris depigmentation. In this article, we report 2 patients carrying known pathogenic variants in the TSC2 gene who exhibited an atypical, unilateral, iris coloboma associated with localized areas of retinal dysembryogenesis.

Neurosurgical treatment of subependymal giant cell astrocytomas in tuberous sclerosis complex: a series of 44 surgical procedures in 31 patients.

BACKGROUND: Subependymal giant cell astrocytomas (SEGA) are benign tumors characteristic of tuberous sclerosis complex (TSC) that may cause hydrocephalus. Various treatments are nowadays available as mTOR inhibitors or surgery. Surgery is still a valid option especially for symptomatic and larger tumors. METHODS: From January 1994 to December 2015, 31 TSC patients harboring SEGA underwent surgery at the Department of Neurosurgery of the Meyer Pediatric Hospital, Florence. Indications for surgery were tumor size and location, growth and cystization/hemorrhage, and hydrocephalus. Clinical data, preoperative and postoperative MRI, recurrence rate, further surgical procedures, and related complications were analyzed. RESULTS: A total of 44 surgeries were performed in 31 TSC patients affected by SEGA, achieving gross total removal (GTR) and subtotal removal (STR), respectively, in 36 and 8 patients. Recurrences occurred in 11 patients; 9 of them underwent further surgical procedures and 2 were treated with mTOR pathway inhibitors. Surgical morbidity and mortality were, respectively, 22.7% and 2.3%. After a mean follow-up of 4.9 years, 90% of patients were tumor-free with good neurological status in 93.3%; twelve (40%) had a ventriculo-peritoneal shunt (VPS) for hydrocephalus. CONCLUSIONS: The present series confirms that the surgical approach, combined with mTOR inhibitors, is still a valid option for the treatment of SEGAs.

Clinical and neuroimaging features of autosomal recessive spastic paraplegia 35 (SPG35): case reports, new mutations, and brief literature review.

Spastic paraplegia 35 (SPG35) is a recessive condition characterized by childhood onset, progressive course, complicated by dystonia, dysarthria, cognitive impairment, and epilepsy. Mutations in the FA2H gene have been described in several families, leading to the proposal of a single entity, named fatty acid hydrolase-associated neurodegeneration (FAHN). Several reports have described a polymorphic radiological picture with white matter lesions of various degrees and a distinct form of neurodegeneration with brain iron accumulation. While we reviewed the pertinent literature, we also report three new patients with SPG35, highlighting the possible absence of white matter lesions even after a long neuroimaging follow-up. Three-dimensional modeling of the mutated proteins was helpful to elucidate the role of the site of mutations and the correlation with the residual enzyme activity as determined in cultured skin fibroblasts.

Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy.

De novo in-frame deletions and duplications in the SPTAN1 gene, encoding the non-erythrocyte αII spectrin, have been associated with severe West syndrome with hypomyelination and pontocerebellar atrophy. We aimed at comprehensively delineating the phenotypic spectrum associated with SPTAN1 mutations. Using different molecular genetic techniques, we identified 20 patients with a pathogenic or likely pathogenic SPTAN1 variant and reviewed their clinical, genetic and imaging data. SPTAN1 de novo alterations included seven unique missense variants and nine in-frame deletions/duplications of which 12 were novel. The recurrent three-amino acid duplication p.(Asp2303_Leu2305dup) occurred in five patients. Our patient cohort exhibited a broad spectrum of neurodevelopmental phenotypes, comprising six patients with mild to moderate intellectual disability, with or without epilepsy and behavioural disorders, and 14 patients with infantile epileptic encephalopathy, of which 13 had severe neurodevelopmental impairment and four died in early childhood. Imaging studies suggested that the severity of neurological impairment and epilepsy correlates with that of structural abnormalities as well as the mutation type and location. Out of seven patients harbouring mutations outside the α/ß spectrin heterodimerization domain, four had normal brain imaging and three exhibited moderately progressive brain and/or cerebellar atrophy. Twelve of 13 patients with mutations located within the spectrin heterodimer contact site exhibited severe and progressive brain, brainstem and cerebellar atrophy, with hypomyelination in most. We used fibroblasts from five patients to study spectrin aggregate formation by Triton-X extraction and immunocytochemistry followed by fluorescence microscopy. αII/ßII aggregates and αII spectrin in the insoluble protein fraction were observed in fibroblasts derived from patients with the mutations p.(Glu2207del), p.(Asp2303_Leu2305dup) and p.(Arg2308_Met2309dup), all falling in the nucleation site of the α/ß spectrin heterodimer region. Molecular modelling of the seven SPTAN1 amino acid changes provided preliminary evidence for structural alterations of the A-, B- and/or C-helices within each of the mutated spectrin repeats. We conclude that SPTAN1-related disorders comprise a wide spectrum of neurodevelopmental phenotypes ranging from mild to severe and progressive. Spectrin aggregate formation in fibroblasts with mutations in the α/ß heterodimerization domain seems to be associated with a severe neurodegenerative course and suggests that the amino acid stretch from Asp2303 to Met2309 in the α20 repeat is important for α/ß spectrin heterodimer formation and/or αII spectrin function.

