Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level.

BACKGROUND: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. METHODS: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. RESULTS: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. CONCLUSION: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients.

Identification of an NF1 Microdeletion with Optical Genome Mapping.

Neurofibromatosis type 1 (NF1) is a clinically heterogeneous neurocutaneous disorder inherited in autosomal dominant manner. Approximately 5-10% of the cases are caused by NF1 microdeletions involving the NF1 gene and its flanking regions. Microdeletions, which lead to more severe clinical manifestations, can be subclassified into four different types (type 1, 2, 3 and atypical) according to their size, the genomic location of the breakpoints and the number of genes included within the deletion. Besides the prominent hallmarks of NF1, patients with NF1 microdeletions frequently exhibit specific additional clinical manifestations like dysmorphic facial features, macrocephaly, overgrowth, global developmental delay, cognitive disability and an increased risk of malignancies. It is important to identify the genes co-deleted with NF1, because they are likely to have an effect on the clinical manifestation. Multiplex ligation-dependent probe amplification (MLPA) and microarray analysis are the primary techniques for the investigation of NF1 microdeletions. However, based on previous research, optical genome mapping (OGM) could also serve as an alternative method to identify copy number variations (CNVs). Here, we present a case with NF1 microdeletion identified by means of OGM and demonstrate that this novel technology is a suitable tool for the identification and classification of the NF1 microdeletions.

A Cross-Sectional Study of the Dermatological Manifestations of Patients with Fabry Disease and the Assessment of Angiokeratomas with Multimodal Imaging.

Fabry disease (FD) is a multisystemic X-linked lysosomal storage disease that presents with angiokeratomas (AKs). Our objective was to investigate the clinical and morphologic features of AKs and to present two experimental techniques, multispectral imaging (MSI) and non-linear microscopy (NLM). A thorough dermatological examination was carried out in our 26 FD patients and dermoscopic images (n = 136) were evaluated for specific structures. MSI was used for the evaluation of AKs in seven patients. NLM was carried out to obtain histology samples of two AKs and two hemangiomas. Although AKs were the most common manifestation, the majority of patients presented an atypical distribution and appearance, which could cause a diagnostic challenge. Dermoscopy revealed lacunae (65%) and dotted vessels (56%) as the most common structures, with a whitish veil present in only 25%. Autofluorescence (405 nm) and diffuse reflectance (526 nm) images showed the underlying vasculature more prominently compared to dermoscopy. Using NLM, AKs and hemangiomas could be distinguished based on morphologic features. The clinical heterogeneity of FD can result in a diagnostic delay. Although AKs are often the first sign of FD, their presentation is diverse. A thorough dermatological examination and the evaluation of other cutaneous signs are essential for the early diagnosis of FD.

Clinical aspects of 22q11.2 microdeletion syndrome / A 22q11.2-microdeletiós szindróma klinikai jellemzoi.

