Development of flow cytometry assay for Wiskott–Aldrich syndrome diagnosis by WASP protein evaluation

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microplatelet thrombocytopenia, eczema, frequent infections and an increased risk of autoimmune disorders and malignant neoplasms. Mutation detection in WAS gene is the gold standard for diagnosis of this disorder. This gene encodes a WASP protein, which works as regulator of cell cytoskeleton and is involved in the transmission of many intracellular signals. Nowadays there is no rapid and reliable method that allows to confirm WAS in a short period of time. Early detection of WAS in patients enables initiation of a donor search and preparation for the HSCT procedure. It also helps to avoid the development of severe and life-threatening conditions during waiting for genetic confirmation of the diagnosis by using pathogenetic therapy. Currently ﬂow cytometry is one of the leading laboratory methods that permits to get the information about the expression of a protein in several hours. The study below describes rapid and reliable based on flow cytometry assay for WAS diagnosis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study included 46 patients with suspected WAS from 2 months to 17 years old. Patients were examined from January 2018 to January 2020. WAS gene defect was confirmed in 35 patients. It was calculated that normal threshold value for WASP expression is 7.07 with sensitivity and specificity 100% and 93.1% respectively. Besides negative correlation between WASP expression index and WAS clinical severity was shown (r = –0.63). This flow cytometry assay can be used for chimerism detection in WAS patients after HSCT. The flow cytometry assay for WASP protein evaluation is rapid, highly sensitive and highly specific. It allows to speed up diagnosis of this disorder.

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Molecular Analysis of WASP Gene - First Report from India.

Blood ◽

10.1182/blood.v110.11.3217.3217 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3217-3217

Author(s):

Rintu Rebecca Jacob ◽

Reena Rajasekar ◽

Eunice S. Edison ◽

Biju George ◽

Vikram Mathews ◽

...

Keyword(s):

Functional Significance ◽

Molecular Basis ◽

Direct Sequencing ◽

Mutation Screening ◽

Clinical Severity ◽

Severe Thrombocytopenia ◽

Molecular Defects ◽

Splice Site Prediction ◽

Increased Risk ◽

Was Gene

Abstract Wiskott Aldrich Syndrome (WAS) is an X-linked recessive disorder associated with severe thrombocytopenia, eczema, bloody diarrhea, profound immunodeficiency and an increased risk of malignancies. More than 200 mutations have been described in the gene encoding the WAS protein (WASP). We report here the molecular basis of WAS in six patients from India. Diagnosis of WAS/X- linked thrombocytopenia (XLT) was based on low platelet count (9000– 1, 25,000/mm3), presence of small platelets (mean: 6.2fl) and detection of WASP by flowcytometry. Patients evaluated in the study could be classified as classical WAS (n=3) and XLT (n=3) depending on the clinical severity of their disease (Zhu et.al 1995). All 12 exons and flanking splice-sites of WAS gene were amplified by polymerase chain reaction (PCR) using primers and PCR conditions adopted with modifications from Kwan et al (PNAS1995;92:4706). Mutation analysis was accomplished by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Abnormal banding pattern was observed only in 4 patients after CSGE analysis. c.995 C>T could not be detected by CSGE but was detected by direct sequencing. The types of molecular defects identified in five patients were as follows: nonsense - 2, deletion - 1 and duplication - 1. Three of these alterations (see table) have been reported previously and are associated with classical WAS. In patients detected to have nonsense mutations, flowcytometric analysis of WASP exhibited high values (table) not consistent with reported western blotting data in these mutations. c.307+10-11dupC was observed in two patients (brothers) with thrombocytopenia, small platelets and mild bleeding. This duplication is not a reported polymorphism. Though this duplication changes the acceptor splice site prediction score from 0.83 to 0.91, its functional significance has not been determined and requires further evaluation. The sixth patient in our study had thrombocytopenia (15,000–45,000/mm3), small platelets (4.8–7.2 fl), mild bleeding, similar family history and normal expression of WASP (86.2%). No mutation has been identified in the coding region of WAS gene in this patient either by CSGE or direct sequencing. Our data shows that the molecular basis for WAS is heterogenous in India. This is the first study evaluating mutations in WAS gene from India. Further mutation screening of patients with WAS will allow the genetic definition, phenotype-genotype corelation, carrier detection and prenatal diagnosis of this condition. Molecular defects observed in WAS patients Patient ID Flow cytometry (%) Mutation *-Reported mutation, ♦-Functional Significance not known PD-22 WASP D1-98.5, WASPB9 - 94.8 c.665C>T, p.Arg211X* PD-16 WASP B9- 6.7 c. 1035-1036 delG, p.Gly314ValfsX120* PD-21 WASP D1-97.2, WASP B9-91.2 c.995 C>T, p. Arg321X* PD-34, PD-35 (siblings) WASP D1-3.6, WASP B9-2; D1-1.8, B9-1 c.307+10-11 dupC♦ PD-28 WASP D1 - 86.2 Mutation not detected

