Is nuchal translucency of 3.0-3.4 mm an indication for cfDNA testing or microarray? - a multicenter retrospective clinical cohort study.

INTRODUCTION: This study aimed to evaluate the occurrence of clinically relevant (sub)microscopic chromosomal aberrations in fetuses with the NT range from 3.0 to 3.4 mm, which would be potentially missed by cfDNA testing. METHODS: A retrospective data analysis of 271 fetuses with NT between 3.0 and 3.4 mm and increased combined test (CT) risk in five cohorts of pregnant women referred for invasive testing and chromosomal microarray was performed. RESULTS: A chromosomal aberration was identified in 18.8% fetuses (1:5; 51/271). In 15% (41/271) of cases trisomy 21, 18, or 13 was found. In 0.7% (2/271) sex chromosome aneuploidy was found. In 1.1% (3/271) of cases, CNV>10Mb was detected, which would potentially also be detected by genome-wide cfDNA testing. The residual risk for missing a submicroscopic chromosome aberration in the presented cohorts is 1.8% (1:54; 5/271). CONCLUSION: Our results indicate that a significant number of fetuses with increased CT risk and presenting NT of 3.0-3.4 mm carry a clinically relevant chromosomal abnormality other than common trisomy. Invasive testing should be offered and counseling on NIPT should include the test limitations that may result in NIPT false negative results in a substantial percentage of fetuses.

Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Cardiovascular Anomalies among 1005 Fetuses Referred to Invasive Prenatal Testing-A Comprehensive Cohort Study of Associated Chromosomal Aberrations.

This retrospective cohort study comprehensively evaluates cardiovascular anomalies (CVAs) and associated extracardiac structural malformations (ECMs) among 1005 fetuses undergoing invasive prenatal testing at a single tertiary Polish center in the context of chromosomal aberrations detected in them by array comparative genomic hybridization (aCGH) and G-band karyotyping. The results of our study show that CVAs are among the most common malformations detected in fetuses undergoing invasive prenatal testing, as they affected 20% of all cases seen in our department. Septal defects predominated among fetuses with numerical aberrations, while conotruncal defects were the most common findings among fetuses with pathogenic copy number variants (CNVs). In 61% of cases, CVAs were associated with ECMs (the diagnosis was confirmed postnatally or in cases of pregnancy termination by means of autopsy). The most common ECMs were anomalies of the face and neck, followed by skeletal defects. In total, pathogenic chromosomal aberrations were found in 47.5% of CVAs cases, including 38.6% with numerical chromosomal aberrations. Pathogenic CNVs accounted for 14.5% of cases with CVAs and normal karyotype. Thus, our study highlights the importance of assessing the anatomy of the fetus, and of the genetic testing (preferably aCGH) that should be offered in all CVA and ECM cases.

Paternal uniparental disomy of chromosome 2 resulting in a concurrent presentation of Crigler-Najjar syndrome type I and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

This is the first report of the concurrent development of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and Crigler-Najjar syndrome type 1 (CNs1) inherited via uniparental disomy of chromosome 2, which are both autosomal recessive pathologies. Through an expanded newborn metabolic panel, a male infant was identified as having an acylcarnitine pattern typical for LCHADD, later confirmed to be caused by a well-characterized pathogenic variant in the HADHA gene located at 2p23. Prolonged non-hematologic jaundice requiring repetitive phototherapy prompted further genetic analysis, leading to the identification of another genetic abnormality consistent with CNs1, which was caused by a novel pathogenic variant in the UGT1A1 gene located at 2q37. The two identified point mutations in chromosome 2 were homozygous and present on separate arms, which indicated potential uniparental disomy. Microarray analysis of the genetic material from the patient and his parents confirmed paternal isodisomy of chromosome 2. Further studies are needed to identify other possible pathogenic variants located on the same defective chromosome, evaluate the combined effect of the two metabolic abnormalities, and plan the best possible treatment and care.

The rs113883650 variant of SLC7A5 (LAT1) gene may alter brain phenylalanine content in PKU.

Functional alteration of the LAT1 amino acid transporter may be responsible for interindividual differences in cerebral phenylalanine content and the lack of intellectual disability in some patients with untreated phenylketonuria. We assessed the effect of the common variant rs113883650 of the SLC7A5 (LAT1) gene on brain phenylalanine content, as measured with use of magnetic resonance spectroscopy. Our results suggest that the presence of this variant could influence the amount of phenylalanine in the brain.

