Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Metabolomics Profiling of Cystic Renal Disease towards Biomarker Discovery.

Cystic renal disease (CRD) comprises a heterogeneous group of genetic and acquired disorders. The cystic lesions are detected through imaging, either incidentally or after symptoms develop, due to an underlying disease process. In this study, we aim to study the metabolomic profiles of CRD patients for potential disease-specific biomarkers using unlabeled and labeled metabolomics using low and high-resolution mass spectrometry (MS), respectively. Dried-blood spot (DBS) and serum samples, collected from CRD patients and healthy controls, were analyzed using the unlabeled and labeled method. The metabolomics profiles for both sets of samples and groups were collected, and their data were processed using the lab's standard protocol. The univariate analysis showed (FDR p < 0.05 and fold change 2) was significant to show a group of potential biomarkers for CRD discovery, including uridine diphosphate, cystine-5-diphosphate, and morpholine. Several pathways were involved in CRD patients based on the metabolic profile, including aminoacyl-tRNA biosynthesis, purine and pyrimidine, glutathione, TCA cycle, and some amino acid metabolism (alanine, aspartate and glutamate, arginine and tryptophan), which have the most impact. In conclusion, early CRD detection and treatment is possible using a metabolomics approach that targets alanine, aspartate, and glutamate pathway metabolites.

Molecular classification of blood and bleeding disorder genes.

The advances and development of sequencing techniques and data analysis resulted in a pool of informative genetic data, that can be analyzed for informing decision making in designing national screening, prevention programs, and molecular diagnostic tests. The accumulation of molecular data from different populations widen the scope of utilization of this information. Bleeding disorders are a heterogeneous group of clinically overlapping disorders. We analyzed the targeted sequencing data from ~1285 Saudi individuals in 17 blood and bleeding disorders genes, to determine the frequency of mutations and variants. We used a replication set of ~5000 local exomes to validate pathogenicity and determine allele frequencies. We identified a total of 821 variants, of these 98 were listed in HGMD as disease related variants and 140 were novel variants. The majority of variants were present in VWF, followed by F5, F8, and G6PD genes, while FGG, FGB, and HBA1 had the lowest number of variants. Our analysis generated a priority list of genes, mutations and novel variants. This data will have an impact on informing decisions for screening and prevention programs and in management of vulnerable patients admitted to emergency, surgery, or interventions with bleeding side effects.

A novel claudin-10 mutation with a unique mechanism in two unrelated families with HELIX syndrome.

HELIX syndrome, characterized by hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia due to claudin-10 (CLDN10) mutations, was recognized in 2017. Here we describe two unrelated Saudi families with this syndrome due to a novel CLDN10 mutation with a unique mechanism of CLDN10 inactivation. The two consanguineous families include 12 affected individuals (three siblings in family 1 and nine members in family 2). They presented with hypokalemia and the above-mentioned features of HELIX syndrome. The underlying mutation was detected by whole exome sequencing, confirmed by Sanger sequencing and functionally indicated by RT-PCR, electrophysiological studies and immunohistochemical staining of transfected HEK293 and MDCK C7 cells, and skin and kidney biopsy tissues. A novel biallelic single nucleotide deletion was identified in exon 5 of CLDN10 (NM_182848.3: c.647delC, p.P216Lfs∗19 for CLDN10a or NM_006984.4: c.653delC, p.P218Lfs∗21 for CLDN10b). The mutation led to frameshift and extension of the original termination codon by nine amino acids with loss of the C-terminus pdz-binding motif. Functional studies showed mRNA degradation and protein retention in intracellular compartments and that the pdz-binding motif is crucial for proper localization of claudin-10 in tight junctions. In the kidney, claudin-10 was replaced by translocation of claudin-2 (proximal tubule) and claudin-19 (thick ascending limb), and in the sweat gland by claudin-3 and occludin. However, these claudins did not functionally compensate for loss of claudin-10. Thus, this novel CLDN10 mutation identified in these two families disrupted the C-terminus pdz-binding motif of claudin-10 causing HELIX syndrome.

Role of gene therapy in Fanconi anemia: A systematic and literature review with future directions.