The application of artificial intelligence to understand the pathophysiological basis of psychogenic nonepileptic seizures.

Psychogenic nonepileptic seizures (PNES) are episodes of paroxysmal impairment associated with a range of motor, sensory, and mental manifestations, which perfectly mimic epileptic seizures. Several patterns of neural abnormalities have been described without identifying a definite neurobiological substrate. In this multicenter cross-sectional study, we applied a multivariate classification algorithm on morphological brain imaging metrics to extract reliable biomarkers useful to distinguish patients from controls at an individual level. Twenty-three patients with PNES and 21 demographically matched healthy controls (HC) underwent an extensive neuropsychiatric/neuropsychological and neuroimaging assessment. One hundred and fifty morphological brain metrics were used for training a random forest (RF) machine-learning (ML) algorithm. A typical complex psychopathological construct was observed in PNES. Similarly, univariate neuroimaging analysis revealed widespread neuroanatomical changes affecting patients with PNES. Machine-learning approach, after feature selection, was able to perform an individual classification of PNES from controls with a mean accuracy of 74.5%, revealing that brain regions influencing classification accuracy were mainly localized within the limbic (posterior cingulate and insula) and motor inhibition systems (the right inferior frontal cortex (IFC)). This study provides Class II evidence that the considerable clinical and neurobiological heterogeneity observed in individuals with PNES might be overcome by ML algorithms trained on surface-based magnetic resonance imaging (MRI) data.

Diagnostic Targeted Resequencing in 349 Patients with Drug-Resistant Pediatric Epilepsies Identifies Causative Mutations in 30 Different Genes.

Targeted resequencing gene panels are used in the diagnostic setting to identify gene defects in epilepsy. We performed targeted resequencing using a 30-genes panel and a 95-genes panel in 349 patients with drug-resistant epilepsies beginning in the first years of life. We identified 71 pathogenic variants, 42 of which novel, in 30 genes, corresponding to 20.3% of the probands. In 66% of mutation positive patients, epilepsy onset occurred before the age of 6 months. The 95-genes panel allowed a genetic diagnosis in 22 (6.3%) patients that would have otherwise been missed using the 30-gene panel. About 50% of mutations were identified in genes coding for sodium and potassium channel components. SCN2A was the most frequently mutated gene followed by SCN1A, KCNQ2, STXBP1, SCN8A, CDKL5, and MECP2. Twenty-nine mutations were identified in 23 additional genes, most of them recently associated with epilepsy. Our data show that panels targeting about 100 genes represent the best cost-effective diagnostic option in pediatric drug-resistant epilepsies. They enable molecular diagnosis of atypical phenotypes, allowing to broaden phenotype-genotype correlations. Molecular diagnosis might influence patients' management and translate into better and specific treatment recommendations in some conditions.

Fast Progression of Cerebellar Atrophy in PLA2G6-Associated Infantile Neuronal Axonal Dystrophy.

Infantile neuronal axonal dystrophy (INAD) is characterized by progressive cerebellar atrophy. MRI has been recommended as a marker of disease progression in cerebellar diseases. We performed a longitudinal brain volumetry study in a couple of bicorial twins with PLA2G6-positive INAD. Brain volumetry was calculated with FreeSurfer software on 3T T1-weighted images acquired at age 28 (t 0) and 36 months (t 1) in patient 1 and at age 22 (t 0) and 31 months (t 1) in patient 2. Data at t 0 were compared to those obtained in 18 control children aged 14-44 months with normal MRI. At t 0, both patients showed markedly lower cerebellar volume compared to controls. At t 1, both patients exhibited a remarkable decrease of cerebellar volume (-25.8% in patient 1; -16.5% in patient 2) and of frontal (-6.8% in patient 1 and -3.3% in patient 2) and occipital (-9.8% in patient 1 and -9.1% in patient 2) cortical GM volume. Our MRI morphometry study indicates that INAD is characterized by a remarkably fast progression of cerebellar atrophy and mild atrophy of the frontal and occipital cortex presumably secondary to deafferentation in the cortical-pons-cerebellum-rubro-thalamus-cortical circuit and visual pathways.