Carriership of the rs113883650/rs2287120 haplotype of the SLC7A5 (LAT1) gene increases the risk of obesity in infants with phenylketonuria.

PURPOSE: Phenylketonuria (PKU) can be effectively treated with the use of a low-phenylalanine diet. However, some patients become overweight despite proper dietary treatment. We hypothesized that this phenomenon could be explained by the presence of specific variants within the genes involved in phenylalanine transport or in the phenylalanine transamination/oxygenation pathway. METHODS: We selected a clinically homogenous group of 100 infants with PKU and assessed their growth patterns in the context of dietary phenylalanine tolerance. Next, within the sample, we performed exome sequencing and assessed a potential relationship between the observed phenotypical variability and the presence of structural variants in a priori selected genes of interest. RESULTS: We detected a highly significant association between overweight and carriership of the rs113883650/rs2287120 haplotype of the SLC7A5 (LAT1) gene, which encodes the main transmembrane transporter of large neutral amino acids and of thyroid hormones. CONCLUSIONS: Our findings suggest a pharmacogenetic effect of the relatively common rs113883650/rs2287120 haplotype of the SLC7A5 gene. This can have practical implications for patients with PKU, since treatment protocols need to be reassessed to better prevent overweight in the carriers of the above variant.

Polymorphisms of SLC19A1 80 G>A, MTHFR 677 C>T, and Tandem TS Repeats Influence Pharmacokinetics, Acute Liver Toxicity, and Vomiting in Children With Acute Lymphoblastic Leukemia Treated With High Doses of Methotrexate.

Introduction: High dose methotrexate (HD-Mtx) is highly effective and significantly improves overall acute lymphoblastic leukemia (ALL) patients survival. The pharmacodynamics of Mtx depends on the polymorphism of genes encoding proteins engaged in the folate metabolism pathway. The aim of the current study is to determine the relationship between variants of folate metabolism-related genes and the frequency of acute toxicities of HD-Mtx. Material and Methods: A group of 133 patients aged 1.5-18.1 years (median: 6.3) was treated in accordance with the ALL-IC-2002 and ALL-IC-2009 protocols. The following polymorphisms were determined: 80 G>A SLC19A1 (solute carrier family 19 member 1; rs1051266) with direct DNA sequencing, as well as 677 C>T MTHFR (methylenetetrahydrofolate reductase; rs1801133) and the tandem repeats of the TS (thymidylate synthase) with PCR technique. HD-Mtx organ toxicities were evaluated based on the laboratory tests results and the National Cancer Institute criteria. Results: In patients with genotypes AA for SLC19A1 and CC or CT for MTHFR Mtx steady state concentrations (Css) and AUCinf were distinctly higher. In patients with genotype 3R/3R for TS initial elimination rate constant was significantly higher (P = 0.003). Patients receiving Mtx at the dose of 5 g/m2 had lower clearance (4.35 vs. 8.92 L/h/m2) as compared to the ones receiving 2 g/m2 that indicates non-linear Mtx elimination at the higher dose. Liver impairment was the most frequently observed toxicity. The homozygous genotype was associated with a significantly higher incidence of hepatic toxicity for both the SLC19A1 (P = 0.037) and TS (P = 0.002). Logistic regression analysis indicated an increased risk of vomiting for the 2R/3R genotype of the TS gene (OR 3.20, 95% CI 1.33-7.68, P = 0.009) and for vomiting and hepatic toxicity for the 3R/3R genotype (vomiting: OR 3.39, 95% CI 1.12-10.23, P = 0.031; liver toxicity: OR 2.28, 95% CI 1.05-4.95, P = 0.038). None of the acute toxicities differed between the analyzed dosing groups. Conclusions: Determination of polymorphisms of SLC19A1, MTHFR, and TS genes might allow for a better prior selection of patients with higher risk of elevated Mtx levels. Our study is the first one to report the increased risk of hepatotoxicity and vomiting in patients with TS polymorphisms.

Transcriptome analysis reveals dysregulation of genes involved in oxidative phosphorylation in a murine model of retinopathy of prematurity.

