The Role of Next-Generation Sequencing in the Management of Patients with Suspected Non-Ischemic Cardiomyopathy after Syncope or Termination of Sudden Arrhythmic Death.

Cardiac arrhythmias and sudden death are frequent in patients with non-ischemic cardiomyopathy and can precede heart failure or additional symptoms where malignant cardiac arrhythmias are mostly the consequence of advanced cardiomyopathy and heart failure. Finding these subgroups and making an early diagnosis could be lifesaving. In our retrospective study, we are presenting arrhythmic types of frequent cardiomyopathies where an arrhythmogenic substrate is less well defined, as in ischemic or structural heart disease. In the period of 2 years, next-generation sequencing (NGS) tests along with standard clinical tests were performed in 208 patients (67 women and 141 men; mean age, 51.2 ± 19.4 years) without ischemic or an overt structural heart disease after syncope or aborted sudden cardiac death. Genetic variants were detected in 34.4% of the study population, with a significant proportion of pathogenic variants (P) (14.4%) and variants of unknown significance (VUS) (20%). Regardless of genotype, all patients were stratified according to clinical guidelines for aggressive treatment of sudden cardiac death with an implantable cardioverter defibrillator (ICD). The P variant identified by NGS serves for an accurate diagnosis and, thus, better prevention and specific treatment of patients and their relatives. Results in our study suggest that targeted sequencing of genes associated with cardiovascular disease is an important addendum for final diagnosis, allowing the identification of a molecular genetic cause in a vast proportion of patients for a definitive diagnosis and a more specific way of treatment. VUS in this target population poses a high risk and should be considered possibly pathogenic in reanalysis.

Identification of potentially pathogenic variants for autism spectrum disorders using gene-burden analysis.

Gene- burden analyses have lately become a very successful way for the identification of genes carrying risk variants underlying the analysed disease. This approach is also suitable for complex disorders like autism spectrum disorder (ASD). The gene-burden analysis using Testing Rare Variants with Public Data (TRAPD) software was conducted on whole exome sequencing data of Slovenian patients with ASD to determine potentially novel disease risk variants in known ASD-associated genes as well as in others. To choose the right control group for testing, principal component analysis based on the 1000 Genomes and ASD cohort samples was conducted. The subsequent protein structure and ligand binding analysis usingI-TASSER package were performed to detect changes in protein structure and ligand binding to determine a potential pathogenic consequence of observed mutation. The obtained results demonstrate an association of two variants-p.Glu198Lys (PPP2R5D:c.592G>A) and p.Arg253Gln (PPP2R5D:c.758G>A) with the ASD. Substitution p.Glu198Lys (PPP2R5D:c.592G>A) is a variant, previously described as pathogenic in association with ASD combined with intellectual disability, whereas p.Arg253Gln (PPP2R5D:c.758G>A) has not been described as an ASD-associated pathogenic variant yet. The results indicate that the filtering process was suitable and could be used in the future for detection of novel pathogenic variants when analysing groups of ASD patients.

Impaired Neurodevelopmental Genes in Slovenian Autistic Children Elucidate the Comorbidity of Autism With Other Developmental Disorders.