Összefoglaló. Bevezetés: A sokszínu tünetspektrummal jellemezheto DiGeorge-szindróma leggyakoribb oka a 22q11.2-microdeletio; incidenciája 1/4000-6000. Célkituzés: A DiGeorge-szindrómára gyanús hazai betegcsoport 22q11.2-microdeletióval társult tüneteinek/panaszainak részletes feltérképezése, a betegség incidenciájának becslése és egy magyarországi 22q11.2-microdeletiós szindróma regiszter létrehozása. Módszer: 2005 és 2019 között a Semmelweis Egyetem II. Gyermekgyógyászati Klinikájára DiGeorge-szindróma gyanújával beutalt és a Veleszületett Rendellenességek Országos Nyilvántartása által regisztrált DiGeorge-szindrómás betegek adatait dolgoztuk fel. A fenotípusjegyeket a Humán Fenotípus Ontológia kódrendszer alapján határoztuk meg. Eredmények: A vizsgálatba 114, igazolt DiGeorge-szindrómás és 113, FISH-vizsgálattal microdeletiót nem hordozó, de klinikailag a DiGeorge-szindróma tüneteit mutató beteget vontunk be. A diagnózis felállításakor a betegek átlagéletkora 5,88 (± 9,66 SD) év volt, eddig a betegek 54,9%-a legalább egy szívmutéten átesett. A betegek leggyakoribb tünetei a kamrai sövényhiány, a mélyen ülo fülek, a gótikus szájpad, a motoros fejlodési elmaradás és a visszatéro fertozések voltak. Megbeszélés: A DiGeorge-szindróma becsült incidenciája hazánkban 1/12 500, közöttük magas a többszörösen veszélyeztetett újszülöttek és a mutéti korrekcióra szorulók aránya. A diagnózis hazánkban 2-3 évvel korábban történik a nemzetközi átlaghoz viszonyítva. Következtetés: A létrehozott regiszterünk alapján Magyarországon a kórkép aluldiagnosztizált. Minden conotruncalis szívfejlodési rendellenesség vagy jelentos kamrai sövényhiány esetén citogenetikai vizsgálat javasolt a DiGeorge-szindróma felmerülo gyanúja miatt. Negatív lelet esetén az atípusos töréspontú microdeletiók azonosítására komparatív genomiális hibridizáció vagy multiplex ligatiofüggo próbaamplifikációs vizsgálat javasolt. A betegek számára multidiszciplináris ellátás szükséges, III-as progresszivitási szintu újszülött intenzív részlegen, gyermekkardiológus és klinikai genetikus részvételével. Orv Hetil. 2022; 163(1): 21-30. INTRODUCTION: The 22q11.2 microdeletion syndrome is the most common cause of DiGeorge syndrome, showing a wide phenotypic spectrum and has an estimated incidence of 1/4000-6000 livebirths. OBJECTIVE: Detailed characterization of the clinical signs/symptoms associated with 22q11.2 deletion, estimation of the national incidence via establishing a Hungarian register. METHOD: Retrospective data between 2005 and 2019 from the 2nd Department of Paediatrics, Semmelweis University and from national database of congenital anomalies were obtained. Phenotypic abnormalities were described using the Human Phenotype Ontology nomenclature. RESULTS: A cohort of 114 DiGeorge patients and 113 patients negative for FISH testing were included. The mean age of patients at diagnosis was 5.88 (± 9.66 SD) years and 54.9% of patients had at least one heart surgery until diagnosis. The main identified symptoms were ventricular septal defect, low-set ears, recurrent infections, high narrow palate and motor development delay. DISCUSSION: The estimated incidence of DiGeorge syndrome in Hungary is 1/12 500 births, the frequency of infants at high risk and in need for surgery is high. Diagnosis is established 2-3 years earlier as compared to the international average. CONCLUSION: Based on the established Hungarian register, the incidence is lower compared to international data. In the case of conotruncal heart anomaly and ventricular septal defects, cytogenetic testing is recommended for the increased probability of DiGeorge syndrome. For second-tier testing, comparative genome hybridization or multiplex ligation-dependent probe amplification are recommended to identify atypical microdeletions. Newborns with DiGeorge syndrome require special care in perinatal intensive centers including pediatric cardiology and genetic counseling. Orv Hetil. 2022; 163(1): 21-30.

Analysis of Fuel Alternative Products Obtained by the Pyrolysis of Diverse Types of Plastic Materials Isolated from a Dumpsite Origin in Pakistan.

The current energy crisis and waste management problems have compelled people to find alternatives to conventional non-renewable fuels and utilize waste to recover energy. Pyrolysis of plastics, which make up a considerable portion of municipal and industrial waste, has emerged as a feasible resolution to both satisfy our energy needs and mitigate the issue of plastic waste. This study was therefore conducted to find a solution for plastic waste management problems, as well as to find an alternative to mitigate the current energy crisis. Pyrolysis of five of the most commonly used plastics, polyethylene terephthalate (PET), high- and low-density polyethylene (HDPE, LDPE), polypropylene (PP), and polystyrene (PS), was executed in a pyrolytic reactor designed utilizing a cylindrical shaped stainless steel container with pressure and temperature gauges and a condenser to cool down the hydrocarbons produced. The liquid products collected were highly flammable and their chemical properties revealed them as fuel alternatives. Among them, the highest yield of fuel conversion (82%) was observed for HDPE followed by PP, PS, LDPE, PS, and PET (61.8%, 58.0%, 50.0%, and 11.0%, respectively). The calorific values of the products, 46.2, 46.2, 45.9, 42.8 and 42.4 MJ/kg for LPDE, PP, HPDE, PS, and PET, respectively, were comparable to those of diesel and gasoline. Spectroscopic and chromatographic analysis proved the presence of alkanes and alkenes with carbon number ranges of C9-C15, C9-C24, C10-C21, C10-C28, and C9-C17 for PP, PET, HDPE, LDPE, and PS, respectively. If implemented, the study will prove to be beneficial and contribute to mitigating the major energy and environmental issues of developing countries, as well as enhance entrepreneurship opportunities by replicating the process at small-scale and industrial levels.