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ISOLATION OF A NOVEL GENE MUTATED IN WISKOTT-ALDRICH SYNDROME

PEDIATRICS ◽

10.1542/peds.96.2.411a ◽

1995 ◽

Vol 96 (2) ◽

pp. 411-411

Author(s):

Sanober Meer ◽

Andrew Liu

Keyword(s):

Genetic Locus ◽

Close Correlation ◽

Gene Replacement ◽

Specific Gene ◽

Severe Thrombocytopenia ◽

Wiskott Aldrich Syndrome ◽

Cdna Sequences ◽

Novel Gene ◽

Increased Risk ◽

Was Gene

Purpose of the Study. Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder associated with severe thrombocytopenia, eczema, bloody diarrhea, B & T cell immunodeficiency and increased risk of malignancies. This study describes the isolation of a novel gene, WAS Protein (WASP) which is mutated in four WAS patients. Methods. The WAS gene had been localized previously by linkage analyses to a >1 Megabase region in chromosome Xp11.22-p11.23. To isolate the WAS gene, a clone contig was constructed in this interval, followed by isolation of cDNA sequences. Results. A novel gene, WASP, was isolated, which is believed to be the main genetic locus in WAS. This is supported by the following findings. First, the gene was isolated from the chromosome Xp11.22-p11.23 interval previously identified as containing the WAS gene. Second, lymphoblasts from two unrelated patients clinically diagnosed with WAS failed to express WASP on Northern blots. Two additional WAS patients were also found to have point substitution mutations of an arginine residue. Finally, WASP is expressed in lymphocytic and megakaryocytic cell lineages, indicating a close correlation between tissue-specific gene expression and the disease manifestations of WAS. Reviewer's Comments. This paper succinctly describes a major breakthrough in the identification of the defective gene in WAS, which is found to be mutated in the four WAS patients studied. This finding brings direction and impetus to research on WAS, provides improved methods for detecting WAS, and brings the possibility of gene replacement therapy for WAS much closer.

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PHENOMENON OF BIMODAL EXPRESSION OF WASP IN PATIENTS WITH WISKOTT–ALDRICH SYNDROME ASSESSED BY FLOW CYTOMETRY

PEDIATRIA Journal named after G N SPERANSKY ◽

10.24110/0031-403x-2021-100-2-31-40 ◽

2021 ◽

Vol 100 (2) ◽

pp. 31-40

Author(s):

D.E. Pershin ◽

◽

E.A. Kulakovskaya ◽

A.L. Khoreva ◽

I.V. Mersiyanova ◽

...

Keyword(s):

Flow Cytometry ◽

Somatic Mutations ◽

Clinical Manifestations ◽

Lymphocyte Subpopulation ◽

Cell Populations ◽

Functional Protein ◽

Wiskott Aldrich Syndrome ◽

Male Patients ◽

Was Gene ◽

Comprehensive Study

Compensatory or revertant somatic mutations (RSM) is a well-known phenomenon in patients with PIs. RSM can lead to the restoration of functional protein expression in some cell populations and thus influence the severity of clinical manifestations in patients with PI. The WAS gene, a mutation that leads to the development of Wiskott–Aldrich syndrome (WAS), is associated with high levels of RSM. In our study, we aimed to describe in detail the RSM phenomenon in patients with WAS, and also to try to study its effect on the severity of the clinical phenotype. Materials and methods of research: a single-center, prospective, open, continuous, non-randomized, comparative study was conducted. The study included 39 male patients with WAS from 2 months and up to 18 years old with mutation in WAS gene, who underwent assessment of WASP expression using flow cytometry. In 2 out of 9 patients, use of magnetic selection made it possible to isolate a population of lymphocytes expressing WASP. Germinal and somatic mutations were identified using Sanger sequencing. To assess severity of clinical manifestations of WAS, the original detailed scoring classification was used. Results: 9 patients had bimodal expression of WASP, which equaled to 23% of all examined. The median age of these patients was 3 years [7 months. – 15 years]. In two patients a presence of RSM was confirmed in WASP-expressing populations. Linear assessment of partially restored WASP expression revealed different patterns of lymphocyte subpopulation involvement in all 9 patients. The effect of RSM on the severity of clinical manifestations of WAS in the studied group of patients was statistically insignificant (p=0,39). Conclusion: according to the data obtained, patients with WAS, demonstrating a bimodal pattern of WASP expression, represent a very heterogeneous group in terms of the severity of clinical manifestations, involvement of cell populations, and the nature of RSM. This work is a methodological prerequisite for further comprehensive study of RSM phenomenon in patients with various PIs.

