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  • Author: Florin Tripon x
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Abstract

Small supernumerary marker chromosome (sSMC) is a rare chromosomal abnormality and is detected in about 0.3% in cases with multiple congenital anomalies (MCA) and/or developmental delay. Different techniques for investigation of cases with MCA and/or developmental delay are available ranging from karyotyping to molecular cytogenetic technique and ultimately multiplex ligation dependent probe amplification (MLPA). Here we present a patient with multiple congenital anomalies for which classical cytogenetic technique was used as a first step in diagnosis and the results being confirmed by MLPA. The karyotype disclosed a sSMC considered to be a fragment of chromosome 22. The MLPA analysis using SALSA MLPA probemix P064-C2 Microdeletion Syndromes-1B confirmed the karyotype results, and according to the manufacturer’s recommendation we performed another confirmation analysis with MLPA probemix P311-B1 Congenital Heart Disease and MLPA probemix P250-B2 DiGeorge. We also suspected an Emanuel syndrome and performed another MLPA analysis with SALSA MLPA probemix P036-E3 Subtelomeres Mix 1 and probemix P070-B3 Subtelomeres Mix 2B for investigation of subtelomeric region that revealed a duplication of 11q25 region and the confirmation was performed using SALSA MLPA probemix P286-B2 Human Telomere-11.

In conclusion, we consider that MLPA is a valuable method for identification of sSMC in children with developmental delay and congenital anomalies. Genetic diagnosis using different molecular techniques, such as MLPA, for increasing accuracy in identification of chromosomal structural aberrations has an important role in clinical diagnosis and in genetic counselling and our case explain the importance of using a specific laboratory technique for each stage of diagnosis.

Abstract

Introduction. We report one elderly patient diagnosed with a rare subtype of acute myeloid leukemia (AML) and also with a very rare fusion gene involving ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) genes.

Material and methods. Clinical examination and routine analysis were performed including peripheral blood smear, immunophenotyping of the peripheral blood by flow cytometry and several molecular analyses.

Results. Peripheral blood smear showed 80% blasts with round and some with convoluted nuclei, with basophilic cytoplasm, identified as monoblast and the majority of cells as promonocytes. Peripheral blood immunophenotyping was consistent with monocytic differentiation. Molecular analysis was negative for FLT3 ITD, FLT3 D835, NPM1, and DNMT3A R882 mutations. Multiplex ligation-dependent probe amplification revealed no copy number aberration. Ligation-dependent reverse transcription polymerase chain reaction (LD-RT-PCR) analysis identified the presence of one gene fusion between ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) genes. The patient had no significant comorbidities, the renal function was normal and Eastern Cooperative Oncology Group performance status was 2 at diagnosis and 1 after treatment. She was treated with decitabine. She became transfusion independent and a reduction of the number of blasts was obtained.

Conclusions. The outcome of our AML patient was favorable but other patients with fusion genes involving ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) should be reported, contributing to a better characterization of the disease, to monitor the minimal residual disease and in the end to more targeted treatment options. LD-RT-PCR represent a valuable multiplex technique for fusion gene analysis.

Abstract

Molecular genetic testing in craniosynostosis leads to the detection of the mutations in the genes encoding fibroblast growth factor receptors (FGFR), providing information about the etiology of the genetic disorder. Muenke syndrome is produced by p.Pro250Arg mutation in FGFR3 gene with evidence of variable expressivity, representing 8% of the syndromic craniosynostoses.

Here, we present the identification of a p.Pro250Arg pathogenic mutation (c.749C>G) in the FGFR3 gene using Multiplex Ligation-dependent Probes Amplification (MLPA) analysis in conjunction with Sanger sequencing in a patient with craniosynostosis and mild intellectual disability. The MLPA analysis detected a reduced signal of the probe, at the site of the c.749C>G mutation, defined by the presence of one allele of C749>G mutation in the FGFR3 gene, exon 7. Sanger sequencing was performed for confirmation and identified heterozygous p.Pro250Arg pathogenic variant (c.749C>G) in exon 7 of the FGFR3.

In conclusion, we assessed the validity and clinical utility of the combined molecular genetic techniques, MLPA analysis, and Sanger sequencing, for craniosynostosis and intellectual disability, improving not only the diagnostic testing but also the genetic counseling and management of the disorder.