Diagnostic difficulties and possibilities of NF1-like syndromes in childhood.

BACKGROUND: Neurofibromatosis type 1 (NF1), which is caused by heterozygous inactivating pathogenic variants in the NF1, has poor phenotypic expressivity in the early years of life and there are numerous conditions, including many other tumor predisposition syndromes, that can mimic its appearance. These are collectively termed NF1-like syndromes and are also connected by their genetic background. Therefore, the NF1's clinical diagnostic efficiency in childhood could be difficult and commonly should be completed with genetic testing. METHODS: To estimate the number of syndromes/conditions that could mimic NF1, we compiled them through an extensive search of the scientific literature. To test the utility of NF1's National Institutes of Health (NIH) clinical diagnostic criteria, which have been in use for a long time, we analyzed the data of a 40-member pediatric cohort with symptoms of the NF1-like syndromes' overlapping phenotype and performed NF1 genetic test, and established the average age when diagnostic suspicion arises. To facilitate timely identification, we compiled strongly suggestive phenotypic features and anamnestic data. RESULTS: In our cohort the utility of NF1's clinical diagnostic criteria were very limited (sensitivity: 80%, specificity: 30%). Only 53% of children with clinically diagnosed NF1 had a detectable NF1 pathogenic variation, whereas 40% of patients without fulfilled clinical criteria tested positive. The average age at first genetic counseling was 9 years, and 40% of children were referred after at least one tumor had already been diagnosed. These results highlight the need to improve NF1-like syndromes' diagnostic efficiency in childhood. We collected the most extensive spectrum of NF1-like syndromes to help the physicians in differential diagnosis. We recommend the detailed, non-invasive clinical evaluation of patients before referring them to a clinical geneticist. CONCLUSIONS: Early diagnosis of NF1-like syndromes can help to prevent severe complications by appropriate monitoring and management. We propose a potential screening, diagnostic and management strategy based on our findings and recent scientific knowledge.

Genotype-Phenotype Associations in Patients With Type-1, Type-2, and Atypical NF1 Microdeletions.

Neurofibromatosis type 1 is a tumor predisposition syndrome inherited in autosomal dominant manner. Besides the intragenic loss-of-function mutations in NF1 gene, large deletions encompassing the NF1 gene and its flanking regions are responsible for the development of the variable clinical phenotype. These large deletions titled as NF1 microdeletions lead to a more severe clinical phenotype than those observed in patients with intragenic NF1 mutations. Around 5-10% of the cases harbor large deletion and four major types of NF1 microdeletions (type 1, 2, 3 and atypical) have been identified so far. They are distinguishable in term of their size and the location of the breakpoints, by the frequency of somatic mosaicism with normal cells not harboring the deletion and by the number of the affected genes within the deleted region. In our study genotype-phenotype analyses have been performed in 17 mostly pediatric patients with NF1 microdeletion syndrome identified by multiplex ligation-dependent probe amplification after systematic sequencing of the NF1 gene. Confirmation and classification of the NF1 large deletions were performed using array comparative genomic hybridization, where it was feasible. In our patient cohort 70% of the patients possess type-1 deletion, one patient harbors type-2 deletion and 23% of our cases have atypical NF1 deletion. All the atypical deletions identified in this study proved to be novel. One patient with atypical deletion displayed mosaicism. In our study NF1 microdeletion patients presented dysmorphic facial features, macrocephaly, large hands and feet, delayed cognitive development and/or learning difficulties, speech difficulties, overgrowth more often than patients with intragenic NF1 mutations. Moreover, neurobehavior problems, macrocephaly and overgrowth were less frequent in atypical cases compared to type-1 deletion. Proper diagnosis is challenging in certain patients since several clinical manifestations show age-dependency. Large tumor load exhibited more frequently in this type of disorder, therefore better understanding of genotype-phenotype correlations and progress of the disease is essential for individuals suffering from neurofibromatosis to improve the quality of their life. Our study presented additional clinical data related to NF1 microdeletion patients especially for pediatric cases and it contributes to the better understanding of this type of disorder.

