Common conditions of use elements. Atomic concepts for consistent and effective information governance.

Myriad policy, ethical and legal considerations underpin the sharing of biological resources, implying the need for standardised and yet flexible ways to digitally represent diverse 'use conditions'. We report a core lexicon of terms that are atomic, non-directional 'concepts of use', called Common Conditions of use Elements. This work engaged biobanks and registries relevant to the European Joint Programme for Rare Diseases and aimed to produce a lexicon that would have generalised utility. Seventy-six concepts were initially identified from diverse real-world settings, and via iterative rounds of deliberation and user-testing these were optimised and condensed down to 20 items. To validate utility, support software and training information was provided to biobanks and registries who were asked to create Sharing Policy Profiles. This succeeded and involved adding standardised directionality and scope annotations to the employed terms. The addition of free-text parameters was also explored. The approach is now being adopted by several real-world projects, enabling this standard to evolve progressively into a universal basis for representing and managing conditions of use.

Lack of guidelines and translational knowledge is hindering the implementation of psychiatric genetic counseling and testing within Europe - A multi-professional survey study.

Genetic research has identified a large number of genetic variants, both rare and common, underlying neurodevelopmental disorders (NDD) and major psychiatric disorders. Currently, these findings are being translated into clinical practice. However, there is a lack of knowledge and guidelines for psychiatric genetic testing (PsychGT) and genetic counseling (PsychGC). The European Union-funded COST action EnGagE (CA17130) network was started to investigate the current implementation status of PsychGT and PsychGC across 35 participating European countries. Here, we present the results of a pan-European online survey in which we gathered the opinions, knowledge, and practices of a self-selected sample of professionals involved/interested in the field. We received answers from 181 respondents. The three main occupational categories were genetic counselor (21.0%), clinical geneticist (24.9%), and researcher (25.4%). Of all 181 respondents, 106 provide GC for any psychiatric disorder or NDD, corresponding to 58.6% of the whole group ranging from 43.2% in Central Eastern Europe to 66.1% in Western Europe. Overall, 65.2% of the respondents reported that genetic testing is offered to individuals with NDD, and 26.5% indicated the same for individuals with major psychiatric disorders. Only 22.1% of the respondents indicated that they have guidelines for PsychGT. Pharmacogenetic testing actionable for psychiatric disorders was offered by 15%. Interestingly, when genetic tests are fully covered by national health insurance, more genetic testing is provided for individuals with NDD but not those with major psychiatric disorders. Our qualitative analyses of responses highlight the lack of guidelines and knowledge on utilizing and using genetic tests and education and training as the major obstacles to implementation. Indeed, the existence of psychiatric genetic training courses was confirmed by only 11.6% of respondents. The question on the relevance of up-to-date education and training in psychiatric genetics on everyday related practice was highly relevant. We provide evidence that PsychGC and PsychGT are already in use across European countries, but there is a lack of guidelines and education. Harmonization of practice and development of guidelines for genetic counseling, testing, and training professionals would improve equality and access to quality care for individuals with psychiatric disorders within Europe.

EMQN: Recommendations for genetic testing in inherited cardiomyopathies and arrhythmias.

Inherited cardiomyopathies and arrhythmias (ICAs) are a prevalent and clinically heterogeneous group of genetic disorders that are associated with increased risk of sudden cardiac death and heart failure. Making a genetic diagnosis can inform the management of patients and their at-risk relatives and, as such, molecular genetic testing is now considered an integral component of the clinical care pathway. However, ICAs are characterised by high genetic and allelic heterogeneity, incomplete / age-related penetrance, and variable expressivity. Therefore, despite our improved understanding of the genetic basis of these conditions, and significant technological advances over the past two decades, identifying and recognising the causative genotype remains challenging. As clinical genetic testing for ICAs becomes more widely available, it is increasingly important for clinical laboratories to consolidate existing knowledge and experience to inform and improve future practice. These recommendations have been compiled to help clinical laboratories navigate the challenges of ICAs and thereby facilitate best practice and consistency in genetic test provision for this group of disorders. General recommendations on internal and external quality control, referral, analysis, result interpretation, and reporting are described. Also included are appendices that provide specific information pertinent to genetic testing for hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies, long QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia.

