In Silico Analyses of All STAT3 Missense Variants Leading to Explore Divergent AD-HIES Clinical Phenotypes.

Autosomal dominant hyper-IgE syndrome (AD-HIES) is linked to dominant negative mutations of the STAT3 protein whose molecular basis for dysfunction is unclear and presenting with a variety of clinical manifestations with only supportive treatment. To establish the relationship between the impact of STAT3 mutations in different domains and the severity of the clinical manifestations, 105 STAT3 mutations were analyzed for their impact on protein stability, flexibility, function, and binding affinity using in Silico approaches. Our results showed that 73% of the studied mutations have an impact on the physicochemical properties of the protein, altering the stability, flexibility and function to varying degrees. In particular, mutations affecting the DNA binding domain (DBD) and the Src Homology 2 (SH2) have a significant impact on the protein structure and disrupt its interaction either with DNA or other STAT3 to form a heterodomain complex, leading to severe clinical phenotypes. Collectively, this study suggests that there is a close relationship between the domain involving the mutation, the degree of variation in the properties of the protein and the degree of loss of function ranging from partial loss to complete loss, explaining the variability of clinical manifestations between mild and severe.

Novel ITGB2 Mutation Is Responsible for a Severe Form of Leucocyte Adhesion Deficiency Type 1.

Leukocyte adhesion deficiency type 1 (LAD1) is a rare autosomal recessive hereditary disorder characterized by recurrent infections, impaired pus formation, delayed wound healing, omphalitis, and delayed separation of the umbilical cord as hallmark features of the disease. It results from mutations in the integrin ß2 subunit gene ITGB2, which encodes the integrin beta chain-2 protein CD18. In this study, we aimed to investigate the case of a five-month-old boy who presented with a clinical phenotype and flow cytometry results suggesting LAD1 disease. Sanger sequencing of all exons and intron boundaries of ITGB2 identified a novel in-frame deletion in exon 7 (ITGB2 c.844_846delAAC, p.Asn282del) in the patient. The p.Asn282del mutation was heterozygous in the child's parents, whereas it was absent in the 96 control individuals from North Africa. This variant was evaluated by two in silico mutation analysis tools, PROVEAN and MutationTaster, which predicted that the mutation was likely to be pathogenic. In addition, molecular modeling with the YASARA View software suggested that this novel mutation may affect the structure of integrin beta-2 and, subsequently, its interaction with integrin alpha-X. In summary, we report a novel pathogenic mutation p.Asn282del associated with LAD1 that expands the mutation diversity of ITGB2 and suggest the combination of flow cytometry and ITGB2 sequencing as a first-line diagnostic approach for LAD disease.

Cytometric analysis and clinical features in a Moroccan cohort with severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is a form of primary immunodeficiency disease (PID). It is characterized by a serious abnormality of the cellular and sometimes humoral system due to a deficiency in development of T cells, B cells and/or NK cells. The early diagnosis of SCID improves the prognosis. Typically, the initial consideration of SCID is made based on low lymphocyte counts. Notwithstanding, the heterogeneity of lymphocyte count presentation makes the diagnosis of SCID a significant challenge. The objective of this cross-sectional retrospective study was to analyze the lymphocyte subpopulation counts along with clinical manifestations within a Moroccan cohort diagnosed as SCID compared to children diagnosed with non-PID diseases. Thirty-five SCID confirmed patients were selected in the period between 2008 and 2018 and compared with non-PID patients. Results of peripheral blood T, B, and NK lymphocyte subpopulation counts were measured by flow cytometry for each SCID subtype. As expected, T cell count was less than 300 cells/µL in most patients with SCID (85.5%). Unexpectedly, significantly higher T cell counts were detected in some patients with a confirmed clinical diagnosis and family history of SCID. 5.7% of our SCID Moroccan cohort had T cell numbers in the range between 300 and 500 cells/µL. 8.7% of our SCID Moroccan cohort had T cell numbers higher than 500 cells/µL. Of the SCID subtypes, the proportion of SCID with B cell deficiencies was highly represented in our cohort. 71.4% of Moroccan SCID patients (25 out of 35 patients) were of T-B-subtype. Furthermore, 40% of the patients (14 out of 35 patients) had a T-B-NK+ profile and 31.4% had a T-B-NK- profile (11 out of 35 patients). The most common clinical manifestations observed in our SCID cohort were pneumonia, failure to thrive, candidiasis, diarrhea, bronchitis and urinary tract infections. Our results not only highlight the relatively frequent presence of atypical SCID in the Moroccan population with unexpectedly high T cell numbers, but also describes the incidence pattern of common SCID subtypes in Morocco. Physicians in Morocco may find this local region-specific difference in SCID important for making improved early diagnosis of this disease.