Autism spectrum disorders (ASD) represent a phenotypically heterogeneous group of patients that strongly intertwine with other neurodevelopmental disorders (NDDs), with genetics playing a significant role in their etiology. Whole exome sequencing (WES) has become predominant in molecular diagnostics for ASD by considerably increasing the diagnostic yield. However, the proportion of undiagnosed patients still remains high due to complex clinical presentation, reduced penetrance, and lack of segregation analysis or clinical information. Thus, reverse phenotyping, where we first identified a possible genetic cause and then determine its clinical relevance, has been shown to be a more efficient approach. WES was performed on 147 Slovenian pediatric patients with suspected ASD. Data analysis was focused on identifying ultrarare or "single event" variants in ASD-associated genes and further expanded to NDD-associated genes. Protein function and gene prioritization were performed on detected clinically relevant variants to determine their role in ASD etiology and phenotype. Reverse phenotyping revealed a pathogenic or likely pathogenic variant in ASD-associated genes in 20.4% of patients, with subsequent segregation analysis indicating that 14 were de novo variants and 1 was presumed compound heterozygous. The diagnostic yield was further increased by 2.7% by the analysis of ultrarare or "single event" variants in all NDD-associated genes. Protein function analysis established that genes in which variants of unknown significance (VUS) were detected were predominantly the cause of intellectual disability (ID), and in most cases, features of ASD as well. Using such an approach, variants in rarely described ASD-associated genes, such as SIN3B, NR4A2, and GRIA1, were detected. By expanding the analysis to include functionally similar NDD genes, variants in KCNK9, GNE, and other genes were identified. These would probably have been missed by classic genotype-phenotype analysis. Our study thus demonstrates that in patients with ASD, analysis of ultrarare or "single event" variants obtained using WES with the inclusion of functionally similar genes and reverse phenotyping obtained a higher diagnostic yield despite limited clinical data. The present study also demonstrates that most of the causative genes in our cohort were involved in the syndromic form of ASD and confirms their comorbidity with other developmental disorders.

Clinical Evaluation of Two Non-Invasive Genetic Tests for Detection and Monitoring of Urothelial Carcinoma: Validation of UroVysion and Xpert Bladder Cancer Detection Test.

A variety of commercially available urinary molecular markers have been introduced for detecting and monitoring urothelial carcinoma (UC). We prospectively evaluated the UroVysionTM Bladder Cancer Kit (FISH) and the Xpert® Bladder Cancer Detection (Xpert) test. Both tests were performed on voided urine samples after negative cystoscopy and negative abdominal ultrasound (US) and/or negative computed tomography urography (CTU). Urine specimens from 156 patients diagnosed with hematuria and suspected of having UC and 48 patients followed up after treatment of UC were analyzed using FISH and Xpert. Among 204 patients, 20 had UC, 11 located in the bladder, six in the ureter, and three in the renal pelvis. FISH had an overall sensitivity (SN) of 78%, a specificity (SP) of 93%, and a negative predictive value (NPV) of 96%. Xpert had an overall SN of 90%, an SP of 85%, and an NPV of 98%. Both tests had high SN, SP, and NPV. The SP of FISH was significantly higher. By using FISH and Xpert in addition to cystoscopy, renal and bladder US, and/or CTU in the diagnostic workup of patients with hematuria and follow-up after transurethral resection of the bladder (TURB), a substantial number of patients (10%) otherwise missed were discovered to have UC.

CNVs and Chromosomal Aneuploidy in Patients With Early-Onset Schizophrenia and Bipolar Disorder: Genotype-Phenotype Associations.

Introduction: Early-onset schizophrenia (EOS) and bipolar disorder (EOB) start before the age of 18 years and have a more severe clinical course, a worse prognosis, and a greater genetic loading compared to the late-onset forms. Copy number variations (CNVs) are an important genetic factor in the etiology of psychiatric disorders. Therefore, this study aimed to analyze CNVs in patients with EOS and EOB and to establish genotype-phenotype relationships for contiguous gene syndromes or genes affected by identified CNVs. Methods: Molecular karyotyping was performed in 45 patients, 38 with EOS and seven with EOB hospitalized between 2010 and 2017. The exclusion criteria were medical or neurological disorders or IQ under 70. Detected CNVs were analyzed according to the standards and guidelines of the American College of Medical Genetics. Result: Molecular karyotyping showed CNVs in four patients with EOS (encompassing the PAK2, ADAMTS3, and ADAMTSL1 genes, and the 16p11.2 microduplication syndrome) and in two patients with EOB (encompassing the ARHGAP11B and PRODH genes). In one patient with EOB, a chromosomal aneuploidy 47, XYY was found. Discussion: Our study is the first study of CNVs in EOS and EOB patients in Slovenia. Our findings support the association of the PAK2, ARHGAP11B, and PRODH genes with schizophrenia and/or bipolar disorder. To our knowledge, this is also the first report of a multiplication of the ADAMTSL1 gene and the smallest deletion of the PAK2 gene in a patient with EOS, and one of the few reports of the 47, XYY karyotype in a patient with EOB.