Need for a precise molecular diagnosis in Beckwith-Wiedemann and Silver-Russell syndrome: what has to be considered and why it is important.

Molecular diagnostic testing of the 11p15.5-associated imprinting disorders Silver-Russell and Beckwith-Wiedemann syndrome (SRS, BWS) is challenging due to the broad spectrum of molecular defects and their mosaic occurrence. Additionally, the decision on the molecular testing algorithm is hindered by their clinical heterogeneity. However, the precise identification of the type of defect is often a prerequisite for the clinical management and genetic counselling. Four major molecular alterations (epimutations, uniparental disomies, copy number variants, single nucleotide variants) have been identified, but their frequencies vary between SRS and BWS. Due to their molecular aetiology, epimutations in both disorders as well as upd(11)pat in BWS are particular prone to mosaicism which might additionally complicate the interpretation of testing results. We report on our experience of molecular analysis in a total cohort of 1448 patients referred for diagnostic testing of BWS and SRS, comprising a dataset from 737 new patients and from 711 cases from a recent study. Though the majority of positively tested patients showed the expected molecular results, we identified a considerable number of clinically unexpected molecular alterations as well as not yet reported changes and discrepant mosaic distributions. Additionally, the rate of multilocus imprinting disturbances among the patients with epimutations and uniparental diploidies could be further specified. Altogether, these cases show that comprehensive testing strategies have to be applied in diagnostic testing of SRS and BWS. The precise molecular diagnosis is required as the basis for a targeted management (e.g. ECG (electrocardiogram) and tumour surveillance in BWS, growth treatment in SRS). The molecular diagnosis furthermore provides the basis for genetic counselling. However, it has to be considered that recurrence risk calculation is determined by the phenotypic consequences of each molecular alteration and mechanism by which the alteration arose. KEY MESSAGES: The detection rates for the typical molecular defects of Beckwith-Wiedemann syndrome or Silver-Russell syndrome (BWS, SRS) are lower in routine cohorts than in clinically well-characterised ones. A broad spectrum of (unexpected) molecular alterations in both disorders can be identified. Multilocus imprinting disturbances (MLID) are less frequent in SRS than expected. The frequency of MLID and uniparental diploidy in BWS is confirmed. Mosaicism is a diagnostic challenge in BWS and SRS. The precise determination of the molecular defects affecting is the basis for a targeted clinical management and genetic counselling.

What should we consider in the case of combined Down- and 47,XY,+i(X)(q10) Klinefelter syndromes? The unique case of a male newborn and review of the literature.

BACKGROUND: Double aneuploidies - especially in combination with structural aberrations - are extremely rare among liveborns. The most frequent association is that of Down (DS) and Klinefelter syndromes (KS). We present the case of a male newborn with a unique 47,XY,+ 21[80%]/48,XY,+i(X)(q10),+ 21[20%] karyotype, hypothesize about his future phenotype, discuss the aspects of management and review the literature. CASE PRESENTATION: The additional association of isochromosome Xq (i(X)(q10)) could be the result of a threefold non-disjunction event. 47,XY,+i(X)(q10) KS is not common and its symptoms differ from the classical KS phenotype. In combined DS and i(X)(q10) KS, the anticipatory phenotype is not simply the sum of the individual syndromic characteristics. This genotype is associated with higher risk for several diseases and certain conditions with more pronounced appearance: emotional and behavioral disorders; poorer mental and physical quality of life; lower muscle mass/tone/strength; connective tissue weakness; muscle hypotonia and feeding difficulties; osteopenia/-porosis with earlier beginning and faster progression; different types of congenital heart diseases; more common occurrence of hypertension; increased susceptibility to infections and female predominant autoimmune diseases; higher risk for hematological malignancies and testicular tumors. CONCLUSIONS: In multiple aneuploidies, the alterations have the potential to weaken or enhance each other, or they may not have modifying effects at all. Prenatal ultrasound signs are not obligatory symptoms of numerous chromosomal anomalies (specifically those involving supernumerary sex chromosomes), therefore combined prenatal screening has pertinence in uncomplicated pregnancies as well.