Gene therapy (GT) has been reported to improve bone marrow function in individuals with Fanconi anemia (FA); however, its clinical application is still in the initial stages. We conducted this systematic review, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, to assess the long-term safety and clinical outcomes of GT in FA patients. Electronic searches from PubMed, Web of Science, Cochrane Library, and Google Scholar were conducted and full texts of articles meeting our inclusion criteria were reviewed. Three clinical trials were included, with a total of nine patients and mean age of 10.7 ±â€¯5.7 years. All patients had lentiviral-mediated GT. A 1-year follow-up showed stabilization in blood lineages, without any serious adverse effects from GT. A metaregression analysis could not be conducted, as very little long-term follow-up data of patients was observed, and the median survival rate could not be calculated. Thus, we can conclude that GT seems to be a safe procedure in FA; however, further research needs to be conducted on the longitudinal clinical effects of GT in FA, for a better insight into its potential to become a standard form of treatment.

Distinctive metabolic profiles between Cystic Fibrosis mutational subclasses and lung function.

INTRODUCTION: Cystic fibrosis (CF) is a lethal multisystemic disease of a monogenic origin with numerous mutations. Functional defects in the cystic fibrosis transmembrane conductance receptor (CFTR) protein based on these mutations are categorised into distinct classes having different clinical presentations and disease severity. OBJECTIVES: The present study aimed to create a comprehensive metabolomic profile of altered metabolites in patients with CF, among different classes and in relation to lung function. METHODS: A chemical isotope labeling liquid chromatography-mass spectrometry metabolomics was used to study the serum metabolic profiles of young and adult CF (n = 39) patients and healthy controls (n = 30). Comparisons were made at three levels, CF vs. controls, among mutational classes of CF, between CF class III and IV, and correlated the lung function findings. RESULTS: A distinctive metabolic profile was observed in the three analyses. 78, 20, and 13 significantly differentially dysregulated metabolites were identified in the patients with CF, among the different classes and between class III and IV, respectively. The significantly identified metabolites included amino acids, di-, and tri-peptides, glutathione, glutamine, glutamate, and arginine metabolism. The top significant metabolites include 1-Aminopropan-2-ol, ophthalmate, serotonin, cystathionine, and gamma-glutamylglutamic acid. Lung function represented by an above-average FEV1% level was associated with decreased glutamic acid and increased guanosine levels. CONCLUSION: Metabolomic profiling identified alterations in different amino acids and dipeptides, involved in regulating glutathione metabolism. Two metabolites, 3,4-dihydroxymandelate-3-O-sulfate and 5-Aminopentanoic acid, were identified in common between the three anlayses and may represent as highly sensitive biomarkers for CF.

Lipidome Alterations Induced by Cystic Fibrosis, CFTR Mutation, and Lung Function.

Cystic fibrosis is a genetic pathology characterized by abnormal accumulation of mucus in the respiratory, gastrointestinal, and reproductive tracts, caused by mutations in the CFTR gene. Although the classical presentation of the condition is well known, there is still a need for a better characterization of metabolic alterations related to cystic fibrosis and different genotypic mutations. We employed untargeted, comprehensive lipidomics of blood serum samples to investigate alterations in the lipid metabolism related to the pathology, mutation classes, and lung function decline. Six unique biomarker candidates were able to independently differentiate diseased individuals from healthy controls with excellent performance. Cystic fibrosis patients showed dyslipidemia for most lipid subclasses, with significantly elevated odd-chain and polyunsaturated fatty acyl lipids. Phosphatidic acids and diacylglycerols were particularly affected by different genotypic mutation classes. We selected a biomarker panel composed of four lipids, including two ceramides, one sphingomyelin, and one fatty acid, which correctly classified all validation samples from classes III and IV. A biomarker panel of five oxidized lipids was further selected to differentiate patients with reduced lung function, measured as predicted FEV1%. Our results indicate that cystic fibrosis is deeply related to lipid metabolism and provide new clues for the investigation of the disease mechanisms and therapeutic targets.

Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis.

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

Comprehensive Genomic Analysis of Noonan Syndrome and Acute Myeloid Leukemia in Adults: A Review and Future Directions.