A mosaic form of microphthalmia with linear skin defects.

BACKGROUND: Microphthalmia with linear skin defects (MLS) syndrome is a rare neurodevelopmental X-dominant disorder. It presents in females as it is normally lethal in males. Three causative genes for MLS syndrome (OMIM 309801) have been identified all taking part in mitochondrial respiratory chain and oxidative phosphorylation. In our case, we describe a newborn with mosaic deletion encompassing HCCS gene resulting in unilateral microphthalmia and facial skin lesions. CASE PRESENTATION: A girl was born with caesarean section at 40 weeks of gestation. Clinical findings revealed anophthalmia of the left eye. The left eyelids were intact, the orbit was empty and the right eye was normal, without any abnormalities. She had typical linear skin defects on the left cheek, one on the left side of the neck, and two on the 3th and 4th fingers of the left hand. The other clinical findings and the neurological exam were normal. US of the brain and EEG were normal. Molecular karyotyping using BlueGnome CytoChip Oligo 4× 180K array was performed detecting an approximately 18% mosaic 3.3 Mb deletion (arr[GRCh37] Xp22.31p22.2(8,622,553_11,887,361)× 1[0.18]). FISH using RPCI11-768H20 BAC clone on cultivated interphase and metaphase lymphocytes was used to confirm the array results. The observed deletion was present in 29% of cells (46,XX,ish del(p22.2p22.31)(RPCI11-768H20)[60/205]). CONCLUSIONS: In this report we present a female proband with MLS syndrome. To our knowledge, there have been only few other cases of mosaic MLS syndrome described in the literature. Our case shows that low grade mosaicism does not preclude full clinical presentation and further supports the critical role of the X inactivation pattern in the development of the clinical findings.

Copy number variant discrepancy resolution using the ClinGen dosage sensitivity map results in updated clinical interpretations in ClinVar.

Conflict resolution in genomic variant interpretation is a critical step toward improving patient care. Evaluating interpretation discrepancies in copy number variants (CNVs) typically involves assessing overlapping genomic content with focus on genes/regions that may be subject to dosage sensitivity (haploinsufficiency (HI) and/or triplosensitivity (TS)). CNVs containing dosage sensitive genes/regions are generally interpreted as "likely pathogenic" (LP) or "pathogenic" (P), and CNVs involving the same known dosage sensitive gene(s) should receive the same clinical interpretation. We compared the Clinical Genome Resource (ClinGen) Dosage Map, a publicly available resource documenting known HI and TS genes/regions, against germline, clinical CNV interpretations within the ClinVar database. We identified 251 CNVs overlapping known dosage sensitive genes/regions but not classified as LP or P; these were sent back to their original submitting laboratories for re-evaluation. Of 246 CNVs re-evaluated, an updated clinical classification was warranted in 157 cases (63.8%); no change was made to the current classification in 79 cases (32.1%); and 10 cases (4.1%) resulted in other types of updates to ClinVar records. This effort will add curated interpretation data into the public domain and allow laboratories to focus attention on more complex discrepancies.

Rare structural variants in the DOCK8 gene identified in a cohort of 439 patients with neurodevelopmental disorders.

Detection of copy number variations (CNVs) is a first-tier clinical diagnostic test for children with neurodevelopmental disorders (NDD), which reveals the genetic cause of the disorder in more than 20%. These are mostly known microdeletion/microduplication syndromes, but variants of unknown clinical significance (VOUS) and ambiguous CNVs can also be detected. An example of the last two are abnormalities in the DOCK8 gene. Conflicting interpretations of CNVs affecting DOCK8 can be found in the literature. Deletions were predicted to have a impact in carriers with variable clinical manifestations, where duplications have been proposed as benign variants. In our study, CNV screening was performed in a cohort involving 439 probands with suspected NDD. We identified known microdeletion/microduplication syndromes in 19% and VOUS CNVs in 8% of patients. Among these, three patients had a CNV encompassing the DOCK8 gene. Although diverse clinical presentations are noted in our three patients, comparison of their phenotypes revealed that abnormalities in cognition and communication, aggressive behaviour and mood swings are common to all of them. Therefore, a clinical relevance, in terms of influencing the psychiatric clinical picture of patients, is proposed for the CNVs disrupting the DOCK8 gene, regardless of whether it is a deletion or duplication.