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Whole Spleen Flow Cytometry Assay

BIO-PROTOCOL ◽

10.21769/bioprotoc.834 ◽

2013 ◽

Vol 3 (15) ◽

Cited By ~ 1

Author(s):

Cathy Yam ◽

Adeline Hajjar

Keyword(s):

Flow Cytometry ◽

Flow Cytometry Assay

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Risk Factors, and Clinical and Etiological Characteristics of Ischemic Strokes in COVID-19-Infected Patients: A Systematic Review of Literature

Cerebrovascular Diseases ◽

10.1159/000514267 ◽

2021 ◽

pp. 1-4

Author(s):

Simone Vidale

Keyword(s):

Risk Factors ◽

Systematic Review ◽

Clinical Features ◽

Case Reports ◽

Clinical Severity ◽

Large Artery ◽

Causal Association ◽

Review Of Literature ◽

Increased Risk ◽

Long Time

Background and Purpose: Coronavirus disease 2019 (COVID-19) infection is an ongoing pandemic and worldwide health emergency that has caused important changes in healthcare systems. Previous studies reported an increased risk of thromboembolic events, including stroke. This systematic review aims to describe the clinical features and etiological characteristics of ischemic stroke patients with COVID-19 infection. Method: A literature search was performed in principal databases for studies and case reports containing data concerning risk factors, clinical features, and etiological characteristics of patients infected with COVID-19 and suffering from stroke. Descriptive and analytical statistics were applied. Results: Overall, 14 articles were included for a total of 93 patients. Median age was 65 (IQR: 55–75) years with prevalence in males. Stroke occurred after a median of 6 days from COVID-19 infection diagnosis. Median National of Institute of Health Stroke Scale (NIHSS) score was 19. Cryptogenic (Cry) strokes were more frequent (51.8%), followed by cardioembolic etiology, and they occurred a long time after COVID-19 diagnosis compared with large-artery atherosclerosis strokes (ptrend: 0.03). The clinical severity of stroke was significantly associated with the severity grade of COVID-19 infection (ptrend: 0.03). Conclusions: Ischemic strokes in COVID-19-infected patients were clinically severe, affecting younger patients mainly with Cry and cardioembolic etiologies. Further multicenter prospective registries are needed to better describe the causal association and the effect of COVID-19 infection on stroke.

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Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽

10.1530/rep-14-0348 ◽

2015 ◽

Vol 149 (4) ◽

pp. 317-327 ◽

Cited By ~ 14

Author(s):

Martyna Łupicka ◽

Gabriel Bodek ◽

Nahum Shpigel ◽

Ehud Elnekave ◽

Anna J Korzekwa

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Mrna Expression ◽

Uterine Horn ◽

Uterine Tissue ◽

Pluripotent Cells ◽

Flow Cytometry Assay ◽

Myometrial Cells ◽

Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

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Current recommendations for clinical surveillance and genetic testing in rhabdoid tumor predisposition: a report from the SIOPE Host Genome Working Group

Familial Cancer ◽

10.1007/s10689-021-00229-1 ◽

2021 ◽

Author(s):

M. C. Frühwald ◽

K. Nemes ◽

H. Boztug ◽

M. C. A. Cornips ◽

D. G. Evans ◽

...

