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.

Molecular characterisation of 36 multilocus imprinting disturbance (MLID) patients: a comprehensive approach.

BACKGROUND: Imprinting disorders (ImpDis) comprise diseases which are caused by aberrant regulation of monoallelically and parent-of-origin-dependent expressed genes. A characteristic molecular change in ImpDis patients is aberrant methylation signatures at disease-specific loci, without an obvious DNA change at the specific differentially methylated region (DMR). However, there is a growing number of reports on multilocus imprinting disturbances (MLIDs), i.e. aberrant methylation at different DMRs in the same patient. These MLIDs account for a significant number of patients with specific ImpDis, and several reports indicate a central role of pathogenic maternal effect variants in their aetiology by affecting the maturation of the oocyte and the early embryo. Though several studies on the prevalence and the molecular causes of MLID have been conducted, homogeneous datasets comprising both genomic and methylation data are still lacking. RESULTS: Based on a cohort of 36 MLID patients, we here present both methylation data obtained from next-generation sequencing (NGS, ImprintSeq) approaches and whole-exome sequencing (WES). The compilation of methylation data did not reveal a disease-specific MLID episignature, and a predisposition for the phenotypic modification was not obvious as well. In fact, this lack of epigenotype-phenotype correlation might be related to the mosaic distribution of imprinting defects and their functional relevance in specific tissues. CONCLUSIONS: Due to the higher sensitivity of NGS-based approaches, we suggest that ImprintSeq might be offered at reference centres in case of ImpDis patients with unusual phenotypes but MLID negative by conventional tests. By WES, additional MLID causes than the already known maternal effect variants could not be identified, neither in the patients nor in the maternal exomes. In cases with negative WES results, it is currently unclear to what extent either environmental factors or undetected genetic variants contribute to MLID.

Enhancing Molecular Testing for Effective Delivery of Actionable Gene Diagnostics.

There is a deep need to navigate within our genomic data to find, understand and pave the way for disease-specific treatments, as the clinical diagnostic journey provides only limited guidance. The human genome is enclosed in every nucleated cell, and yet at the single-cell resolution many unanswered questions remain, as most of the sequencing techniques use a bulk approach. Therefore, heterogeneity, mosaicism and many complex structural variants remain partially uncovered. As a conceptual approach, nanopore-based sequencing holds the promise of being a single-molecule-based, long-read and high-resolution technique, with the ability of uncovering the nucleic acid sequence and methylation almost in real time. A key limiting factor of current clinical genetics is the deciphering of key disease-causing genomic sequences. As the technological revolution is expanding regarding genetic data, the interpretation of genotype-phenotype correlations should be made with fine caution, as more and more evidence points toward the presence of more than one pathogenic variant acting together as a result of intergenic interplay in the background of a certain phenotype observed in a patient. This is in conjunction with the observation that many inheritable disorders manifest in a phenotypic spectrum, even in an intra-familial way. In the present review, we summarized the relevant data on nanopore sequencing regarding clinical genomics as well as highlighted the importance and content of pre-test and post-test genetic counselling, yielding a complex approach to phenotype-driven molecular diagnosis. This should significantly lower the time-to-right diagnosis as well lower the time required to complete a currently incomplete genotype-phenotype axis, which will boost the chance of establishing a new actionable diagnosis followed by therapeutical approach.

Clinical evaluation of rare copy number variations identified by chromosomal microarray in a Hungarian neurodevelopmental disorder patient cohort.

BACKGROUND: Neurodevelopmental disorders are genetically heterogeneous pediatric conditions. The first tier diagnostic method for uncovering copy number variations (CNVs), one of the most common genetic etiologies in affected individuals, is chromosomal microarray (CMA). However, this methodology is not yet a routine molecular cytogenetic test in many parts of the world, including Hungary. Here we report clinical and genetic data of the first, relatively large Hungarian cohort of patients whose genetic testing included CMA. METHODS: Clinical data were retrospectively collected for 78 children who were analyzed using various CMA platforms. Phenotypes of patients with disease-causing variants were compared to patients with negative results using the chi squared/Fisher exact tests. RESULTS: A total of 30 pathogenic CNVs were identified in 29 patients (37.2%). Postnatal growth delay (p = 0.05564), pectus excavatum (p = 0.07484), brain imaging abnormalities (p = 0.07848), global developmental delay (p = 0.08070) and macrocephaly (p = 0.08919) were more likely to be associated with disease-causing CNVs. CONCLUSION: Our results allow phenotypic expansion of 14q11.2 microdeletions encompassing SUPT16H and CHD8 genes. Variants of unknown significance (n = 24) were found in 17 patients. We provide detailed phenotypic and genetic data of these individuals to facilitate future classification efforts, and spotlight two patients with potentially pathogenic alterations. Our results contribute to unraveling the diagnostic value of rare CNVs.