Acute myeloid leukemia (AML) in the setting of Noonan syndrome (NS) has been reported before without clear guidelines for treatment or prognosis in these subgroups of patients, most likely due to its rarity and incomplete understanding of the pathogenesis of both diseases. In the current era of next-generation sequencing-based genomic analysis, we can better identify patients with NS with more accurate AML-related prognostic markers. Germline mutations in PTPN11 are the most common cause of NS. Somatic mutations in NPM1 occur frequently in AML. Here, we describe a young adult patient with a novel combined germline PTPN11 and somatic NPM1, IDH1,and BCL6 mutations who presented with fatal AML. In addition, a 50.5-Mb interstitial deletion of 7q21.11-q33 in tumor DNA was detected by chromosomal microarray analysis. While mutations in the transcriptional repressor BCL6 are known to contribute to the pathogenesis of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), its novel identification in this patient suggests an expanded role in aggressive AML. The identification of key molecular aberrations including the overexpression of SHP2, which drives leukemogenesis and tumorigenesis, has led to the development of novel investigational targeted SHP2 inhibitors.

Dried Blood Spot-Based Metabolomic Profiling in Adults with Cystic Fibrosis.

Mucoviscidosis of the respiratory, gastrointestinal, and genitourinary tracts is the major pathology in patients with cystic fibrosis (CF), a lethal monogenic panethnic and multisystemic disease most commonly identified in Caucasians. Currently, the measurement of immuno reactive trypsinogen in dry blood spots (DBSs) is the gold-standard method for initial newborn screening for CF, followed by targeted CF transmembrane regulator (CFTR) mutation analysis, and ultimate confirmation with abnormally elevated sweat chloride. Previous metabolomics studies in patients with CF reported on different biomarkers such as breath 2-aminoacetophenone produced during acute and chronic infection in human tissues, including the lungs of CF patients. Herein, we used liquid and gas chromatography-mass spectrometry-based targeted metabolomics profiling to identify potentially reliable, sensitive, and specific biomarkers in DBSs collected from 69 young and adult people including CF patients (n = 39) and healthy control (n = 30). A distinctive metabolic profile including 26 significantly differentially expressed metabolites involving amino acids, glycolysis, mitochondrial and peroxisomal metabolism, and sorbitol pathways was identified. Specifically, the osmolyte (sorbitol) was remarkably downregulated in CF patients compared to healthy controls indicating perturbation in the sorbitol pathway, which may be responsible for the mucoviscidosis seen in patients with CF. The significance of our findings is supported by the clinical utility of inhaled mannitol and hypertonic saline in patients with CF. The systemic administration of sorbitol in such patients may confer additional benefits beyond the respiratory system, especially in those with misfolded CFTR proteins.

Outcome of hematopoietic stem cell transplantation (HCT) from HLA-matched related donor for Fanconi anemia (FA) in adolescents and adults: a retrospective study by Eastern Mediterranean Blood and Marrow Transplantation Group (EMBMT).

Hematopoietic Stem Cell Transplantation (HSCT) is the only potentially curative treatment option for the hematologic complications that occur in patients with Fanconi anemia (FA). In this study, we present a retrospective multicenter analysis from the Eastern Mediterranean Blood and Marrow Transplantation Group (EMBMT) of matched related donor HSCT for FA in adolescents and adults transplanted between 1988 and 2015. Forty-five patients received HSCT with a median age at transplant of 18 years, the interquartile range (IQR) (15-23.5); 25 (55.6%) patients were females and 20 (44.4%) were males. Conditioning regimen was fludarabine-based in 29 (64.4%) patients, irradiation-based in five (11.1%) patients, and the remaining patients received other combinations. Indication for HSCT was bone marrow failure in 39 (86.7%) and myelodysplastic syndrome in six (13.3%) patients. Stem cell source was bone marrow in 22 (48.9%), peripheral blood in 20 (44.4%), umbilical cord blood in one (2.2%), and combination of bone marrow and cord blood in two (4.4%) patients. Twenty-seven (60%) patients engrafted and five (11.1%) had primary engraftment failure. The median time to neutrophil engraftment was 14 days (range 10-21 days); median time for platelet engraftment was 17 days (10-33 days). The probability of developing grade II-IV acute GVHD for all patients was 7.0% and chronic GVHD 36.6%. No new malignancies were reported. The OS probability was 53.6% (95% CI, 38.3-68.9%) with a median follow-up of 13 months (95% CI, 1-240). Our HLA-matched related HSCT results in AYA patients with FA compare favorably with other reported international registry data.

Hypomorphic variants in AK2 reveal the contribution of mitochondrial function to B-cell activation.

