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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

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.

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.

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.

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.

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.

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.

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.

Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long-term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM-CD105(+) cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 × 10(5) ± 0.9 × 10(5) CD105(+) cells from 10 small volume (10-25 ml) BM samples achieving an enrichment >100-fold in MSC. Seeding 2 × 10(3) immunoselected cells/cm(2) we were able to produce 2.5 × 10(8) ± 0.7 × 10(8) MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29(+), CD90(+), CD73(+), CD105(+), CD45(-); they suppressed allogeneic T-cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM-CD105(+) cells.

Sarcoidosis is a complex disease with autoimmune basis and still unknown etiology. We have screened for mutations in the cystic fibrosis conductance regulator (CFTR) gene and genotyped single-nucleotide polymorphisms in the tumor necrosis factor (TNF), interferon alpha-10 (IFNA10), IFNA17, and interferon gamma (IFNG) genes in 89 Greek patients with sarcoidosis and 212 control subjects to detect possible association between them and the risk for developing sarcoidosis. We have found a statistically significant increase (p = 6.1 x 10(-8)) of CFTR mutation carriers in the population of patients with sarcoidosis versus the control population. A difference was also noted within the group of patients with sarcoidosis where the ones with CFTR mutations suffered more frequently from dyspnea than those without (p = 5 x 10(-6)). Our study did not reproduce the associations previously noted with the TNF, IFNA10, IFNA17, and IFNG genes, which highlights the genetic complexity of the disorder and is in agreement with previous studies showing that CFTR might be an important factor in the clinical course of the disease.

Autism is a neurodevelopmental disorder characterized by clinical, etiologic, and genetic heterogeneity. During the last decade, predisposing genes and genetic loci were under investigation. Recently, mutations in two X-linked neuroligin genes, neuroligin 3 (NLGN3) and neuroligin 4 (NLGN4), have been implicated in the pathogenesis of autism. In our ongoing survey, we screened 169 patients with autism for mutations linked with autism. In the preliminary study of specific exons of NLGN3 and NLGN4 genes, we identified the p.K378R substitution (c.1597 AG) in exon 5 of the NLGN4 gene in a patient who was found to have mild autism and normal IQ at 3 years of age. The same mutation has previously been found in a patient with autism. It is important that, for the first time, a specific mutation in neuroligins is confirmed in a molecular screen in another homogeneous ethnic population. This finding further contributes to consideration of neuroligins as probable candidate genes for future molecular genetic studies, suggesting that a defect of synaptogenesis may predispose to autism.

We investigated the hypothesis that coinheritance of the common A(TA)(n)TAA promoter mutation at the UGT1A1 locus associated with Gilbert syndrome is a risk factor for gallstone formation in a homogeneous adult population, by conducting a case-control study that included 198 adult patients with cholelithiasis and 152 healthy controls both of Greek origin. Three genotypes were found: 7/7 (17.8% in controls and 23.3% in patients), 6/7 (33.5% in controls and 46.5% in patients), and normal homozygous 6/6 (48.7% in controls and 30.3% in patients). The Gilbert UGT1A1 genotypes 6/7 and 7/7 show significant association (odds ratio 2.225, 95% confidence interval 1.373-3.605, p=0.001, and odds ratio 2.101, 95% confidence interval 1.171-3.770, p=0.013, respectively) with cholelithiasis risk. This association supports the theory that genetic factors are responsible for a fraction of symptomatic gallstone disease; however, further studies are required in different ethnic groups to fully elucidate the involvement of Gilbert syndrome in gallstone disease.

Objectives Wilson disease (WD) has a wide spectrum of clinical manifestations. Affected children may be entirely asymptomatic and the diagnosis problematic. Herein we present the clinical and laboratory characteristics of 57 children with WD and point out the diagnostic difficulties in a pediatric population. Patients and methods Clinical and laboratory data were collected from 57 consecutive children with WD. Evaluation included detailed physical examination, conventional laboratory testing, genetic analysis, and liver biopsy. Results The mean age at diagnosis was 9.27 +/- 3.62 years (range 4 months-18 years). Twenty patients were symptomatic, 19 were referred because of abnormal liver function test results and/or hepatomegaly, and 18 received their diagnoses after family screening. Twenty-two patients had both Kayser-Fleischer ring and decreased serum ceruloplasmin levels, 13 had urinary copper excretion after penicillamine challenge >1600 microg/24 hours, and 3 had liver copper content >250 microg/g dry weight. Of the remaining 19 patients, 17 had both low serum ceruloplasmin 75 microg/24 hours before, or >1000 microg/24 hours after penicillamine challenge. In 2 patients with equivocal cases who had serum ceruloplasmin 26 mg/dL, the diagnosis was confirmed by genetic analysis. No correlation was found between specific mutations and the disease phenotypic expression. Chelating therapy was well tolerated, and the outcome was satisfactory. Conclusions WD in children may be obscure and requires extensive investigation to establish the diagnosis. Genetic analysis is needed in equivocal cases.

