Emmanouel Kanavakis

Wilson disease in children: analysis of 57 cases.

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 μg/24 hours, and 3 had liver copper content >250 μg/g dry weight. Of the remaining 19 patients, 17 had both low serum ceruloplasmin ≤20 mg/dL and increased urinary copper excretion, >75 μg/24 hours before, or >1000 μg/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.

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study.

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.

The complex SNP and CNV genetic architecture of the increased risk of congenital heart defects in Down syndrome

Congenital heart defect (CHD) occurs in 40% of Down syndrome (DS) cases. While carrying three copies of chromosome 21 increases the risk for CHD, trisomy 21 itself is not sufficient to cause CHD. Thus, additional genetic variation and/or environmental factors could contribute to the CHD risk. Here we report genomic variations that in concert with trisomy 21, determine the risk for CHD in DS. This case-control GWAS includes 187 DS with CHD (AVSD = 69, ASD = 53, VSD = 65) as cases, and 151 DS without CHD as controls. Chromosome 21-specific association studies revealed rs2832616 and rs1943950 as CHD risk alleles (adjusted genotypic P-values <0.05). These signals were confirmed in a replication cohort of 92 DS-CHD cases and 80 DS-without CHD (nominal P-value 0.0022). Furthermore, CNV analyses using a customized chromosome 21 aCGH of 135K probes in 55 DS-AVSD and 53 DS-without CHD revealed three CNV regions associated with AVSD risk (FDR ≤ 0.05). Two of these regions that are located within the previously identified CHD region on chromosome 21 were further confirmed in a replication study of 49 DS-AVSD and 45 DS- without CHD (FDR ≤ 0.05). One of these CNVs maps near the RIPK4 gene, and the second includes the ZBTB21 (previously ZNF295) gene, highlighting the potential role of these genes in the pathogenesis of CHD in DS. We propose that the genetic architecture of the CHD risk of DS is complex and includes trisomy 21, and SNP and CNV variations in chromosome 21. In addition, a yet-unidentified genetic variation in the rest of the genome may contribute to this complex genetic architecture.

Cell-free DNA levels in acute myocardial infarction patients during hospitalization

Objectives — The objectives of this study were to investigate cell-free DNA daily concentration changes following an acute myocardial infarction (AMI) and to assess any correlations with complications during hospitalization. Methods and results — Serial cell-free DNA level determinations were performed by quantitative Real-Time PCR in 47 AMI patients once daily during hospitalization (235 samples) and once in 100 healthy subjects. Cell-free DNA concentrations are significantly higher in patients throughout hospitalization compared to healthy subject levels (2.644 (SE 0.0952) vs. 1.519 (SE 0.0566), P < 0.001). The median maximum cell-free DNA concentration was 3.5-fold higher (Mann Whitney P = 0.0035) in 20/47 patients with complicated post AMI course – group I – (1719.7, range 117.32-4996212.1 GenEq/ml plasma) compared with 27/47 patients without complications – group II – (492.9, range 56.43-4715.15 GenEq/ml plasma). Substantial differences exist between cell-free DNA concentrations measured on tpre (the day before the complication) and tc (the day the complication occurred) as well as tpost (the day after the complication) in group I whereby cell-free DNA rises significantly in tc and remains elevated in tpost (tpre vs. tc, 2.445 vs. 2.965, P = 0.0171 and tpre vs. tpost 2.445 vs. 2.913, P = 0.023). Conclusions — Cell-free DNA concentrations were elevated in AMI patients compared to healthy control subjects, rise significantly when complications occur and have a potential clinical value in monitoring patient progress during hospitalization.

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms

BackgroundAlthough, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy.MethodsOverall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients’ clinical characteristics and pathological measures.ResultsIn several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied.ConclusionsDeeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry’s disease (FD), steroid 21-hydroxylase deficiency (21-HD), and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations, which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases where other techniques have failed.

A study of a single variant allele (rs1426654) of the pigmentation-related gene SLC24A5 in Greek subjects.

Abstract:  The SLC24A5 gene, the human orthologue of the zebrafish golden gene, has been shown to play a key role in human pigmentation. In this study, we investigate the prevalence of the variant allele rs1426654 in a selected sample of Greek subjects. Allele‐specific polymerase chain reaction was performed in peripheral blood samples from 158 attendants of a dermatology outpatient service. The results were correlated with pigmentary traits and MC1R genotype. The vast majority of subjects (99%) were homozygous for the Thr111 allele. Only two subjects from the control group (1.26%) were heterozygous for the alanine and threonine allele. Both of these Thr111/Ala111 heterozygotes carried a single polymorphism of MC1R (one with the V92M variant and another with the V60L variant). Following reports of the rs1426654 polymorphism reaching fixation in the European population, our study of Greek subjects showed a prevalence of the Thr111 allele, even among subjects with darker skin pigmentation or phototype.

Psychomotor development of children born after preimplantation genetic diagnosis and parental stress evaluation

BackgroundThe increasing number of children conceived following preimplantation genetic diagnosis (PGD) necessitates the evaluation of their motor and cognitive development. The primary study objective was to evaluate the physical, developmental, and neurological outcome of children born after PGD in Greece. In addition, the secondary study objective was to compare the stress levels regarding parental roles between parents of PGD children and those of naturally conceived children.MethodsA cross-sectional study design was applied. The study population consisted of 31 children (aged 2 months to 7.5 years) born after PGD analysis and their parents. The developmental evaluation of children included a detailed physical evaluation and cognitive assessment with the Bayley Scales of Infant Development. The parent stress index was applied to evaluate comparative parental stress levels between those parents of PGD children and those of naturally conceived healthy children.ResultsHigh rates of caesarean deliveries, increased incidence of prematurity, multiples and low-birth weight were observed among the 31 PGD children. Overall, 24 of the 31 PGD children had cognitive skills within normal range [general developmental quotient (GDQ): 86–115], while 6 children had lower levels of cognitive skills (GDQ<85). With regard to parental stress, PGD parents reported lower levels of parenting stress as compared to parents of naturally conceived children (P<0.01).ConclusionsThe enhanced frequency of poor cognitive and motor skills as well as low parental stress necessitates early detection and intervention for developmental delays among PGD children.

Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis

Abstract Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ARTs) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of Human Endogenous Retroviruses (HERV)-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3. p57KIP2 and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57KIP2 and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57KIP2 and IGF2/H19 genes differentially methylated regions had low coverage in transposable element (TE) sequences, the respective one of DLK1/MEG3 was characterized by an almost 2-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that TEs may be affecting the establishment of genomic imprinting under stress conditions.

Cystic Fibrosis Conductance Regulator, Tumor Necrosis Factor, Interferon Alpha-10, Interferon Alpha-17, and Interferon Gamma Genotyping as Potential Risk Markers in Pulmonary Sarcoidosis Pathogenesis in Greek Patients

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.