STARR with Contour Transtar for Obstructed Defecation Syndrome: Long-Term Results.

INTRODUCTION: Obstructed defecation syndrome (ODS) is a widespread and disabling syndrome. With this study, we want to evaluate the long-term results of stapled transanal rectal resection (STARR) performed with Contour Transtar device in the treatment for ODS. A re-evaluation of 113 patients subjected to STARR from June 2007 to January 2010 was conducted. METHODS: All the patients treated for symptomatic ODS with STARR with Contour Transtar were included in the study. We re-evaluate all patients treated in the study period with clinical examination and specific questionnaire to verify the stability of the functional results and the satisfaction at 5 years from surgery. Constipation was graded using the Agachan-Wexner constipation score; eventual use of aids to defecate and patient satisfaction were assessed preoperatively, 6 months and 5 years after surgery. Long-term complications were also investigated. RESULTS: Constipation intensity decreased from the preoperative value of 15.8 (±4.9) to 5.2 (±3.9) (p < 0.0001) at 6 months and remained stable after 5 years (7.4 ± 4.1; p < 0.01). Patients who use laxatives and enema decrease from 74 (77%) and 27 (28%) to only 16 (17%; p < 0.001) and 5 (5%; p < 0.001), respectively, at 5-year follow-up. None continue to help themselves with digitations after surgery. Also the satisfaction rate remained stable (3.64 vs 3.81) during the 5 years of the study. CONCLUSION: The long-term results have demonstrated the efficacy of the STARR with Contour Transtar in treating ODS and the stability over time of the defecatory improvements. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov NCT02971332.

Psychoactive substances belonging to the amphetamine class potently activate brain carbonic anhydrase isoforms VA, VB, VII, and XII.

Identifying possible new biological activities of psychoactive substances belonging to various chemical classes may lead to a better understanding of their mode of action and side effects. We report here that amines structurally related to amphetamine, a widely used psychoactive substance, such as amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in important physiological functions. Of the 11 investigated human (h) isoforms, the widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII, and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to their activation of these enzymes.

Advances in new psychoactive substances identification: the U.R.I.To.N. Consortium.

Identification of new psychoactive substances (NPS) in biological and non-biological samples represents a hard challenge for forensic toxicologists. Their great chemical variety and the speed with which new NPS are synthesised and spread make stringent the need of advanced tools for their detection based on multidisciplinary approaches. For this reason, in August 2016, the "Unit of Research and Innovation in Forensic Toxicology and Neuroscience of Addiction" (U.R.I.To.N.) was founded by the Forensic Toxicology Division of the University of Florence. In this Research Unit, various professionals (i.e. forensic toxicologists, chemists, physicians) collaborate to study all the aspects of drugs of abuse, especially NPS. Herein, we describe the multidisciplinary approach comprising liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), gas chromatography hyphenated to mass spectrometry (GC-MS) and solution nuclear magnetic resonance analysis that allowed the identification of three NPS such as 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 2-amino-1-(4-bromo-2,5-dimethoxyphenyl)ethan-1-one (bk-2C-B), and 3-(2-aminopropyl)indole (α-methyltryptamine) in seized materials.

Alcohol, caffeine, and nicotine consumption in adolescents: hair analysis versus self-report.

BACKGROUND: Clinical reliability of self-reported data for alcohol, caffeine, and nicotine consumptions is lacking, particularly in adolescents. OBJECTIVES: To compare a self-report questionnaire and hair analysis to assess the reliability and effectiveness of the self-report. METHODS: A cross-sectional study on 14-15-year-old Italian students (n = 874, 38% males, 62% females) was performed comparing self-reported data to hair analysis. The latter quantified hair concentrations of caffeine, nicotine, cotinine, ethyl glucuronide (EtG), and fatty acid ethyl esters (FAEEs) using mass spectrometry. RESULTS: Concordance between self-report and hair testing ranged from good to poor across substances and levels of use: poor for heavy alcohol intake (EtG: k = 0.36, 20 positive cases by hair analysis, false negative by self-report, 2.3% of total sample; FAEE k = 0.31, 25 positive cases, 2.9% of total sample); fair to poor for active smokers (k = 0.40, 125 positive cases, 14.3% of total sample); and moderate for caffeine (k = 0.57, 56 positive cases, 6.4% of total sample). CONCLUSIONS: Epidemiological studies on alcohol, caffeine, and nicotine consumption in adolescents may benefit from the inclusion of toxicological analysis on hair samples to overcome the under-reporting phenomenon of questionnaires and detect more cases of problematic substance use.

