Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children.

PURPOSE: Leukocyte adhesion deficiency (LAD) represents a rare group of inherited inborn errors of immunity (IEI) characterized by bacterial infections, delayed umbilical stump separation, and autoimmunity. This single-center study aimed at describing the clinical, immunological, and molecular characterizations of 34 LAD-I Egyptian pediatric patients. METHODS: Details of 34 patients' personal medical history, clinical and laboratory findings were recorded; Genetic material from 28 patients was studied. Mutational analysis was done by Sanger sequencing. RESULTS: Omphalitis, skin and soft tissue infections with poorly healing ulcers, delayed falling of the umbilical stump, and recurrent or un-resolving pneumonia were the most common presentations, followed by chronic otitis media, enteropathy, periodontitis; and recurrent oral thrush. Persistent leukocytosis and neutrophilia were reported in all patients, as well as CD18 and CD11b deficiency. CD18 expression was < 2% in around 90% of patients. Sixteen different pathological gene variants were detected in 28 patients who underwent ITGß2 gene sequencing, of those, ten were novel and six were previously reported. Three families received a prenatal diagnosis. Patients were on antimicrobials according to culture's results whenever available, and on prophylactic Trimethoprim-Sulfamethoxazole 5 mg/kg once daily, with regular clinical follow up. Hematopoietic stem cell transplantation (HSCT) was offered for 4 patients. However due to severity of the disease and delay in diagnosis, 58% of the patients passed away in the first 2 years of life. CONCLUSION: This study highlights the importance of early diagnosis and distribution of ITGß2 gene mutation in Egyptian children. Further molecular studies, however, remain a challenging necessity for better disease characterization in the region.

Integrins in Health and Disease-Suitable Targets for Treatment?

Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the ß2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant ß (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on ß2 integrins that are specifically expressed by leukocytes. The pathophysiological role of ß2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of ß2 integrins in vivo employed mice with a constitutive knockout of all ß2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of ß2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of ß2 integrins by our group has enabled the dissection of cell-specific roles of ß2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate ß2 integrin activity for therapeutic applications.

A novel leukocyte adhesion deficiency type III mutation manifests functional importance of the compact FERM domain in kindlin-3.

BACKGROUND: Leukocyte adhesion deficiency III (LAD-III) is a rare autosomal recessive syndrome characterized by functional deficiencies of platelets and leukocytes that occurs due to mutations in the FERMT3 gene encoding kindlin-3. Kindlin-3 is a FERM domain-containing adaptor protein that is essential in integrin activation. We have previously demonstrated that the FERM domain of kindlin-3 is structurally compact and plays an important role in supporting integrin activation in a mouse model. The impact of destabilizing the compact FERM domain in kindlin-3 on the development of LAD-III in humans remains uncertain. OBJECTIVES: To use primary cells from a patient with LAD-III to validate the role of the compact FERM domain in kindlin-3 function in platelets and leukocytes. METHODS: The patient is a 4-year-old girl who since infancy has displayed clinical features of LAD-III. Patient platelets and leukocytes were functionally analyzed, and structural analysis of the kindlin-3 variant was conducted. RESULTS: We identified a novel homozygous missense mutation in the FERMT3 (c.412G>A, p.E138K) FERM domain. Substantially reduced levels of kindlin-3 were detected in the proband's platelets and leukocytes. Functional evaluation verified that integrin αIIbß3-mediated platelet activation, spreading, and aggregation and ß2-integrin-mediated neutrophil adhesion and spreading were significantly compromised. Structural analysis revealed that this newly identified E138K substitution in kindlin-3 destabilizes the compacted FERM domain, resulting in poor expression of kindlin-3 in blood cells and subsequent LAD-III. CONCLUSION: We have identified a novel missense mutation and verified the functional significance of the compact kindlin-3 FERM domain in supporting integrin functions in platelets and leukocytes.

Clinical and immunological characteristics of 69 leukocyte adhesion deficiency-I patients.