FBN1 gene mutations in 26 Hungarian patients with suspected Marfan syndrome or related fibrillinopathies.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder mainly affecting the cardiovascular, ocular and musculo-skeletal systems. FBN1 gene mutations lead to MFS and related connective tissue disorders. In this work we described clinical and molecular data of 26 unrelated individuals with suspected MFS who were referred for FBN1 mutation analysis. FBN1 gene sequencing was performed by next generation sequencing and Sanger sequencing methods. We identified 23 causal or potentially causal (including variants of uncertain significance) FBN1 variants, seven of them was novel (˜30%). About 30% of the cases were sporadic. FBN1 mutations were associated with MFS in the majority of the patients, in two cases with severe and early onset manifestation of the syndrome. Missense mutations were detected in 69.6% (16/23), the majority of them were located in one of the cbEGF motifs and ˜70% of them substituted conserved cystein residues. Small deletions/duplications were identified in 13% of the cases (3/23), while splice site variants were detected in 17.4% (4/23). In three unrelated patients a low frequency recurrent silent variant (c.3294C > T (p.Asp1098=) was identified. FBN1 mRNA analysis showed that the mutation does not lead to aberrant splicing, based on available data the mutation was classified as benign.

Pharmacokinetics, pharmacodynamics, and safety of moss-aGalactosidase A in patients with Fabry disease.

Moss-aGalactosidase A (moss-aGal) is a moss-derived version of human α-galactosidase developed for enzyme replacement therapy in patients with Fabry disease. It exhibits a homogenous N-glycosylation profile with >90% mannose-terminated glycans. In contrast to mammalian cell produced α-galactosidase, moss-aGal does not rely on mannose-6-phosphate receptor mediated endocytosis but targets the mannose receptor for tissue uptake. We conducted a phase 1 clinical trial with moss-aGal in six patients with confirmed diagnosis of Fabry disease during a 28-day schedule. All patients received a single dose of 0.2 mg/kg moss-aGal by i.v.-infusion. Primary endpoints of the trial were safety and pharmacokinetics; secondary endpoints were pharmacodynamics by analyzing urine and plasma Gb3 and lyso-Gb3 concentrations. In all patients, the administered single dose was well tolerated. No safety issues were observed. Pharmacokinetic data revealed a stable nonlinear profile with a short plasma half-life of moss-aGal of 14 minutes. After one single dose of moss-aGal, urinary Gb3 concentrations decreased up to 23% 7 days and up to 60% 28 days post-dose. Plasma concentrations of lyso-Gb3 decreased by 3.8% and of Gb3 by 11% 28 days post-dose. These data reveal that a single dose of moss-aGal was safe, well tolerated, and led to a prolonged reduction of Gb3 excretion. As previously shown, moss-aGal is taken up via the mannose receptor, which is expressed on macrophages but also on endothelial and kidney cells. Thus, these data indicate that moss-aGal may target kidney cells. After these promising results, phase 2/3 clinical trials are in preparation.

[Steroid 21-hydroxylase deficiency, the most frequent cause of congenital adrenal hyperplasia]. / Szteroid-21-hidroxiláz-deficientia, a congenitalis adrenalis hyperplasia leggyakoribb oka.

