Case report: The success of empagliflozin therapy for glycogen storage disease type 1b

IntroductionGlycogen storage disease type 1b (GSD-1b) is characterized by neutropenia and neutrophil dysfunction generated by the accumulation of 1,5-anhydroglucitol-6-phosphate in neutrophils. Sodium-glucose co-transporter 2 inhibitors, such as empagliflozin, facilitate the removal of this toxic metabolite and ameliorate neutropenia-related symptoms, including severe infections and inflammatory bowel disease (IBD). Our case series presents the treatment of three pediatric GSD-1b patients with empagliflozin over a follow-up of three years; the most extended reported follow-up period to date.Cases descriptionA retrospective analysis of empagliflozin treatment of three pediatric GSD-1b patients (two male and one female; ages at treatment initiation: 4.5, 2.5 and 6 years) was performed. Clinical and laboratory data from a symmetrical period of up to three years before and after the therapy introduction was reported. Data on the clinical course of the treatment, IBD activity, the need for antibiotic treatment and hospitalizations, neutrophil count and function, and markers of inflammation were assessed. Prior the introduction of empagliflozin, patients had recurrent oral mucosa lesions and infections, abdominal pain, and anemia. During empagliflozin treatment, the resolution of aphthous stomatitis, termination of abdominal pain, reduced frequency and severity of infections, anemia resolution, increased appetite, and improved wound healing was observed in all patients, as well as an increased body mass index in two of them. In a patient with IBD, long-term deep remission was confirmed. An increased and stabilized neutrophil count and an improved neutrophil function enabled the discontinuation of G-CSF treatment in all patients. A trend of decreasing inflammation markers was detected.ConclusionsDuring the three-year follow-up period, empagliflozin treatment significantly improved clinical symptoms and increased the neutrophil count and function, suggesting that targeted metabolic treatment could improve the immune function in GSD-1b patients

Prikaz primera prvega uspešnega genskega zdravljenja slovenskega bolnika z mukopolisaharidozo tipa I v tujini

Mukopolisaharidoze so skupina lizosomskih bolezni kopičenja. Njihova skupna značilnost je pomanjkanje delovanja encimov, ki razgrajujejo glikozaminoglikane, polisaharide, ki se povezujejo s proteoglikani in tvorijo zunajcelični matriks. Ker ni encimov, ki bi glikozaminoglikane razgrajevali, se ti kopičijo v lizosomih in povzročijo njihovo okvaro in zato se okvarijo drugi celični organeli, celice in končno organi. Klinična slika je široka, od nevro-kognitivnega upada, skeletno-mišičnih deformacij in tipičnih obraznih sprememb. Ključno je zgodnje prepoznavanje bolezni, čeprav s trenutno dostopnimi načini zdravljenja bolezni ne moremo ozdraviti, lahko pa le upočasnimo njen potek, kar je najbolj učinkovito v fazi bolezni še pred pojavom simptomov. Obetavni način zdravljenja je gensko zdravljenje, ki nakazuje možnost ozdravitve bolezni. Predstavljamo primer dečka, pri katerem je bil prepoznan zgodnji kognitivni upad in za bolezen tipične spremembe. Napoten je bil v terciarno ustanovo, kjer je bila diagnoza potrjena. Deček je bil julija 2018 zdravljen z eksperimentalnim genskim zdravljenjem v tujini (bolnišnica San Raffaele, Milano, Italija). V opisanem primeru gre po našem vedenju za prvo uspešno izpeljano gensko zdravljenje pri slovenskih bolnikih ter za enega prvih primerov genskega zdravljenja mukopolisaharidoze tipa I v svetovnem merilu. Gensko zdravljenje s tem postaja del nove klinične stvarnosti, kar predstavlja pomemben mejnik za naš prostor. Zaradi naprednih zdravljenj bi bilo v prihodnosti smiselno uvesti presejalno testiranje novorojenčkov za MPS tipa I, ki se pri nas ali v Evropi doslej še ne izvaja. Do tedaj pa ostaja najpomembnejši predpogoj za uspešno zdravljenje zgodnja klinična prepoznava bolezni in napotitev v terciarno zdravstveno ustanovo