BACKGROUND: The gene AK2 encodes the phosphotransferase adenylate kinase 2 (AK2). Human variants in AK2 cause reticular dysgenesis, a severe combined immunodeficiency with agranulocytosis, lymphopenia, and sensorineural deafness that requires hematopoietic stem cell transplantation for survival. OBJECTIVE: We investigated the mechanisms underlying recurrent sinopulmonary infections and hypogammaglobulinemia in 15 patients, ranging from 3 to 34 years of age, from 9 kindreds. Only 2 patients, both of whom had mildly impaired T-cell proliferation, each had a single clinically significant opportunistic infection. METHODS: Patient cells were studied with next-generation DNA sequencing, tandem mass spectrometry, and assays of lymphocyte and mitochondrial function. RESULTS: We identified 2 different homozygous variants in AK2. AK2G100S and AK2A182D permit residual protein expression, enzymatic activity, and normal numbers of neutrophils and lymphocytes. All but 1 patient had intact hearing. The patients' B cells had severely impaired proliferation and in vitro immunoglobulin secretion. With activation, the patients' B cells exhibited defective mitochondrial respiration and impaired regulation of mitochondrial membrane potential and quality. Although activated T cells from the patients with opportunistic infections demonstrated impaired mitochondrial function, the mitochondrial quality in T cells was preserved. Consistent with the capacity of activated T cells to utilize nonmitochondrial metabolism, these findings revealed a less strict cellular dependence of T-cell function on AK2 activity. Chemical inhibition of ATP synthesis in control T and B cells similarly demonstrated the greater dependency of B cells on mitochondrial function. CONCLUSIONS: Our patients demonstrate the in vivo sequelae of the cell-specific requirements for the functions of AK2 and mitochondria, particularly in B-cell activation and antibody production.

Metabolomics Distinguishes DOCK8 Deficiency from Atopic Dermatitis: Towards a Biomarker Discovery.

Bi-allelic mutations in the dedicator of cytokinesis 8 (DOCK8) are responsible for a rare autosomal recessive primary combined immunodeficiency syndrome, characterized by atopic dermatitis, elevated serum Immunoglobulin E (IgE) levels, recurrent severe cutaneous viral infections, autoimmunity, and predisposition to malignancy. The molecular link between DOCK8 deficiency and atopic skin inflammation remains unknown. Severe atopic dermatitis (AD) and DOCK8 deficiency share some clinical symptoms, including eczema, eosinophilia, and increased serum IgE levels. Increased serum IgE levels are characteristic of, but not specific to allergic diseases. Herein, we aimed to study the metabolomic profiles of DOCK8-deficient and AD patients for potential disease-specific biomarkers using chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS). Serum samples were collected from DOCK8-deficient (n = 10) and AD (n = 9) patients. Metabolomics profiling using CIL LC-MS was performed on patient samples and compared to unrelated healthy controls (n = 33). Seven metabolites were positively identified, distinguishing DOCK8-deficient from AD patients. Aspartic acid and 3-hydroxyanthranillic acid (3HAA, a tryptophan degradation pathway intermediate) were up-regulated in DOCK8 deficiency, whereas hypotaurine, leucyl-phenylalanine, glycyl-phenylalanine, and guanosine were down-regulated. Hypotaurine, 3-hydroxyanthranillic acid, and glycyl-phenyalanine were identified as potential biomarkers specific to DOCK8 deficiency. Aspartate availability has been recently implicated as a limiting metabolite for tumour growth and 3HAA; furthermore, other tryptophan metabolism pathway-related molecules have been considered as potential novel targets for cancer therapy. Taken together, perturbations in tryptophan degradation and increased availability of aspartate suggest a link of DOCK8 deficiency to oncogenesis. Additionally, perturbations in taurine and dipeptides metabolism suggest altered antixidation and cell signaling states in DOCK8 deficiency. Further studies examining the mechanisms underlying these observations are necessary.

Mutational profile and genotype/phenotype correlation of non-familial pheochromocytoma and paraganglioma.