Abstract

We assume that the CRISPR Cas9 theory must be delimited by applicability, because the consequences of long term DNA manipulation remain unknown. Moreover, the irreversibility of this procedure should instigate researchers to reserved opinions.

Usefulness as well as benefits of CRISPR Cas9 made it one of the most popular and used genome editing technique. But with its huge potential, ethical and safety concerns emerge. Therefore, before continuing research in this direction we should have a well organized system that is able to make that differentiation between research and reproduction. However we truly believe in the future of genetic engineering and with the CRISPR-Cas9 system we expect that the opportunity of treating now so called incurable diseases arises. Time is all we need.

Abstract

Background: Nowadays, cytogenetics and molecular genetics, but not only, are mandatory in acute myeloid leukemia (AML) management, as a consequence of their impact on AML pathogenesis, classification, risk-stratification, prognosis and treatment. Objective: The aim of our study was to present our algorithm for the analysis of copy number changes, aneuploidies and somatic mutations focusing on a rare AML case positive for four somatic mutations. Methods: Cytogenetic analysis, Multiplex Ligationdependent Probe Amplification (MLPA) analysis, somatic mutation analysis (for FLT3 ITD, FLT3 D835, DNMT3A R882 and NPM1 c.863_864ins) by using several PCR techniques and also next-generation sequencing (NGS) analysis were performed. Results: Cytogenetic analysis did not reveal structural or numerical chromosomal anomalies. The patient’s DNA showed no copy number changes or aberrations (CNAs) following the MLPA analysis. By using several molecular technologies we found four mutations: FLT3-ITD, FLT3 D835 (c.2504A>T, D835V), DNMT3A R882C, and NPM1 c.863_864insTCTG. Challenges, benefits, applications and the limitations of each molecular technique used for the investigation of the mentioned mutation, and not only, are also described. Conclusion: All these techniques can be useful in the diagnosis of AML patients, each of them covering the limits of the other technique. New strategies for a positive, fast, accurate and reliable diagnosis are mandatory in cases with AML.

Abstract

Objective: The aim of the current study was to investigate possible associations between catalase C262T (CAT C262T), glutathione peroxidase 1 Pro198Leu (GPX1 Pro198Leu), manganese superoxide dismutase Ala16Val (MnSOD Ala16Val) gene polymorphisms and non-Hodgkin Lymphoma risk (NHL) in a Romanian population and the five-year overall survival rate of the NHL patients.

Methods: We included in this case-control study 406 individuals, divided into two groups: the control group (n=315) and the patients group (n=91). The DNA was extracted from peripheral blood and amplified using specific techniques.

Results: The variant homozygous genotype of GPX1 Pro198Leu represents a risk factor for NHL development and no associations regarding the risk for NHL were found for MnSOD Ala16Val and CAT C262T gene polymorphisms. Two of the studied polymorphisms were associated with the overall survival rate thus: negative association regarding MnSOD Ala16Val, associated with higher overall survival rate and a positive one regarding CAT C262T, associated with lower overall survival rate.

Conclusions: According to our results, the mentioned polymorphisms may be considered as susceptible markers of the five-year overall survival rate for NHL patients. Future studies with a larger number of patients are needed to confirm our results.

Abstract

The aim of our study was to evaluate the association between variant genotype of angiotensinogen (AGT) c.-58A>C, lifestyle factors and clinical factors and corporeal extension of gastric inflammatory and preneoplastic lesions.

Methods: Our study included 209 subjects who underwent a complete set of gastric biopsies, followed by genotyping. They were included to study inflammatory gastric changes and preneoplastic lesions and were grouped according to the localization of changes.

Results: No significant statistical associations were noticed between AGT c.-58A>C genotypes and the corporeal extension of the inflammation or preneoplastic injury groups. Extending preneoplastic lesions to the gastric body was associated with smoking habits (p=0.01) and additionally, there was a significant association between nicotine consumption and the body extension of preneoplastic lesions (p=0.01). The use of acenocoumarol was frequently associated with the progression of histological lesions to preneoplastic lesions (p=0.01). Compared with the wild-type AA genotype, the combined genotypes AA+CC of AGT c.-58A>C were significantly associated with the progression of inflammatory gastric lesions’ according to the regular ingested doses of nonsteroidal anti-inflammatory drugs (NSAIDs).