The value of semi-automatic ultrasound measurement of carotid artery intima-media thickness in the evaluation of vascular health in children and adolescents

Uvod. Zgodnja opredelitev otrok, ki imajo povišano tveganje za zgodnji razvoj ateroskleroze je neobhodno potrebno za nacrtovanje ukrepov primarne preventive za ohranjanje žilnega zdravja. V tem doktorskem delu je orisana patofiziologija razvoja ateroskleroze pri otrocih, s poudarkom na kohortah otrok s kronicnimi boleznimi, ki prispevajo k zgodnjim zapletom ateroskleroze. Predstavljene so dostopne metode ocene žilnega zdravja, s poudarkom na ultrazvocni oceni perifernega ožilja. Izmed teh smo za oceno žilnega zdravja v izbrani populaciji izbrali neinvazivni biomarker napredovanja ateroskleroze, debelino intime-medie karotidne arterije. Testirali smo novo, polavtomatsko radiofrekvencno ultrazvocno metodo meritve cIMT, RFQIMT, ki je predstavljena kot primerna za uporabo pri klinicnem delu tudi za specialiste ne-radioloških in ne-kardioloških ved. Metoda za zdravo evropsko otroško populacijo ni imela znanih referencnih vrednosti debeline intime medie karotidne arterije. Cilji. Oceniti debelino intime medie karotidne arterije z radiofrekvencno ultrazvocno metodo, ki nudi programsko vodeno meritev (RF-Quality Intima-Media Thickness RFQIMT, Esaote®, Genova, Italija). Meritev smo opravili na zdravi kohorti kavkazijskih otrok starih od 6 do 18 let. Dodaten cilj je bil opredeliti vpliv spola, starosti, telesne višine, pubertetnega statusa, debelosti, krvnega tlaka in nizke telesne zmogljivosti na debelino intime medie karotidne arterije. Metode in Preiskovanci. Z vprašalnikom smo pridobili podatke o starosti, morebitnih kronicnih boleznih in uporabi zdravil ter pubertetnem statusu. Antropometricne meritve ter meritve krvnega tlaka so bile izvedene po ustaljenih postopkih. Sledil je izracun opisne statistike za starost, spol, pubertetni status antropometricne mere, oceno debelosti, krvnega tlaka, fizicne zmogljivosti in njihovih SDS vrednosti. Normativne vrednosti cIMT za RFQIMT metodo je bil izracunan z metodo LMS. Vpliv spremenljivk na cIMT je bil ocenjen z ANOVA testom. Vpliv neodvisnih spremenljivk na vrednost cIMT smo ocenili z modelom multiple linearne regresije. Narejen je bil sistematski pregled objavljenih normativnih vrednosti cIMT v literature, vrednosti so bile primerjane z izdelanimi normative te študije. Statisticna analiza je bila narejena z uporabo programa R-project 3.6.0 software (https://cran.r-project.org). Hipoteze. cIMT izmerjena z uporabo RFQIMT metode narašca s starostjo, linearno rastjo in pubertetnim razvojem pri otrocih in mladostnikih starosti 6 do 18 let. Spol pomembno vpliva na debelino cIMT izmerjene z RFQIMT metodo. Normativne vrednosti cIMT izmerjene z RFQIMT metodo so primerljive z normativnimi vrednostmi objavljenimi v literaturi, ki so bile izmerjene z uporabo drugih validiranih ultrazvocnih metod. cIMT izmerjena z RFQIMT metodo je višja že pri otrocih in mladostnikih ob prisotnih dejavnikih tveganja: debelosti, višjem krvnem tlaku ter nizki fizicni zmogljivosti. Rezultati. 1137 otrok z normalno telesno težo ter vrednostmi krvnega tlaka (decki: N = 512povprecna starost 12.04 ± 3.52 let, deklice: N = 625, povprecna starost 12.98 ± 3.83 let) je bilo vkljucenih v izracun normativnih vrednosti cIMT za spol, starost ter telesno višino z uporabo LMS metode. Normativne vrednosti so predstavljene v percentilnih krivuljah, tabelah ter LMS tabeli loceno po spolu, starosti in telesni višini in so pripravljene za nadaljnjo klinicno uporabo. cIMT narašca s starostjo, višino, obsegom bokov ter ITM in je višji pri deckih. Debelost, krvni tlak in fizicna zmogljivost lahko vplivajo na višje vrednosti cIMT že v opisani kohorti otrok in mladostnikov. Zakljucki. RFQIMT metoda omogoca meritev cIMT že od 6. leta dalje. Vrednost cIMT zavisi od spola, starosti in telesne višine. Vpliv debelosti, višjega krvnega tlaka in slabe fizicne zmogljivosti na hitrejši potek zgodnjih stopenj ateroskleroze se kaže že v otroški dobi.Background. Early identification of children at risk of atherosclerosis (AS) is paramount for implementing primary preventive measures addressing vascular health. This dissertation presents the background of the pathophysiology of AS development in children, with the cohorts of children carrying