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Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual's life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis.

Although MSCs grant pronounced potential for cell therapies, several factors, such as their heterogeneity restrict their use. To overcome these limitations, iMSCs (MSCs derived from induced pluripotent stem cells (iPSCs) have attracted attention. Here, we analyzed the transcriptome of MSCs, iPSCs and iMSCs derived from healthy individuals and osteoarthritis (OA) patients and explored miRNA-mRNA interactions during these transitions. We performed RNA-seq and gene expression comparisons and Protein-Protein-Interaction analysis followed by GO enrichment and KEGG pathway analyses. MicroRNAs’ (miRNA) expression profile using miRarrays and differentially expressed miRNA’s impact on regulating iMSCs gene expression was also explored. Our analyses revealed that iMSCs derivation from iPSCs favors the expression of genes conferring high proliferation, differentiation, and migration properties, all of which contribute to a rejuvenated state of iMSCs compared to primary MSCs. Additionally, our exploration of the involvement of miRNAs in this rejuvenated iMSCs transcriptome concluded in twenty-six miRNAs that, as our analysis showed, are implicated in pluripotency. Notably, the identified here interactions between hsa-let7b/i, hsa-miR-221/222-3p, hsa-miR-302c, hsa-miR-181a, hsa-miR-331 with target genes HMGA2, IGF2BP3, STARD4, and APOL6 could prove to be the necessary tools that will convey iMSCs into the ideal mean for cell therapy in osteoarthritis.

PURPOSE: To investigate whether preimplantation genetic testing for aneuploidy (PGT-A) improves the clinical outcome in patients with advanced maternal age (AMA), recurrent miscarriages (RM), and recurrent implantation failure (RIF). METHODS: Retrospective cohort study from a single IVF center and a single genetics laboratory. One hundred seventy-six patients undergoing PGT-A were assigned to three groups: an AMA group, an RM group, and a RIF group. Two hundred seventy-nine patients that did not undergo PGT-A were used as controls and subgrouped similarly to the PGT-A cohort. For the PGT-A groups, trophectoderm biopsy was performed and array comparative genomic hybridization was used for PGT-A. Clinical outcomes were compared with the control groups. RESULTS: In the RM group, we observed a significant decrease of early pregnancy loss rates in the PGT-A group (18.1% vs 75%) and a significant increase in live birth rate per transfer (50% vs 12.5%) and live birth rate per patient (36% vs 12.5%). In the RIF group, a statistically significant increase in the implantation rate per transfer (69.5% vs 33.3%) as well as the live birth rate per embryo transfer (47.8% vs 19%) was observed. In the AMA group, a statistically significant reduction in biochemical pregnancy loss was observed (3.7% vs 31.5%); however, live birth rates per embryo transfer and per patient were not significantly higher than the control group. CONCLUSION: Our results agree with recently published studies, which suggest caution in the universal application of PGT-A in women with infertility. Instead, a more personalized approach by choosing the right candidates for PGT-A intervention should be followed.

Genetics of epilepsy are highly heterogeneous and complex. Lesions detected involve genes encoding various types of channels, transcription factors, and other proteins implicated in numerous cellular processes, such as synaptogenesis. Consequently, a wide spectrum of clinical presentations and overlapping phenotypes hinders differential diagnosis and highlights the need for molecular investigations toward delineation of underlying mechanisms and final diagnosis. Characterization of defects may also contribute valuable data on genetic landscapes and networks implicated in epileptogenesis. This study reports on genetic findings from exome sequencing (ES) data of 107 patients with variable types of seizures, with or without additional symptoms, in the context of neurodevelopmental disorders. Multidisciplinary evaluation of ES, including ancillary detection of copy number variants (CNVs) with the ExomeDepth tool, supported a definite diagnosis in 59.8% of the patients, reflecting one of the highest diagnostic yields in epilepsy. Emerging advances of next-generation technologies and ‘in silico’ analysis tools offer the possibility to simultaneously detect several types of variations. Wide assessment of variable findings, specifically those found to be novel and least expected, reflects the ever-evolving genetic landscape of seizure development, potentially beneficial for increased opportunities for trial recruitment and enrollment, and optimized, even personalized, medical management.

Introduction: Cystic fibrosis (CF) is the most common life-shortening recessive genetic disease in Caucasians, affecting primarily the lungs. The objective of our study was to investigate potential ophthalmologic involvement in adult patients with CF. Methods: Fifty adult patients with cystic fibrosis and 60 age- and sex-matched controls underwent complete ophthalmologic examination including tear-film Break-Up Time (BUT), Macular Thickness, and peripapillary Retinal Nerve Fiber Layer (pRNFL) thickness measurements using Spectral Domain-OCT. Results: CF patients had significantly lower nasal-inferior pRNFL thickness (median 82 IQR 67–102 vs 92.5 IQR 82–107, p = 0.005) and lower percentage of normal tear Break-Up Time (56.0% vs 96.7%, p = 0.001) than healthy controls. All CF patients with BUT Conclusions: Our study is, to the best of our knowledge, the largest ophthalmologic study of patients with cystic fibrosis. We found that CF patients had significantly decreased inferior-quadrant peripapillary retinal nerve fiber layer thickness and decreased tear-film break-up time compared to controls. We highlight the importance of careful regular ophthalmologic assessment and follow-up of these patients.

