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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.

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.

Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome) is an autosomal recessive disorder caused by the deficit of the arylsulfatase B (ARSB) enzyme, which leads to dermatan sulfate pathological storage, resulting in a wide spectrum of clinical phenotypes. To date more than 130 different mutations were reported, most of them being restricted to individual families. We here report the first study on the ARSB gene mutations in MPS VI patients of Turkish ethnogeographic origin. On the whole we analyzed 13 unrelated families recruited from 3 different Turkish clinical centers, for a total of 52 subjects, including patients, parents, and siblings. The molecular characterization of ARSB gene in these subjects lead to the identification of eight different mutations (6 missense mutations and two single-nucleotide deletions) one of which novel: c.532C>G (p.H178D). We characterized seven different genotypes, all homozygous except one. The analysis highlighted c.962T>C (p.L321P) as the most frequently detected mutation in the group of patients examined and the c.1072G>A (p.V358M) as the most frequent polymorphism. All parents and 50% of the healthy siblings analyzed carried in a heterozygous condition the mutation identified in the affected relative. The high number of homozygotes reported in this study reflects the high degree of consanguinity of the Turkish population, being the parents of most of the patients here examined, first-degree cousins. As consanguineous marriages are an integral part of the Turkish society, carriers identification accompanied by genetic counseling in families at risk is the eligible approach to minimize the effects of consanguinity in this population.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We studied eight kindreds with gyrate atrophy of choroid and retina (GA), a rare autosomal recessive disorder caused by mutations of the OAT gene, encoding the homoexameric enzyme ornithine-delta-aminotransferase. We identified four novel and five previously reported mutations. Missense alleles were expressed in yeast strain carrying a deletion of the orthologous of human OAT. All mutations markedly reduced enzymatic activity. However, the effect on the yeast growth was variable, suggesting that some mutations retain residual activity, below the threshold of the enzymatic assay. Mutant proteins were either highly unstable and rapidly degraded, or failed to assemble to form the active OAT hexamer. Where possible, fibroblast analysis confirmed these data. We found no correlation between the residual enzymatic activity and the age of onset, or the severity of symptoms. Moreover, the response to B6 was apparently not related to the specific mutations carried by patients. Overall these data suggest that other factors besides the specific OAT genotype modulate (GA) phenotype in patients. Finally, we found that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator known to increase mitochondrial biogenesis, markedly stimulates OAT expression, thus representing a possible treatment for a subset of GA patients with hypomorphic alleles., (© 2012 WILEY PERIODICALS, INC.)

It is known that the bone marrow (BM) CD133(+) cells play an important role in the hematopoietic compartment, but this is not their only role. The cells indeed can take part in vascular reconstitution when they become endothelial cells (EC), in skeletal muscle fiber regeneration when there is a switch in muscle precursors, and to cardiomyocyte phenotypic conversion when differentiating in cardiomyocytes-like cells. While the role in hematopoiesis and vasculogenesis of the selected cells is well established, their ability to differentiate along multiple non-EC lineages has not yet been fully elucidated. The goal of this study is to assert whether human CD133(+)BM-derived cells are able to differentiate in vitro, besides to blood cells, cell lineages pertinent to the mesoderm germ layers. To this end, we isolated CD133(+) cells using a clinically approved methodology and compared their differentiation potential to that of hematopoietic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) obtained from the same BM samples. In our culture conditions, CD133 expression was consistently decreased after passage 2, as well as the expression of the stemness markers c-kit and OCT4, whereas expression of Stage Specific Embryonic Antigen 4 (SSEA4) remained consistent in all different conditions. Expanded CD133 were also positive for HLA-ABC, but negative for HLA-DR, in accordance with what has been previously reported for MSCs. Moreover, CD133(+) cells from human BM demonstrated a wide range of differentiation potential, encompassing not only mesodermal but also ectodermal (neurogenic) cell lineages. CD133 antigen could be potentially used to select a cell population with similar characteristics as MSCs for therapeutic applications.

Subtelomeric rearrangements are one of the main causes of multiple congenital anomalies and mental retardation, and they are detected in 5% of patients. We report on a 6.5-year-old boy with mental retardation, dysmorphic features, and behavioral problems, who revealed 1q44-qter trisomy and 22q13.3-qter monosomy due to a maternal cryptic translocation t(1;22). We compared the clinical and cytogenetic data of our patient with those of another case presenting a pure 22qter monosomy and with those of all 1qter trisomy cases reported in the international literature. To the best of our knowledge, the subterminal 1q trisomy found in the present case has been reported in only 12 patients to date (including five familial cases). This report aims to contribute to our understanding of 1q44-qter trisomy.

Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, beta-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4(+), CD8(+) T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability.