risk factors for early vascular aging. Known methods of vascular health evaluation focusing on ultrasound technology, which evaluates changes in the vascular wall of the peripheral vascular tree, are briefly introduced. Among these methods, a noninvasive biomarker of AS progression, carotid intima–media thickness (cIMT), was chosen to evaluate vascular wall properties in the study population. A new semi-automatic technology, RFQIMT, is claimed to be useful for non-radiology and non-cardiology specialists in clinical practice, and it was tested. However, this method has no known reference values for the European healthy pediatric population. Objectives. To evaluate the normative values of cIMT, the radiofrequency-based software-guided technique RF-Quality Intima–Media Thickness (RFQIMT) was used in a healthy cohort of Caucasian children 6 to 18 years old. In addition, the correlation of sex, age, height, pubertal status, obesity, blood pressure, and low physical fitness with cIMT value was analyzed. Methods and Subjects. Healthy volunteers 6 to 18 years old were enrolled in the study with parental consent. Individuals with chronic illness were excluded. Age, chronic illness, medication use, and pubertal status were acquired with a questionnaire. Anthropometric and blood pressure measurements were obtained using standardized methods. Descriptive statistics for age, sex, pubertal developmental stage, anthropometrics, obesity measures, blood pressure, and physical fitness and their standard deviation scores were calculated. Sex-, age-, and height-specific normative values of cIMT for the RFQIMT method were calculated using the LMS method for the non-obese and non-hypertensive sub-cohort (cohort A) of children. A systematic literature review of published normal cIMT values in children was carried out using the PRISMA methodology, and the normative values identified were compared to those in this study. The association of covariates was assessed with a one-way ANOVA test on a total healthy cohort (cohort B). In a sub-cohort with available physical fitness data and obesity scores from the SLOfit database (cohort C), the influence of physical fitness and obesity on cIMT progression was studied. A multiple linear regression model was used to analyze the effects of independent variables on cIMT. Statistical analysis was performed using R-project 3.6.0 software (https://cran.r-project.org) in all statistical calculations and analysis. Hypotheses. cIMT measured using the RFQIMT method increases with age, linear growth, and pubertal maturation in children and adolescents 6 to 18 years old. Sex influences the cIMT value measured by the RFQIMT method. The normative values of cIMT using the RFQIMT method in children and adolescents 6 to 18 years old are comparable to the normative values of cIMT published in the literature that were established using other validated manual and semi-automatic methods of cIMT measurement. cIMT measured using the RFQIMT method is already higher in children and adolescents in the presence of risk factors: obesity, higher blood pressure, and low physical fitness. Results. A total of 1,241 children were enrolled in the study. Of these, 1,202 healthy Caucasian children and adolescents 6 to 18 years old were included in the evaluation. From these, sub-cohort A of 1,137 non-obese normotensive children (males: n = 512mean age 12.04 ± 3.52 years, females: n = 625, mean age 12.98 ± 3.83 years) were included in the calculation of sex, age, and height-specific normative cIMT using the LMS method. Normative values of cIMT measured using the RFQIMT method are presented in the form of percentile charts, percentile tables, and LMS tables, separately for sex, age, and height for further clinical use. cIMT increased with age, height, hip circumference, and BMI, and it was was higher in males. Obesity, blood pressure, and poor physical fitness were associated with higher cIMT values for cohort C, for which the data from the SLOfit dataset were available. Conclusions. The RFQIMT technique provided reliable measurements of cIMT for sex-, age-, and height-specific normative cIMT values in children 6 to 18 years old. In addition, the correlation of obesity, high blood pressure, and poor physical fitness with higher cIMT values can already be seen in the pediatric population