Pathogenic KMT2E variants underly O'Donnell-Luria-Rodan syndrome, a recently described neurodevelopmental disorder characterized by global developmental delay, variable degrees of intellectual disability, and subtle facial dysmorphism. Less common findings include autism, seizures, gastrointestinal (GI) problems, and abnormal head circumference. Occurrence of mostly truncating variants as well as the similar phenotype observed in individuals with deletions spanning KMT2E suggest haploinsufficiency of this gene as a common mechanism for the disorder, while a gain-of-function or dominant-negative effect cannot be ruled out for some missense variants. Deletions reported in the literature encompass several additional known or presumed haploinsufficient genes, thus leading to more complex phenotypes. Here, we describe a male with antenatal onset hydronephrosis, hypotonia, global developmental delay, prominent GI symptoms as well as facial dysmorphism. Chromosomal microarray revealed a 239-kb de novo microdeletion spanning KMT2E and LHFPL3. Clinical presentation of our proband, harboring one of the smallest deletions of the region confirms the core features of this disorder, suggests GI symptoms as a prominent finding in affected individuals while expanding the phenotypic spectrum to abnormalities of the urinary tract.

Coffin-Siris Syndrome 4 is an autosomal dominant congenital malformation syndrome caused by heterozygous mutations in the SMARCA4 gene with its main features being intellectual disability, developmental delay, behavioral abnormalities, and hypoplastic or absent fifth fingernails and fifth distal phalanges. Here, we report a young woman with developmental delay, moderate intellectual disability, and bilateral sensorineural hearing loss, referred for genetic testing. High-resolution chromosomal microarray analysis identified a 428-kb deletion in chromosome 19 which included the SMARCA4 gene. We conclude that haploinsufficiency of SMARCA4 may be a valid pathophysiological mechanism leading to milder Coffin-Siris syndrome phenotypes.

A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 (HSD17B1) and Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 (BAK1) and KH Domain Containing 1 Pseudogene 1 (KHDC1P1) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. Abbreviations: AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm

BACKGROUND/OBJECTIVES:Optic-pathway-glioma(OPG) represents the most common central-nervous tumor in children with neurofibromatosis type-1(NF1), occurring at an incidence of 15-20%. It is estimated that 1/3 of NF1 patients(pts) with OPG will need treatment. DESIGN-METHODS:We performed a retrospective-review of all NF1-pts examined in the First Hellenic Multidisciplinary-Clinic – Center of Expertise for Neurocutaneous-disorders. Gender, age, MRI-radiological and ophthalmological findings, presence of OPG, management and outcome were analyzed. RESULTS:Since the establishment of the Clinic in 2016, 198pts with clinical diagnosis of NF1 based on NIH1988-criteria were evaluated and of them, 165(73 females, median age:5.5y, range:0.3-17.1y), who had imaging studies were included in this analysis. Eighty three pts(50.3%) had NF1-positive genetic-testing and 45NF1-family-history(27.3%). Imaging-findings from optic pathway were found in 55/165pts(28females). Percentage of pts with findings were 51.7% for 10y, respectively. The median age of their first brain-MRI imaging was 2.82y. Upon 1stMRI-imaging, 70.9% presented thickness of the optic nerves(ON)(25,4%bilateral, 20% optic chiasm,18.1% right ON, 10.0% left ON), 14.5%ON-tortuosity, 38.1%OPG(43,5% in the optic-chiasm) and 34.5% contrast enhancement. Of notice, 14pts presented an OPG after a median follow-up time of 1.79y. According to LGG2004-protocol indications for treatment, only 15/55pts had to be treated(27,2%, 5pts with family history, 33.3% between 5-10y). Severe vision-loss with need for immediate start of treatment upon 1stMRI imaging was found in 4pts, of whom 75% had family-history and first evaluation after the 5th year of age. Of notice, only 2pts

Neurofibromatosis Type 1 (NF1) is caused by mutations of the NF1 gene. The aim of this study was to identify the genetic causes underlying the disease, attempt possible phenotype/genotype correlations and add to the NF1 mutation spectrum. A screening protocol based on genomic DNA was established in 168 patients, encompassing sequencing of all coding exons and adjoining introns using a custom targeted next generation sequencing protocol and subsequent confirmation of findings with Sanger sequencing. MLPA was used to detect deletions/duplications and positive findings were confirmed by RNA analysis. All novel findings were evaluated according to ACMG Standards and guidelines for the interpretation of sequence variants with the aid of in-silico bioinformatic tools and family segregation analysis. A germline variant was identified in 145 patients (86%). In total 49 known and 70 novel variants in coding and non-coding regions were identified. Seven patients carried whole or partial gene deletions. NF1 patients, present with high phenotypic variability even in cases where the same germline disease causing variant has been identified. Our findings will contribute to a better knowledge of the genetic causes and the phenotypic expression related to the disease.

The clinical spectrum of Aspergillus fumigatus diseases in cystic fibrosis (CF) patients, including allergic bronchopulmonary aspergillosis (ABPA) and Aspergillus fumigatus chronic colonization, has recently gained attention due to its association with the progression of lung disease. Our aim was to examine whether there is a difference on pathogenic variant frequencies of the CFTR gene between CF patients with ABPA and those with A. fumigatus chronic colonization.Greek CF patients diagnosed with ABPA and/or A. fumigatus chronic colonization were grouped according to their CFTR genotype. Patients with "minimal" CFTR function were defined as carrying a combination of class I or II pathogenic variants, while patients with "residual" function as carrying at least one class III, IV, V or VI pathogenic variant.Fifty-four CF patients were included and all except one were defined as having "minimal" CFTR function. Among the 108 CFTR alleles, 69 (63.9%) of pathogenic variants belonged to class II, and 32 (29.6%) to class I. Five patients had a history of both ABPA and A. fumigatus chronic colonization. No significant difference was detected among patients diagnosed only with ABPA (n = 29) and those who had only a positive history of A. fumigatus chronic colonization (n = 20). The median age of ABPA diagnosis was significantly lower than the median age of A. fumigatus chronic colonization (P = 0.011), while no significant difference was detected on median FEVNo significant differences were detected in the type of CFTR pathogenic variants among patients with ABPA and those with A. fumigatus colonization. Similar studies should be performed in larger CF populations of different ethnic origin to further confirm our results.