BACKGROUND: In order to support the comprehensive classification of Leukocyte Adhesion Deficiency-I (LAD-I) severity by simultaneous screening of CD11a/CD18, this study assessed clinical, laboratory, and genetic findings along with outcomes of 69 LAD-I patients during the last 15 years. METHODS: Sixty-nine patients (40 females and 29 males) with a clinical phenotype suspected of LAD-I were referred to Immunology, Asthma, and Allergy research institute, Tehran, Iran between 2007 and 2022 for further advanced immunological screening and genetic evaluations as well as treatment, were enrolled in this study. RESULTS: The diagnosis median age of the patients was 6 months. Delayed umbilical cord separation was found in 25 patients (36.2%). The median diagnostic delay time was 4 months (min-max: 0-82 months). Forty-six patients (66.7%) were categorized as severe (CD18 and/or CD11a: below 2%); while 23 children (33.3%) were in moderate category (CD18 and/or CD11a: 2%-30%). During the follow-ups, 55.1% of children were alive with a mortality rate of 44.9%. Skin ulcers (75.4%), omphalitis (65.2%), and gingivitis (37.7%) were the most frequent complaints. Genetic analysis of the patients revealed 14 previously reported and three novel pathogenic mutations in the ITGB2 gene. The overall survival of patients with and without hematopoietic stem cell transplantation was 79.3% and 55.6%, respectively. CONCLUSION: Physicians' awareness of LAD-I considering delayed separation of umbilical cord marked neutrophilic leukocytosis, and variability in CD11 and CD18 expression levels, and genetic analysis leads to early diagnosis and defining disease severity. Moreover, the prenatal diagnosis would benefit families with a history of LAD-I.

Pediatric pyoderma gangrenosum associated with leukocyte adhesion deficiency type 1: A case report and review of the literature.

Pyoderma gangrenosum is a rare neutrophilic dermatosis characterized by painful skin ulcers with necrotic, undermined margins. In severe cases, particularly in pediatric patients, work-up for an associated autoimmune, inflammatory, malignant, or genetic disorder should be considered based on the clinical presentation. We report a unique case of pediatric pyoderma gangrenosum with a leukemoid reaction, secondary to an autosomal recessive leukocyte adhesion deficiency type 1.

Clinical and Osteopetrosis-Like Radiological Findings in Patients with Leukocyte Adhesion Deficiency Type III.

BACKGROUND: Leukocyte and platelet integrin function defects are present in leukocyte adhesion deficiency type III (LAD-III) due to mutations in FERMT3. Additionally, osteoclast/osteoblast dysfunction develops in LAD-III. AIM: To discuss the distinguishing clinical, radiological, and laboratory features of LAD-III. METHODS: This study included the clinical, radiological, and laboratory characteristics of twelve LAD-III patients. RESULTS: The male/female ratio was 8/4. The parental consanguinity ratio was 100%. Half of the patients had a family history of patients with similar findings. The median age at presentation and diagnosis was 18 (1-60) days and 6 (1-20) months, respectively. The median leukocyte count on admission was 43,150 (30,900-75,700)/µL. The absolute eosinophil count was tested in 8/12 patients, and eosinophilia was found in 6/8 (75%). All patients had a history of sepsis. Other severe infections were pneumonia (66.6%), omphalitis (25%), osteomyelitis (16.6%), gingivitis/periodontitis (16%), chorioretinitis (8.3%), otitis media (8.3%), diarrhea (8.3%), and palpebral conjunctiva infection (8.3%). Four patients (33.3%) received hematopoietic stem cell transplantation (HSCT) from HLA-matched-related donors, and one deceased after HSCT. At initial presentation, 4 (33.3%) patients were diagnosed with other hematologic disorders, three patients (P5, P7, and P8) with juvenile myelomonocytic leukemia (JMML), and one (P2) with myelodysplastic syndrome (MDS). CONCLUSION: In LAD-III, leukocytosis, eosinophilia, and bone marrow findings may mimic pathologies such as JMML and MDS. In addition to non-purulent infection susceptibility, patients with LAD-III exhibit Glanzmann-type bleeding disorder. In LAD-III, absent integrin activation due to kindlin-3 deficiency disrupts osteoclast actin cytoskeleton organization. This results in defective bone resorption and osteopetrosis-like radiological changes. These are distinctive features compared to other LAD types.

A Novel Deletion in FERMT3 Causes LAD-III in a Turkish Family.

Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.

Hematologically important mutations: Leukocyte adhesion deficiency (second update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the ß subunit (CD18) of the ß2 integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Lea and Leb blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells, involved in the regulation of ß integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.

Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Understanding the Role of LFA-1 in Leukocyte Adhesion Deficiency Type I (LAD I): Moving towards Inflammation?

LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the ITGB2 (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.

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.

Defining the mild variant of leukocyte adhesion deficiency type II (SLC35C1-congenital disorder of glycosylation) and response to l-fucose therapy: Insights from two new families and review of the literature.

Leukocyte adhesion deficiency type II (LAD II, also known as SLC35C1-congenital disorder of glycosylation) is an autosomal recessive disorder characterized by growth and cognitive impairment, peripheral neutrophilia, recurrent infections, and the Bombay blood phenotype. A subset of patients with a milder presentation has been described with short stature and developmental delay but minimal immune and hematologic features. Some patients with LAD II benefit from oral fucose therapy, though this has not been previously studied in patients with milder disease. In this study, we describe two new patients from separate families with the milder variant of LAD II and review the published literature on this rare disorder. We demonstrate improvement in speech and cognition, CD15 expression, and core fucosylation of serum glycoproteins after 27 months of oral fucose supplementation in one patient. These patients further support the stratification of this disorder into distinct subtypes, a classical severe and an attenuated variant, and provide preliminary evidence of benefit of fucose therapy in the latter group.

A novel ITGB2 variant with long survival in patients with leukocyte adhesion defect type-I.

Leukocyte adhesion deficiency is an autosomal recessive primary immunodeficiency that has been divided into three types: LAD1 (beta-2 integrin (CD18) family deficiency/defect), LAD2 (absence of fucosylated carbonhydrate ligands for selectins) and LAD3 (defective activation of all beta integrins). However, recently LAD4 has been described in cystic fibrosis patients, with a defect in integrin activation reported in monocytes. LAD-I is the most common type and prevalence of 1 in 1,000,000 live births. Clinical features of LAD patients are recurrent bacterial and fungal infections, omphalitis with delayed umbilical stump separation, significant leukocytosis especially neutrophilia during infection periods, impaired pus formation, and delayed traumatic or surgical wound healing. Flow cytometry is considered a useful tool for rapid diagnosis of the disease. The study of CD18 and CD11 (a, b, c) expression patterns in peripheral blood leukocytes helps to distinguish different phenotypes of LAD-I. In general, patients with ≥ 2% CD18 expression tend to have a less severe infection and often survive until adulthood, whereas < 2% CD18 expression often results in death in infancy. In this case report, three siblings, 10, 15, and 17 years old, diagnosed with leukocyte adhesion defect type 1 in adolescence age group, are presented.

Clinical and laboratory findings in patients with leukocyte adhesion deficiency type I: A multicenter study in Turkey.

Leukocyte adhesion deficiency type I is a rare primary immunodeficiency disorder characterized by mutations in the ITGB2 gene encoding CD18. We present clinical and immunological features of 15 patients with leukocyte adhesion deficiency type 1 (LAD-1). Targeted next-generation sequencing was performed with either a primary immunodeficiency gene panel comprising 266 genes or a small LAD-panel consisting of five genes for genetic analysis. To measure the expression level of integrins on the leukocyte surface, flow cytometry analysis was performed. The median age of the patients at diagnosis was 3 (1-48) months. Eleven (73%) of the 15 patients had a LAD-1 diagnosis in their first 6 months and 14 (93%) patients had consanguineous parents. Delayed separation of the umbilical cord was present in 80% (n = 12) of the patients in our cohort, whereas omphalitis was observed in 53% (n = 8) of the patients. Leukocytosis with neutrophil predominance was observed in 73% (n = 11) patients. Nine distinct variants in the ITGB2 gene in 13 of the 15 patients with LAD-1 were characterized, two of which (c.305_306delAA and c.779_786dup) are novel homozygous mutations of ITGB2. Four unrelated patients from Syria had a novel c.305_306delAA mutation that might be a founder effect for patients of Syrian origin. Four (27%) patients underwent hematopoietic stem cell transplantation. Two patients died because of HSCT complications and the other two are alive and well. Early differential diagnosis of the patients is critical in the management of the disease and genetic evaluation provides a basis for family studies and genetic counseling.