Clinical and Immunological Features of 96 Moroccan Children with SCID Phenotype: Two Decades' Experience.

Severe combined immunodeficiency (SCID) is a heterogeneous group of primary immunodeficiency diseases (PIDs) characterized by a lack of autologous T lymphocytes. This severe PID is rare, but has a higher prevalence in populations with high rates of consanguinity. The epidemiological, clinical, and immunological features of SCIDs in Moroccan patients have never been reported. The aim of this study was to provide a clinical and immunological description of SCID in Morocco and to assess changes in the care of SCID patients over time. This cross-sectional retrospective study included 96 Moroccan patients referred to the national PID reference center at Casablanca Children's Hospital for SCID over two decades, from 1998 to 2019. The case definition for this study was age < 2 years, with a clinical phenotype suggestive of SCID, and lymphopenia, with very low numbers of autologous T cells, according to the IUIS Inborn Errors of Immunity classification. Our sample included 50 male patients, and 66% of the patients were born to consanguineous parents. The median age at onset and diagnosis were 3.3 and 6.5 months, respectively. The clinical manifestations commonly observed in these patients were recurrent respiratory tract infection (82%), chronic diarrhea (69%), oral candidiasis (61%), and failure to thrive (65%). The distribution of SCID phenotypes was as follows: T-B-NK+ in 44.5%, T-B-NK- in 32%, T-B+NK- in 18.5%, and T-B+NK+ in 5%. An Omenn syndrome phenotype was observed in 15 patients. SCID was fatal in 84% in the patients in our cohort, due to the difficulties involved in obtaining urgent access to hematopoietic stem cell transplantation, which, nevertheless, saved 16% of the patients. The autosomal recessive forms of the clinical and immunological phenotypes of SCID, including the T-B-NK+ phenotype in particular, were more frequent than those in Western countries. A marked improvement in the early detection of SCID cases over the last decade was noted. Despite recent progress in SCID diagnosis, additional efforts are required, for genetic confirmation and particularly for HSCT.

Age-stratified pediatric reference values of lymphocytes in the Moroccan population.