Identification of alternative transcripts of NSD1 gene in Sotos Syndrome patients and healthy subjects.

NSD1 gene (Nuclear Receptor Binding SET Domain Protein 1) encodes a methyltransferase that plays an important role in embryonic development. NSD1 is implicated in the transcription and methylation of histone H3 at lysine 36 (H3-K36), but the molecular mechanisms involved in these processes remain largely unknown. Pathogenic variants of NSD1 gene lead to Sotos syndrome, and have also been detected in some type of cancers, such as acute myeloid leukemia. In this study we have investigated NSD1 mRNA expression in fibroblast cell lines obtained from 14 Sotos patients and from 8 healthy controls. In addition to the expected NSD1 canonical transcript (isoform 1), we identified two additional, not yet reported, short NSD1 mRNA isoforms: NSD1 Δ5Δ7 (isoform 2) and NSD1 Δ19-23 (isoform 3), both in healthy subjects and in Sotos patients. We also show that NSD1 mutations in patients can be associated with a decreased level of NSD1 mRNA, as expected. Moreover, one patient, bearing the NSD1 variant c.6010-10G > A, expressed an additional shorter transcript derived from an aberrant splicing event. These results may provide a basis to elucidate the impact of different NSD1 pathogenic variants on the heterogeneity of phenotype associated with Sotos syndrome.

CAPRIN1 haploinsufficiency causes a neurodevelopmental disorder with language impairment, ADHD and ASD.

We describe an autosomal dominant disorder associated with loss-of-function variants in the Cell cycle associated protein 1 (CAPRIN1; MIM*601178). CAPRIN1 encodes a ubiquitous protein that regulates the transport and translation of neuronal mRNAs critical for synaptic plasticity, as well as mRNAs encoding proteins important for cell proliferation and migration in multiple cell types. We identified 12 cases with loss-of-function CAPRIN1 variants, and a neurodevelopmental phenotype characterized by language impairment/speech delay (100%), intellectual disability (83%), attention deficit hyperactivity disorder (82%) and autism spectrum disorder (67%). Affected individuals also had respiratory problems (50%), limb/skeletal anomalies (50%), developmental delay (42%) feeding difficulties (33%), seizures (33%) and ophthalmologic problems (33%). In patient-derived lymphoblasts and fibroblasts, we showed a monoallelic expression of the wild-type allele, and a reduction of the transcript and protein compatible with a half dose. To further study pathogenic mechanisms, we generated sCAPRIN1+/- human induced pluripotent stem cells via CRISPR-Cas9 mutagenesis and differentiated them into neuronal progenitor cells and cortical neurons. CAPRIN1 loss caused reduced neuronal processes, overall disruption of the neuronal organization and an increased neuronal degeneration. We also observed an alteration of mRNA translation in CAPRIN1+/- neurons, compatible with its suggested function as translational inhibitor. CAPRIN1+/- neurons also showed an impaired calcium signalling and increased oxidative stress, two mechanisms that may directly affect neuronal networks development, maintenance and function. According to what was previously observed in the mouse model, measurements of activity in CAPRIN1+/- neurons via micro-electrode arrays indicated lower spike rates and bursts, with an overall reduced activity. In conclusion, we demonstrate that CAPRIN1 haploinsufficiency causes a novel autosomal dominant neurodevelopmental disorder and identify morphological and functional alterations associated with this disorder in human neuronal models.

NSD1 Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint.