Precocious puberty in a girl with 3-methylglutaconic aciduria type 1 (3-MGA-I) due to a novel AUH gene mutation

3-methylglutaconic aciduria type 1 (3-MGA-I) (MIM ID #250950) is an ultra-rare, autosomal recessive organic aciduria, resulting from mutated AUH gene, leading to the deficient 3-methylglutaconyl-CoA hydratase (3-MGH). Only around 40 cases are previously reported, caused by a spectrum of 10 mutations. The clinical spectrum of 3-MGA-I in children is heterogeneous, varying from asymptomatic individuals to mild neurological impairment, speech delay, quadriplegia, dystonia, choreoathetoid movements, severe encephalopathy, psychomotor retardation, basal ganglia involvement. Early dietary treatment with leucine restriction and carnitine supplementation may be effective in improving neurological state in pediatric patients with 3- MGA-I. We presented a girl with 3-MGA-I due to novel AUH gene mutation (homozygous variant c.330 + 5G > A) and confirmed by almost undetectable 3-MGH-enzyme activity, who initially presented with central precocious puberty at an early age of 4.5 years. Precocious puberty might be associated with the 3-MGA-I, as is reported previously in some other metabolic disorders that result in pathologic accumulation of metabolites or toxic brain damage. Therapy with GnRH agonist triptorelin effectively arrested pubertal development

Optimizing the Phenylalanine Cut-Off Value in a Newborn Screening Program

Phenylketonuria (PKU) was the first disorder for which newborn screening (NBS) was introduced in the early 1960s. Slovenia started the NBS program for PKU in 1979, and the fluorimetric method was implemented in 1992, with a phenylalanine (Phe) cut-off set at 120 mol/L. This value has been in use for almost thirty years and has never been revised. We aimed to analyze the DBS samples and review the data from a large nationwide cohort of newborns to optimize the cut-off values for HFA screening to minimize the number of false positives while maintaining the highest level of sensitivity by detecting all those who needed to be treated. In the first prospective part of the study, we analyzed samples of all newborns in Slovenia in 2019 and 2020, and in the second retrospective part, we reviewed data from all known patients with hyperphenylalaninemia (HFA) in Slovenia born from 2000 to 2018. We defined true screening-positive cases as those that required a low-Phe diet. The sensitivity, specificity and positive predictive values of the modeling elevation of the Phe cut-off value from 120 µmol/L to 200 µmol/L were assessed. The number of recalls at the cut-off of 120 µmol/L was 108 out of 37,784 samples at NBS (2019–2020). Six newborns were defined as true positives and 102 samples as false positives. If the cut-off value was adjusted to 160 µmol/L, only 12 samples exceeded it and all six true positive newborns would be detected. Among the 360,000 samples collected at the NBS between 2000 and 2018, 72 HFA patients in need of a low-Phe diet were found. All the diagnosed cases would have been detected if the cut-off was set to 160 µmol/L. We demonstrated in a large group of newborns (400,000 in 20 years) that using the fluorimetric approach, a cut-off value of 160 µmol/L, rather than 120 mol/L, is safe and that there were no missing true positive patients who required treatment. By increasing the cut-off, this method becomes more precise, resulting in a significantly reduced rate of false positives and thus being less burdensome on both families and the healthcare system

Comparison of Tandem Mass Spectrometry and the Fluorometric Method—Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland–Altman analysis indicated a bias of −38.9% (−23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM

