The kindlin family member 3 [kindlin-3, or KIND3; preferred name FERMT3 (Uniprot accession Q86UX7)] is a cytosolic “β-integrin adapter protein” expressed mainly in hematopoietic cells (1, 2). Deficiencies in FERMT3 have been linked to the autosomal recessive “leukocyte adhesion deficiency, type III” (LAD3, MIM#612840), where all the β-integrins (β₁, β₂, and β₃) are defective (3–8). This entity is a joint of LAD1-like leukocyte dysfunction (persistent leukocytosis plus infections, typically without pus formation) and Glanzmann thrombasthenia-like platelet dysfunction (mucocutaneous bleeding) (9). Individuals with this condition present in early infancy with severe bleeding and frequent infections (10).

The FERM central domain (residues 258 to 558; InterPro accession: Q86UX7) of FERMT3 binds to and activates platelet integrin α_IIbβ₃ (CD41/CD61). This receptor is activated by ADP, epinephrine, collagen, arachidonic acid, and thrombin. Activated receptors then bind fibrinogen, von Willebrand factor (vWF), fibronectin, and vitronectin (11–13). FERM domain is also essential for activating integrins β₁ and β₂ on the surface of granulocytes and lymphocytes (14–16). FERM defects, thus, demonstrate the essential role of F-actin-rich structures in the podosomes of lymphocytes, granulocytes and platelets (17, 18).

Only very few children with LAD3 have been reported in the literature. Thus, clinical descriptions of new patients are important to define the disease phenotype. We present here a toddler with novel compound heterozygous variants in FERMT3 (MIM#607901). This report features his characteristics and natural history. Its purpose is to enhance the recognition of this disorder and perform a structural bioinformatics analysis of his variations.

This 18-month-old male toddler was born at term to asymptomatic, non-consanguineous parents (from Kerala, India). The pregnancy and delivery were uneventful. His birth weight was 2.9 kg. He had no siblings; the mother was gravida 2, para 1, abortion (spontaneous) 1. His paternal grandmother had severe menorrhagia, requiring multiple transfusions. Otherwise, the family history was negative.

He was born in the United Arab Emirates and received the BCG (Bacillus Calmette–Guérin) vaccine [0.05 mL, intradermal injection in the upper left arm (manufactured by Serum Institute of India Pvt. Ltd., India)] at birth (Figure 1, left upper panel). His postnatal period was complicated by a delayed umbilical cord separation for about 3 weeks. He presented at 2 months of age with petechial rash over his arms and seborrheic dermatitis over his scalp. Starting at 8 months of age, he had recurrent episodes of severe nose bleeding; consequently, several blood counts were requested. He had 10 admissions since birth; six for severe epistaxis, two for work-up of marked leukocytosis, one for the bone marrow procedure, and one for a significant skin rash (eczema-like). His overall management included two red cell transfusions (over a 4 month period), three platelet transfusions, and tranexamic acid (used only for a short time period).

He was never febrile; his temperatures were ≤37.2°C. On two occasions, his blood cultures (requested because of the marked leukocytosis) grew Micrococcus luteus and Staphylococcus haemolyticus, which were considered skin flora contaminants. His physical examination was remarkable for the skin findings of dryness, bruises (petechiae and purpura), and a few scars, as shown in Figure 1 (middle and right upper panel). He was uncircumcised. His growth and development were appropriate for age.

Investigations were requested because of the unexplained persistent leukocytosis. His white blood cell differential counts are shown in Figures 2A,B. Persistent marked leukocytosis (mainly lymphocytosis, monocytosis, and eosinophilia) were evident. In addition, “left shift” (neutrophil bands, metamyelocytes, and myelocytes) was a frequent finding (Figures 3A,B). His chest radiograph was remarkable for a notably prominent thymus (reported as a “round opacity at the mediastinum”), Figure 1 (lower panels). Findings on the abdominal ultrasound and echocardiogram were normal. Serum ferritin was ≤20 μg/L and C-reactive protein (CRP) ≤ 1.8 mg/L. Serum immunoglobulins (IgA, IgG, and IgM) were normal. At 9 months of age, his lymphocyte subset was: CD3 T cells [11,002/μL (41%), reference values (10th−90th centiles) = 1,900–5,900], CD4 T cells [6,423/μL (24%), reference values = 1,400–4,300], CD8 T cells [3,785/μL (14%), reference values = 500–1,700], CD19 B cells [14,206/μL (53%), reference values = 610–2,600], and NK cells (426/μL, 2%). Bone marrow examination was normal; the megakaryocytes were adequate in number and had normal morphology (Figures 3C,D). Leukocyte RNA was negative for leukemia fusion gene transcripts (MDx Leukemia Fusion Gene Q30 screen; QuanDx, Menlo Park, CA, USA).