Congenital adrenal hyperplasia is a group of genetic diseases due to the disablement of 7 genes; one of them is steroid 21-hydroxylase deficiency. The genes of congenital adrenal hyperplasia encode enzymes taking part in the steroidogenesis of adrenal gland. Steroid 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations of the steroid 21-hydroxylase gene. The mutations of steroid 21-hydroxylase gene cause 95% of the congenital adrenal hyperplasia cases. Although the non-classic steroid 21-hydroxylase deficiency with mild symptoms is seldom diagnosed, the classic steroid 21-hydroxylase deficiency may lead to life-threatening salt-wasting and adrenal crises due to the insufficient aldosterone and cortisol serum levels. The classic type requires life-long steroid replacement which may result in cushingoid side effects, and typical comorbidities may be also developed. The patients' quality of life is decreased, and their mortality is much higher than that of the population without steroid 21-hydroxylase deficiency. The diagnosis, consequences and the patients' life-long clinical care require a multidisciplinary approach: the specialists in pediatrics, internal medicine, endocrinology, laboratory medicine, genetic diagnostics, surgery, obstetrics-gynecology and psychology need to work together. Orv Hetil. 2018; 159(7): 269-277.

Targeted Next Generation Sequencing Approach in Patients Referred for Silver-Russell Syndrome Testing Increases the Mutation Detection Rate and Provides Decisive Information for Clinical Management.

OBJECTIVE: To investigate the contribution of differential diagnoses to the mutation spectrum of patients referred for Silver-Russell syndrome (SRS) testing. STUDY DESIGN: Forty-seven patients referred for molecular testing for SRS were examined after exclusion of one of the SRS-associated alterations. After clinical classification, a targeted next generation sequencing approach comprising 25 genes associated with other diagnoses or postulated as SRS candidate genes was performed. RESULTS: By applying the Netchine-Harbinson clinical scoring system, indication for molecular testing for SRS was confirmed in 15 out of 47 patients. In 4 out of these 15 patients, disease-causing variants were found in genes associated with other diagnoses. These patients carried mutations associated with Bloom syndrome, Mulibrey nanism, KBG syndrome, or IGF1R-associated short stature. We could not detect any pathogenic mutation in patients with a negative clinical score. CONCLUSIONS: Some of the differential diagnoses detected in the cohort presented here have a major impact on clinical management. Therefore, we emphasize that the molecular defects associated with these clinical pictures should be excluded before the clinical diagnosis "SRS" is made. Finally, we could show that a broad molecular approach including the differential diagnoses of SRS increases the detection rate.

Cardiac rehabilitation programme as a non-pharmacological platelet inhibitory tool in acute coronary syndrome survivors.

Background Acute coronary syndrome is associated with platelet hyperactivity, which in its persistent form, promotes recurrent thrombotic events. Complex cardiac rehabilitation after acute coronary syndrome improves clinical outcome; however, its effect on platelet hyperactivity is unknown. Design and methods We enrolled 84 acute coronary syndrome patients on dual antiplatelet therapy, who underwent a new complex cardiac rehabilitation programme (NovaCord physiotherapy, lifestyle counselling, strict diet, stress management and regular coaching) and 51 control acute coronary syndrome patients with traditional cardiac rehabilitation. Platelet functionality was determined at enrolment and at three months follow-up by aggregometry, serum platelet-derived growth factor levels, total- and platelet-derived microvesicle counts (PMV; CD41a+/CD61+, CD62P+). Results Platelet aggregation parameters and platelet-derived growth factor levels were significantly decreased in the complex cardiac rehabilitation group at three months (1 µg/ml collagen, median (interquartile range): 22 (10-45) vs 14 (7.5-25.5)%, p = 0.0015; 2 µg/ml collagen: 36 (22-60) vs 26.5 (16-37)%, p = 0.0019; 1.25 µM adenosine-diphosphate: 4.5 (1-10) vs 1 (0-3)%, p = 0.0006; 5 µM adenosine-diphosphate: 27 (16-38) vs 22 (12-31)%, p = 0.0078; epinephrine: 33 (15-57) vs 27 (12-43)%, p = 0.01; platelet-derived growth factor: 434.6 (256.0-622.7) vs 224.8 (148.5-374.1) pg/ml, p = 0.0001). In contrast, these changes were absent or did not reach statistical significance in the traditional cardiac rehabilitation group. Platelet-derived microvesicle counts were significantly decreased in both groups, while total microvesicle count was significantly reduced only in the complex cardiac rehabilitation group (median (interquartile range): 3945.5 (2138-5661) vs 1739 (780-2303) count/µl; p = 0.0001). Conclusions Platelet hyperactivity three months after acute coronary syndrome significantly decreased in patients undergoing complex cardiac rehabilitation. Besides dual antiplatelet therapy, effective management and comprehensive control of cardiovascular risk factors might represent a new, non-pharmacological approach to influence platelet functionality.