We have been investigating the functional consequences of rare disease-associated amino acid substitutions in the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with "severe" pancreatic insufficient cystic fibrosis (CF) and R1070W with "mild" pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in nonpolarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized Madin Darby canine kidney (MDCK) cell lines that express either wild-type or mutant CFTR from the same genomic integration site. Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a "severe" phenotype. Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had "mild" disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems can greatly assist the interpretation of the disease-causing potential of rare missense mutations.

Genetic variation in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to play a role in breast cancer. To determine the possible contribution of genetic variation in the ESR1 (ER-alpha), ESR2 (ER-beta) and AR genes in breast cancer risk the -1174(TA)(7-27), c. 1092+3607(CA)(10-26) and c. 172(CAG)(6-40) repeat variants were studied in a case-control study of 79 women with sporadic breast cancer and 155 controls. No significant difference was observed in the frequency distribution of -1174(TA)(7-27) in the ESR1 gene between patients and controls, while a significant difference was observed for repeat polymorphisms c. 1092+3607(CA)(10-26) in the ESR2 gene and c. 172(CAG)(6-40) in the AR gene (p0.0001). A significantly decreased odds ratio (OR) for breast cancer risk was observed in individuals having the LL and the SL genotypes for both the ESR2 (OR=0.010, 95% CI 0.003-0.036, p0.001; OR=0.013, 95% CI 0.004-0.040, p0.0001, respectively) and the AR gene (OR=0.040, 95% CI 0.011-0.138, p0.0001; OR=0.189, 95% CI 0.10-0.359, p0.0001, respectively), compared to SS genotype. The protective effect of these genotypes remained evident even after adjustment for various risk factors (BMI, age, age at menarche and menopause, family history). In conclusion, an association for breast cancer risk between short (SS) alleles for the repeat variants of the ESR2 and AR genes was found in women of Greek descent.

Uridine diphospho-glucuronosyltransferase 1 (UGT1A1) is involved in estradiol glucuronidation, which may play a central role in the etiology of breast cancer. A common insertion/deletion polymorphism in the TATAA-box of the promoter region of UGT1A1 results in decreased initiation of transcription, and has been associated with breast cancer risk in different ethnic groups. In the present study, the role of the above genetic variation at the UGT1A1 locus in breast cancer susceptibility was investigated in a homogeneous population. Our case-control study included 136 women with breast cancer and 186 healthy female controls of Greek origin. The polymorphism A(TA)nTAA in the promoter region of UGT1A1 gene was studied using the Fragment Analysis Software of an automated DNA sequencer and three genotypes (homozygous 7/7, heterozygous 6/7, and normal homozygous 6/6) were identified. No significant associations were observed between the 7/7 genotype and breast cancer risk, indicating that further studies in Caucasian women are needed to elucidate the role of UGT1A1 polymorphism in breast cancer risk.

Background There are three distinct subtypes of Trichorhinophalangeal syndrome (TRPS); TRPS type I, TRPS type II and TRPS type III. Features common to all three subtypes include sparse, slowly growing scalp hair, laterally sparse eyebrows, a bulbous tip of the nose (pear-shaped), and protruding ears. Langer–Giedion syndrome (LGS) or TRPS type II is a contiguous gene syndrome on 8q24.1, involving loss of functional copies of the TRPS1 and EXT1 genes. We report a male patient that was referred to the Department of Medical Genetics due to hypotonia and dysmorphic facial features. Results Cytogenetic and array- Comparative Genomic Hybridization (aCGH) analysis revealed that the patient was a carrier of an interstitial deletion at 8q23.1-q24.12 of 12,5 Mb. Parental karyotype indicated that the father carried an apparently balanced insertion: 46, ΧΥ, der(10)ins(10;8)(q22;q23q24). Conclusions This is the first report of an apparently balanced insertion including chromosomes 8 and 10 contributing to the etiology of LGS/ TRPS type II. Τimely diagnosis of parental balanced chromosomal rearrangements can reduce the risk of subsequent miscarriages as well as abnormal offspring.

Contains fulltext : 152777.pdf (Publisher’s version ) (Open Access) We describe an X-linked genetic syndrome associated with mutations in TAF1 and manifesting with global developmental delay, intellectual disability (ID), characteristic facial dysmorphology, generalized hypotonia, and variable neurologic features, all in male individuals. Simultaneous studies using diverse strategies led to the identification of nine families with overlapping clinical presentations and affected by de novo or maternally inherited single-nucleotide changes. Two additional families harboring large duplications involving TAF1 were also found to share phenotypic overlap with the probands harboring single-nucleotide changes, but they also demonstrated a severe neurodegeneration phenotype. Functional analysis with RNA-seq for one of the families suggested that the phenotype is associated with downregulation of a set of genes notably enriched with genes regulated by E-box proteins. In addition, knockdown and mutant studies of this gene in zebrafish have shown a quantifiable, albeit small, effect on a neuronal phenotype. Our results suggest that mutations in TAF1 play a critical role in the development of this X-linked ID syndrome.

