NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

Divergent and dynamic activity of endogenous retroviruses in burn patients and their inflammatory potential

Genes constitute ~ 3% of the human genome, whereas human endogenous retroviruses (HERVs) represent ~ 8%. We examined post-burn HERV expression in patients' blood cells, and the inflammatory potentials of the burn-associated HERVs were evaluated. Buffy coat cells, collected at various time points from 11 patients, were screened for the expression of eight HERV families, and we identified their divergent expression profiles depending on patient, HERV, and time point. The population of expressed HERV sequences was patient-specific, suggesting HERVs' inherent genomic polymorphisms and/or differential expression potentials depending on characteristics of patients and courses of injury response. Some HERVs were shared among the patients, while the others were divergent. Interestingly, one burn-associated HERV gag gene from a patient's genome induced IL-6, IL-1β, Ptgs-2, and iNOS. These findings demonstrate that injury stressors initiate divergent HERV responses depending on patient, HERV, and disease course and implicate HERVs as genetic elements contributing to polymorphic injury pathophysiology.     

Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma

We previously demonstrated high expression of primary-microRNA BIC (pri-miR-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral-blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT-PCR (qRT-PCR) and of miR-155 using Northern blotting. Both BIC RNA ISH and qRT-PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein-Barr virus (EBV)-positive cases] compared to HL and normal controls. In agreement with these findings, no miR-155 was observed in BL tissues. EBV-negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV-positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.     

Unmanipulated donor lymphocytes for EBV-related PTLD after T-cell depleted HLA-haploidentical transplantation

Epstein-Barr virus (EBV)-related post-transplantation lymphoproliferative disorder (PTLD) is a life-threatening complication in patients given T-cell-depleted hematopoietic stem cell transplantation from an HLA-haploidentical relative (haplo-HSCT). We report the case of a child who developed severe EBV-related PTLD after haplo-HSCT from his mother. Despite receiving the anti-CD20 monoclonal antibody, the patient presented with intestinal obstruction due to huge abdominal lymphadenopathy, hematemesis, and nodulary pulmonary lesions. Histology showed that the lesions were due to CD20-/CD19+ large neoplastic B cells. The patient underwent double intestinal resection with partial abdominal lymphadenectomy and then received 3 monthly doses of donor-derived unmanipulated mononuclear cells. The initial dose of CD3+ cells was 3?10(5)/kg recipient body weight. The 2 additional doses consisted of 5?10(5) CD3+ cells/kg. No sign or symptom attributable to graft-versus-host disease was observed, and the patient completely cleared EBV-related lesions. The child was disease-free for 13 months after the first lymphocyte infusion. This case demonstrates that repeated infusions of controlled numbers of donor CD3+ cells cure EBV-related PTLD in haplo-HSCT without inducing graft-versus-host disease.     

Relationship between X-inactivation and clinical involvement in Fabry heterozygotes. Eleven novel mutations in the α-galactosidase A gene in the Czech and Slovak population

We have identified 21 different -galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.     

Reliability of quantitative EEG (qEEG) measures and LORETA current source density at 30 days

There is a growing interest for using quantitative EEG and LORETA current source density in clinical and research settings. Importantly, if these indices are to be employed in clinical settings then the reliability of these measures is of great concern. Neuroguide (Applied Neurosciences) is sophisticated software developed for the analyses of power, and connectivity measures of the EEG as well as LORETA current source density. To date there are relatively few data evaluating topographical EEG reliability contrasts for all 19 channels and no studies have evaluated reliability for LORETA calculations. We obtained 4 min eyes-closed and eyes-opened EEG recordings at 30-day intervals. The EEG was analyzed in Neuroguide and FFT power, coherence and phase was computed for traditional frequency bands (delta, theta, alpha and beta) and LORETA current source density was calculated in 1 Hz increments and summed for total power in eight regions of interest (ROI). In order to obtain a robust measure of reliability we utilized a random effects model with an absolute agreement definition. The results show very good reproducibility for total absolute power and coherence. Phase shows lower reliability coefficients. LORETA current source density shows very good reliability with an average 0.81 for ECB and 0.82 for EOB. Similarly, the eight regions of interest show good to very good agreement across time. Implications for future directions and use of qEEG and LORETA in clinical populations are discussed.     

Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease

Abstract

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy.

Methods

We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY.

Results

We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related – most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction.

Conclusion

Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.

     

Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS

The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon-gamma-producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7- CD27- compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1-polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.