Summary Advancing age has a negative impact on female fertility. As implantation rates decline during the normal maternal life course, age-related, embryonic factors are altered and our inability to monitor these factors in an unbiased genome-wide manner in vivo has severely limited our understanding of early human embryo development and implantation. Our high-throughput methodology uses trophectoderm samples representing the full spectrum of maternal reproductive ages with embryo implantation potential examined in relation to trophectoderm transcriptome dynamics and reproductive maternal age. Potential embryo-endometrial interactions were tested using trophectoderm sampled from young women, with the receptive uterine environment representing the most ‘fertile’ environment for successful embryo implantation. Potential roles for extracellular exosomes, embryonic metabolism and regulation of apoptosis were revealed. These biomarkers are consistent with embryo-endometrial crosstalk/developmental competency, serving as a mediator for successful implantation. Our data opens the door to developing a diagnostic test for predicting implantation success in women undergoing fertility treatment., Graphical abstract, Highlights • Trophectoderm transcriptome profiles are altered with maternal age • Trophectoderm transcriptome profiles can delineate maternal reproductive biological age • A genome-wide methodology reveals potential embryo-endometrial communication • Exosome-related expression that could mediate implantation success decreases with aging, Embryology; Omics

Mesenchymal stromal cells (MSCs) can be derived from a wide range of fetal and adult sources including pluripotent stem cells (PSCs). The properties of PSC-derived MSCs need to be fully characterized, in order to evaluate the feasibility of their use in clinical applications. PSC-MSC proliferation and differentiation potential in comparison with bone marrow (BM)-MSCs is still under investigation. The objective of this study was to determine the proliferative and chondrogenic capabilities of both human induced pluripotent stem cell (hiPSC-) and embryonic stem cell (hESC-) derived MSCs, by comparing them with BM-MSCs.MSCs were derived from two hiPSC lines (hiPSC-MSCs), the well characterized Hues9 hESC line (hESC-MSCs) and BM from two healthy donors (BM-MSCs). Proliferation potential was investigated using appropriate culture conditions, with serial passaging, until cells entered into senescence. Differentiation potential to cartilage was examined after in vitro chondrogenic culture conditions.BM-MSCs revealed a fold expansion of 1.18x10⁵ and 2.3x10⁵ while the two hiPSC-MSC lines and hESC-MSC showed 5.88x10¹⁰, 3.49x10⁸ and 2.88x10⁸, respectively. Under chondrogenic conditions, all MSC lines showed a degree of chondrogenesis. However, when we examined the formed chondrocyte micromasses by histological analysis of the cartilage morphology and immunohistochemistry for the chondrocyte specific markers Sox9 and Collagen II, we observed that PSC-derived MSC lines had formed pink rather than hyaline cartilage, in contrast to BM-MSCs.In conclusion, MSCs derived from both hESCs and hiPSCs had superior proliferative capacity compared to BM-MSCs, but they were inefficient in their ability to form hyaline cartilage.

Introduction A new emerging role of nitric oxide (NO) in the aetiology of osteoarthritis (OA) has been reported. Inducible NO synthase (iNOS), produced by chondrocytes, is the major source of NO in the osteoarthritic cartilage. The aim of this study is to evaluate the potential association between the -1173C/T (rs9282799), -1026 C/A (rs 2779249) and -954G/C (rs1800482) single nucleotide polymorphisms (SNPs) in the promoter of the iNOS gene (NOS2A) and the incidence of knee OA in Greek population. Methods Ninety-six patients with primary knee OA were included in the study along with 44 controls. Genotypes were identified using polymerase chain reaction (PCR) and DNA sequencing techniques. Allelic and genotypic frequencies were compared between patients and controls. Results None of the -1173C/T, -1026 C/A and -954G/C SNPs were detected in the studied population, either in patients or controls. However, another SNP was identified at the site -1056 at the promoter region, where the initial G allele was substituted by the T allele. Interestingly, the TT genotype was completely absent in controls, but was detected in six patients with a 6.2% observed frequency. The difference between patients and controls was not statistically significant (p-value = 0.18). In male OA patients, the observed frequency of the TT genotype was higher (28.6%) in comparison to the 0% of the male controls (p-value = 0.1). The frequency of the G allele was 0.82 in controls and 0.78 in OA patients (p-value = 0.53). Conclusions The present study demonstrates that the 954G/C, -1026C/A, -1056G/T and -1173C/T SNPs of the NOS2A gene are not a risk factor for primary knee OA in Greek population. Moreover, -954G/C, -1026C/A and -1173C/T are rare, if not completely absent, in the Greek population. Additional research is mandatory in order to investigate the association of these SNPs with OA in different ethnic populations.

Paternal contributions to the zygote are thought to extend beyond delivery of the genome and paternal RNAs have been linked to epigenetic transgenerational inheritance in different species. In addition, sperm–egg fusion activates several downstream processes that contribute to zygote formation, including PLC zeta-mediated egg activation and maternal RNA clearance. Since a third of the preimplantation developmental period in the mouse occurs prior to the first cleavage stage, there is ample time for paternal RNAs or their encoded proteins potentially to interact and participate in early zygotic activities. To investigate this possibility, a bespoke next-generation RNA sequencing pipeline was employed for the first time to characterise and compare transcripts obtained from isolated murine sperm, MII eggs and pre-cleavage stage zygotes. Gene network analysis was then employed to identify potential interactions between paternally and maternally derived factors during the murine egg-to-zygote transition involving RNA clearance, protein clearance and post-transcriptional regulation of gene expression. Ourin silicoapproach looked for factors in sperm, eggs and zygotes that could potentially interact co-operatively and synergistically during zygote formation. At least five sperm RNAs (Hdac11,Fbxo2,Map1lc3a,Pcbp4andZfp821) met these requirements for a paternal contribution, which with complementary maternal co-factors suggest a wider potential for extra-genomic paternal involvement in the developing zygote.