Impact of prenatal screening on the prevalence of Down syndrome in Slovenia.

OBJECTIVES: To evaluate the impact of prenatal screening and genetic testing for trisomy 21 (T21) on the prevalence of T21 in Slovenia. DESIGN AND SETTING: Data about all prenatally and postnatally confirmed cases of T21 in Slovenia between 1981 and 2012 were collected retrospectively from all genetic laboratories in Slovenia. The expected number of babies with T21 according to maternal age was calculated. MAIN OUTCOME MEASURES: The primary outcomes measures were number of fetuses and newborn infants with T21 diagnosed prenatally and postnatally and the impact of advances in screening and genetic diagnostics on the prevalence of newborns with T21 in Slovenia. RESULTS: Despite a significantly increased mean maternal age from 25.4 years in year 1981 to 30.3 years in year 2012 the prevalence of newborn infants with T21 was 0.51 per 1000 births compared to 0.55 per 1000 births, respectively. The prevalence of prenatally diagnosed cases increased from 0.03 per 1000 births to 2.06 per 1000. The detection rate of T21 in year 2012 was 78,9%. The total number of prenatal invasive procedures (chorionic villous sampling and amniocenteses) carried out during that period was rising until 2002, since when it is stable at around 7%. CONCLUSION: The advancement and implementation of screening tests and prenatal diagnostic procedures in Slovenia caused an important improvement in the efficiency of the prenatal detection of T21.

Predictive value of paroxysmal EEG abnormalities for future epilepsy in focal febrile seizures.

PURPOSE OF THE STUDY: To reassess the predictive role of clinical parameters and epileptiform paroxysmal EEG abnormalities for subsequent epilepsy in patients with febrile seizures. PATIENTS AND METHODS: 179 patients with febrile seizures were included in a prospective study investigating the impact of some clinical parameters and EEG abnormalities that could be important for future epilepsy. EEGs were performed in afebrile patients after hospital discharge. The follow-up period from the first presentation ranged from 2.1 to 9.2 years (mean, 6.6 years). The correlation between the development of epileptic seizures and the presence of epileptiform EEG abnormalities in the two groups was evaluated with the Mann-Whitney and chi-square test. Statistical significance was set at p<0.05. RESULTS: Febrile seizures occurred more than once in 58 (32.5%) patients, with one recurrence in 32 (17.9%) patients and multiple recurrences in 26 (14.5%) patients. The incidence of paroxysmal abnormalities was 16.8%. Of these, 15 patients (50%) showed generalized paroxysms only, while in 15 patients (50%), focal abnormalities were found. Epilepsy developed in 12 patients (6.7%). There were 27 patients with clinically focal features of the first febrile seizure, five (18.5%) of whom developed epilepsy. With focal EEG abnormalities included, the incidence of epilepsy increased to 50%. CONCLUSION: Generalized EEG discharges in patients with febrile seizures are not predictive of later epilepsy, but focal discharges are.

A coalescence of two syndromes in a girl with terminal deletion and inverted duplication of chromosome 5.