Prevalence of nine genetic defects in Chinese Holstein cattle.

Worldwide use of elite sires has caused inbreeding accumulation and high frequencies of genetic defects in dairy cattle populations. In recent years, several genetic defect genes or haplotypes have been identified in Holstein cattle. A rapid and reliable microfluidic chip with Kompetitive allele-specific PCR (KASP) assay was developed in our previous study for the detection of heterozygotes at eight genetic defect loci of bovine leukocyte adhesion deficiency (BLAD), Brachyspina syndrome (BS), complex vertebral malformation (CVM), Holstein haplotype 1 (HH1), Holstein haplotype 3 (HH3), Holstein haplotype 4 (HH4), Holstein haplotype 5 (HH5) and haplotype for cholesterol deficiency (HCD). This study aimed to extend that assay to include a newly identified genetic defect of Holstein haplotype 6 (HH6) and to estimate the frequencies of carriers for each of the nine genetic defects in six Chinese Holstein herds. Of the 1633 cows, carrier frequencies of the genetic defects were 6.92%, 5.76%, 4.46%, 4.30%, 3.62%, 2.94%, 1.86% and 0.37% for HH1, HH3, CVM, HH5, HCD, BS, HH6 and BLAD, respectively. No carrier was found for HH4. Notably, 27.43% of cows carried at least one genetic defect, while 2.27% and 0.12% of cows carried double and triple genetic defect alleles, respectively. The existence of genetic defects calls for routine molecular testing and effective management of genetic defects by avoiding carrier-to-carrier mating in production herds and eliminating or at least reducing the frequency of the defective alleles through marker-assisted selection in breeding herds.

Incidental diagnosis of leukocyte adhesion deficiency type II following ABO typing.

Individuals with the Bombay phenotype (Oh) in the ABO blood group system do not express the H, A, and B antigens but have no clinical symptoms. Bombay phenotype with clinical symptoms has been described in leukocyte adhesion deficiency type II (LAD II), a fucosylation disorder caused by mutations in SLC35C1. Only few LAD II patients have been described so far. Here we describe an additional patient, a 22-year old male, born to unrelated parents, presenting with inflammatory skin disease, periodontitis, growth, and mental retardation, admitted to the department of dentistry for treatment under general anesthesia. Pre-operative routine investigations revealed the presence of the Bombay phenotype (Oh). Genomic sequencing identified two novel triplet deletions of the SLC35C1 gene. Functional investigations confirmed the diagnosis of LAD II. Therapy with oral fucose led to the disappearance of the chronic skin infections and improvements in behavior and attention span.

Primary Immunodeficiencies With Defects in Innate Immunity: Focus on Orofacial Manifestations.

The field of primary immunodeficiencies (PIDs) is rapidly evolving. Indeed, the number of described diseases is constantly increasing thanks to the rapid identification of novel genetic defects by next-generation sequencing. PIDs are now rather referred to as "inborn errors of immunity" due to the association between a wide range of immune dysregulation-related clinical features and the "prototypic" increased infection susceptibility. The phenotypic spectrum of PIDs is therefore very large and includes several orofacial features. However, the latter are often overshadowed by severe systemic manifestations and remain underdiagnosed. Patients with impaired innate immunity are predisposed to a variety of oral manifestations including oral infections (e.g., candidiasis, herpes gingivostomatitis), aphthous ulcers, and severe periodontal diseases. Although less frequently, they can also show orofacial developmental abnormalities. Oral lesions can even represent the main clinical manifestation of some PIDs or be inaugural, being therefore one of the first features indicating the existence of an underlying immune defect. The aim of this review is to describe the orofacial features associated with the different PIDs of innate immunity based on the new 2019 classification from the International Union of Immunological Societies (IUIS) expert committee. This review highlights the important role played by the dentist, in close collaboration with the multidisciplinary medical team, in the management and the diagnostic of these conditions.

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1.

ß2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of ß2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18-/- mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18-/- mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18-/- mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18-/- mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.