A unique haplotype of RCCX copy number variation: from the clinics of congenital adrenal hyperplasia to evolutionary genetics.

There is a difficulty in the molecular diagnosis of congenital adrenal hyperplasia (CAH) due to the c.955C>T (p.(Q319*), formerly Q318X, rs7755898) variant of the CYP21A2 gene. Therefore, a systematic assessment of the genetic and evolutionary relationships between c.955C>T, CYP21A2 haplotypes and the RCCX copy number variation (CNV) structures, which harbor CYP21A2, was performed. In total, 389 unrelated Hungarian individuals with European ancestry (164 healthy subjects, 125 patients with non-functioning adrenal incidentaloma and 100 patients with classical CAH) as well as 34 adrenocortical tumor specimens were studied using a set of experimental and bioinformatic methods. A unique, moderately frequent (2%) haplotypic RCCX CNV structure with three repeated segments, abbreviated to LBSASB, harboring a CYP21A2 with a c.955C>T variant in the 3'-segment, and a second CYP21A2 with a specific c.*12C>T (rs150697472) variant in the middle segment occurred in all c.955C>T carriers with normal steroid levels. The second CYP21A2 was free of CAH-causing mutations and produced mRNA in the adrenal gland, confirming its functionality and ability to rescue the carriers from CAH. Neither LBSASB nor c.*12C>T occurred in classical CAH patients. However, CAH-causing CYP21A2 haplotypes with c.955C>T could be derived from the 3'-segment of LBSASB after the loss of functional CYP21A2 from the middle segment. The c.*12C>T indicated a functional CYP21A2 and could distinguish between non-pathogenic and pathogenic genomic contexts of the c.955C>T variant in the studied European population. Therefore, c.*12C>T may be suitable as a marker to avoid this genetic confound and improve the diagnosis of CAH.

Long time enzyme replacement therapy stabilizes obstructive lung disease and alters peripheral immune cell subsets in Fabry patients.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder, causing accumulation of globotriaosylceramid in different organs. Glycolipids are activators of different immune cell subsets the resulting inflammation is responsible for organ damage. Pulmonary involvement leads to airway inflammation; however, data on severity, as well as the effect of enzyme replacement therapy on lung function parameters and changes in peripheral immune cell subsets on lung involvement are sparse. METHODS: Seven Fabry patients and four carriers underwent detailed clinical examinations screening for pulmonary manifestations. Repetitive measurements were performed on five patients on ERT (average follow-up 5 years). Patients with Fabry disease and control volunteers were included into peripheral blood cell measurements. RESULTS: Lung involvement was present in all patients. Symptoms suggestive for lung disease were mild, however, obstructive ventilatory disorder, dominantly affecting small airways accompanied by hyperinflation was demonstrated in all affected patients. ERT resulted in small improvement of FEV1 in most treated patients. Decreased ratio of myeloid DC, Th17 cells while increase in T helper (Th)1 cells, and no change in Th2 and regulatory T (Treg) cells were detected in Fabry patients. CONCLUSIONS: Fabry disease results mainly in mild symptoms related to lung involvement, characterized by moderate non-reversible obstructive ventilatory disorder. Stabilization of airway obstruction during follow-up was observed using ERT in most patients, emphasizing the importance of this treatment in respect of pulmonary manifestations. Changes of immune cell subsets in the peripheral blood might play a role in inflammatory process, including small airways in Fabry patient's lung.

Fast and robust next-generation sequencing technique using ion torrent personal genome machine for the screening of neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational "hot spots." There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.

[Flow cytometry in the diagnosis of hemophagocytic lymphohistiocytosis in a case with fatal outcome]. / Az áramlási citometria jelentosége a haemophagocytás lymphohistiocytosis diagnosztikájában egy fatális kimenetelu eset bemutatása kapcsán.