Microdeletions in 1q21 and 8q12.1 depict two additional molecular subgroups of Silver-Russell syndrome like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetic disorder characterized by intrauterine and postnatal growth restriction, relative macrocephaly at birth, body asymmetry and typical facial features. Clinical and molecular heterogeneity is described in SRS. Common causes are loss of methylation of the imprinting center 1 in 11p15 and maternal uniparental disomy of chromosome 7. Other genetic alterations include disturbances of imprinted regions in 14q32, 7q32 and 11p15 as well as submicroscopic deletions and duplications. Single nucleotide variants in genes like IGF2, HMGA2, PLAG1, CDKN1C have also been identified in patients with SRS phenotypes. However, routine molecular diagnostics usually focus on 11p15 and chromosome 7, while less frequent causes are not systematically addressed. RESULTS: Here we report two patients with SRS features in which molecular karyotyping revealed microdeletions in 1q21 and 8q12.1 respectively. In a 3.5-year-old girl with postnatal growth restriction, feeding difficulties, relative macrocephaly and distinct SRS features a 2 Mb deletion in 1q21.1q21.2 was identified. Our second case is a 1.5-year-old boy with intrauterine and postnatal growth restriction, feeding difficulties and distinct facial features with a 77 kb deletion in 8q12.1 affecting PLAG1 as the only protein-encoding gene with known function. CONCLUSIONS: The 1q21 region has not yet been assigned as an SRS region, although six patients with the same deletion and SRS features including relative macrocephaly have been described before. This new case adds to the evidence that distal 1q21 should be annotated as an SRS candidate region. The PLAGL1 alteration is the smallest deletion in 8q12.1 ever reported in a patient with SRS phenotype and it finally confirms that PLAG1 is the SRS causing gene in 8q12.1. To increase the diagnostic yield in patients with suspected SRS, we recommend both molecular karyotyping and next generation sequencing-based approaches.

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.

Deletion of 16q22.2q23.3 in a Boy with a Phenotype Reminiscent of Silver-Russell Syndrome.

A 15-month-old boy presented with growth and global developmental delay, feeding difficulties, sleep disturbance and several minor anomalies, including a large anterior fontanel, relative macrocephaly, and a triangular face. Clinical suspicion prompted genetic investigations for Silver-Russell syndrome and related disorders. SNP array analysis led to the diagnosis of an approximately 10-Mb large deletion of the long arm in chromosome 16q22.2q23.3. Interstitial deletions of 16q show a wide variability of related features; however, considering the differences in size and location of the deletions in the known patients, the phenotypic overlap is surprising. Here, we report a novel microdeletion, compare the proband with data from scientific literature and international databases, and discuss possible diagnostic implications.

Chromosome 2q14.3 microdeletion encompassing CNTNAP5 gene in a patient carrying a complex chromosomal rearrangement.

We report a patient with loss of chromosome region 2q14.3 encompassing exon 1 of the gene CNTNAP5. The deletion occurred in association with a de novo complex chromosomal rearrangement, characterized by routine G-banding, fluorescence in situ hybridization and microarray analysis. The presented patient's phenotype is dominated by severe early childhood weight gain, severe speech delay and behavioural problems. To our knowledge, a few similar patients have been reported previously. CNTNAP5 is a member of the neurexin gene family and is associated with autism spectrum disorder and potentially other behavioural and neurodevelopmental disorders. Recent data point to its possible role in obesity and/or metabolism. The phenotype of the herein presented pediatric patient corroborates CNTNAP5's pathogenic role in human disease.

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.

One test for all: whole exome sequencing significantly improves the diagnostic yield in growth retarded patients referred for molecular testing for Silver-Russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is an imprinting disorder which is characterised by severe primordial growth retardation, relative macrocephaly and a typical facial gestalt. The clinical heterogeneity of SRS is reflected by a broad spectrum of molecular changes with hypomethylation in 11p15 and maternal uniparental disomy of chromosome 7 (upd(7)mat) as the most frequent findings. Monogenetic causes are rare, but a clinical overlap with numerous other disorders has been reported. However, a comprehensive overview on the contribution of mutations in differential diagnostic genes to phenotypes reminiscent to SRS is missing due to the lack of appropriate tests. With the implementation of next generation sequencing (NGS) tools this limitation can now be circumvented. MAIN BODY: We analysed 75 patients referred for molecular testing for SRS by a NGS-based multigene panel, whole exome sequencing (WES), and trio-based WES. In 21/75 patients a disease-causing variant could be identified among them variants in known SRS genes (IGF2, PLAG1, HMGA2). Several patients carried variants in genes which have not yet been considered as differential diagnoses of SRS. CONCLUSIONS: WES approaches significantly increase the diagnostic yield in patients referred for SRS testing. Several of the identified monogenetic disorders have a major impact on clinical management and genetic counseling.

Evaluation of Organic Wastes as Substrates for Rearing Zophobas morio, Tenebrio molitor, and Acheta domesticus Larvae as Alternative Feed Supplements.