About 30%-40% of patients with pheochromocytoma (PCC) and paraganglioma (PGL) have underlying germline mutations in certain susceptibility genes despite absent family history of these tumors. Here, we present mutational profile of 101 such patients with PCC/PGL (PPGL) from the highly consanguineous population of Saudi Arabia. Results: Of 101 cases with PPGL, 37/101 (36.6%) had germline mutations. Mutations were detected in 30 cases by PCR and direct Sanger sequencing and in 7 additional cases by NGS. The most commonly mutated gene was SDHB (21/101 cases, 20.8%) and the most common SDHB mutation was c.268C>T, p.R90X occurring in 12/21 (57%) cases. Mutations also occurred in SDHC (4/101, 3.96%), SDHD (3/101, 3%), VHL (2/101, 2%) and MAX (2/101, 2%) genes. The following genes were mutated in 1 patient each (1%), RET, SDHA, SDHAF2, TMEM127 and NF1. Metastatic PPGL occurred in 6/21 cases (28.6%) with SDHB mutations and in 1 case with SDHAF2 mutation. Patients and Methods: DNA was isolated from peripheral blood (53 patients) or from non-tumorous formalin fixed paraffin embedded (FFPE) tissue (48 patients). PCR and direct Sanger sequencing of RET, SDHx, VHL, MAX and TMEM127 genes were performed. Cases without mutations were subjected to whole exome sequencing using next generation sequencing (NGS). Conclusion: About 37% of PPGL without family history of such tumors harbor germline mutations. The most commonly mutated gene is SDHB followed by SDHC, SDHD, VHL, MAX and rarely RET, SDHA, SDHAF2, TMEM127 and NF1. SDHB mutations were associated with metastatic PPGL in more than a quarter of cases.

Proteomic Analysis of Morphologically Changed Tissues after Prolonged Dexamethasone Treatment.

Prolonged dexamethasone (Dex) administration leads to serious adverse and decrease brain and heart size, muscular atrophy, hemorrhagic liver, and presence of kidney cysts. Herein, we used an untargeted proteomic approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous identification of changes in proteomes of the major organs in Sprague-Dawley (SD rats post Dex treatment. The comparative and quantitative proteomic analysis of the brain, heart, muscle, liver, and kidney tissues revealed differential expression of proteins (n = 190, 193, 39, 230, and 53, respectively) between Dex-treated and control rats. Functional network analysis using ingenuity pathway analysis (IPA revealed significant differences in regulation of metabolic pathways within the morphologically changed organs that related to: (i) brain-cell morphology, nervous system development, and function and neurological disease; (ii) heart-cellular development, cellular function and maintenance, connective tissue development and function; (iii) skeletal muscle-nucleic acid metabolism, and small molecule biochemical pathways; (iv) liver-lipid metabolism, small molecular biochemistry, and nucleic acid metabolism; and (v) kidney-drug metabolism, organism injury and abnormalities, and renal damage. Our study provides a comprehensive description of the organ-specific proteomic profilesand differentially altered biochemical pathways, after prolonged Dex treatement to understand the molecular basis for development of side effects.

Metabolomics toward personalized medicine.

Metabolomics, which is the metabolites profiling in biological matrices, is a key tool for biomarker discovery and personalized medicine and has great potential to elucidate the ultimate product of the genomic processes. Over the last decade, metabolomics studies have identified several relevant biomarkers involved in complex clinical phenotypes using diverse biological systems. Most diseases result in signature metabolic profiles that reflect the sums of external and internal cellular activities. Metabolomics has a major role in clinical practice as it represents >95% of the workload in clinical laboratories worldwide. Many of these metabolites require different analytical platforms, such as Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), and Ultra Performance Liquid Chromatography (UPLC), while many clinically relevant metabolites are still not routinely amenable to detection using currently available assays. Combining metabolomics with genomics, transcriptomics, and proteomics studies will result in a significantly improved understanding of the disease mechanisms and the pathophysiology of the target clinical phenotype. This comprehensive approach will represent a major step forward toward providing precision medical care, in which individual is accounted for variability in genes, environment, and personal lifestyle. In this review, we compare and evaluate the metabolomics strategies and studies that focus on the discovery of biomarkers that have "personalized" diagnostic, prognostic, and therapeutic value, validated for monitoring disease progression and responses to various management regimens.

Quantitative profiling of cytokines and chemokines in DOCK8-deficient and atopic dermatitis patients.

