Chimerism Monitoring by Short Tandem Repeat (STR) Markers in Allogeneic Stem Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell (allo-HSC) transplantation is used in the treatment of malignant hematological diseases. An important tool in monitoring post-transplantation evolution is represented by the percentage of donor's blood cells found in recipient's blood, known as chimersim. This is useful in predicting the graft rejection and the risk of disease relapse. In this study, we present the importance of multiplex STR markers in chimerism monitoring of a 8 year old girl diagnosed with acute lymphoblastic leukemia (ALL). METHODS: In the pre-transplant stage, saliva on buccal swabs and blood samples in EDTA were collected from the donor and recipient and used as reference samples. The DNA extraction from saliva and blood samples was done using the Pure Link Genomic DNA kit (Invitrogen, USA). For the DNA quantification, the Quantifiler Human DNA kit (Applied Biosystems, USA) was used on an ABI 7500 Real-time PCR system (Applied Biosystems, USA). Amplification of the STR markers was performed using the AmpFLSTR NGM SElect kit (Applied Biosystems, USA) on a ProFlex PCR System. The PCR products were separated and detected on an ABI 3500 Genetic Analyzer (Applied Biosytems, USA). RESULTS: One month post-transplantation of HSC, a mixed chimerism (MC) containing 38% of donor's cells was obtained from a bone marrow aspiration sample. On the 45th day, a new transplantation was performed. On the 15th day after 2nd transplantation, a MC with 91% donor's cells was obtained. On the 21st day after the 2nd transplantation, a complete chimerism (CC) with 100% donor's cells was obtained. CONCLUSIONS: Chimerism monitoring is useful in identifying those patients in risk for relapse or graft rejection.

Inflammatory gene expression profiles in Crohn's disease and ulcerative colitis: a comparative analysis using a reverse transcriptase multiplex ligation-dependent probe amplification protocol.

BACKGROUND AND AIMS: Cytokines and their receptors play a critical role in the pathogenesis of the inflammatory bowel disease (IBD). The aim of this study was to investigate the expression profiles of inflammatory genes in inflamed and non-inflamed colonic tissue samples in patients with Crohn's disease (CD) and ulcerative colitis (UC), and to identify molecular signatures for different IBD phenotypes. METHODS: Seventy-one patients diagnosed with IBD (38 CD, 33 UC) and 15 non-IBD controls have been included in the study. For each patient, biopsy samples were obtained during colonoscopy from inflamed (L) and healthy (N) mucosa. We investigated by commercially available reverse-transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) kit the mRNA expression of a set of 40 genes involved in inflammation: cytokines, chemokines, receptors, signal transduction molecules and transcription factors. RESULTS: In L biopsies from patients with CD, higher expression levels were found for IL-4 (p=0.009) and IL-12p35 (p=0.0005), whereas in L biopsy samples from patients with UC higher expression levels were found for IL-8 (p=0.03), chemokines SCYA3 (p=0.05), SCYA4 (p=0.01) and glutathione S-transferase P1 (p=0.01). In N biopsies of patients with CD higher expression levels were found for IL-1R (p=0.01) and IL-12p35 (p=0.007), whereas in N biopsies of patients with UC higher expression levels were found for IL-15 (p=0.009) and SCYA8 (p=0.001). The logistic regression analysis has indicated that low expression levels of IL-2 and IL-10, together with higher ASCA IgG titers were independently associated with penetrating/stricturing CD. CONCLUSIONS: RT-MLPA is a sensitive and effective method for the evaluation of the profiles of inflammatory genes in IBD, with potential future applications for diagnosis, phenotypic stratification and targeted therapy.

Spontaneous recurrent mutations and a complex rearrangement in the MECP2 gene in the light of current models of mutagenesis.

Mutations in the methyl-CpG-binding protein 2 (MECP2) gene are associated with Rett syndrome (RTT). The MECP2 gene has some unique characteristics: (1) it is mainly affected by de novo mutations, due to recurrent independent mutational events in a defined "hot spot" regions or positions; (2) complex mutational events along a single allele are frequently found in this gene; (3) most mutations arise on paternal X chromosome. The recurrent point mutations involve mainly CpG dinucleotides, where C>T transitions are explained by methylation-mediated deamination. The complex mutational events might be explained by the genomic architecture of the region involving the MECP2 gene. The finding that most spontaneous mutations arise on paternal X-chromosome supports the higher contribution of replication-mediated mechanism of mutagenesis. We present 9 types of mutations in the MECP2 gene, detected in a group of 22 Bulgarian and 6 Romanian classical RTT patients. Thirteen patients were clarified on molecular level (46.4%). The point mutations in our sample account for 61.5%. One intraexonic deletion was detected in the present study (7.7%). One novel insertion c.321_322insGAAG, p.(Lys107_Leu108insGluAlafs2*) was found (7.7%). Large deletions and complex mutations account for 23%. A novel complex mutational event c.[584_624del41insTT; 638delTinsCA] was detected in a Romanian patient. We discuss different types of the MECP2 mutations detected in our sample in the light of the possible mechanisms of mutagenesis. Complex gene rearrangements involving a combination of deletions and insertions have always been most difficult to detect, to specify precisely and hence to explain in terms of their underlying mutational mechanisms.