X-linked myotubular myopathy (XLMTM) is a rare inherited neuromuscular disorder associated with mutations in the MTM1 gene on the Xq28 region. We report a severely affected girl with XLMTM, caused by maternally inherited 661 kb Xq28 microduplication identified by chromosomal microarray analysis and confirmed also on DNA from muscle biopsy with a custom-designed X-chromosome-specific microarray. X-inactivation analysis revealed a skewed inactivation pattern on the proband's muscle biopsy. Muscle biopsy histopathology was indicative of increased variability in fiber diameter, marked and diffuse endomysial proliferation of adipose and connective tissues, as well as predominance of type 1 fibers.

Given the complexity of neurocutaneous syndromes, a multidisciplinary approach has been advocated in order to provide optimum care.Subjects and Methods: Retrospective analysis of a cohort of 157 pa...

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases, such as necrotizing enterocolitis (NEC), gastrointestinal graft versus host disease (GVHD), and the primary acute phase of chronic inflammatory bowel disease (CIBD), exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study, we comparatively assess the efficacy of human MSCs derived from bone marrow (BM), umbilical cord blood (UCB), human embryonic stem cells (ESCs), or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs), UCB (UCB-MSCs), ESCs (ESC-MSCs), or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group, significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment.

Acute Lymphoblastic Leukemia (ALL) is a malignancy of the immature lymphoid cells mainly associated with numerical and structural chromosomal aberrations. The current standard for profiling the diverse genetic background comprises a combination of conventional karyotype and FISH analysis for the most common translocations, albeit with many limitations. Chromosomal Microarray Analysis (CMA) is a high throughput whole genome method that is gradually implemented in routine clinical practice, but not many studies have compared the two methods. Here we aim to investigate the added benefits of utilizing the high resolution 2 x 400 K G3 CGH + SNP CMA platform in routine diagnostics of pediatric ALL. From the 29 bone marrow samples that were analyzed, CMA identified clinically relevant findings in 83%, while detecting chromosomal aberrations in 75% of the patients with normal conventional karyotype. The most common finding was hyperdiploidy (20%), and the most common submicroscopic aberration involved CDKN2A/B genes. The smallest aberration detected was a 9 kb partial NF1 gene duplication. The prognosis of the patients when combining conventional cytogenetics and CMA was either changed or enhanced in 66% of the cases. A rare duplication possibly indicative of a cryptic ABL1-NUP214 fusion gene was found in one patient. We conclude that CMA, when combined with conventional cytogenetic analysis, can significantly enhance the genetic profiling of patients with pediatric ALL in a routine clinical setting.

Genome‐wide studies in tumor cells have indicated that chromatin‐modifying proteins are commonly mutated in human cancers. The lysine‐specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination‐mediated double‐strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

Background Antley-Bixler syndrome (ABS) is an exceptionally rare craniosynostosis syndrome that can be accompanied by disordered steroidogenesis, and is mainly caused by mutations in the POR gene, inherited in an autosomal recessive manner. Here we report the prenatal and postmortem findings of three sibling fetuses with ABS as a result of compound heterozygosity of a paternal submicroscopic deletion and a maternal missense mutation in the POR gene. Methods Prenatal ultrasound and postmortem examination were performed in three sibling fetuses with termination of pregnancy at 22, 23, and 17 weeks of gestation, respectively. Molecular analysis of fetus 2 and 3 included (a) bidirectional sequencing of exon 8 of the POR gene after amplification of the specific locus by polymerase chain reaction, to detect single nucleotide variants (SNVs) and (b) high resolution comparative genomic hybridization (CGH) positive single nucleotide polymorphism array CGH (aCGH) analysis to detect copy number variants (CNVs), copy neutral areas of loss of heterozygosity and uniparental disomy. Results The diagnosis of ABS was suggested by the postmortem examination findings. The combination of the POR gene molecular analysis and aCGH revealed a compound heterozygous genotype of a maternal SNV (p.A287P) and a paternal CNV (NC_000007.13:g.(?_75608488)_(75615534_?)del). Conclusion To the best of our knowledge, these sibling fetuses add to the few reported cases of ABS, caused by a combination of a SNV and a CNV in the POR gene. The detailed description of the pathologic and radiographic findings of second trimester fetuses affected with ABS adds novel knowledge concerning the early ABS phenotype, in lack of previous relevant reports. Birth Defects Research (Part A) 106:536–541, 2016. © 2016 Wiley Periodicals, Inc.

Background Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. Methods A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. Results In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. Conclusions The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples.

Chromosomal microarray analysis (CMA) is currently considered a first-tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High-resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X-chromosome while the duplications, of which 7 on the X-chromosome, were rarer (22/65, 33.8%). Fifty-one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.