The number of circulating lymphocytes is altered in a number of diseases including either increase (lymphocytosis) or decrease (lymphocytopenia). Therefore, the assessment of total blood lymphocyte numbers and the relative distribution of lymphocyte subsets is a critical front-line tool in the clinical diagnosis of a number of diseases, including pediatric diseases and disorders. However, the interpretation of this data requires comparison of patient's results to reliable reference values. Blood lymphocyte subpopulation numbers are also subject to genetic polymorphisms, immunogenic and environmental factors and vary greatly between populations. While the best practice reference values should be established within local representative populations of healthy subjects, to date, Caucasian reference values are used in Morocco due to the absence of indigenous reference values. Potential differences in blood lymphocyte subpopulation reference values between Caucasian versus Moroccan populations can adversely affect the diagnosis of pediatric and childhood diseases and disorders such as primary immunodeficiency (PID) in Morocco. OBJECTIVE: The aim of this study was to establish the age-stratified normal reference values of blood lymphocyte subsets for the pediatric Moroccan population. METHODS: We measured the concentration of lymphocyte subpopulations by flow cytometry from 83 Moroccan healthy subjects stratified into 5 age groups of 0-1, 1-2, 2-6, 6-12 and > 12-18 (adult). RESULTS: The absolute and relative amounts of the main lymphocyte subsets of T-cells, B cells and Natural Killer (NK) cells were measured and compared to previously described reference values from Cameroonian, Turkish, American and Dutch populations. Additionally, we also observed an age-related decline in the absolute population sizes of lymphocyte subsets within our study group. Relative proportions of CD3+CD4+ helper T lymphocytes decreased with increasing age and by 12 years-adult age, both proportions of CD3+CD4+ helper T lymphocytes and CD3+CD8+ cytotoxic T lymphocytes, as well as CD3-CD19+ B lymphocytes were also decreased. Finally, we compared the median values and range of our Moroccan study group with that of published results from Cameroon, Turkey, USA and Netherlands and observed significant differences in median and mean values of absolute number and relative proportions of lymphocyte subsets especially at 0-1 years and 1-2 years age groups. Above age 12 years, the Moroccan values were lower. For NK cells, the Moroccan values are also lower. CONCLUSIONS: The results of this study have a significant impact in improving the threshold values of the references intervals routinely used in the diagnosis of paediatric diseases such as PIDs or mother-to-child transmitted HIV within the Moroccan population.

Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations.

In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variants sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 68 recurrent mutations, including ten hotspot non-synonymous mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed that certain genotypes are specific to geographic locations. We also identified co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. The phylogentic analysis identified two major Clades C1 and C2 harboring mutations L3606F and G614D, respectively and both emerging for the first time in China. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets. We have also created an inclusive unified database (http://covid-19.medbiotech.ma) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.

Targeting the GRP78-Dependant SARS-CoV-2 Cell Entry by Peptides and Small Molecules.

The global burden of infections and the rapid spread of viral diseases show the need for new approaches in the prevention and development of effective therapies. To this end, we aimed to explore novel inhibitor compounds that can stop replication or decrease the viral load of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is currently no approved treatment. Besides using the angiotensin-converting enzyme (ACE2) receptor as a main gate, the CoV-2 can bind to the glucose-regulating protein 78 (GRP78) receptor to get into the cells to start an infection. Here, we report potential inhibitors comprising small molecules and peptides that could interfere with the interaction of SARS-CoV-2 and its target cells by blocking the recognition of the GRP78 cellular receptor by the viral Spike protein. These inhibitors were discovered through an approach of in silico screening of available databases of bioactive peptides and polyphenolic compounds and the analysis of their docking modes. This process led to the selection of 9 compounds with optimal binding affinities to the target sites. The peptides (satpdb18674, satpdb18446, satpdb12488, satpdb14438, and satpdb28899) act on regions III and IV of the viral Spike protein and on its binding sites in GRP78. However, 4 polyphenols such as epigallocatechin gallate (EGCG), homoeriodictyol, isorhamnetin, and curcumin interact, in addition to the Spike protein and its binding sites in GRP78, with the ATPase domain of GRP78. Our work demonstrates that there are at least 2 approaches to block the spread of SARS-CoV-2 by preventing its fusion with the host cells via GRP78.

Genomic Diversity and Hotspot Mutations in 30,983 SARS-CoV-2 Genomes: Moving Toward a Universal Vaccine for the "Confined Virus"?