BACKGROUND: Retinal gene expression pattern is severely altered after exposition to hyperoxia in mice with oxygen-induced retinopathy (OIR), a common model of retinopathy of prematurity. Gene ontology and signaling pathway analyses may add new insights into a better understanding of the pathogenesis of this disease. METHODS: Seven-day-old C57BL/6J mice (n = 60) were exposed to 75% oxygen for 5 days and then recovered in room air. The controls (n = 60) were kept in the normoxic conditions. Retinas were harvested immediately following hyperoxia, during the phase of maximal neovascularization, and at the time of neovascularization regression. The retinal RNA samples were evaluated for gene expression using mouse gene expression microarrays. DAVID annotation tools were used for gene ontology and pathway analyses. RESULTS: The most significantly enriched signaling pathways during the neovascularization phase of OIR were: focal adhesion; ECM-receptor interaction; PI3K-Akt; oxidative phosphorylation; and Alzheimer's, Parkinson's and Huntington's disease signaling pathways. Genes involved in apoptosis, cell proliferation, cell differentiation, and immune responses were associated with neovascularization regression. CONCLUSIONS: Performed analyses revealed the possible involvement of various signaling pathways in OIR pathomechanism, mostly specific to the OIR phase. Dysregulation of genes involved in oxidative phosphorylation may have an impact on neovascularization development.

Pulmonary vascular disease is evident in gene regulation of experimental bronchopulmonary dysplasia.

Objective: To examine the gene expression regarding pulmonary vascular disease in experimental bronchopulmonary dysplasia in young mice. Premature delivery puts babies at risk of severe complications. Bronchopulmonary dysplasia (BPD) is a common complication of premature birth leading to lifelong affection of pulmonary function. BPD is recognized as a disease of arrested alveolar development. The disease process is not fully described and no complete cure or prevention is known. The focus of interest in the search for treatment and prevention of BPD has traditionally been at airspace level; however, the pulmonary vasculature is increasingly acknowledged in the pathology of BPD. The aim of the investigation was to study the gene expression in lungs with BPD with regards to pulmonary vascular disease (PVD).Methods: We employed a murine model of hyperoxia-induced BPD and gene expression microarray technique to determine the mRNA expression in lung tissue from young mice. We combined gene expression pathway analysis and analyzed the biological function of multiple single gene transcripts from lung homogenate to study the PVD relevant gene expression.Results: There were n = 117 significantly differentially regulated genes related to PVD through down-regulation of contractile elements, up- and down-regulation of factors involved in vascular tone and tissue-specific genes. Several genes also allowed for pinpointing gene expression differences to the pulmonary vasculature. The gene Nppa coding for a natriuretic peptide, a potent vasodilator, was significantly down-regulated and there was a significant up-regulation of Pde1a (phosphodiesterase 1A), Ptger3 (prostaglandin e receptor 3), and Ptgs1 (prostaglandin-endoperoxide synthase one).Conclusion: The pulmonary vasculature is affected by the arrest of secondary alveolarization as seen by differentially regulated genes involved in vascular tone and pulmonary vasculature suggesting BPD is not purely an airspace disease. Clues to prevention and treatment may lie in the pulmonary vascular system.

Short- and long-term impact of hyperoxia on the blood and retinal cells' transcriptome in a mouse model of oxygen-induced retinopathy.

BACKGROUND: We aimed to identify global blood and retinal gene expression patterns in murine oxygen-induced retinopathy (OIR), a common model of retinopathy of prematurity, which may allow better understanding of the pathogenesis of this severe ocular prematurity complication and identification of potential blood biomarkers. METHODS: A total of 120 C57BL/6J mice were randomly divided into an OIR group, in which 7-day-old pups were maintained in 75% oxygen for 5 days, or a control group. RNA was extracted from the whole-blood mononuclear cells and retinal cells on days 12, 17, and 28. Gene expression in the RNA samples was evaluated with mouse gene expression microarrays. RESULTS: There were 38, 1370 and 111 genes, the expression of which differed between the OIR and control retinas on days 12, 17, and 28, respectively. Gene expression in the blood mononuclear cells was significantly altered only on day 17. Deptor and Nol4 genes showed reduced expression both in the blood and retinal cells on day 17. CONCLUSION: There are sustained marked changes in the global pattern of gene expression in the OIR mice retinas. An altered expression of Deptor and Nol4 genes in the blood mononuclear cells requires further investigation as they may indicate retinal neovascularization.