Background Retinal dystrophies are a clinically and genetically heterogeneous group of disorders which affect more than two million people worldwide. The present study focused on the role of the ABCA4 gene in the pathogenesis of hereditary retinal dystrophies (autosomal recessive Stargardt disease, autosomal recessive cone-rod dystrophy, and autosomal recessive retinitis pigmentosa) in patients of Greek origin. Materials and methods Our cohort included 26 unrelated patients and their first degree healthy relatives. The ABCA4 mutation screening involved Sanger sequencing of all exons and flanking regions. Evaluation of novel variants included sequencing of control samples, family segregation analysis and characterization by in silico prediction tools. Twenty five patients were also screened for copy number variations by array-comparative genomic hybridization. Results Excluding known disease-causing mutations and polymorphisms, two novel variants were identified in coding and non-coding regions of ABCA4. Array-CGH analysis revealed two partial deletions of USH2A and MYO3A in two patients with nonsyndromic autosomal recessive retinitis pigmentosa. Conclusions The ABCA4 mutation spectrum in Greek patients differs from other populations. Bioinformatic tools, segregation analysis along with clinical data from the patients seemed to be crucial for the evaluation of genetic variants and particularly for the discrimination between causative and non-causative variants., Highlights • Sixteen known pathological genetic variants were identified in ABCA4 gene in Greek patients with retinal dystrophies. • Two novel variants were found in patients with Stargardt’s disease and cone-rod dystrophy respectively. • Two reported mutations in Stargardt's patients were identified in retinitis pigmentosa and cone-rod dystrophy patients. • The mutations p.Gly1961Glu and p.Ala1038Val, which are common in other populations, where also found in our cohort consisted of 26 Greek patients. • Array-comparative genome hybridization revealed large deletions in two out of the 25 cases studied.

In the current setting, we attempted to verify and validate miRNA candidates relevant to pediatric primary brain tumor progression and outcome, in order to provide data regarding the identification of novel prognostic biomarkers. Overall, 26 resected brain tumors were studied from children diagnosed with pilocytic astrocytomas (PAs) (n = 19) and ependymomas (EPs) (n = 7). As controls, deceased children who underwent autopsy and were not present with any brain malignancy were used. The experimental approach included microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression profiles of miR-15a and miR-24-1. The multiparameter analyses were performed with MATLAB. Matching differentially expressed miRNAs were detected in both PAs and EPs, following distinct comparisons with the control cohort; however, in several cases, they exhibited tissue-specific expression profiles. On correlations between miRNA expression and EP progression or outcome, miR-15a and miR-24-1 were found upregulated in EP relapsed and EP deceased cases when compared to EP clinical remission cases and EP survivors, respectively. Taken together, following several distinct associations between miRNA expression and diverse clinical parameters, the current study repeatedly highlighted miR-15a and miR-24-1 as candidate oncogenic molecules associated with inferior prognosis in children diagnosed with ependymoma.

Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

The potential use of patient-specific induced pluripotent stem cells (hiPSCs) in the study and treatment of hematological diseases requires the setup of efficient and safe protocols for hiPSC generation. We aimed to adopt a reprogramming method for large-scale production of integration-free patient-specific hiPSC-lines in our stem cell processing laboratory, which supports a pediatric hematopoietic stem cell transplant unit located at a tertiary care children's hospital. We describe our 5-year experience in generation of hiPSC-lines from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) using synthetic mRNAs encoding reprogramming factors. We generated hiPSC-lines from pediatric patients with β-Thalassemia, Sickle Cell Anemia, Blackfan-Diamond Anemia, Severe Aplastic Anemia, DOCK8 Immunodeficiency and 1 healthy control. After optimization of the reprogramming procedure, average reprogramming efficiency of BM-MSCs was 0.29% (range 0.25–0.4). The complete reprogramming process lasted 14–16 days. Three to five hiPSC-colonies per sample were selected, expanded to 5 culture passages and then frozen. The whole procedure took an average time of 1.8 months (range 1.6–2.2). The hiPSC-lines expressed embryonic stem cell markers and exhibited pluripotency. This mRNA reprogramming method can be applicable in a hematopoietic stem cell culture lab setting and would be useful for the clinical translation of patient-specific hiPSCs.

The high incidence of cystic fibrosis (CF) is due to the frequency of the c.1521_1523delCTT variant in the cystic fibrosis transmembrane conductance regulator (CFTR), but its age and origin are uncertain. This gap limits attempts to shed light on the presumed heterozygote selective advantage that accounts for the variant's high prevalence among Caucasian Europeans and Europe-derived populations. In addition, explaining the nature of heterozygosity to screened individuals with one c.1521_1523delCTT variant is challenging when families raise questions about these issues. To address this gap, we obtained DNA samples from 190 patients bearing c.1521_1523delCTT and their parents residing in geographically distinct European populations plus a Germany-derived population in the USA. We identified microsatellites spanning CFTR and reconstructed haplotypes at 10 loci to estimate the time/age of the most recent common ancestor (tMRCA) with the Estiage program. We found that the age estimates differ between northwestern populations, where the mean tMRCA values vary between 4600 and 4725 years, and the southeastern populations where c.1521_1523delCTT seems to have been introduced only about 1000 years ago. The tMRCA values of Central Europeans were intermediate. Thus, our data resolve a controversy by establishing an early Bronze Age origin of the c.1521_1523delCTT allele and demonstrating its likely spread from northwest to southeast during ancient migrations. Moreover, taking the archeological record into account, our results introduce a novel concept by suggesting that Bell Beaker folk were the probable migrating population responsible for the early dissemination of c.1521_1523delCTT in prehistoric Europe.

Inducible nitric oxide synthase (iNOS) is the enzyme responsible for the production of nitric oxide (NO), a major proinflammatory and destructive mediator in osteoarthritis (OA). Areas covered: This is a comprehensive review of the recent literature on the involvement of iNOS in osteoarthritis and its potential to be used as a target for OA treatment. Evidence from in vitro, in vivo and human studies was systematically collected using medical search engines. Preclinical studies have focused on the effect of direct and indirect iNOS inhibitors in both animal and human tissues. Apart from direct inhibitors, common pharmacological agents, herbal and dietary medicines as well as hyperbaric oxygen, low level laser and low intensity pulsed ultrasound have been shown to exhibit a chondroprotective effect by inhibiting the expression of iNOS. Expert opinion: Data support the further investigation of iNOS inhibitors for the treatment of OA in human studies and clinical trials. Indirect iNOS inhibitors such as interleukin 1 inhibitors also need to be studied in greater detail. Finally, human studies need to be conducted on the herbal and dietary medicines and on the non-invasive, non-pharmacological treatments.