An increasing amount of evidence indicates the critical role of the NSD1 gene in Sotos syndrome (SoS), a rare genetic disease, and in tumors. Molecular mechanisms affected by NSD1 mutations are largely uncharacterized. In order to assess the impact of NSD1 haploinsufficiency in the pathogenesis of SoS, we analyzed the gene expression profile of fibroblasts isolated from the skin samples of 15 SoS patients and of 5 healthy parents. We identified seven differentially expressed genes and five differentially expressed noncoding RNAs. The most upregulated mRNA was stratifin (SFN) (fold change, 3.9, Benjamini−Hochberg corrected p < 0.05), and the most downregulated mRNA was goosecoid homeobox (GSC) (fold change, 3.9, Benjamini−Hochberg corrected p < 0.05). The most upregulated lncRNA was lnc-C2orf84-1 (fold change, 4.28, Benjamini−Hochberg corrected p < 0.001), and the most downregulated lncRNA was Inc-C15orf57 (fold change, −0.7, Benjamini−Hochberg corrected p < 0.05). A gene set enrichment analysis reported the enrichment of genes involved in the KRAS and E2F signaling pathways, splicing regulation and cell cycle G2/M checkpoints. Our results suggest that NSD1 is involved in cell cycle regulation and that its mutation can induce the down-expression of genes involved in tumoral and neoplastic differentiation. The results contribute to defining the role of NSD1 in fibroblasts for the prevention, diagnosis and control of SoS.

Regarding the rights and duties of Clinical Laboratory Geneticists in genetic healthcare systems; results of a survey in over 50 countries.

Specialists of human genetic diagnostics can be divided into four groups: Medical Geneticists (MDG), Genetic Nurses and/or Counsellors (GN/GC), Clinical Laboratory Geneticists (CLG) and Laboratory Genetics Technicians (LGT). While the first two groups are in direct patient contact, the work of the latter two, of equal importance for patient care, are often hidden as they work behind the scenes. Herein the first study on the rights and duties of CLGs is presented. We present the results of a survey performed in 35 European and 18 non-European countries with 100 participating specialists. A national CLG title is available in 60% of European countries, and in 77% of the surveyed European countries a CLG can be the main responsible head of the laboratory performing human genetic tests. However, in only 20% of European countries is a lab-report valid with only a CLGs' signature - even though the report is almost always formulated by the CLG, and an interpretation of the obtained results in a clinical context by the CLG is expected in nearly 90% of European countries. Interestingly, CLGs see patients in 30% of European countries, and are also regularly involved in student education. Overall, the CLG profession includes numerous duties, which are quite similar in all regions of the world. Strikingly, the CLG's rights and responsibilities of leading a lab, or signing a report are regulated differently according to country specific regulations. Overall, the CLG is a well-recognized profession worldwide and often working within a multidisciplinary team of human genetic diagnostics professionals.

Prognostic value of chromosomal imbalances, gene mutations, and BAP1 expression in uveal melanoma.

Uveal melanoma (UM) exhibits recurring chromosomal abnormalities and gene driver mutations, which are related to tumor evolution/progression. Almost half of the patients with UM develop distant metastases, predominantly to the liver, and so far there are no effective adjuvant therapies. An accurate UM genetic profile could assess the individual patient's metastatic risk, and provide the basis to determine an individualized targeted therapeutic strategy for each UM patient. To investigate the presence of specific chromosomal and gene alterations, BAP1 protein expression, and their relationship with distant progression free survival (DPFS), we analyzed tumor samples from 63 UM patients (40 men and 23 women, with a median age of 64 years), who underwent eye enucleation by a single cancer ophthalmologist from December 2005 to June 2016. UM samples were screened for the presence of losses/gains in chromosomes 1p, 3, 6p, and 8q, and for mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX. BAP1 protein expression was detected by immunohistochemistry (IHC). Multivariate analysis showed that the presence of monosomy 3, 8q gain, and loss of BAP1 protein were significantly associated to DPFS, while BAP1 gene mutation was not, mainly due to the presence of metastatic UM cases with negative BAP1 IHC and no BAP1 mutation detected by Sanger sequencing. Loss of BAP1 protein expression and monosomy 3 represent the strongest predictors of metastases, and may have important implications for implementation of patient surveillance, properly designed clinical trials enrollment, and adjuvant therapy.