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.European Journal of Human Genetics advance online publication, 27 May 2015; doi:10.1038/ejhg.2015.99.

WOS: 000230868400013, PubMed ID: 15930910, Trigonocephaly and Wilson's disease (WD) are two different entities. The former is a type of craniosynostosis that occurs because of fusion of the metopic suture and the latter, also called hepatolenticular degeneration, is caused by an accumulation of copper in tissues all over the body because of failure of copper excretion. No single gene has been identified for trigonocephaly whereas the ATP7B gene has been shown to be responsible for Wilson's disease. Here we present two siblings born to non-consanguineous parents who both presented with trigonocephaly, Wilson's disease and facial dysmorphism. In addition, the female has renal agenesis and the male has a history of undescended testis. Karyotypes were normal and no mutation of the ATP7B gene has been identified in the patients or their parents.

PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of beta-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.

Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. Methods: As a model, we designed a protocol based on the LightCycler™ technology to screen for a spectrum of β-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of β-globin gene mutations in the Greek population, including IVSII-745C→G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G→A, IVSI-5G→A, IVSI-6T→C, IVSI-110G→A, and Cd39 C→T. Results: The protocol, standardized by analysis of 100 β-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek β-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations ΗbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five β-globin gene mutations from other population groups (IVSI-1G→T, IVSI-5G→C, IVSI-116T→G, Cd37 TGG→TGA, and Cd41/42 −TCTT). Conclusions: The described LightCycler system protocol can rapidly screen for many β-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

More than 200 Wilson disease (WD) disease-causing mutations have been defined to date. Missense mutations are largely prevalent while splice-site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT-PCR method we performed the molecular characterization of four consensus splice-site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122-8T>G, 2866-6T>G, and 3061-12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype.

Cystic Fibrosis (CF) is one of the commonest autosomal recessive genetic diseases that show a high carrier frequency amongst Caucasian populations. Although there has been tremendous progress in the available therapies, compared to the past, the disease is still associated with significant morbidity and mortality. Because of the severe clinical manifestations and the shortened life expectancy of patients, population based carrier screening, to identify heterozygous carrier couples at risk of having affected children, has been recommended. We review the history of population based carrier screening for CF, focusing on the screening guidelines, the current requirements and the difficulties of implementation, considering especially the heterogeneity of CF-causing mutations.

A 4.5 years old male with myoclonic epilepsy on Valproic acid (VPA) monotherapy, developed an acute pancreatitis. The discontinuation of VPA and substitution with Levetiracetam was followed by clinical improvement but a relapse of the pancreatitis was noted one month later. The investigation excluded a structural abnormality but revealed a heterozygous CTFR mutation. The contribution of the CTFR mutation on this VPA-induced recurrent pancreatitis cannot be ignored.

Sarcoidosis is a multisystemic granulomatous disease of unknown etiology that primarily affects adults between the ages of 20 and 40 years old. It is characterized by the activation of Th1 lymphocytes resulting in the production of inflammatory cytokines and the formation of noncaseating epithelioid cell granulomas in affected tissues. The lungs and lymphatic system are the ones most frequently affected. The disease usually presents spontaneous remission in the first two years and, in a few patients, the disease progresses to pulmonary fibrosis or other fatal complications depending on the affected organ. The pathogenesis of sarcoidosis is still not clearly defined, and is considered an interaction between the environment and risk alleles in many genes. The present case control study consisted of 146 Greek patients with sarcoidosis and 90 healthy volunteers from the same ethnic group. The coding and neighboring intronic regions of the BTNL2 gene were sequenced and risk alleles were compared amongst the two groups. Thirty-seven different variants were detected from which 12 were synonymous substitutions and 25 non-synonymous. With the help of in silico tools (SIFT, PolyPhen, PROVEAN, PMut and EX_SKIP), 13 variants were classified as possible pathological risk variants including 4 novel ones. The most common risk variants contributing to phenotypic modulation of sarcoidosis were p.S360G and p.S334L, with the latter contributing to a more severe disease stage with extra-pulmonary manifestations such as skin granulomas and relapses being more common., Highlights • Thirteen pathological risk variants were discovered in BTNL2 gene in sarcoidosis patients. • The common risk variant p.S360G was found in cases (37.67%) and controls (30%). • The 2nd most common variant p.S334L was found in cases (16.43%) and controls (12.22%). • Six novel variants were detected with 4 deemed pathological. • Coinheritance of common and novel variants affected the final clinical phenotype.