Adriano Angioni

Anatomic patterns of conotruncal defects associated with deletion 22q11

Purpose: Patients with cardiovascular malformations (CVMs) and deletion 22q11 from our series were studied in order to (1) analyze the association with dysmorphic features and noncardiac anomalies, (2) identify specific cardiac patterns and the distinctive association with additional CVMs.Methods: From 1993 to 2000, 931 patients with CVM (95 with a clinical diagnosis of DiGeorge/velocardiofacial syndrome (DG/VCFS), 208 with different genetic syndromes, 628 without dysmorphic features) underwent accurate cardiac assessment, clinical and phenotypical examination, and screening for deletion 22q11 by fluorescence in situ hybridization (FISH).Results: Deletion 22q11 was detected in 88 of the total patients, and in 87 of the 95 patients with a clinical diagnosis of DG/VCFS. Only one patient among the 628 without dysmorphic features had deletion 22q11. Conotruncal heart defects were the most common CVMs, often presenting in association with additional anomalies in four areas of the cardiovascular system: (1) the aortic arch can be right sided, cervical, double, and the subclavian artery can be aberrant, (2) the pulmonary arteries can present discontinuity, diffuse hypoplasia, discrete stenosis, defect of arborization and major aortopulmonary collateral arteries (MAPCA), (3) the infundibular septum can be malaligned, hypoplastic, or absent, (4) the semilunar valves can be bicuspid, severely dysplastic, insufficient, or stenotic.Conclusion: In subjects with deletion 22q11 CVM is virtually always associated with one or more noncardiac anomalies. Deletion 22q11 is exceptionally rare in children with nonsyndromic CVMs. Specific patterns of CVMs are observed in patients with deletion 22q11, including (1) anomalies of the aortic arch, (2) anomalies of the pulmonary arteries and of the pulmonary blood supply, (3) defects of the infundibular septum, (4) malformations of the semilunar valves. These additional CVMs may influence the surgical treatment of these patients.

Constitutional trisomy 8 as first mutation in multistep carcinogenesis: Clinical, cytogenetic, and molecular data on three cases

Three patients, with constitutional trisomy 8 mosaicism (CT8M), who developed a malignancy are reported. The diagnoses were refractory anaemia, acute lymphoblastic leukaemia, and idiopathic myelofibrosis. In the child with acute leukaemia, the CT8M was diagnosed at birth due to severe dysmorphisms and malformations; the other two patients showed a milder phenotype, and the CT8M was diagnosed only after the finding of trisomy 8 in neoplastic cells. The review of eight similar, previously reported cases and the clinical, cytogenetic, and molecular studies performed in our patients led us to make the following observations: (1) CT8M predisposes to neoplasms, preferentially to myelo‐ or lymphoproliferative diseases; (2) a gene dosage effect for glutathione reductase in red blood cells was seen in two of our patients; (3) the wide phenotypic variation of CT8M was confirmed: trisomy 8 in neoplastic cells of phenotypically near‐normal cases may be misinterpreted as acquired; and (4) molecular studies suggested a postzygotic origin of the trisomy in our three cases, with the supernumerary chromosome being of paternal origin in one case and of maternal origin in the other two. We postulate that the trisomy 8 in neoplasms may often occur by mitotic nondisjunction in an early embryonic multipotent cell and that what is usually interpreted as an acquired trisomy 8 may in fact be CT8M. The constitutional trisomy 8 would act as a pathogenetically important first mutation in multistep carcinogenesis. Whenever trisomy 8 is found in malignancies, the patient should be reevaluated clinically to exclude CT8M, and CT8M patients should be monitored for the possible development of malignancies. Genes Chromosom Cancer 17:94–101 (1996).

The etiology of acute recurrent pancreatitis in children: A challenge for pediatricians

Objectives: To assess specific etiologies of acute recurrent pancreatitis at a single Italian pediatric cystic fibrosis (CF) center. Methods: We studied, retrospectively, 78 young patients (39 female subjects; mean age at diagnosis, 8.8 ± 5.1 years) affected by acute recurrent episodes of pancreatitis, remained etiologically undiagnosed at first-level assessment. All patients were submitted to endoscopic retrograde cholangiopancreatography to exclude biliopancreatic malformations and tested for CF by a sweat chloride test. Most patients also were studied for the research of CFTR, PRSS1, and SPINK1 gene mutations. Results: A high percentage of family history for chronic pancreatitis was observed (20.5%). The sweat test identified 8 subjects (10.3%) with classic CF (2 patients) or at risk for CF (6 patients). Genetic analysis showed mutations in CFTR, SPINK1, and PRSS1 genes in 39.6%, 7.1%, and 4.5% of patients, respectively. A biliopancreatic malformation was diagnosed in 15 patients (19.2%). We also observed biliary lithiasis (5 patients [6.5%]), congenital pancreatic polycystosis (2 patients), a case of dyslipidemia, and 1 patient with a posttransplantation, drug-induced pancreatitis. Conclusions: Recurrent pancreatitis in children has several etiologies. Genetic testing confirms the high frequency of CFTR mutations. This suggests that it is of some value to identify patients with late-onset CF and CFTR-related disorders.

Diagnosis of Noonan syndrome and related disorders using target next generation sequencing

BackgroundNoonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies.MethodsIn this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL.ResultsAll expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing.ConclusionHere we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.