The distinct clinical features of giant cell tumor of bone in pagetic and non-pagetic patients are associated with genetic, biochemical and histological differences.

Giant Cell Tumor of Bone (GCT) is a tumor characterized by neoplastic mesenchymal stromal cells and a high number of osteoclast-like multinucleated giant cells. Rarely, GCT could arise in bones affected by Paget's disease of bone (GCT/PDB). Although it is already known that GCT/PDB and GCT show a different clinical profile regarding the age-onset and skeletal localization, our deep clinical comparison between the two GCT/PDB and GCT cohorts, permitted us to identify additional differences (e.g. focality, ALP serum levels, the 5-year survival rate and the familial recurrence), strongly suggesting a different molecular basis. Accordingly, driver somatic mutations in H3F3A and IDH2 were described in GCT patients, while we recently identified a germline mutation in ZNF687 as the genetic defect of GCT/PDB patients. Here, we detected H3F3A mutations in our GCT cohort, confirming its molecular screening as the elected diagnostic tool, and then we excluded the two-hit in H3F3A and IDH2 as the trigger event for the GCT/PDB development. Importantly, we also identified an alternative biochemical profile with GCT/PDB not exhibiting the up-regulation of the GCT marker FGFR2IIIc. Finally, our histological analysis also showed a different appearance of the two forms of the tumor, with GCT/PDB showing a higher number of osteoclast-like giant cells (twice), with an abnormal number of nuclei per cell, corroborating its different behaviour in terms of neoplastic properties. We demonstrated that the distinct clinical features of pagetic and conventional GCT are associated with different genetic background, resulting in a specific biochemical and histological behaviour of the tumour.

European registration process for Clinical Laboratory Geneticists in genetic healthcare.

Tremendous progress in genetics and genomics led to a wide range of healthcare providers, genetic tests, and more patients who can benefit from these developments. To guarantee and improve the quality of genetic testing, a unified European-based registration for individuals qualified in biomedicine was realized. Therefore a Europe-wide recognition of the profession 'European registered Clinical Laboratory Geneticist (ErCLG)' based on a syllabus of core competences was established which allows for harmonization in professional education. The 'European Board of Medical Genetics division - Clinical Laboratory Geneticist' provides now since 3 years the possibility to register as an ErCLG. Applicants may be from all European countries and since this year also from outside of Europe. Five subtitles reflect the exact specialty of each ErCLG, who can reregister every 5 years. A previously not possible statistics based on ~300 individuals from 19 countries as holders of an ErCLG title provides interesting insights into the professionals working in human genetics. It could be substantiated that there are around twice as many females than males and that a PhD title was achieved by 80% of registered ErCLGs. Also most ErCLGs are still trained as generalists (66%), followed by such ErCLGs with focus on molecular genetics (23%); the remaining are concentrated either on clinical (6%), tumor (4%) or biochemical genetics (1%). In conclusion, besides MDs and genetic counselors/nurses an EU-wide recognition system for Clinical Laboratory Geneticist has been established, which strengthens the status of specialists working in human genetic diagnostics in Europe and worldwide.

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq™ Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status.

ABCB4 mutations in adult patients with cholestatic liver disease: impact and phenotypic expression.