A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS.

Fatty acid ethyl esters (FAEEs) and ethyl-glucuronide (EtG) in meconium have been widely studied as biomarkers of maternal alcohol consumption during pregnancy. Many analytical approaches have been proposed for their analysis, mostly consisting of separated extraction procedures requiring the use of two meconium aliquots. This study aimed to validate a new analytical procedure for the simultaneous extraction of FAEEs and EtG from a meconium aliquot through a single solid-phase extraction (SPE) applied to 242 anonymized samples of meconium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl-palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetonitrile, and a single SPE performed by means of aminopropyl columns. FAEEs were eluted with hexane, followed by EtG elution with water. Both the mixtures were dried, recovered, and analyzed by liquid chromatography-tandem mass spectrometry using C8 (FAEEs) and C18 (EtG) columns. Transitions were: m/z 257 → 57,88, Myr; m/z 262 → 57,88, Myr-d5; m/z 285 → 57, 72, Pal; m/z 290 → 57,258, Pal-d5; m/z 311 → 72,114, Ole; m/z 316 → 72,265, Ole-d5; m/z 257 → 57,72 Ste; m/z 318 → 57,286, Ste-d5; m/z 221 → 75,85, EtG; m/z 226 → 75,85, EtG-d5. Lower limit of quantification range was 10-15 ng/g for FAEEs and 10 ng/g for EtG. Linearity was evaluated for different concentration ranges; the mean coefficients of determination (R (2)) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respectively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficiency was 93 % for EtG and 75-85 % for FAEEs.

Does ghost ileostomy have a role in the laparoscopic rectal surgery era? A randomized controlled trial.

BACKGROUND: Anastomotic leakage following anterior rectal resection is the most important and most commonly faced complication of laparoscopy and open surgery. To prevent this complication, the construction of a preventing stoma is usually adopted. It is not easy to decide whether to construct a protective stoma in patients with a medium risk of anastomotic leakage. In these patients, ghost ileostomy (GI), a pre-stage ileostomy that can be externalized and opened if needed, has proved useful. We conducted a prospective, randomized, controlled study to evaluate the advantages of GI in laparoscopic rectal resection. METHODS: All patients with surgical indications for laparoscopic rectal resection who were at medium risk for anastomotic leakage from January 2007 to January 2013 were included and were randomly divided in 2 groups. All of the patients were subjected to laparoscopic anterior rectal resection with the performance of GI (group A) or without the construction of any protective stoma (group B). The presence and severity of clinically evident postoperative anastomotic leakage and other postoperative complications and reinterventions were investigated. RESULTS: Of the 55 patients allocated to group A, 3 experienced anastomotic leakage compared with 4 in group B. The patients with GI experienced a lower severity of anastomotic leakage and shorter hospitalization compared with the patients in group B. None of the patients with GI and anastomotic leakage required laparotomy to treat the dehiscence. CONCLUSIONS: The use of GI in laparoscopic rectal resections in patients at medium risk for anastomotic leakage was useful because it allowed for the avoidance of stoma creation in all of the patients, thus reducing the number of stomas performed, improving the quality of life of the patients and preserving, in most cases, the benefits gained by laparoscopy.

Vertical extraventricular functional hemispherotomy: a new variant for hemispheric disconnection. Technical notes and results in three patients.