BACKGROUND: Rearrangements involving chromosome 5p often result in two syndromes, Cri-du-chat (CdC) and Trisomy 5p, caused by a deletion and duplication, respectively. The 5p15.2 has been defined as a critical region for CdC syndrome; however, genotype-phenotype studies allowed isolation of particular characteristics such as speech delay, cat-like cry and mental retardation, caused by distinct deletions of 5p. A varied clinical outcome was also observed in patients with Trisomy 5p. Duplications of 5p10-5p13.1 manifest themselves in a more severe phenotype, while trisomy of regions distal to 5p13 mainly causes mild and indistinct features. Combinations of a terminal deletion and inverted duplication of 5p are infrequent in literature. Consequences of these chromosomal rearrangements differ, depending on size of deletion and duplication in particular cases, although authors mainly describe the deletion as the cause of the observed clinical picture. CASE PRESENTATION: Here we present a 5-month-old Slovenian girl, with de novo terminal deletion and inverted duplication of chromosome 5p. Our patient presents features of both CdC and Trisomy 5. The most prominent features observed in our patient are a cat-like cry and severe malformations of the right ear. CONCLUSION: The cat-like cry, characteristic of CdC syndrome, is noted in our patient despite the fact that the deletion is not fully consistent with previously defined cat-like cry critical region in this syndrome. Features like dolichocephaly, macrocephaly and ear malformations, associated with duplication of the critical region of Trisomy 5p, are also present, although this region has not been rearranged in our case. Therefore, the true meaning of the described chromosomal rearrangements is discussed.

An evaluation of SOX2 and hTERC gene amplifications as screening markers in oral and oropharyngeal squamous cell carcinomas.

BACKGROUND: Oral and oropharyngeal squamous cell carcinomas (OSCC) are among the most common cancers. The poor survival rate among oral cancer patients can be attributed to several factors, one of them being lack of early detection. A key approach to this problem would be to detect potentially malignant lesion at their early stage. Using the FISH technique, oral brush cytology slides can be an easy and rapid screening approach for malignant cell detection. The present study was designed to detect hTERC and SOX2 amplifications in OSSC exfoliative tumor cells and evaluate whether those two gene amplifications might serve as a supportive biomarker in early detection and diagnosis of oral and oropharyngeal SCC. RESULTS: Brush biopsies were collected from exophytic and exulcerated oral and oropharyngeal lesions of the oral cavity of 71 patients and 22 healthy controls. FISH techniques using a TERC-specific DNA probe and a SOX2 DNA specific probe both combined with a centromere 3-specific control probe was performed on the cytology slides. A 100 squamous epithelial cell nuclei of the smears per slide were analysed. As abnormal FISH pattern were considered amplified and polyploid patterns.From 71 brush biopsies of oropharynx and other locations in oral cavity analysed by FISH 49 were considered to be abnormal (69%). The over representation of polyploidy and/or TERC/SOX2 amplification in tumour samples was statistically significant when compared to controls (p = 0.01). CONCLUSION: SOX2 and TERC gene amplifications are common in all squamous cell carcinomas and their detection in early stages could be crucial for early detection and more accurate prognosis. Our study strongly suggests that early detection by FISH on cytobrushed samples could be a possible non-invasive screening method even before a tissue biopsy is performed.

Submicroscopic interstitial deletion of chromosome 11q22.3 in a girl with mild mental retardation and facial dysmorphism: Case report.

BACKGROUND: Except for terminal deletions that lead to Jacobsen syndrome, interstitial deletions involving the long arm of chromosome 11 are not frequently reported. A clinically distinct phenotype is usually observed in these cases, and no clear genotype-phenotype correlation is proposed. RESULTS: Here we present a case study of a 5-year-old girl with de novo submicroscopic deletion of chromosome 11q22.3 with mild mental retardation and facial dysmorphism. A standard cytogenetic analysis did not reveal any structural aberrations. In contrary, array-CGH analysis indicated a small deletion of 11q22.3. DISCUSSION: To our knowledge, this is the smallest 11q22.3 deletion reported in literature, containing nine RefSeq genes. Although none of the deleted genes are obvious candidates for the features observed in our patient, genes CUL5 and SLN could play a key role in the features described.

Is the JAK2 V617F mutation a hallmark for different forms of thrombosis?