BACKGROUND: The ABCB4 gene encodes the MDR3 protein. Mutations of this gene cause progressive familial intrahepatic cholestasis type 3 (PFIC3) in children, but their clinical relevance in adults remains ill defined. The study of a well-characterized adult patient series may contribute to refining the genetic data regarding cholangiopathies of unknown origin. Our aim was to evaluate the impact of ABCB4 mutations on clinical expression of cholestasis in adult patients. METHODS: We consecutively evaluated 2602 subjects with hepatobiliary disease. Biochemical evidence of a chronic cholestatic profile (CCP) with elevated serum gamma-glutamyltransferase activity or diagnosis of intrahepatic cholestasis of pregnancy (ICP) and juvenile cholelithiasis (JC) were inclusion criteria. The personal/family history of additional cholestatic liver disease (PFH-CLD), which includes ICP, JC, or hormone-induced cholestasis, was investigated. Mutation screening of ABCB4 was carried out in 90 patients with idiopathic chronic cholestasis (ICC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), ICP, and JC. RESULTS: Eighty patients had CCP. PSC and ICC patients with PFH-CLD had earlier onset of disease than those without it (p = 0.003 and p = 0.023, respectively). The mutation frequency ranged from 50% (ICP, JC) to 17.6% (PBC). Among CCP patients, presence or absence of PFH-CLD was associated with ABCB4 mutations in 26.8 vs 5.1% (p = 0.013), respectively; in the subset of ICC and PSC patients, the corresponding figures were 44.4 vs 0% (p = 0.012) and 28.6 vs 8.7% (p = 0.173). CONCLUSIONS: Cholangiopathies attributable to highly penetrant ABCB4 mutant alleles are identifiable in a substantial proportion of adults that generally have PFH-CLD. In PSC and ICC phenotypes, patients with MDR3 deficiency have early onset of disease.

Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature.

Leri-Weill dyschondrosteosis is a pseudoautosomal dominantly-inherited skeletal dysplasia ascribed to haploinsufficiency of the SHOX gene caused by deletions, point mutations, or partial duplications of the gene, or to heterozygous deletions upstream or downstream of the intact SHOX gene involving conserved non-coding cis-regulatory DNA elements that show enhancer activity. Recently, two SHOX conserved non-coding element duplications, one upstream and one downstream, were reported in patients referred with idiopathic short stature. To further evaluate the role of these duplications in SHOX-related disorders, we describe seven patients (five with Leri-Weill dyschondrosteosis and two with short stature) all of whom have duplications of part of the upstream or downstream conserved non-coding element regions, identified by multiplex ligation-dependent probe amplification. In addition, we show data from 32 patients with an apparently identical downstream duplication that includes a proposed putative regulatory element (identified by multiplex ligation-dependent probe amplification or array comparative genome hybridization), which results in a variable phenotype from normal to mild Leri-Weill dyschondrosteosis. These additional data provide further evidence that duplications of upstream and downstream long range cis-regulatory DNA elements can result in a SHOX-related phenotype.

ELOVL5 mutations cause spinocerebellar ataxia 38.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis.

Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance.

PURPOSE: To dissect the genetics of benign familial epilepsies of the first year of life and to assess the extent of the genetic overlap between benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS), and benign familial infantile seizures (BFIS). METHODS: Families with at least two first-degree relatives affected by focal seizures starting within the first year of life and normal development before seizure onset were included. Families were classified as BFNS when all family members experienced neonatal seizures, BFNIS when the onset of seizures in family members was between 1 and 4 months of age or showed both neonatal and infantile seizures, and BFIS when the onset of seizures was after 4 months of age in all family members. SCN2A, KCNQ2, KCNQ3, PPRT2 point mutations were analyzed by direct sequencing of amplified genomic DNA. Genomic deletions involving KCNQ2 and KCNQ3 were analyzed by multiple-dependent probe amplification method. KEY FINDINGS: A total of 46 families including 165 affected members were collected. Eight families were classified as BFNS, 9 as BFNIS, and 29 as BFIS. Genetic analysis led to the identification of 41 mutations, 14 affecting KCNQ2, 1 affecting KCNQ3, 5 affecting SCN2A, and 21 affecting PRRT2. The detection rate of mutations in the entire cohort was 89%. In BFNS, mutations specifically involve KCNQ2. In BFNIS two genes are involved (KCNQ2, six families; SCN2A, two families). BFIS families are the most genetically heterogeneous, with all four genes involved, although about 70% of them carry a PRRT2 mutation. SIGNIFICANCE: Our data highlight the important role of KCNQ2 in the entire spectrum of disorders, although progressively decreasing as the age of onset advances. The occurrence of afebrile seizures during follow-up is associated with KCNQ2 mutations and may represent a predictive factor. In addition, we showed that KCNQ3 mutations might be also involved in families with infantile seizures. Taken together our data indicate an important role of K-channel genes beyond the typical neonatal epilepsies. The identification of a novel SCN2A mutation in a family with infantile seizures with onset between 6 and 8 months provides further confirmation that this gene is not specifically associated with BFNIS and is also involved in families with a delayed age of onset. Our data indicate that PRRT2 mutations are clustered in families with BFIS. Paroxysmal kinesigenic dyskinesia emerges as a distinctive feature of PRRT2 families, although uncommon in our series. We showed that the age of onset of seizures is significantly correlated with underlying genetics, as about 90% of the typical BFNS families are linked to KCNQ2 compared to only 3% of the BFIS families, for which PRRT2 represents the major gene.