Therapy-type related long-term outcomes in mucopolysaccaridosis type II (Hunter syndrome)

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a rare, X-linked recessive multisystem lysosomal storage disease due to iduronate-2-sulfatase enzyme deficiency. We presented three unrelated Slovenian patients with the severe form of MPS II that received three different management approaches: natural course of the disease without received specific treatment, enzyme replacement therapy (ERT), and hematopoietic stem cell transplantation (HSCT). The decision on the management depended on disease severity, degree of cognitive impairment, and parent’s informed decision. The current benefits of MPS II treatments are limited. The lifelong costly intravenous ERT brings significant benefits but the patients with severe phenotypes and neurological involvement progress to cognitive decline and disability regardless of ERT, as demonstrated in published reviews and our case series. The patient after HSCT was the only one of the three cases reported to show a slowly progressing cognitive development. The type of information from the case series is insufficient for generalized conclusions, but with advanced myeloablative conditioning, HSCT may be a preferred treatment option in early diagnosed MPS II patients with the severe form of the disease and low disease burden at the time of presentation

Expanded newborn screening program in Slovenia using tandem mass spectrometry and confirmatory next generation sequencing genetic testing

In the last two decades, the introduction of tandem mass spectrometry in clinical laboratories has enabled simultaneous testing of numerous acylcarnitines and amino acids from dried blood spots for detecting many aminoacidopathies, organic acidurias and fatty acid oxidation disorders. The expanded newborn screening was introduced in Slovenia in September 2018. Seventeen metabolic diseases have been added to the pre-existing screening panel for congenital hypothyroidism and phenylketonuria, and the newborn screening program was substantially reorganized and upgraded

Newborn Screening in a Pandemic—Lessons Learned

The COVID-19 pandemic affected many essential aspects of public health, including newborn screening programs (NBS). Centers reported missing cases of inherited metabolic disease as a consequence of decreased diagnostic process quality during the pandemic. A number of problems emerged at the start of the pandemic, but from the beginning, solutions began to be proposed and implemented. Contingency plans were arranged, and these are reviewed and described in this article. Staff shortage emerged as an important issue, and as a result, new work schedules had to be implemented. The importance of personal protective equipment and social distancing also helped avoid disruption. Staff became stressed, and this needed to be addressed. The timeframe for collecting bloodspot samples was adapted in some cases, requiring reference ranges to be modified. A shortage of essential supplies and protective equipment was evident, and laboratories described sharing resources in some situations. The courier system had to be adapted to make timely and safe transport possible. Telemedicine became an essential tool to enable communication with patients, parents, and medical staff. Despite these difficulties, with adaptations and modifications, some centers evaluated candidate conditions, continued developments, or began new NBS. The pandemic can be regarded as a stress test of the NBS under real-world conditions, highlighting critical aspects of this multidisciplinary system and the need for establishing local, national, and global strategies to improve its robustness and reliability in times of shortage and overloaded national healthcare systems

Clinical and genetic characteristics of a patient with phosphoribosyl pyrophosphate synthetase 1 deficiency and a systematic literature review

Phosphoribosylpyrophosphate synthetase 1 (PRSI) is an enzyme involved in nucleotide metabolism. Pathogenic variants in the PRPS1 are rare and PRS-I deficiency can manifest as three clinical syndromes: X-linked non-syndromic sensorineural deafness (DFN2), X-linked Charcot-Marie-Tooth neuropathy type 5 (CMTX5) and Arts syndrome. We present a Slovenian patient with PRS-I enzyme deficiency due to a novel pathogenic variant – c.424G > A (p.Val142Ile) in the PRPS1 gene, who presented with gross motor impairment, severe sensorineural deafness, balance issues, ataxia, and frequent respiratory infections. In addition, we report the findings of a systemic literature review of all described male cases of Arts syndrome and CMTX5 as well as intermediate phenotypes. As already proposed by other authors, our results confirm PRS-I deficiency should be viewed as a phenotypic continuum rather than three separate syndromes because there are multiple reports of patients with an intermediary clinical presentation