Studies have focused on identifying combinations of insects and organic waste to optimise bio-conversion. Here, the effects of different diets (10% chicken feed complemented with 90% vegetable waste, garden waste, cattle manure, or horse manure) on growth and survival rates, and nutritional value of Zophobas morio and Tenebrio molitor larvae, and Acheta domesticus were investigated. Compared with chicken feed, organic waste decreased the individual larval weight, although green waste showed fewer negative effects than the manure. The macronutrient concentrations in garden waste were moderate compared with chicken feed, and vegetable waste was the poorest diet in terms of nutrient concentration, as revealed by the principal component analysis (PCA). There was no difference in weight between larvae reared on garden waste and those reared on vegetable waste. Tenebrio molitor and A. domesticus showed the maximum growth rates at 71-101 and 36-66 days of age at 22.5 ± 2.5 °C, respectively. The PCA showed that the protein and fibre concentrations were inversely proportional to fat concentration in the larvae. Acheta domesticus was rich in proteins, whereas Z. morio and T. molitor were rich in fat. Feeding nutrient-poor diets resulted in a lower protein and a higher fat concentration in the larvae.

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.

Clinical and genetic findings in Hungarian pediatric patients carrying chromosome 16p copy number variants and a review of the literature.

The short arm of chromosome 16 (16p) is enriched for segmental duplications, making it susceptible to recurrent, reciprocal rearrangements implicated in the etiology of several phenotypes, including intellectual disability, speech disorders, developmental coordination disorder, autism spectrum disorders, attention deficit hyperactivity disorders, obesity and congenital skeletal disorders. In our clinical study 73 patients were analyzed by chromosomal microarray, and results were confirmed by fluorescence in situ hybridization or polymerase chain reaction. All patients underwent detailed clinical evaluation, with special emphasis on behavioral symptoms. 16p rearrangements were identified in 10 individuals. We found six pathogenic deletions and duplications of the recurrent regions within 16p11.2: one patient had a deletion of the distal 16p11.2 region associated with obesity, while four individuals had duplications, and one patient a deletion of the proximal 16p11.2 region. The other four patients carried 16p variations as second-site genomic alterations, acting as possible modifying genetic factors. We present the phenotypic and genotypic results of our patients and discuss our findings in relation to the available literature.

Frequency of KCNQ1 variants causing loss of methylation of Imprinting Centre 2 in Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by disturbances of the chromosomal region 11p15.5. The most frequent molecular finding in BWS is loss of methylation (LOM) of the Imprinting Centre 2 (IC2) region on the maternal allele, which is localised in intron 10 of the KCNQ1 gene. In rare cases, LOM of IC2 has been reported in families with KCNQ1 germline variants which additionally cause long-QT syndrome (LQTS). Thus, a functional link between disrupted KCNQ1 transcripts and altered IC2 methylation has been suggested, resulting in the co-occurrence of LQTS and BWS in case of maternal inheritance. Whereas these cases were identified by chance or in patients with abnormal electrocardiograms, a systematic screen for KCNQ1 variants in IC2 LOM carriers has not yet been performed. RESULTS: We analysed 52 BWS patients with IC2 LOM to determine the frequency of germline variants in KCNQ1 by MLPA and an amplicon-based next generation sequencing approach. We identified one patient with a splice site variant causing premature transcription termination of KCNQ1. CONCLUSIONS: Our study strengthens the hypothesis that proper KCNQ1 transcription is required for the establishment of IC2 methylation, but that KCNQ1 variants cause IC2 LOM only in a small number of BWS patients.

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.

Similar Cause, Different Phenotype: SOX9 Enhancer Duplication in a Family.

INTRODUCTION: 46,XX ovotesticular disorder of sex development (DSD), as defined by the Chicago consensus in 2006, is characterized by histologically confirmed testicular and ovarian tissue in an individual with a 46,XX karyotype and a wide phenotypic spectrum from female to male appearance. CASE PRESENTATION: We report the case of two 46,XX sex determining region Y (SRY) gene-negative siblings and their 46,XY father with an approximately 150 kilobase pair (kbp) duplication upstream of SOX9 (SRY-box 9) gene's transcriptional start site on chromosome 17 (chr17), which involved SOX9's minimal critical 46,XX sex reversal region. This duplication is sufficient to trigger male development in the absence of Y-chromosomal material and can lead to various degrees of masculinization in 46,XX individuals by overexpression of SOX9. Based on anamnestic information and pedigree analysis, another possible carrier of this copy number variation (CNV) could have been the father's sister. DISCUSSION: By comparing the duplications of our two sibling patients and previously reported similar cases, we suggest that the small differences between their breakpoints could alternatively modify the inner structure and functioning of SOX9'stopologically associated domain (TAD) due to the differing fine TAD arrangements. Our data support the phenotypic modularity impact - incomplete penetrance and variable expressivity - of very similar but non-identical CNVs, which are possibly inherited across three generations.