Conclusion: The AGT c.-58A>C polymorphism is not associated with extension of the gastric lesions. In accordance with nicotine and alcohol consumption, the acenocoumarol co-treatment and multiple cardiac pathologies are associated with the corporeal progression of these injuries. The age below 70 years and NSAIDs treatment for the patients with heterozygous AC genotype and variant homozygous CC genotype for the mentioned SNP have been associated with the corporeal extension of gastric inflammation.

Abstract

Introduction: The widespread use of sevoflurane as an induction and maintenance volatile agent of general anesthesia demostrates an increased safety profile. Sevoflurane contact with CO2 absorbents lead to the occurrence of toxic compounds such as Compund A and Compound B. Among the side efffects of Sevoflurane remember the renal toxic effect much discussed in the literature but still unresolved. In previous research we have demonstrated the glomerular protein changes as a result of exposure to Sevoflurane. In the current study we intend to monitor the changes in blood urea nitrogen and serum creatinine after exposure to Sevoflurane.

Material and method: We included in our study 90 patients who were anesthetized in the Department of Anesthesiology of the County Mure Hospital during 01.10.2009-01.10.2014. They had normal values for blood urea nitrogen and serum creatinine and had no preoperative proteinuria. Serum and urine samples were taken preoperatively and at 24 and 72 hours postanesthetic and were analyzed in the laboratory. Proteinuria was determined by spectrophotometry.

Results: After protein quantitative determination by spectrophotometry and statistical anaysis we obtained significant differences by comparing the average preoperative/24 hours total protein (p<0.0001) and 24/72 hours (p<0.0001). There are no significant statistical differences by comparing the blood urea nitrogen at the three intervals (p<0.53) and no statistical changes for mean serum creatinine (p<0.18).

Conclusions: Changes in glomerular filtered proteins following exposure to Sevoflurane demonstrate its toxic effect on glomerular tubules. Lack of perioperative significant wich is why we recommend determining perioperative urinary protein as a marker of glomerular damage.

Abstract

Congenital inferior vena cava anomalies have a reduced frequency in general population, many times being an asymptomatic finding. Patients caring such anomalies are at risk to develop deep vein thrombosis. In this paper, we present 2 siblings with deep venous thrombosis and inferior vena cava abnormalities, with a symptomatic onset at similar age. The inferior vena cava abnormality was documented by an angio-CT in each case. The thrombophilic workup was negative. Patients were treated with conservative therapy: low molecular weight heparin anticoagulants converted later to oral anticoagulant with resolution of symptoms and disappearance of the thrombus. Finally, in the absence of any risk factor in a young patient admitted with deep vein thrombosis investigations to exclude inferior vena cava anomalies are mandatory.

Abstract

Introduction: In approximately 96% of probands, the diagnosis of Treacher Collins Syndrome (TCS) is confirmed by molecular genetic tests. These tests can detect heterozygous mutation of TCOF1 gene (coding treacle protein) and variants of POLR1D gene (coding RNA polymerase I subunit D) with autosomal dominant inheritance, or biallelic variants of POLR1C gene (coding RNA polymerase I subunit C) and POLR1D with autosomal recessive inheritance.

Case presentation: We present a neonate proband with family history of clinical features suggestive for TCS. Our patient was investigated for copy number changes (CNCs) of TCOF1 gene using SALSA MLPA P310-B3 TCOF1 probemix to perform Multiplex Ligation-dependent Probe Amplification (MLPA), the results being normal. Dysmorphic features revealed “bird-like” face with trigonocephaly, craniosynostosis, hypoplastic supraorbital rims, underdeveloped zygomas, mandibular hypoplasia and retrognathia (mandibulofacial dysostosis). Other clinical features, like abnormal position and structure of the external ears (microtia, with a bilateral low-set ears, crumpled and malformed pinnae and aural atresia), were also observed.

Conclusion: Taking into account our results, and also data found in literature, we consider that all TCS cases, but in particular patients with specific TCS features and without CNCs, require additional investigations using sequencing techniques.