Comparative two time-point proteome analysis of the plasma from preterm infants with and without bronchopulmonary dysplasia.

BACKGROUND: In this study, we aimed to analyze differences in plasma protein abundances between infants with and without bronchopulmonary dysplasia (BPD), to add new insights into a better understanding of the pathogenesis of this disease. METHODS: Cord and peripheral blood of neonates (≤ 30 weeks gestational age) was drawn at birth and at the 36th postmenstrual week (36 PMA), respectively. Blood samples were retrospectively subdivided into BPD(+) and BPD(-) groups, according to the development of BPD. RESULTS: Children with BPD were characterized by decreased afamin, gelsolin and carboxypeptidase N subunit 2 levels in cord blood, and decreased galectin-3 binding protein and hemoglobin subunit gamma-1 levels, as well as an increased serotransferrin abundance in plasma at the 36 PMA. CONCLUSIONS: BPD development is associated with the plasma proteome changes in preterm infants, adding further evidence for the possible involvement of disturbances in vitamin E availability and impaired immunological processes in the progression of prematurity pulmonary complications. Moreover, it also points to the differences in proteins related to infection resistance and maintaining an adequate level of hematocrit in infants diagnosed with BPD.

Immune System Regulation Affected by a Murine Experimental Model of Bronchopulmonary Dysplasia: Genomic and Epigenetic Findings.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common cause of abrupted lung development after preterm birth. BPD may lead to increased rehospitalization, more severe and frequent respiratory infections, and life-long reduced lung function. The gene regulation in lungs with BPD is complex, with various genetic and epigenetic factors involved. OBJECTIVES: The aim of this study was to examine the regulatory relation between gene expression and the epigenome (DNA methylation) relevant for the immune system after hyperoxia followed by a recovery period in air using a mouse model of BPD. METHODS: Newborn mice pups were subjected to an immediate hyperoxic condition from birth and kept at 85% O2 levels for 14 days followed by a 14-day period in room air. Next, mice lung tissue was used for RNA and DNA extraction with subsequent microarray-based assessment of lung transcriptome and supplementary methylome analysis. RESULTS: The immune system-related transcriptomeregulation was affected in mouse lungs after hyperoxia. A high proportion of genes relevant in the immune system exhibited significant expression alterations, e.g., B cell-specific genes central to the cytokine-cytokine receptor interaction, the PI3K-AKT, and the B cell receptor signaling pathways. The findings were accompanied by significant DNA hypermethylation observed in the PI3K-AKT pathway and immune system-relevant genes. CONCLUSIONS: Oxygen damage could be partly responsible for the increased susceptibility and abnormal response to respiratory viruses and infections seen in premature babies with BPD through dysregulated genes.

An iTRAQ-Based Quantitative Proteomic Analysis of Plasma Proteins in Preterm Newborns With Retinopathy of Prematurity.

Purpose: Retinopathy of prematurity (ROP) is a vision-threatening complication of a premature birth, in which the etiology still remains unclear. Importantly, the molecular processes that govern these effects can be investigated in a perturbed plasma proteome composition. Thus, plasma proteomics may add new insights into a better understanding of the pathogenesis of this disease. Methods: The cord and peripheral blood of neonates (≤30 weeks gestational age) was drawn at birth and at the 36th postmenstrual week (PMA), respectively. Blood samples were retrospectively subdivided into ROP(+) and ROP(-) groups, according to the development of ROP. Results: The quantitative analysis of plasma proteome at both time points revealed 30 protein abundance changes between ROP(+) and ROP(-) groups. After standardization to gestational age, children who developed ROP were characterized by an increased C3 complement component and fibrinogen level at both analyzed time points. Conclusions: Higher levels of the complement C3 component and fibrinogen, present in the cord blood and persistent to 36 PMA, may indicate a chronic low-grade systemic inflammation and hypercoagulable state that may play a role in the development of ROP.

Hyperoxia induces epigenetic changes in newborn mice lungs.

