The Italian National External Quality Assessment Program in Cytogenetics: 4 years of activity (2013-2016) following the introduction of poor performance criteria

Background. Italian External Quality Assessment (IEQA) Program in Cytogenetics, established in 2001 by the Istituto Superiore di Sanità (ISS), covers both Constitutional and Oncohaematological diagnosis. In 2013, performance criteria were defined and adopted. In this paper, we present the data from the first 4 years of activity (2013-2016) following the introduction of performance criteria. Methods. The enrollment is voluntary, fee-based and open to both public and private Italian laboratories. The scheme is annual and retrospective; a national panel of experts assess technical, analytical and interpretative performance. Results. Overall, 95 distinct Italian laboratories participated in different Cytogenetics IEQA schemes over the 2013-2016 years and most of the laboratories took part in Constitutional diagnosis. General hospitals and local health centers represented 40% of the total participants and the percentage of laboratories from Northern Regions was more than 45% of total participants throughout the 4-year period. As regards the performance evaluation, on average, 11, 9 and 23% of participants were marked as poor performers in Prenatal, Postnatal and Oncohaematological schemes, respectively. With regard to critical errors, ISCN nomenclature in Prenatal and Postnatal schemes, and interpretation in Oncohaematological diagnosis, were identified as main issues. On the other hand, karyotype errors and inadequate analysis decreased strongly, over the 4 years, in Constitutional and Oncohaematological diagnosis, respectively. Conclusions. Our data show that the introduction of poor performance encourages laboratories to address critical issues, and the IEQA participation helps to improve quality in cytogenetic testing. 

Urine-Derived Epithelial Cells as a New Model to Study Renal Metabolic Phenotypes of Patients with Glycogen Storage Disease 1a

Glycogen storage diseases (GSDs) represent a model of pathological accumulation of glycogen disease in the kidney that, in animal models, results in nephropathy due to abnormal autophagy and mitochondrial function. Patients with Glycogen Storage Disease 1a (GSD1a) accumulate glycogen in the kidneys and suffer a disease resembling diabetic nephropathy that can progress to renal failure. In this study, we addressed whether urine-derived epithelial cells (URECs) from patients with GSD1a maintain their biological features, and whether they can be used as a model to study the renal and metabolic phenotypes of this genetic condition. Studies were performed on cells extracted from urine samples of GSD1a and healthy subjects. URECs were characterized after the fourth passage by transmission electron microscopy and immunofluorescence. Reactive oxygen species (ROS), at different glucose concentrations, were measured by fluorescent staining. We cultured URECs from three patients with GSD1a and three healthy controls. At the fourth passage, URECs from GSD1a patients maintained their massive glycogen content. GSD1a and control cells showed the ciliary structures of renal tubular epithelium and the expression of epithelial (E-cadherin) and renal tubular cells (aquaporin 1 and 2) markers. Moreover, URECs from both groups responded to changes in glucose concentrations by modulating ROS levels. GSD1a cells were featured by a specific response to the low glucose stimulus, which is the condition that more resembles the metabolic derangement of patients with GSD1a. Through this study, we demonstrated that URECs might represent a promising experimental model to study the molecular mechanisms leading to renal damage in GSD1a, due to pathological glycogen storage

Caldesmon over-expression in type 1 diabetic nephropathy

Substantial evidence supports a genetic susceptibility to develop nephropathy in type 1 diabetes and a key pathogenic role of actin cytoskeleton dysfunction in this complication. We previously reported that many cytoskeletal proteins were either up- or down-regulated in fibroblast cells from type 1 diabetic (T1DM) patients with nephropathy. The gene of one of these proteins, caldesmon, lies in a chromosomal region linked to nephropathy and its promoter region contains a single nucleotide polymorphism that is associated with nephropathy. Hence, we analyzed caldesmon gene and protein expression in cultured fibroblasts from T1DM patients with and without nephropathy and from control subjects. Caldesmon gene was studied in cells cultured under normal glucose levels by quantitative real-time RT-PCR. Caldesmon protein isoforms were quantified both under normal and high glucose conditions by two-dimensional electrophoresis. Caldesmon gene was over-expressed in fibroblasts from diabetic patients with nephropathy, in comparison to both those from diabetic patients without nephropathy and those from controls. We quantified six caldesmon protein isoforms, two of them were increased whereas another one was decreased only in fibroblasts from diabetic patients with nephropathy. None of these isoforms showed any difference in their relative abundance in response to high glucose. Variable results in response to high glucose were observed in the expression of other proteins in the three experimental groups. Our data lend further support to an involvement of caldesmon in the susceptibility to diabetic nephropathy in type 1 diabetes, independently from environmental glucose levels