Background MicroRNAs (miRNAs) in circulation have emerged as promising biomarkers. In this study, we aimed to identify a circulating miRNA signature for osteoarthritis (OA) patients and in combination with bioinformatics analysis to evaluate the utility of selected differentially expressed miRNAs in the serum as potential OA biomarkers. Methods Serum samples were collected from 12 primary OA patients, and 12 healthy individuals were screened using the Agilent Human miRNA Microarray platform interrogating 2549 miRNAs. Receiver Operating Characteristic (ROC) curves were constructed to evaluate the diagnostic performance of the deregulated miRNAs. Expression levels of selected miRNAs were validated by quantitative real-time PCR (qRT-PCR) in all serum and in articular cartilage samples from OA patients (n = 12) and healthy individuals (n = 7). Bioinformatics analysis was used to investigate the involved pathways and target genes for the above miRNAs. Results We identified 279 differentially expressed miRNAs in the serum of OA patients compared to controls. Two hundred and five miRNAs (73.5%) were upregulated and 74 (26.5%) downregulated. ROC analysis revealed that 77 miRNAs had area under the curve (AUC) > 0.8 and p

Background High Resolution Melting (HRM) Analysis is a validated, robust, low-cost, high throughput CF screening method. Here, we report the development and retrospective evaluation of the diagnostic value of a novel multiplex HRM, genotyping and haplotyping method for CF prenatal diagnosis (generic HRM/haplotyping). Methods 80 study samples from 20 carrier couples referred for PND (whole blood in EDTA and CVS or amniotic fluid) were genotyped retrospectively using the suggested protocol. Results All DNA samples (variable sources, extraction methods and unknown concentrations) were successfully amplified by the 1st and 2nd round PCR. The Se, Sp, NPV and PPV for the generic HRM/haplotyping method are calculated at 100%. Conclusions This generic protocol for PND using HRM, facilitates the simultaneous analysis of DNA samples from various sources in a fast, robust and efficient way. It can be easily adapted and applied for any genetic condition.

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Introduction To determine the miRNA expression profile in placentas complicated by Preeclampsia (PE) and compare it to uncomplicated pregnancies. Methods Sixteen placentas from women with PE, [11 with early onset PE (EOPE) and 5 with late onset PE (LOPE)], as well as 8 placentas from uncomplicated pregnancies were analyzed using miRNA microarrays. For statistical analyses the MATLAB® simulation environment was applied. The over-expression of miR-518a-5p was verified using Quantitative Real-Time Polymerase Chain Reaction. Results Forty four miRNAs were found dysregulated in PE complicated placentas. Statistical analysis revealed that miR-431, miR-518a-5p and miR-124* were over-expressed in EOPE complicated placentas as compared to controls, whereas miR-544 and miR-3942 were down-regulated in EOPE. When comparing the miRNA expression profile in cases with PE and PE-growth restricted fetuses (FGR), miR-431 and miR-518a-5p were found over-expressed in pregnancies complicated by FGR. Discussion Since specific miRNAs can differentiate EOPE and LOPE from uncomplicated placentas, they may be considered as putative PE-specific biomarkers. MiR-518a-5p emerged as a potential diagnostic indicator for EOPE cases as well as for PE-FGR complicated placentas, indicating a potential link to the severity of the disease.

SummaryIn characterizing a disease, it is common to search for dysfunctional genes by assaying the transcriptome. The resulting differentially expressed genes are typically assessed for shared features, such as functional annotation or co-expression. While useful, the reliability of these systems methods is hard to evaluate. To better understand shared disease signals, we assess their replicability by first looking at gene-level recurrence and then pathway-level recurrence along with co-expression signals across six pedigrees of a rare homogeneous X-linked disorder, TAF1 syndrome. We find most differentially expressed genes are not recurrent between pedigrees, making functional enrichment largely distinct in each pedigree. However, we find two highly recurrent “functional outliers” (CACNA1I and IGFBP3), genes acting atypically with respect to co-expression and therefore absent from a systems-level assessment. We show this occurs in re-analysis of Huntington’s disease, Parkinson’s disease and schizophrenia. Our results suggest a significant role for genes easily missed in systems approaches.

ALOX5AP (5-lipoxygenase) has been recognized as a susceptibility gene for stroke. Using a case-control design, the whole coding and adjoining intronic regions of ALOX5AP were sequenced to study the role of SNPs and their interplay with other risk factors in Greek patients with stroke. Patients (n=213) were classified by the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Their mean age of was 58.9 ± 14.64, comprising 145 males. The control group consisted of 210 subjects, ethnicity, sex and age matched, with no stroke history. Risk factors (hyperlipidemia, hypertension, atrial fibrillation, migraine, CAD, diabetes, smoking and alcohol consumption) were assessed as confounding factors and comparisons were done using logistic regression analysis. SNPs rs4769055, rs202068154 and rs3803277 located in intronic regions of the gene and according to in silico programs EX_SKIP and HSF possibly affecting splicing of exons 1 and 2 of ALOX5AP, showed significantly different frequencies between patients and controls. The genotype frequencies of rs4769055: AA, of rs202068154: AC and of rs3803277: CA were significantly higher (p

Osteoarthritis is the most common form of arthritis with still unknown pathogenic etiology and considerable contribution of genetic factors. One of the mechanisms of cartilage degradation in osteoarthritis is enzymatic proteolysis of the extracellular matrix by metalloproteinases. MMP-1, produced by chondrocytes and synovial cells, is a major proteinase of the MMPs family. The present study aims at evaluating the association of MMP1 gene -1607 1G/2G (rs1799750) polymorphism with primary knee osteoarthritis in the Greek population. One hundred fifty five patients with primary symptomatic knee osteoarthritis participated in the study along with 139 controls. Genotypes were determined using PCR-RLFP technique. Allelic and genotypic frequencies were compared between both study groups. There was no significant association between MMP1 -1607 1G/2G polymorphism and knee osteoarthritis, in crude analysis; however, after multiple logistic regression analysis, 1G/2G was associated with reduced odds of knee osteoarthritis by 75% in males, compared to genotypes 1G/1G + 2G/2G, adjusting for age and BMI (adjusted OR: 0.25, 95% CI: 0.069, 0.910, p = 0.035). The present study shows that MMP1 -1607 1G/2G (rs1799750) polymorphism might be a risk factor for knee osteoarthritis susceptibility in the Greek population. Further investigations are needed to confirm this association in the pathogenesis of osteoarthritis.