BACKGROUND/AIMS: The association between venous thrombosis outside the splanchnic area as well arterial thromboembolism and the JAK2 V617F mutation, an important marker for chronic myeloproliferative neoplasms (MPN), is not completely clear. METHODS: Four hundred forty-four patients with venous thrombosis of the lower/upper limbs and/or pulmonary embolism and 60 patients with ischemic stroke were screened for the JAK2 V617F mutation, factor V Leiden, and factor II G20210A. RESULTS: The JAK2 V617F mutation was detected in 1.4% of patients with venous thrombosis and in 3.3% of patients with ischemic stroke. Because 6 out of 2,430 control individuals with no medical history of venous thrombosis, stroke, or MPN were positive for the JAK2 V617F mutation, a significant association was observed (OR 5.53, CI 1.77-17.2, p = 0.0053 for venous thrombosis; OR 13.9, CI 2.75-70.5, p = 0.0145 for stroke). CONCLUSION: We provide evidence of the association between the JAK2 V617F mutation and different forms of thrombosis. This association is comparable with the association between inherited risk factors (factor V Leiden and factor II G20210A) and thrombotic events, but with a much lower prevalence of the mutation. Finally, the JAK2 V617F mutation is not absent from the general population despite being considered somatic and an acquired genetic variation.

Screening of TERC gene amplification as an additional genetic diagnostic test in detection of cervical preneoplastic lesions.

TERC gene amplification was investigated as a possible diagnostic marker for use in routine cytological screening to improve the accuracy of conventional screening procedures in detection of cervical preneoplastic lesions. Cervical smears were screened and classified as low-grade or high-grade squamous intraepithelial lesions (LSIL or HSIL). A fluorescence in situ hybridization procedure using a TERC-specific DNA probe was performed on the same specimens and TERC gene copy number was evaluated. More than two signals per cell were defined as TERC positive. In cervical smears graded after conization as cervical intraepithelial neoplasia grade 1 (CIN 1), no TERC-positive cases were found in either LSIL or HSIL, and no TERC amplification was found in LSIL cases with histological results CIN 1 and CIN 2. Amplifications of the TERC gene first appeared in HSIL cases with CIN 2 histology. In the CIN 3 group, TERC-positive cases were present in both LSIL and HSIL; in these, there were no statistically significant differences between TERC-positive and TERC-negative cases. Statistically significant differences in TERC-positive cases were found between LSIL and HSIL without regard to the CIN grade. From the results obtained, it can be concluded that TERC gene amplifications inevitably lead to a high risk of CIN 3 in both LSIL and HSIL after cytological smear examination. A high CIN grade is not necessarily correlated with TERC amplification, but a positive TERC result certainly demands a high CIN classification.

Subtelomeric chromosome rearrangements in children with idiopathic mental retardation: applicability of three molecular-cytogenetic methods.

AIM: To identify cryptic subtelomeric rearrangement, a possible cause of idiopathic mental retardation by means of multiprobe telomere fluorescent in situ hybridization (T-FISH). METHODS: Hundred patients (median age 3.0 years) with mental retardation and dysmorphic features were screened using specific T-FISH probes. Multiplex ligation-dependent probe amplification and comparative genomic hybridization were used for the confirmation of results. RESULTS: Telomere fluorescent in situ hybridization revealed 11 subtelomeric abnormalities in 10 patients (10%; 95% CI, 5.0-17.5). Four of these had only a deletion of subtelomere 2q, which was apparently a normal variant. Among 6 true aberrations (6%; 95% CI, 2.5-12.5) we found 2 de novo subtelomeric deletions and 4 unbalanced subtelomeric rearrangements (one de novo). All clinically significant subtelomeric rearrangements were confirmed by multiplex ligation-dependent probe amplification. Comparative genomic hybridization was used to investigate the whole genome of patients in whom a subtelomeric anomaly was found, confirming some, but not all subtelomeric rearrangements. CONCLUSION: Telomere fluorescent in situ hybridization and multiplex ligation-dependent probe amplification are both very useful and interchangeable methods for detection of unbalanced chromosome rearrangements, but T-FISH also detects balanced rearrangements. In our experiment the resolution power of comparative genomic hybridization was too low for subtelomeric screening compared with T-FISH and multiplex ligation-dependent probe amplification.