Hemophagocytic lymphohistiocytosis is a multisystem inflammation, generated by the uncontrolled and excessive activation of cytotoxic T lymphocytes and natural killer cells. Severe immunodeficiency and generalized macrophage activation can often be detected in the background of this life threatening disorder. It is classified as a primary immunodeficiency. Functional abnormalities of the perforin protein or defects in granule secretory mechanisms are caused by gene mutations in most cases. Diagnostic criteria of hemophagocytic lymphohistiocytosis are the following: fever, splenomegaly, cytopenias affecting at least two of the 3 lineages in peripheral blood, hypertriglyceridemia and hyperferritinemia, elevated serum level of soluble interleukin-2 receptor (sCD25), hypofibrinogenemia, hemophagocytosis in bone marrow and decreased cytotoxic T cell and natural killer cell activity. In this case report the authors summarize the utility of functional flow cytometry in the diagnosis of hemophagocytic lymphohistiocytosis. Using flow cytometry, elevated intracellular perforin content, decreased killing activity of cytotoxic T cells and natural killer cells, and impaired cell surface expression of CD107a (LAMP1 protein) from in vitro stimulated blood lymphocytes were detected. Abnormal secretion of perforin was also demonstrated. Genetic testing revealed mutation of the MUNC 13-4 gene, which confirmed the base of the abnormal flow cytometric findings. This case report demonstrates the value of functional flow cytometry in the rapid diagnosis of genetically determined hemophagocytic lymphohistiocytosis, a condition in which early diagnosis is critical for optimal management. The authors emphasize the significance of functional flow cytometry in the differential diagnosis of immunodeficiencies.

Multiple splice variants of EWSR1-ETS fusion transcripts co-existing in the Ewing sarcoma family of tumors.

PURPOSE: The Ewing sarcoma family of tumors (EFT) is characterized by fusions of the EWSR1 gene on chromosome 22q12 with either one of the genes encoding members of the ETS family of transcription factors, in the majority of cases FLI1 or ERG. Many alternative EWSR1-ETS gene fusions have been encountered, due to variations in the locations of the EWSR1 and ETS genomic breakpoints. The resulting heterogeneity in EWSR1-ETS fusion transcripts may further be increased by the occurrence of multiple splice variants within the same tumor. Here we present a retrospective study designed to detect all of the EWSR1-FLI1 and EWSR1-ERG fusion transcripts in a series of 23 fresh frozen EFT tissues. METHODS: RT-PCR and nested fluorescent multiplex PCR were used to amplify EWSR1-FLI1 and EWSR1-ERG transcripts from EFT tissues. Fusion transcripts were identified by laser-induced fluorescent capillary electrophoresis and confirmed by sequence analysis. RESULTS: Nine different EWSR1-FLI1 fusion transcripts and one EWSR1-ERG fusion transcript were identified in 21 out of 23 fresh frozen EFT tissue samples. In five cases multiple fusion transcripts were found to coexist in the same tumor sample. We additionally reviewed previous reports on twelve cases with multiple EWSR1-ETS fusion transcripts. CONCLUSIONS: Alternative splicing may frequently affect the process of EFT-associated fusion gene transcription and, as such, may significantly contribute to the pathogenic role of EFT-associated chromosome translocations. In a considerable number of cases this may result in multiple splice variants within the same tumor.

Six cases of rare gene amplifications and multiple copy of fusion gene in childhood acute lymphoblastic leukemia.

Cytogenetic aberrations are very important factors in risk assessment of childhood hematological malignancies. We report six childhood acute lymphoid leukemia (ALL) cases with rare cytogenetic aberrations: five with RUNX1, ABL1 or MLL proto-oncogene amplification and one case of multiple copies of ETV6/RUNX1 fusion genes. The simultaneous presence of two adverse genetic aberrations is of special interest: ETV6-RUNX1 fusion gene is associated with good prognosis and intrachromosomal amplification of the homologue RUNX1 gene is associated with poor prognosis. We also report a patient with MLL amplification, a unique finding in childhood T-ALL. Report of these subtle rearrangements contributes to our understanding of diagnostic and prognostic significance of these rare cytogenetic abnormalities.