Electroclinical presentation and genotype-phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations.

PURPOSE: Unverricht-Lundborg disease (EPM1A) is frequently due to an unstable expansion of a dodecamer repeat in the CSTB gene, whereas other types of mutations are rare. EPM1A due to homozygous expansion has a rather stereotyped presentation with prominent action myoclonus. We describe eight patients with five different compound heterozygous CSTB point or indel mutations in order to highlight their particular phenotypical presentations and evaluate their genotype-phenotype relationships. METHODS: We screened CSTB mutations by means of Southern blotting and the sequencing of the genomic DNA of each proband. CSTB messenger RNA (mRNA) aberrations were characterized by sequencing the complementary DNA (cDNA) of lymphoblastoid cells, and assessing the protein concentrations in the lymphoblasts. The patient evaluations included the use of a simplified myoclonus severity rating scale, multiple neurophysiologic tests, and electroencephalography (EEG)-polygraphic recordings. To highlight the particular clinical features and disease time-course in compound heterozygous patients, we compared some of their characteristics with those observed in a series of 40 patients carrying the common homozygous expansion mutation observed at the C. Besta Foundation, Milan, Italy. KEY FINDINGS: The eight compound heterozygous patients belong to six EPM1A families (out of 52; 11.5%) diagnosed at the Laboratory of Genetics of the Galliera Hospitals in Genoa, Italy. They segregated five different heterozygous point or indel mutations in association with the common dodecamer expansion. Four patients from three families had previously reported CSTB mutations (c.67-1G>C and c.168+1_18del); one had a novel nonsense mutation at the first exon (c.133C>T) leading to a premature stop codon predicting a short peptide; the other three patients from two families had a complex novel indel mutation involving the donor splice site of intron 2 (c.168+2_169+21delinsAA) and leading to an aberrant transcript with a partially retained intron. The protein dose (cystatin B/ß-actin) in our heterozygous patients was 0.24 ± 0.02, which is not different from that assessed in patients bearing the homozygous dodecamer expansion. The compound heterozygous patients had a significantly earlier disease onset (7.4 ± 1.7 years) than the homozygous patients, and their disease presentations included frequent myoclonic seizures and absences, often occurring in clusters throughout the course of the disease. The seizures were resistant to the pharmacologic treatments that usually lead to complete seizure control in homozygous patients. EEG-polygraphy allowed repeated seizures to be recorded. Action myoclonus progressively worsened and all of the heterozygous patients older than 30 years were in wheelchairs. Most of the patients showed moderate to severe cognitive impairment, and six had psychiatric symptoms. SIGNIFICANCE: EPM1A due to compound heterozygous CSTB mutations presents with variable but often markedly severe and particular phenotypes. Most of our patients presented with the electroclinical features of severe epilepsy, which is unexpected in homozygous patients, and showed frequent seizures resistant to pharmacologic treatment. The presence of variable phenotypes (even in siblings) suggests interactions with other genetic factors influencing the final disease presentation.

Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties.

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII® (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.