Supplemental oxygen exposure is a risk factor for the development of bronchopulmonary dysplasia (BPD). Reactive oxygen species may damage lung tissue, but hyperoxia also has the potential to alter genome activity via changes in DNA methylation. Understanding the epigenetic potential of hyperoxia would enable further improvement of the therapeutic strategies for BPD. Here we aimed to identify hyperoxia-related alterations in DNA methylation, which could affect the activity of crucial genetic pathways involved in the development of hyperoxic lung injury. Newborn mice (n = 24) were randomized to hyperoxia (85% O2) or normoxia groups for 14 days, followed by normoxia for the subsequent 14 days. The mice were sacrificed on day 28, and lung tissue was analyzed using microarrays developed for the assessment of genome methylation and expression profiles. The mean DNA methylation level was higher in the hyperoxia group than the normoxia group. The analysis of specific DNA fragments revealed hypermethylation of > 1000 gene promoters in the hyperoxia group, confirming the presence of the DNA-hypermethylation effect of hyperoxia. Further analysis showed significant enrichment of the TGF-ß signaling pathway (p = 0.0013). The hypermethylated genes included Tgfbr1, Crebbp, and Creb1, which play central roles in the TGF-ß signaling pathway and cell cycle regulation. Genome expression analysis revealed in the hyperoxia group complementary downregulation of genes that are crucial for cell cycle regulation (Crebbp, Smad2, and Smad3). These results suggest the involvement of the methylation of TGF-ß pathway genes in lung tissue reaction to hyperoxia. The data also suggest that hyperoxia may be a programming factor in newborn mice.

Comparison of whole genome expression profile between preterm and full-term newborns.

OBJECTIVES: Evaluate the time dependent expression of genes in preterm neonates and verify the influence of ontogenic maturation and the environmental factors on the gene expression after birth. MATERIAL AND METHODS: The study was carried out on 20 full-term newborns and 62 preterm newborns (mean birth weight = 1002 [g] (SD: 247), mean gestational age = 27.2 weeks (SD: 1.9)). Blood samples were drawn from all the study participants at birth and at the 36th week postmenstrual age from the preterm group to assess whole genome expression in umbilical cord blood and in peripheral blood leukocytes, respectively. (SurePrint G3 Human Gene Expression v3, 8x60K Microarrays (Agilent)). RESULTS: A substantial number of genes was found to be expressed differentially at the time of birth and at 36 PMA in comparison to the term babies with more genes being down-regulated than up-regulated. However, the fold change in the majority of cases was < 2.0. Extremely preterm and very preterm infants were characterized by significantly down-regulated cytokine and chemokine related pathways. The number of down-regulated genes decreased and number of up-regulated genes increased at 36 PMA vs. cord blood. There were no specific gene expression pathway profiles found within the groups of different gestational ages. CONCLUSIONS: Preterm delivery is associated with a different gene expression profile in comparison to term delivery. The gene expression profile changes with the maturity of a newborn measured by the gestational age.

Human Induced Pluripotent Stem Cell-Derived Microvesicles Transmit RNAs and Proteins to Recipient Mature Heart Cells Modulating Cell Fate and Behavior.

Microvesicles (MVs) are membrane-enclosed cytoplasmic fragments released by normal and activated cells that have been described as important mediators of cell-to-cell communication. Although the ability of human induced pluripotent stem cells (hiPSCs) to participate in tissue repair is being increasingly recognized, the use of hiPSC-derived MVs (hiPSC-MVs) in this regard remains unknown. Accordingly, we investigated the ability of hiPSC-MVs to transfer bioactive molecules including mRNA, microRNA (miRNA), and proteins to mature target cells such as cardiac mesenchymal stromal cells (cMSCs), and we next analyzed effects of hiPSC-MVs on fate and behavior of such target cells. The results show that hiPSC-MVs derived from integration-free hiPSCs cultured under serum-free and feeder-free conditions are rich in mRNA, miRNA, and proteins originated from parent cells; however, the levels of expression vary between donor cells and MVs. Importantly, we found that transfer of hiPSC components by hiPSC-MVs impacted on transcriptome and proteomic profiles of target cells as well as exerted proliferative and protective effects on cMSCs, and enhanced their cardiac and endothelial differentiation potential. hiPSC-MVs also transferred exogenous transcripts from genetically modified hiPSCs that opens new perspectives for future strategies to enhance MV content. We conclude that hiPSC-MVs are effective vehicles for transferring iPSC attributes to adult somatic cells, and hiPSC-MV-mediated horizontal transfer of RNAs and proteins to injured tissues may be used for therapeutic tissue repair. In this study, for the first time, we propose a new concept of use of hiPSCs as a source of safe acellular bioactive derivatives for tissue regeneration.