The COVID-19 pandemic has been ongoing since its onset in late November 2019 in Wuhan, China. Understanding and monitoring the genetic evolution of the virus, its geographical characteristics, and its stability are particularly important for controlling the spread of the disease and especially for the development of a universal vaccine covering all circulating strains. From this perspective, we analyzed 30,983 complete SARS-CoV-2 genomes from 79 countries located in the six continents and collected from 24 December 2019, to 13 May 2020, according to the GISAID database. Our analysis revealed the presence of 3206 variant sites, with a uniform distribution of mutation types in different geographic areas. Remarkably, a low frequency of recurrent mutations has been observed; only 169 mutations (5.27%) had a prevalence greater than 1% of genomes. Nevertheless, fourteen non-synonymous hotspot mutations (>10%) have been identified at different locations along the viral genome; eight in ORF1ab polyprotein (in nsp2, nsp3, transmembrane domain, RdRp, helicase, exonuclease, and endoribonuclease), three in nucleocapsid protein, and one in each of three proteins: Spike, ORF3a, and ORF8. Moreover, 36 non-synonymous mutations were identified in the receptor-binding domain (RBD) of the spike protein with a low prevalence (<1%) across all genomes, of which only four could potentially enhance the binding of the SARS-CoV-2 spike protein to the human ACE2 receptor. These results along with intra-genomic divergence of SARS-CoV-2 could indicate that unlike the influenza virus or HIV viruses, SARS-CoV-2 has a low mutation rate which makes the development of an effective global vaccine very likely.

Genome Sequences of Six SARS-CoV-2 Strains Isolated in Morocco, Obtained Using Oxford Nanopore MinION Technology.

Here, we report the draft genome sequences of six severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains. SARS-CoV-2 is responsible for the COVID-19 pandemic, which started at the end of 2019 in Wuhan, China. The isolates were obtained from nasopharyngeal swabs from Moroccan patients with COVID-19. Mutation analysis revealed the presence of the spike D614G mutation in all six genomes, which is widely present in several genomes around the world.

Moroccan lymphocyte subsets reference ranges: age, gender, ethnicity, and socio-economic factors dependent differences.

Lymphocyte subsets reference ranges are helpful for a precise diagnosis and therapy of various diseases. We attempted in the current study to establish Moroccan lymphocyte reference range and reveal age, gender, ethnicity, income, and instructional levels dependent differences. Lymphocyte subsets percentage and absolute count were determined by 4-color flow cytometry in a population study of 145 adults Moroccan healthy volunteers. Analysis showed significant age-dependent changes. Age was associated with a decrease of naïve CD4+ and CD8+ T cells and an increase of memory CD4+ or CD8+ T cells. Activated CD4+ CD38+ and CD8+ CD38+ T cells, Treg as well as NK cell showed age-dependent alterations. In contrast, B cells remained unchanged. A higher percentage of CD3+ and CD4+ T cells was observed in females while CD8+, B and NK cells count were higher in men. Ethnicity, instructional levels, and personal income seem to not influence lymphocyte subsets reference values. This study provides reference ranges for lymphocyte subsets of healthy Moroccan adults. These results can be used for other North African (Maghrebian) countries considering their geographic, ethnic, economic, and cultural similarities.

[A rare cause of exertional dry cough: agenesis of the left pulmonary artery associated with pulmonary hypoplasia]. / Cause rare d'une toux sèche à l'effort: agénésie de l'artère pulmonaire gauche avec hypoplasie pulmonaire.

Agenesis of the left pulmonary artery associated with hypoplasia of the ipsilateral lung is a rare congenital malformation in children; it can be discovered fortuitously or because of the presence of recurrent respiratory infections. Diagnosis is based on thoracic angioscanner. Treatment is essentially conservative. We report the case of a 6-year old child with agenesis of the left pulmonary artery associated with hypoplasia of the ipsilateral lung detected because of exertional dry cough.