Telomere shortening and telomere position effect in mild ring 17 syndrome

BackgroundRing chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations.ResultsThe results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family.ConclusionsSubtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

Kabuki syndrome: clinical and molecular diagnosis in the first year of life

Objective To review the clinical and molecular genetic characteristics of 16 patients presenting a suspected diagnosis of Kabuki syndrome (KS) in the first year of life, to evaluate the clinical handles leading to a prompt diagnosis of KS in newborns. Clinical diagnosis of KS can be challenging during the first year of life, as many diagnostic features become evident only in subsequent years. Methods All patients were clinically investigated by trained clinical geneticists. A literature review was performed using the Pubmed online database and diagnostic criteria suggested by DYSCERNE–Kabuki Syndrome Guidelines (2010) were used (a European Network of Centres of Expertise for Dysmorphology, funded by the European Commission Executive Agency for Health and Consumers (DG Sanco), Project 2006122). Molecular analysis of the known causative genes of KS, KMT2D/MLL2 and KDM6A, was performed through MiSeq-targeted sequencing platform. All mutations identified were validated by Sanger sequencing protocols. Results Mutations in KMT2D gene were identified in 10/16 (62%) of the patients, whereas none of the patients had KDM6A mutations. Facial dysmorphisms (94%), feeding difficulties (100%) and hypotonia (100%) suggested the clinical diagnosis of KS. No significative differences in terms of facial features were noticed between mutation positive and negative patients of the cohort. Brachydactyly, joint laxity and nail dysplasia were present in about 80% of the patients. Other congenital anomalies were most commonly present in the mutated group of patients, including left-sided cardiac abnormalities, skeletal, renal and anorectal malformations and hypertricosis. Conclusions We present an overview of patients with KS diagnosed during the first year of life. Early diagnosis is serviceable in terms of clinical management and for targeted genetic counselling.

CIZ gene rearrangements in acute leukemia: report of a diagnostic FISH assay and clinical features of nine patients

CIZ gene rearrangements in acute leukemia: report of a diagnostic FISH assay and clinical features of nine patients

Biological, functional and genetic characterization of bone marrow-derived mesenchymal stromal cells from pediatric patients affected by acute lymphoblastic leukemia

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p<0.001) and ability to support in vitro hematopoiesis (p = 0.04) as compared with HD-MSCs, while they had similar capacity to inhibit in vitro mitogen-induced T-cell proliferation (p = N.S.). ALL-MSCs showed neither the typical translocations carried by the leukemic clone (when present), nor other genetic abnormalities acquired during ex vivo culture. Our findings indicate that ALL-MSCs display reduced ability to proliferate and to support long-term hematopoiesis in vitro. ALL-MSCs isolated at diagnosis do not differ from those obtained during treatment.

Syndromic non-compaction of the left ventricle: Associated chromosomal anomalies

Non‐compaction of the left ventricle (NCLV) is a cardiomyopathy characterized by prominent left ventricular trabeculae and deep intertrabecular recesses. Associated extracardiac anomalies occur in 14–66% of patients of different series, while chromosomal anomalies were reported in sporadic cases. We investigated the prevalence of chromosomal imbalances in 25 syndromic patients with NCLV, using standard cytogenetic, subtelomeric fluorescent in situ hybridization, and array‐comparative genomic hybridization (CGH) analyses. Standard chromosome analysis disclosed an abnormality in three (12%) patients, including a 45,X/46,XX mosaic, a 45,X/46,X,i(Y)(p11) mosaic, and a de novo Robertsonian 13;14 translocation in a child affected by hypomelanosis of Ito. Cryptic chromosome anomalies were found in six (24%) cases, including 1p36 deletion in two patients, 7p14.3p14.1 deletion, 18p subtelomeric deletion, 22q11.2 deletion associated with velo‐cardio‐facial syndrome, and distal 22q11.2 deletion, each in one case. These results recommend accurate clinical evaluation of patients with NCLV, and suggest that chromosome anomalies occur in about one third of syndromic NCLV individuals, without metabolic/neuromuscular disorder. Array‐CGH analysis should be included in the diagnostic protocol of these patients, because different submicroscopic imbalances are causally associated with this disorder and can pinpoint candidate genes for this cardiomyopathy.

Interstitial Insertion of AF10 into the ALL1 Gene in a Case of Infant Acute Lymphoblastic Leukemia

The ALL1 gene at 11q23 is a promiscuous gene participating in chromosomal abnormalities of acute leukemias with 1 of over 30 potential partner genes. Among these, the AF10 gene at band 10p12 has been recently cloned and characterized. Acute leukemias with the ALL1/AF10 chimeric gene frequently show heterogeneity in the breakpoints on 10p, as well as complex insertion (10;11) as a result of complex molecular mechanisms leading to the ALL1/AF10 fusion. In this context, we report the first description of an infant acute lymphoblastic leukemia with an interstitial insertion of the AF10 gene into the 11q23 band, resulting in the transcription of the ALL1/AF10 fusion product. Furthermore, we show how different diagnostic tools such as molecular, cytogenetic, and fluorescence in situ hybridization (FISH) analyses should be combined to resolve complex situations in the 11q23 setting.