PURPOSE: Hemispherectomy and disconnective hemispherotomy are the most effective epilepsy surgical procedures for the treatment of epilepsy due to hemispheric pathologies such as Sturge-Weber syndrome, diffuse hemispheric cortical dysplasia, and posttraumatic and postischemic focal lesions. Disconnective hemispherotomy is nowadays preferred to reduce surgical morbidity in term of early and late complications (i.e., cerebral superficial hemosiderosis). Despite the number of existing technical variants conceived to further reduce the amount of brain tissue to be removed, postoperative hydrocephalus still persists and may account for an average incidence of 15-41% according to different series and reviews. A new variant of disconnective vertical hemispherotomy we termed vertical extraventricular parasagittal hemispherotomy is described aiming to further reduce the amount of removed brain tissue and so the risk of postoperative hydrocephalus in favor of a pure hemispheric disconnection. METHODS: Three patients affected by drug-resistant epilepsy due to different hemispheric pathologies (posttraumatic epilepsy, Sturge-Weber syndrome, diffuse hemispheric cortical dysplasia) were considered to be candidates for vertical extraventricular parasagittal hemispherotomy disconnective based on presurgical evaluation protocol. The oldest patient was 15 years old, the two youngest were both 2 years old. RESULTS: None of the patients experienced early and late surgical complications. After a mean follow-up of 36 months (range 12-60 months), two patients were seizure free, one relapsed seizures 18 months later. Postoperative hydrocephalus never occurred. CONCLUSION: Vertical extraventricular parasagittal hemispherotomy may be an efficacious and less invasive technique as it consists in a pure disconnection of the hemisphere with less amount of brain tissue removed and a theoretical reduced risk of postoperative hydrocephalus.

Does the removal of retained staples really improve postoperative chronic sequelae after transanal stapled operations?

BACKGROUND: Transanal stapled procedures are increasingly being used. Several postoperative complications can be referred to their application, including those related to the presence of retained staples at the level of the staple line. OBJECTIVE: This study was conducted to assess whether the removal of the retained staples is a useful approach to improve some of the most common postoperative complications of these surgical techniques. DESIGN: This is a retrospective study. SETTINGS: The study was conducted at the One-Day Surgery Unit of St. Andrea Hospital. PATIENTS: All of the patients who underwent a stapled transanal procedure from January 2003 to December 2011 were included in the study. Patients included in the study were followed postoperatively for 1 year after surgery to identify the presence of retained staples. INTERVENTIONS: If identified, the retained staples were removed endoscopically or transanally. MAIN OUTCOME MEASURES: After the staple removal, patients were followed with biweekly office visit for 2 months to evaluate the progression of symptoms. RESULTS: From the 566 patients included in the study, 165 experienced postoperative complications, and in 66 of these cases, retained staples were found and removed. With the removal of retained staples, symptoms were almost all resolved or improved. In only 1 case did the retained staples removal not modify the symptoms. LIMITATIONS: The study design may have introduced potential selection bias. In addition, the study was limited by the lack of a specific questionnaire for the evaluation of symptoms improvement. CONCLUSIONS: The removal of the retained staples is an efficacious and safe procedure to solve or improve postoperative complications and should be always considered.

Enhancing the sensitivity of the LC-MS/MS detection of propofol in urine and blood by azo-coupling derivatization.

Propofol is a low-polarity, volatile molecule that is difficult for an electrospray ion source (ESI) to ionize in either negative ion mode (NIM) or positive ion mode (PIM), which hampers its detection via liquid chromatography-mass spectrometry. The aim of the present study was to use a new derivatization agent to improve ionization efficiency and to develop an efficient liquid chromatography-multiple mass spectrometry (LC-MS/MS) determination of propofol in urine and blood, taking advantage of an electrophilic aromatic substitution. An azo-coupling reaction with a diazonium salt from aniline was performed to introduce a protonation site into the molecule. The diazonium salt was generated by aniline in water solution by HCl and sodium nitrite; derivatization was achieved by stirring a mixture of the diazonium salt and propofol in sodium hydroxide solution for 30 min below 5 °C. A liquid-liquid extraction with dichloromethane and ethyl acetate was performed to obtain the azo derivative (molecular composition: C18H22ON2; molecular weight: 282 Da) in high yield. The compound provided very high ionization yields in both PIM and NIM ESI, and the protonated or deprotonated molecule gave intense signals. The transitions m/z 283 → 77, 241 and m/z 281 → 176, 161 were chosen for the PIM and NIM, respectively, in order to develop quantitative methods of detecting propofol in urine and blood via triple-quadrupole LC-MS/MS. These methods proved to be highly sensitive, with limits of quantification of 0.4 pg/mL and 0.1 ng/mL obtained in the NIM when analyzing 1 mL of urine and 100 µL of blood, respectively.

Generation of a human induced pluripotent stem cell line from a patient with GM3 synthase deficiency using self-replicating RNA vector.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.