Purpose: We evaluated the experiences of 19 Italian laboratories concerning 241 small supernumerary marker chromosomes (sSMCs) with the aim of answering questions arising from their origin from any chromosome, their variable size and genetic content, and their impact on the carrier's phenotype., Methods: Conventional protocols were used to set up the cultures and chromosome preparations. Both commercial and homemade probes were used for the fluorescent in situ hybridization analyses., Results: A total of 113 of the 241 sSMCs were detected antenatally, and 128 were detected postnatally. There were 52 inherited and 172 de novo cases. Abnormal phenotype was present in 137 cases (57%), 38 of which were antenatally diagnosed. A mosaic condition was observed in 87 cases (36%). In terms of morphology, monocentric and dicentric bisatellited marker chromosomes were the most common, followed by monocentric rings and short-arm isochromosomes. The chromosomes generating the sSMCs were acrocentric in 132 cases (69%) and non-acrocentric chromosomes in 60 cases (31%); a neocentromere was hypothesized in three cases involving chromosomes 6, 8, and 15., Conclusion: The presented and published data still do not allow any definite conclusions to be drawn concerning karyotype-phenotype correlations. Only concerted efforts to characterize molecularly the sSMCs associated or not with a clinical phenotype can yield results suitable for addressing karyotype-phenotype correlations in support of genetic counseling.

Alveolar rhabdomyosarcoma (ARMS) is associated with the specific chromosomal translocation (2;13)(q35;q14) or its rarer variant t(1;13)(p36;q14), which produces the fusion gene PAX7-FKHR. Here we describe the human cell line RC2, derived from an ARMS, which harbors a cryptic t(1;13)(p36;q14) and concomitantly shows amplification of the PAX7-FKHR fusion gene and of the MYCN oncogene. The t(1;13) and MYCN oncogene were studied by standard cytogenetic analysis and molecular techniques. The reverse transcriptase polymerase chain reaction demonstrated the expression of PAX7-FKHR mRNA in RC2 cells, although karyotype analysis failed to demonstrate a t(1;13)(p36;q14) chromosomal translocation or a derivative 13 chromosome. Double minute chromosomes were detected in all the metaphases studied. Fluorescence in situ hybridization analysis revealed multiple copies of the PAX7-FKHR fusion gene localized exclusively on a subset of double minutes, whereas multiple copies of MYCN were identified on other double minute chromosomes. Southern-blot analysis demonstrated that RC2 cells contain approximately 20 copies of the MYCN oncogene. So far no continuous RMS cell line carrying the t(1;13)(p36;q14) has been described, and PAX7-FKHR and MYCN amplifications have always been reported to occur separately in rhabdomyosarcoma (RMS). The availability of an ARMS cell line that harbors the t(1;13)(p36;q14) constitutes a useful tool for further understanding the role of the PAX7-FKHR fusion gene in RMS oncogenesis and may improve knowledge of the possible relation between PAX7-FKHR and MYCN amplification.

The authors report on cytogenetic results of six patients with hypomelanosis of Ito. Karyotypes from peripheral lymphocytes prometaphases and skin fibroblasts metaphases were normal. A review of the literature revealed no specific chromosomal abnormality but a close association between mosaicism and HI syndrome. The X-chromosome was involved in 53% of the abnormal cases.

We report a case of nuchal cystic hygroma with spontaneous resolution detected by ultrasound examination at 13 weeks' gestation. Fetal karyotype and amniotic fluid alpha-fetoprotein levels were normal. Extreme caution in evaluating this situation is stressed.

A structured pre-planned form was used to collect data on age distribution and morbidity of Down Syndrome (DS). Survival rates are also reported. The incidence of autoimmune disease among DS individuals was higher than in the normal population. Alopecia (7%), atopic dermatitis (35%), and autoimmune thyroiditis were the most frequently reported conditions, but diabetes and ulcerative colitis were also observed.

A study of three new cases with different trisomies involving chromosome 9 and a review of about 100 cases of partial trisomy 9 reported in the literature, suggested some cytogenetical and clinical correlations and lead us to propose the nomenclature of Rethore's syndrome type 1 and type 2.

Proximal extra material in the long arm of chromosome 15, has been described in individuals with different phenotypes (isolated mental retardation, multiple malformations, repeated miscarriages), and with apparently normal phenotype, in which cytogenetic analysis was invariably carried out on the basis of clinical indications. The paper describes a child with mental retardation, and his father, who both had proximal extra material in 15q. Caution is advised in the study of karyotype-phenotype correlation.

Cytogenetic investigation was carried out on 231 female patients referred for suspected sex chromosome abnormality. Cases were classified into five groups according to reason for referral and chromosome abnormality frequency was estimated. The overall frequency of abnormal karyotypes was 38.5%. The rate of positive identification of chromosome abnormality ranges from 0 in patients with secondary amenorrhoea to 80% in those with Turner phenotype. Our data demonstrate that the indications for referral of female patients with suspected sex chromosome abnormality are not only primary amenorrhoea alone or short stature and primary amenorrhoea without Turner stigmata, but also short stature of unknown etiology without any additional anomaly during childhood.