Constitutional microdeletions affecting 3q13.2q13.31 are rare and attempts for genotype–phenotype correlations have only recently been made in a cohort of 28 patients. The major phenotypic features of this rare syndrome are hypotonia, developmental delay, and facial anomalies. In this study, we report on a male infant with a novel reciprocal 3.671 Mb microduplication at the genomic region 3q13.2q13.31 associated with dysmorphic features and multiple congenital anomalies. The current patient was investigated by high-resolution array comparative genomic hybridization (aCGH). This is the first report of a microduplication 3q13.2q13.31 that shares a lot of common clinical features with those carrying the microdeletion. The 3q13.2q13.31 duplicated region in our patient contains nine dosage sensitive genes, amongst them the genes ATG3, CCDC80, KIAA2018, NAA50, ZDHHC23, DRD3, ZBTB20, GAP43, LSAMP. As it is the case for many other well-described reciprocal deletion/duplication syndromes, some have very different clinical features (Williams–Beuren deletion syndrome, WBS/WBS triplication) [Somerville et al. (2005); N Engl J Med 353:1694–1701], while others share similar phenotypic features (22q11.2 microdeletion/microduplication) [Portnoi (2009); Eur J Med Genet 52:88–93]. In conclusion, we describe the main phenotypic features of a possibly novel microduplication 3q13.2q13.31 syndrome. Additionally five of the dosage-sensitive genes and BOC gene are suggested to be responsible for the main phenotypic features. Evaluation of multiple patients with the microduplication is needed for full delineation of this syndrome. © 2013 Wiley Periodicals, Inc.

A 28-month-old girl with dysmorphic craniofacial features, microcephaly, hypotonia, psychomotor retardation, failure to thrive and gastrointestinal problems was referred for clinical evaluation. Array-CGH analysis revealed one of the smallest de novo microdeletions on chromosome 16q21q22.1, 2.03 Mb in size. Advanced molecular analysis contributes to more precise genotype-phenotype correlation and accurate definition of the breakpoints in the deleted/duplicated regions.

BackroundMicrocephaly can either be isolated or it may coexist with other neurological entities and/or multiple congenital anomalies, known as syndromic microcephaly. Although many syndromic cases can be classified based on the characteristic phenotype, some others remain uncertain and require further investigation. The present study describes the application of array-comparative genomic hybridization (array-CGH) as a diagnostic tool for the study of patients with clinically unknown syndromic microcephaly.MethodsFrom a cohort of 210 unrelated patients referred with syndromic microcephaly, we applied array-CGH analysis in 53 undiagnosed cases. In all the 53 cases except one, previous standard karyotype was negative. High-resolution 4 × 180K and 1 × 244K Agilent arrays were used in this study.ResultsIn 25 out of the 53 patients with microcephaly among other phenotypic anomalies, array-CGH revealed copy number variations (CNVs) ranging in size between 15 kb and 31.6 Mb. The identified CNVs were definitely causal for microcephaly in 11/53, probably causal in 7/53, and not causal for microcephaly in 7/53 patients. Genes potentially contributing to brain deficit were revealed in 16/53 patients.ConclusionsArray-CGH contributes to the elucidation of undefined syndromic microcephalic cases by permitting the discovery of novel microdeletions and/or microduplications. It also allows a more precise genotype-phenotype correlation by the accurate definition of the breakpoints in the deleted/duplicated regions.

The cyclin-dependent kinase inhibitor p21(WAF1/Cip1) is the prototype downstream effector of the tumor suppressor protein p53. Yet, evidence from human cancer and mice models, imply that p21(WAF1/Cip1), under certain conditions, can exercise oncogenic activity. The mechanism behind this behavior is still obscure. Within this context we unexpectedly noticed, predominantly in p53 mutant human cancers, that a subset of highly atypical cancerous cells expressing strongly p21(WAF1/Cip1) demonstrated also signs of proliferation. This finding suggests either tolerance to high p21(WAF1/Cip1) levels or that p21(WAF1/Cip1) per se guided a selective process that led to more aggressive off-springs. To address the latter scenario we employed p21(WAF1/Cip1)-inducible p53-null cellular models and monitored them over a prolonged time period, using high-throughput screening means. After an initial phase characterized by stalled growth, mainly due to senescence, a subpopulation of p21(WAF1/Cip1) cells emerged, demonstrating increased genomic instability, aggressiveness and chemo-resistance. At the mechanistic level unremitted p21(WAF1/Cip1) production “saturates” the CRL4(CDT2) and SCF(Skp2) ubiquitin ligase complexes reducing the turn-over of the replication licensing machinery. Deregulation of replication licensing triggered replication stress fuelling genomic instability. Conceptually, the above notion should be considered when anti-tumor strategies are designed, since p21(WAF1/Cip1) responds also to p53-independent signals, including various chemotherapeutic compounds.