X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

PURPOSE: X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. METHODS: Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. RESULTS: We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. CONCLUSIONS: This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

Inherited and acquired immunodeficiencies underlying tuberculosis in childhood.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) and a few related mycobacteria, is a devastating disease, killing more than a million individuals per year worldwide. However, its pathogenesis remains largely elusive, as only a small proportion of infected individuals develop clinical disease either during primary infection or during reactivation from latency or secondary infection. Subacute, hematogenous, and extrapulmonary disease tends to be more frequent in infants, children, and teenagers than in adults. Life-threatening primary TB of childhood can result from known acquired or inherited immunodeficiencies, although the vast majority of cases remain unexplained. We review here the conditions conferring a predisposition to childhood clinical diseases caused by mycobacteria, including not only M.tb but also weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria. Infections with weakly virulent mycobacteria are much rarer than TB, but the inherited and acquired immunodeficiencies underlying these infections are much better known. Their study has also provided genetic and immunological insights into childhood TB, as illustrated by the discovery of single-gene inborn errors of IFN-γ immunity underlying severe cases of TB. Novel findings are expected from ongoing and future human genetic studies of childhood TB in countries that combine a high proportion of consanguineous marriages, a high incidence of TB, and an excellent clinical care, such as Iran, Morocco, and Turkey.

Chronic granulomatous disease in Morocco: genetic, immunological, and clinical features of 12 patients from 10 kindreds.

PURPOSE: Chronic granulomatous disease (CGD) is characterized by an inability of phagocytes to produce reactive oxygen species (ROS), which are required to kill some microorganisms. CGD patients are known to suffer from recurrent bacterial and/or fungal infections from the first year of life onwards. From 2009 to 2013, 12 cases of CGD were diagnosed in Morocco. We describe here these Moroccan cases of CGD. METHODS: We investigated the genetic, immunological and clinical features of 12 Moroccan patients with CGD from 10 unrelated kindreds. RESULTS: All patients were children suffering from recurrent bacterial and/or fungal infections. All cases displayed impaired NADPH oxidase activity in nitroblue tetrazolium (NBT), dihydrorhodamine (DHR) or 2',7' dichlorofluorescein diacetate (DCFH-DA) assays. Mutation analysis revealed the presence of four different mutations of CYBB in four kindreds, a recurrent mutation of NCF1 in three kindreds, and a new mutation of NCF2 in three patients from a single kindred. A large deletion of CYBB gene has detected in a patient. The causal mutation in the remaining one kindred was not identified. CONCLUSION: The clinical features and infectious agents found in these patients were similar to those in CGD patients from elsewhere. The results of mutation analysis differed between kindreds, revealing a high level of genetic and allelic heterogeneity among Moroccan CGD patients. The small number of patients in our cohort probably reflects a lack of awareness of physicians. Further studies on a large cohort are required to determine the incidence and prevalence of the disease, and to improve the description of the genetic and clinical features of CGD patients in Morocco.

IL-12Rß1 deficiency in two of fifty children with severe tuberculosis from Iran, Morocco, and Turkey.

BACKGROUND AND OBJECTIVES: In the last decade, autosomal recessive IL-12Rß1 deficiency has been diagnosed in four children with severe tuberculosis from three unrelated families from Morocco, Spain, and Turkey, providing proof-of-principle that tuberculosis in otherwise healthy children may result from single-gene inborn errors of immunity. We aimed to estimate the fraction of children developing severe tuberculosis due to IL-12Rß1 deficiency in areas endemic for tuberculosis and where parental consanguinity is common. METHODS AND PRINCIPAL FINDINGS: We searched for IL12RB1 mutations in a series of 50 children from Iran, Morocco, and Turkey. All children had established severe pulmonary and/or disseminated tuberculosis requiring hospitalization and were otherwise normally resistant to weakly virulent BCG vaccines and environmental mycobacteria. In one child from Iran and another from Morocco, homozygosity for loss-of-function IL12RB1 alleles was documented, resulting in complete IL-12Rß1 deficiency. Despite the small sample studied, our findings suggest that IL-12Rß1 deficiency is not a very rare cause of pediatric tuberculosis in these countries, where it should be considered in selected children with severe disease. SIGNIFICANCE: This finding may have important medical implications, as recombinant IFN-γ is an effective treatment for mycobacterial infections in IL-12Rß1-deficient patients. It also provides additional support for the view that severe tuberculosis in childhood may result from a collection of single-gene inborn errors of immunity.