Genetic analysis of the paired box transcription factor (PAX8) gene in a cohort of Polish patients with primary congenital hypothyroidism and dysgenetic thyroid glands.

BACKGROUND: The morphological and biochemical phenotype of PAX8 mutation in patients with congenital hypothyroidism (CH) is variable. The contribution of mutations in PAX8 gene in children with CH and dysgenetic thyroid glands still remains a subject of interest for researchers. PATIENTS AND METHODS: Some 48 children (37 girls and 11 boys) with CH associated with thyroid ectopy (n=22), agenesis (n=10), hypoplasia (n=6), or thyroid dysgenesis of unknown cause (n=10) were enrolled. The study participants were born in south-eastern Poland in the years 1993-2012 and were selected for neonatal mass screening for CH. DNA was extracted from peripheral blood samples using Master Pure DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA). The 12 exons of the PAX8 gene along with their exon-intron boundaries were amplified and sequenced by the Sanger method. Capillary electrophoresis was run on ABI 3500 (Applied Biosystems, Carlsbad, CA, USA). RESULTS: Novel heterozygous transition in exon 3 (c.68G>A) was detected in a 3-year-old girl with a thyroid hypoplasia. This substitution was not identified in the patient's parents (de novo event). Additionally, a novel genetic variant in 3'UTR region of exon 12 (c.*416C>T) occurred in a 3-year-old boy with ectopic thyroid tissue and concomitant congenital urogenital malformation. This heterozygous variant was also detected in other healthy family members. Thirteen well-described single nucleotide polymorphisms were revealed in the PAX8 gene. CONCLUSIONS: The study reports on the occurrence of two novel heterozygous substitutions in the PAX8 gene. Estimation of the contribution of the revealed c.68G>A variant to the etiology of CH in a girl with hypoplastic thyroid requires further functional analysis.

Identification of deletions in children with congenital hypothyroidism and thyroid dysgenesis with the use of multiplex ligation-dependent probe amplification.

INTRODUCTION: Thyroid dysgenesis (TD) is the most common cause of congenital hypothyroidism (CH). Important genetic factors possibly contributing to TD etiologies include mutations of thyroid transcription factors and TSHR-encoding genes. OBJECTIVE: Our objective was to determine multiplex ligation-dependent probe amplification (MLPA) utility in detecting the copy number changes in patients with CH and TD. METHODS: The study included 45 children from southeastern Poland selected via already established neonatal screening for CH. Genomic DNA was extracted from peripheral blood samples and used in MLPA analysis. Genetic variations were analyzed within selected fragments of the PAX8, FOXE1, NKX2-1, thyroid stimulating hormone receptor (TSHR), and TPO genes. RESULTS: Three heterozygous deletion types in probe hybridization regions were identified for the following genes: PAX8 (exon 7), TSHR (exon 2), and FOXE1 (exon 1). Monoallelic deletions were identified in 5/45 TD subjects. CONCLUSIONS: MLPA is a useful tool for copy number changes detection and might both improve and expand genetic analysis for CH and TD.

Development and maturation of the immune system in preterm neonates: results from a whole genome expression study.

To expand the knowledge about the consecutive expression of genes involved in the immune system development in preterm neonates and to verify if the environment changes the gene expression after birth we conducted a prospective study that included three cohorts: (A) extremely (gestational age (GA): 23-26 weeks; n = 41), (B) very (GA: 27-29 weeks; n = 39), and (C) moderately preterm infants (GA: 30-32 weeks; n = 33). Blood samples were drawn from the study participants on the 5th and 28th day of life (DOL). The mRNA samples were evaluated for gene expression with the use of GeneChip Human Gene 1.0ST microarrays. Differential expression analysis revealed small subsets of genes that presented positive or negative monotone trends in both the 5th (138 genes) and 28th DOL (308 genes) in the three subgroups of patients. Based on pathway enrichment analysis, we found that most of the pathways that revealed a positive monotone trend were involved in host immunity. The most significantly GA dependent pathways were T-cell receptor signaling pathway and intestinal immune network for IgA production. Overall 4431 genes were differentially expressed between the 5th and 28th DOL. Despite differences in gestational age, patients with the same postconceptional age have a very similar expression of genes.