High resolution oligonucleotide array Comparative Genome Hybridization technology (array-CGH) has greatly assisted the recognition of the 1p36 contiguous gene deletion syndrome. The 1p36 deletion syndrome is considered to be one of the most common subtelomeric microdeletion syndromes and has an incidence of ~1 in 5000 live births, while respectively the "pure" 1p36 microduplication has not been reported so far. We present seven new patients who were referred for genetic evaluation due to Developmental Delay (DD), Mental Retardation (MR), and distinct dysmorphic features. They all had a wide phenotypic spectrum. In all cases previous standard karyotypes were negative. Array-CGH analysis revealed five patients with interstitial 1p36 microdeletion (four de novo and one maternal) and two patients with de novo reciprocal duplication of different sizes. These were the first reported "pure" 1p36 microduplication cases so far. Three of our patients carrying the 1p36 microdeletion syndrome were also found to have additional pathogenetic aberrations. These findings (del 3q27.1; del 4q21.22-q22.1; del 16p13.3; dup 21q21.2-q21.3; del Xp22.12) might contribute to the patients' severe phenotype, acting as additional modifiers of their clinical manifestations. We review and compare the clinical and array-CGH findings of our patients to previously reported cases with the aim of clearly delineating more accurate genotype-phenotype correlations for the 1p36 syndrome that could allow for a more precise prognosis.

BACKGROUND Cat-Eye syndrome (CES) with teratoma has not been previously reported. We present the clinical and molecular findings of a 9-month-old girl with features of CES and also a palpable midline neck mass proved to be an extragonadal mature teratoma, additionally characterized by array comparative genomic hybridization (aCGH). RESULTS High resolution oligonucleotide-based aCGH confirmed that the supernumerary marker chromosome (SMC) derived from chromosome 22, as was indicated by molecular cytogenetic analysis with fluorescence in situ hybridization (FISH). Additionally, aCGH clarified the size, breakpoints, and gene content of the duplication (dup 22q11.1q11.21; size:1.6 Mb; breakpoints: 15,438,946-17,041,773; hg18). The teratoma tissue was also tested with aCGH, in which the CES duplication was not found, but the analysis revealed three aberrations: del Xp22.3 (108,864-2788,689; 2.7 Mb hg18), dup Yp11.2 (6688,491-7340,982; 0.65 Mb, hg18), and dup Yq11.2q11.23 (12,570,853-27,177,133; 14.61 Mb, hg18). These results indicated 46 XY (male) karyotype of the teratoma tissue, making this the second report of mature extragonadal teratoma in a female neonate, probably deriving from an included dizygotic twin of opposite sex (fetus in fetu). CONCLUSIONS Our findings extend the phenotypic spectrum of CES syndrome, a disorder with clinical variability, pointing out specific dosage-sensitive genes that might contribute to specific phenotypic features.

Clinical characteristics of patients are not always related to specific syndromes. Array-comparative genomic hybridization (aCGH) is used to detect submicroscopic copy number variants within the genome not visible by conventional karyotyping. The clinical application of aCGH has helped the genetic diagnosis of patients with unexplained developmental delay/intellectual disability, autism spectrum disorders, with or without multiple congenital anomalies. Since 2008, we have implemented aCGH with the 244K and 4 × 180K Agilent platform on 334 patients with various degrees of developmental delay/intellectual disability, seizures, autism spectrum disorders, multiple congenital anomalies and normal previous conventional karyotype. Many of the patients had also received a variety of other genetic tests (Fragile X syndrome, Rett syndrome, single FISH tests or metabolic screens), which were normal. Clinically significant submicroscopic imbalances with aCGH were detected in 84 (∼25.15%) patients. aCGH is proving to be a powerful tool for the identification of novel chromosomal syndromes, thus allowing accurate prognosis and phenotype-genotype correlations.

Ring chromosomes are rare cytogenetic findings and are mostly associated with an abnormal phenotype. We report on the prenatal diagnosis of a ring chromosome 10 in a fetus in which talipes equinovarus was incidentally found during routine obstetric ultrasound at 22 weeks of gestation. Amniocentesis was undertaken and cytogenetic analysis revealed a de novo non-mosaic apparently stable ring chromosome 10 replacing one of the two homologs. Multiplex Ligation-dependent Probe Amplification (MLPA) revealed subtelomeric deletions in both the short and long arm of chromosome 10. Analysis with high resolution micro-array based comparative genomic hybridization (array-CGH), defined the ring chromosome as del 10p15.3-p14 (12.59 Mb in size) and del 10q26.3 (4.22 Mb in size) and revealed the genes that are deleted. After elected termination of the pregnancy at 27th week of gestation a detailed autopsy of the fetus allowed for genotype-phenotype correlations. To our knowledge, this is the first case of a de novo ring chromosome 10 which is reported during prenatal diagnosis and is thoroughly investigated with array CGH and autopsy study.

The 22q13 deletion syndrome or Phelan-McDermid syndrome is a neurodevelopmental disorder associated with developmental delay, hypotonia, delayed or absent speech, autistic-like behavior, normal to accelerated growth and dysmorphic faces. We report the occurrence of central precocious puberty in a boy diagnosed with Phelan-McDermid syndrome. At the age of 1 year, our patient presented with increased testicular volume for his age, bone age advancement and growth acceleration. Stimulated gonadotropin levels demonstrated a premature activation of the hypothalamic-pituitary-gonadal (HPG) axis. Central precocious puberty was treated with gonadotropin-releasing hormone (GnRH) analog. Molecular diagnosis with array-comparative genomic hybridization (CGH) revealed a major deletion of 5.8 Mb at the 22q13 chromosomal region and a 25 kb duplication at the 9q34.3 region that included the NOTCH-1 gene. On the background of 22q13 deletion syndrome and data from animals on the effect of abnormal NOTCH-1 gene expression on kisspeptin neuron formation, we discuss the probable role of Notch signaling in the premature activation of the HPG axis.