Ring 17 syndrome: First clinical report without intellectual disability

Ring chromosomes are rare abnormalities caused by the fusion of the telomeric regions. Three-ring chromosome syndromes (Cr 20, Cr 17 and Cr 14) cause epilepsy with variable phenotypes. In ring 17 patients with mild phenotype, some authors have shown an epilepsy syndrome similar to that of ring 20. We report the first case of a girl with ring chromosome 17 and a normal neurological and general cognitive profile. She had had, from 9 years old, focal pharmacoresistant epilepsy associated with episodes of non-convulsive status epilepticus with mainly autonomic features. Cytogenetic analysis revealed an abnormal karyotype characterised by the presence of de novo ring chromosome 17 in 19% of metaphases. The array CGH(100 KB) did not show any genetic deletion. The clinical and epilepsy phenotype was, to a certain degree, similar to that of ring 20 syndrome

De novo trisomy 20p characterized by array comparative genomic hybridization: Report of a novel case and review of the literature.

We report on a boy with speech delay, mental retardation, motor clumsiness, hyperactivity, dysmorphic facial features, brachytelephalangy and short stature. Electrocardiogram, echocardiography, renal ultrasound, electroencephalogram, fundoscopic exam and auditory brainstem responses were all normal. Brain magnetic resonance imaging showed a left temporal arachnoid cyst and a small pineal gland cyst. High resolution karyotype and FISH analysis detected a de novo duplication of the short arm of chromosome 20. A molecular characterization of the chromosomal anomaly was performed by array-CGH, confirming a 17.98 Mb duplication of the short arm of chromosome 20 associated with a small duplication on chromosome 3p, that was shown to be maternally inherited. This is one of the few cases of de novo trisomy 20p with extensive workup, characterization at molecular level and close follow-up from the neonatal period to age 30 months. We also compared the phenotype of our patient with that previously reported in literature, therefore contributing to better define the trisomy 20p syndrome and helping pediatricians and geneticists to better counsel families about the developmental prognosis of these children

Glycolytic enzyme expression and pyruvate kinase activity in cultured fibroblasts from type 1 diabetic patients with and without nephropathy.

Since type 1 diabetes mellitus (T1DM) patients with nephropathy (DN+) are insulin-resistant, we aimed to identify (new) potential molecular sites involved in the alterations of glucose metabolism in these patients. We examined the expression of glycolytic enzymes in cultured fibroblasts from T1DM(DN+) patients as compared to those from T1DM patients without nephropathy (DN-) and from controls. Pyruvate kinase (PK) activity was also determined. Human skin fibroblasts were grown in normal glucose (6 mM). RNAs and proteins were analyzed, respectively, using cRNA microarray and two-dimensional electrophoresis followed by identification with mass spectrometry. PK activity was measured using a spectrophotometric assay. As compared to controls, increases in the gene expression of hexokinase, phosphoglucomutase, phosphofructokinase, aldolase and triosephosphate isomerase were found in T1DM(DN+) patients, but not in T1DM(DN-) patients. In T1DM(DN+) patients, the protein analysis showed an altered expression of three glycolytic enzymes: triosophosphate isomerase, enolase and PK. In addition, PK activity in fibroblasts from T1DM(DN+) patients was lower than that in T1DM(DN-) and in controls. In conclusion, this study reports novel alterations of enzymes involved in glucose metabolism that may be associated with the pathophysiology of insulin resistance and of renal damage in T1DM(DN+) patients