BACKGROUND: Hyper-IgE syndromes (HIES) are a clinically overlapping, heterogeneous group of inborn errors of immunity characterized by elevated serum IgE level, eosinophilia, atopy, and immune dysregulation. Deficiency of DOCK8 protein is potentially a life-threatening autosomal recessive HIES and only curable with bone marrow transplantation. Hence, the diagnosis of DOCK8 deficiency is critical and should be sought at an early stage to initiate definitive therapy. METHODS: Serum samples from patients with DOCK8 deficiency and atopic dermatitis were profiled on a cytokine/chemokine panel for potential differential expression. RESULTS: CXCL10 and TNF-A were upregulated in DOCK8 patients when compared to AD, possibly contributing toward increased susceptibility to infections and cancer. In contrast, epidermal growth factor (EGF) was significantly downregulated in a subgroup of DOCK8-deficient and AD patients, while IL-31 expression was comparable between both DOCK8-deficient and AD cohorts, possibly contributing toward pruritus seen in both groups. CONCLUSION: This comprehensive cytokine profile in HIES patients reveals distinctive biomarkers that differentiate between the DOCK8-deficient and AD patients. The unique expression profile of various inflammatory cytokines in patients with DOCK8 deficiency vs atopic dermatitis likely reflects disease-specific perturbations in multiple cellular processes and pathways leading to a predisposition to infections and allergies seen in these patients. These data agree with the role for EGF replacement therapy in EGF-deficient individuals with AD as well as DOCK8 deficiency through a potential shared pathway. In addition, these novel biomarkers may be potentially useful in distinguishing DOCK8 deficiency from AD allowing early-targeted treatment options.

Multiplexed detection of DOCK8, PGM3 and STAT3 proteins for the diagnosis of Hyper-Immunoglobulin E syndrome using gold nanoparticles-based immunosensor array platform.

Multiplexed biosensors hold great promise for early diagnosis of diseases where the detection of multiple biomarkers is required. Hyper Immunoglobulin E syndromes (HIES) are rare primary immunodeficiency disorders associated with mutations either in the signal transducer and activator of transcription 3 (STAT3), dedicator of cytokinesis 8 DOCK8) or phosphoglucomutase 3 (PGM3) genes. Yet, the diagnosis of HIES is challenged by the complexity of the existing laboratory assays. Here, we report for the first time the development of a multiplexed electrochemical immunosensor for the simultaneous detection of DOCK8, STAT3 and PGM3 proteins. The immunosensor was constructed on carbon array electrodes that were first modified by electrodeposition of gold nanoparticles (AuNPs). The array electrodes were then used to immobilize specific antibodies for the three proteins after the functionalization of the electrodes with cysteamine/glutaraldehyde linkers. The simultaneous detection of the DOCK8, PGM3 and STAT3 proteins was successfully realized by the immunosensor with respective limits of detections of 3.1, 2.2 and 3.5 pg/ml. The immunosensor has shown good sensitivity as well as selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and Duchenne Muscular Dystrophy (DMD). Moreover, the immunosensor was successfully applied in human serum samples showing capability to distinguish the HIES from the control samples.

Carbon nanofiber-based multiplexed immunosensor for the detection of survival motor neuron 1, cystic fibrosis transmembrane conductance regulator and Duchenne Muscular Dystrophy proteins.

Simultaneous and point-of-care detection of multiple protein biomarkers has significant impact on patient care. Spinal Muscular Atrophy (SMA), Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well known progressive hereditary disorders associated with increased morbidity as well as mortality. Therefore, rapid detection of biomarkers specific for these three disorders in newborns offers new opportunities for early diagnosis, delaying symptoms and effective treatment. Here, we report the development of a disposable carbon nanofiber (CNF)-based electrochemical immunosensor for simultaneous detection of survival motor neuron 1 (SMN1), cystic fibrosis transmembrane conductance regulator (CFTR) and DMD proteins. The CNF-modified array electrodes were first functionalized by electroreduction of carboxyphenyl diazonium salt. Then, the immunosensor was fabricated by the covalent immobilization of the three antibodies on the working electrodes of the array sensor via carbodiimide (EDC/NHS) chemistry. Simultaneous detection of CFTR, DMD and SMN1 was achieved with high sensitivity and detection limits of 0.9 pg/ml, 0.7 pg/ml and 0.74 pg/ml, respectively. The multiplexed immunosensor has also shown strong selectivity against non-specific proteins. Moreover, high recovery percentage was obtained when the immunosensor was applied in spiked whole blood samples. This voltammetric immunosensor offers cost effective, easy to use, rapid and high throughput potential screening method for these three hereditary disorders using only few drops of blood.