A comparative proteomic study of sera in paediatric systemic lupus erythematosus patients and in healthy controls using MALDI-TOF-TOF and LC MS–A pilot study

BACKGROUND: Paediatric systemic lupus erythematosus (pSLE) exhibits an aggressive clinical phenotype with severe complications and overall poor prognosis. The aim of this study was to analyse differential expression of low molecular weight (LMW) serum protein molecules of pSLE patients with active disease in comparison to sera of healthy age matched controls. Further, some of the differential expressed spots were characterised and identified by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and liquid chromatography (LC-MS). METHODS: 2D-PAGE was performed using pooled sera of active pSLE and age matched healthy controls. Gels were silver-stained and differentially expressed protein spots were detected by automated image master platinum 2D software. 79 ± 17 protein spots were detected for control gels and 78 ± 17 protein spots for patient gels. Of these eleven protein spots were selected randomly and characterized by MALDI-TOF MS (five protein spots) and LC MS (six protein spots) techniques. RESULTS: Out of the 11 protein spots, 5 protein spots were significantly upregulated viz., leiomodin 2 (LMOD2); epidermal cytokeratin 2; immunoglobulin kappa light chain variable region; keratin 1 and transthyretin (TTR). Three protein spots were significantly down regulated e.g., apolipoprotein A1 (APOA1); chain B human complement component C3c; campath antibody antigen complex. Two protein spots (complement component C3; retinol binding protein (RBP) were found to be expressed only in disease and one protein spot cyclohydrolase 2 was only expressed in controls. CONCLUSIONS: We conclude that 2-D maps of patients with active pSLE and controls differ significantly. In this pilot study, using proteomic approach we have identified differential expressed proteins (of LMW) e.g., RBP, LMOD 2, TTR, Component C3c Chain B and APO A1. However, in future, further studies need to confirm the physiological and pathological role of these proteins in similar cohorts of pSLE

Investigation of the causal etiology in a patient with T-B+NK+ immunodeficiency

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A, suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants

A comparative proteomic study of sera in paediatric systemic lupus erythematosus patients and in healthy controls using MALDI-TOF-TOF and LC MS–A pilot study

Abstract Background Paediatric systemic lupus erythematosus (pSLE) exhibits an aggressive clinical phenotype with severe complications and overall poor prognosis. The aim of this study was to analyse differential expression of low molecular weight (LMW) serum protein molecules of pSLE patients with active disease in comparison to sera of healthy age matched controls. Further, some of the differential expressed spots were characterised and identified by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and liquid chromatography (LC-MS). Methods 2D-PAGE was performed using pooled sera of active pSLE and age matched healthy controls. Gels were silver-stained and differentially expressed protein spots were detected by automated image master platinum 2D software. 79 ± 17 protein spots were detected for control gels and 78 ± 17 protein spots for patient gels. Of these eleven protein spots were selected randomly and characterized by MALDI-TOF MS (five protein spots) and LC MS (six protein spots) techniques. Results Out of the 11 protein spots, 5 protein spots were significantly upregulated viz., leiomodin 2 (LMOD2); epidermal cytokeratin 2; immunoglobulin kappa light chain variable region; keratin 1 and transthyretin (TTR). Three protein spots were significantly down regulated e.g., apolipoprotein A1 (APOA1); chain B human complement component C3c; campath antibody antigen complex. Two protein spots (complement component C3; retinol binding protein (RBP) were found to be expressed only in disease and one protein spot cyclohydrolase 2 was only expressed in controls. Conclusions We conclude that 2-D maps of patients with active pSLE and controls differ significantly. In this pilot study, using proteomic approach we have identified differential expressed proteins (of LMW) e.g., RBP, LMOD 2, TTR, Component C3c Chain B and APO A1. However, in future, further studies need to confirm the physiological and pathological role of these proteins in similar cohorts of pSLE.</p

Newborn screening for SCID identifies patients with ataxia telangiectasia.