Keyword(s):

Working Group ◽

Soft Part ◽

Genetic Disorders ◽

Panel Discussion ◽

Rhabdoid Tumor ◽

Host Genome ◽

Cancer Surveillance ◽

Malignant Neoplasms ◽

Pathogenic Variants ◽

Increased Risk

AbstractThe rhabdoid tumor (RT) predisposition syndromes 1 and 2 (RTPS1 and 2) are rare genetic conditions rendering young children vulnerable to an increased risk of RT, malignant neoplasms affecting the kidney, miscellaneous soft-part tissues, the liver and the central nervous system (Atypical Teratoid Rhabdoid Tumors, ATRT). Both, RTPS1&2 are due to pathogenic variants (PV) in genes encoding constituents of the BAF chromatin remodeling complex, i.e. SMARCB1 (RTPS1) and SMARCA4 (RTPS2). In contrast to other genetic disorders related to PVs in SMARCB1 and SMARCA4 such as Coffin-Siris Syndrome, RTPS1&2 are characterized by a predominance of truncating PVs, terminating transcription thus explaining a specific cancer risk. The penetrance of RTPS1 early in life is high and associated with a poor survival. However, few unaffected carriers may be encountered. Beyond RT, the tumor spectrum may be larger than initially suspected, and cancer surveillance offered to unaffected carriers (siblings or parents) and long-term survivors of RT is still a matter of discussion. RTPS2 exposes female carriers to an ill-defined risk of small cell carcinoma of the ovaries, hypercalcemic type (SCCOHT), which may appear in prepubertal females. RT surveillance protocols for these rare families have not been established. To address unresolved issues in the care of individuals with RTPS and to propose appropriate surveillance guidelines in childhood, the SIOPe Host Genome working group invited pediatric oncologists and geneticists to contribute to an expert meeting. The current manuscript summarizes conclusions of the panel discussion, including consented statements as well as non-evidence-based proposals for validation in the future.

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X-linked thrombocytopenia caused by a mutation in the Wiskott-Aldrich syndrome (WAS) gene that disrupts interaction with the WAS protein (WASP)-interacting protein (WIP)

Experimental Hematology ◽

10.1016/s0301-472x(02)01023-8 ◽

2003 ◽

Vol 31 (2) ◽

pp. 150-158 ◽

Cited By ~ 23

Author(s):

Jennifer N Luthi ◽

Manish J Gandhi ◽

Jonathan G Drachman

Keyword(s):

Interacting Protein ◽

Wiskott Aldrich Syndrome ◽

Was Gene

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Occupational class inequalities in disability retirement after hospitalisation

Scandinavian Journal of Public Health ◽

10.1177/1403494817726618 ◽

2017 ◽

Vol 46 (3) ◽

pp. 331-339 ◽

Cited By ~ 4

Author(s):

Olli Pietiläinen ◽

Mikko Laaksonen ◽

Eero Lahelma ◽

Aino Salonsalmi ◽

Ossi Rahkonen

Keyword(s):

Cardiovascular Diseases ◽

Mental Disorders ◽

Confidence Interval ◽

Musculoskeletal Disorders ◽

Hazard Ratio ◽

Malignant Neoplasms ◽

Occupational Class ◽

Disability Retirement ◽

Increased Risk ◽

Professional Class

Aims: This study aimed to investigate whether hospitalisation is associated with increased risk of disability retirement differently across four occupational classes. Methods: 170,510 employees of the City of Helsinki, Finland were followed from 1990 to 2013 using national registers for hospitalisations and disability retirement. Increases in the risk of disability retirement after hospitalisation for any cause, cardiovascular diseases, musculoskeletal disorders, mental disorders, malignant neoplasms, respiratory diseases and injuries were assessed across four occupational classes: professional, semi-professional, routine non-manual and manual, using competing risks models. Results: In general, hospitalisation showed a slightly more increased risk of disability retirement in the lower ranking occupational classes. Hospitalisation among women for mental disorders showed a more increased risk in the professional class (hazard ratio 14.73, 95% confidence interval 12.67 to 17.12) compared to the routine manual class (hazard ratio 7.27, 95% confidence interval 6.60 to 8.02). Occupational class differences were similar for men and women. The risk of disability retirement among women increased most in the routine non-manual class after hospitalisation for musculoskeletal disorders and injuries, and most in the professional class after hospitalisation for cardiovascular diseases. The corresponding risks among men increased most in the two lowest ranking classes after hospitalisation for injuries. Conclusions: Ill-health as measured by hospitalisation affected disability retirement in four occupational classes differently, and the effects also varied by the diagnostic group of hospitalisation. Interventions that tackle work disability should consider the impact of ill-health on functioning while taking into account working conditions in each occupational class.

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