The electrocardiographic abnormalities in highly trained athletes compared to the genetic study related to causes of unexpected sudden cardiac death.

BACKGROUND: Electrocardiograms in elite endurance athletes sometimes show bizarre patterns suggestive of inherited channelopathies (Brugada syndrome, long QTc, catecholaminergic polymorphic ventricular tachycardia) and cardiomyopathies (arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy) responsible for unexpected sudden cardiac death. Among other methods, genetic analyses are required for correct diagnosis. OBJECTIVE: To correlate 12-lead electrocardiographic patterns suggestive of inherited channelopathies and cardiomyopathies to specific genetic analyses. DESIGN: Prospective study (2004-2007) of screening 12-lead ECG tracings in standard position and higher intercostal spaces V1 to V3 precordial leads, performed in athletes and normal sedentary subjects aged match. Genetic analyses of subjects with ECG abnormalities suggested inherited channelopathies and cardiomyopathies. SETTING: All cardiologic exams and electrocardiograms were performed at "Prof. Dr. C.C. Iliescu" National Institute of Cardiovascular Diseases (Bucharest, Romania). The genetic studies were done at "Mina Minovici" National Institute of Forensic Medicine (Bucharest, Romania). PARTICIPANTS: 347 elite endurance athletes (seniors--190, juniors--157), mean age of 20; 200 subjects mean age of 21, belonging to the control group of 505 normal sedentary population. RESULTS: Seniors. RSR' (V1 to V3) pattern, in 45 cases (23.68%), 5 of them with questionable Brugada sign (elevated J wave and "coved" ST segment, < 2 mm in one lead, V1. Typically, Brugada 1 sign was found in one case (0.52%) with no SCN5A abnormalities. One athlete (0.52%) had normal ECG and exon1 SCN5A duplication. MRI confirmed three arrhythmic right ventricular cardiomypathy epsilon waves (1.57%), in one case. ST-segment elevation myocardial injury like in V1-V3 precordial leads in 34 athletes (17.89%). Genetic analyses-no gene mutations. Juniors. Upright J wave was found in 43 cases (27.38%). Convex ST segment elevation in V1-V3/V4, in 39 cases (24.84%). Bifid T wave with two distinct peaks was found in 39 cases (24.84%), 5 of them with mild prolonged QTc (0.48"-0.56") and KCN genes mutations. Nine (5.73%) of the elevated ST segment juniors had questionable Brugada sign, two of which with KCN (n=1) and SCN5A (n=1) gene mutations. Ajmaline provocative test was negative in 4 and was refused by 5 subjects. CONCLUSION: Bizarre QRS, ST-T patterns suggestive of abnormal impulse conduction in the right ventricle, including the right outflow tract, associated with prolonged QTc interval in some cases were observed in highly trained endurance athletes. The genetic analyses, negative in most athletes, identified surprising mutations in SCN5A and KCN genes in some cases.

[MLPA technique--principles and use in practice]. / Tehnica MLPA. Principii si utilitate practica.

MLPA (Multiplex Ligation-dependent Probe Amplification) is a recently introduced method, based on PCR principle, useful for the detection of different genetic abnormalities (aneuploidies, gene deletions/duplications, subtelomeric rearrangements, methylation status etc). The technique is simple, reliable and cheap. We present this method to discuss its importance for a modern genetic service and to underline its multiple advantages.

Allele frequencies of 13 short tandem repeat (STR) loci in the Romanian population.

A population study on 13 tetra- and pentameric STR loci (D3S1358, VWA, D8S1179, D21S11, D18S51, D16S539, D2S1338, D19S433, THO1, FGA, ACTBP2 (SE33), Penta D and Penta E) was performed with Romanians from the Bucharest area.

Y-chromosomal STR haplotypes in a Romanian population sample.

A set of seven STR-loci mapping on the male-specific region of the human Y chromosome (DYS19, DYS385, DYS389 I/II, DYS390, DYS391, DYS392, DYS393) were typed by means of two multiplex PCR reactions and capillary electrophoresis in a Romanian population sample of 104 unrelated males. Among the 97 different haplotypes observed, 92 were unique, while 5 occurred more than once. The observed haplotype diversity was 0.989.