PURPOSE: Severe combined immunodeficiency (SCID) is characterized by failure of T lymphocyte development and absent or very low T cell receptor excision circles (TRECs), DNA byproducts of T cell maturation. Newborn screening for TRECs to identify SCID is now performed in several states using PCR of DNA from universally collected dried blood spots (DBS). In addition to infants with typical SCID, TREC screening identifies infants with T lymphocytopenia who appear healthy and in whom a SCID diagnosis cannot be confirmed. Deep sequencing was employed to find causes of T lymphocytopenia in such infants. METHODS: Whole exome sequencing and analysis were performed in infants and their parents. Upon finding deleterious mutations in the ataxia telangiectasia mutated (ATM) gene, we confirmed the diagnosis of ataxia telangiectasia (AT) in two infants and then tested archival newborn DBS of additional AT patients for TREC copy number. RESULTS: Exome sequencing and analysis led to 2 unsuspected gene diagnoses of AT. Of 13 older AT patients for whom newborn DBS had been stored, 7 samples tested positive for SCID under the criteria of Californias newborn screening program. AT children with low neonatal TRECs had low CD4 T cell counts subsequently detected (R = 0.64). CONCLUSIONS: T lymphocytopenia in newborns can be a feature of AT, as revealed by TREC screening and exome sequencing. Although there is no current cure for the progressive neurological impairment of AT, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers

Newborn screening for SCID identifies patients with ataxia telangiectasia.

Severe combined immunodeficiency (SCID) is characterized by failure of T lymphocyte development and absent or very low T cell receptor excision circles (TRECs), DNA byproducts of T cell maturation. Newborn screening for TRECs to identify SCID is now performed in several states using PCR of DNA from universally collected dried blood spots (DBS). In addition to infants with typical SCID, TREC screening identifies infants with T lymphocytopenia who appear healthy and in whom a SCID diagnosis cannot be confirmed. Deep sequencing was employed to find causes of T lymphocytopenia in such infants.Whole exome sequencing and analysis were performed in infants and their parents. Upon finding deleterious mutations in the ataxia telangiectasia mutated (ATM) gene, we confirmed the diagnosis of ataxia telangiectasia (AT) in two infants and then tested archival newborn DBS of additional AT patients for TREC copy number.Exome sequencing and analysis led to 2 unsuspected gene diagnoses of AT. Of 13 older AT patients for whom newborn DBS had been stored, 7 samples tested positive for SCID under the criteria of California's newborn screening program. AT children with low neonatal TRECs had low CD4 T cell counts subsequently detected (R = 0.64).T lymphocytopenia in newborns can be a feature of AT, as revealed by TREC screening and exome sequencing. Although there is no current cure for the progressive neurological impairment of AT, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers

Newborn screening for SCID identifies patients with ataxia telangiectasia.

Severe combined immunodeficiency (SCID) is characterized by failure of T lymphocyte development and absent or very low T cell receptor excision circles (TRECs), DNA byproducts of T cell maturation. Newborn screening for TRECs to identify SCID is now performed in several states using PCR of DNA from universally collected dried blood spots (DBS). In addition to infants with typical SCID, TREC screening identifies infants with T lymphocytopenia who appear healthy and in whom a SCID diagnosis cannot be confirmed. Deep sequencing was employed to find causes of T lymphocytopenia in such infants.Whole exome sequencing and analysis were performed in infants and their parents. Upon finding deleterious mutations in the ataxia telangiectasia mutated (ATM) gene, we confirmed the diagnosis of ataxia telangiectasia (AT) in two infants and then tested archival newborn DBS of additional AT patients for TREC copy number.Exome sequencing and analysis led to 2 unsuspected gene diagnoses of AT. Of 13 older AT patients for whom newborn DBS had been stored, 7 samples tested positive for SCID under the criteria of California's newborn screening program. AT children with low neonatal TRECs had low CD4 T cell counts subsequently detected (R = 0.64).T lymphocytopenia in newborns can be a feature of AT, as revealed by TREC screening and exome sequencing. Although there is no current cure for the progressive neurological impairment of AT, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers

Sequencing of a central nervous system tumor demonstrates cancer transmission in an organ transplant

Four organ transplant recipients from an organ donor diagnosed with anaplastic pleomorphic xanthoastrocytoma developed fatal malignancies for which the origin could not be confirmed by standard methods. We identified the somatic mutational profiles of the neoplasms using next-generation sequencing technologies and tracked the relationship between the different samples. The data were consistent with the presence of an aggressive clonal entity in the donor and the subsequent proliferation of descendent tumors in each recipient. Deleterious mutations in,,,, and, and a chromosomal deletion spanning thegenes, were shared between the recipients' lesions. In addition to demonstrating that DNA sequencing tracked a donor/recipient cancer transmission, this study established that the genetic profile of a donor tumor and its potential aggressive phenotype could have been determined before transplantation was considered. As the genetic correlates of tumor invasion and metastases become better known, adding genetic profiling by DNA sequencing to the data considered for transplant safety should be considered