Hemostatic evaluation was also requested because of the severe epistaxis. His platelet counts and secondary hemostasis [activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), thrombin time (TT), and serum fibrinogen] were normal. Platelet function screen (performed using Dade® PFA Collagen/EPI and Collagen/ADP Test Cartridges, Siemens Healthcare Diagnostics GmbH; Marburg, Germany) showed prolonged collagen-epinephrine and collagen-ADP closure times > 300 s (normal, ≤ 164 s). Von Willebrand factor (vWF) antigen was 59.6% (normal, 50–200) and activity 46.7% (normal, 50–200); these tests were performed using Innovance® VWF Ac and VWF Ag (Siemens Healthcare Diagnostics GmbH; Marburg, Germany). The results of vWF were normal as his blood group was “O”; even though, the red blood cell phenotype may have partially contributed to his bleeding tendency. Platelet aggregation studies (performed using the Bio/Data Corporation product; Horsham, PA, USA) revealed severely impaired aggregations in response to all agonists, suggesting a defect in the inside-out activation of integrin α_IIbβ₃ (Table 1). Spontaneous platelet aggregation, a pathologic finding, was not present.

Flow cytometry revealed a normal expression of the leukocyte receptors CD11a (integrin, alpha L), CD11b (integrin, alpha M), CD11c (integrin, alpha X), and CD18 (integrin β₂). It also revealed a normal expression (99%) of the platelet receptors CD41 (integrin α_IIb), CD61 (integrin β₃), CD42a (GPIX), and CD42b (GPlb-α). These tests were performed using monoclonal antibodies from Becton, Dickinson and Company, BD Biosciences; San Jose, California, USA (see Supplementary Material).

In view of his abnormal platelet function profile and marked leukocytosis, “diagnostic exome sequencing test” was requested (mainly considering the probability of LAD3). Two novel compound heterozygous variants were found: (1) Non-sense (stop-gain, resulting in a truncated product) NM_178443.2(FERMT3):c.1800G>A, p.Trp600^* (inherited from the mother), and (2) Non-stop (stop-loss, resulting in an elongated polypeptide) NM_178443.2(FERMT3):c.2001del p.^*668Glufs^*106 (inherited from the father).

The non-sense variant FERMT3:p.Trp600^* leads to a premature truncation of the protein and the loss of 68 residues from the C-terminal (Figure 4A). This truncation results in the loss of two α-helices and a β-sheet, comprising of three β-strands (Figure 4B). Computational prediction scores from multiple predictors [combined annotation dependent depletion (CADD): 46; likelihood ratio test (LRT): 0.8433; MutationTaster: 0.81] obtained from Ensembl Variant Effect Predictor (VEP; https://www.ensembl.org/Tools/VEP) (20) suggest that this variant is “deleterious,” while Varsome (21) flags it as “pathogenic.” The Trp600^* variation shortens the F3 subdomain of FERMT3. The F3 subdomain is a part of the integrin-binding pocket of FERMT3 and this variation is, therefore, expected to inhibit the ability of FERMT3 to bind to integrins.

The p.^*668Glufs^*106 non-stop variant results in an extended polypeptide with an additional 105 amino acids, a 16% extension to the wild-type sequence. Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov) (22) searches did not identify any homologous sequences or structures. Secondary structure prediction with PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/) (23) indicated that this region could potentially harbor six α-helices (Figure 4C). While secondary structures have been predicted in the extended sequence of p.^*668Glufs^*106 (Figure 4C), it is unclear if this extended part will cooperatively fold to produce a functional domain. Nonetheless, even if it does, its close proximity to the integrin-binding pocket of the F3 subdomain, located near the C-terminal of the protein, is likely to disrupt its ability to bind to integrins.

Pathogenic variants of FERMT3 (MIM#607901) cause LAD3 (MIM#612840). GnomAD v2.1.1 lists 1,112 variations in FERMT3¹. Of these, 372 are start-loss, stop-loss, inframe deletion, inframe insertion, frameshift, or non-sense mutations (6, 24). Variations that map on to the FERM (F: 4.1 protein, E: ezrin, R: radixin, and M: moesin) and PH (pleckstrin homology) domains of FERMT3 are shown in Figure 4D.

As indicated above, LAD3 is joint defects of LAD1 (MIM#116920)-like immune deficiency and Glanzmann thrombasthenia (MIM#273800)-like bleeding. The defect involves the intracellular protein that interacts with β-integrins in hematopoietic cells, including granulocytes, lymphocytes, macrophages, and platelets. The predominant phenotypes result from failures to activate β₁ and β₂ integrins on the surface of granulocytes and lymphocytes and β₃ integrin (α_IIbβ₃) on the surface of platelets (13, 14). As previously shown, integrins α₂β₁ and α_IIbβ₃ support the irreversible adhesive interactions of platelets with collagen and VWF; these receptors also support the platelet recruitment and spreading (25). Integrin β₁ (CD29) also pairs with other α subunits, such as α₄β₁ (a vascular cell adhesion protein 1, VCAM-1, receptor), α₅β₁ (a fibronectin receptor), and α₆β₁ (a laminin receptor) (26). Integrin β₂ (CD18), on the other hand, is exclusively expressed on the surface of leukocytes. Its pairing with one of a number of distinct α subunits (e.g., α_M, or CD11b) enables cell-cell interactions, such as the binding to intercellular adhesion molecule 1 (ICAM-1) on an activated endothelial cell thus mediating neutrophil adhesion and spreading (27). Similarly, integrin β₃ (CD61) was shown to pair with the α_v subunit; the resulting receptor (α_vβ₃) binds the secreted protein vitronectin and mediates cell adhesion, spreading and signaling (28). Defective “inside-to-outside” signalings explain the hallmarks of the entity of granulocytosis (innate immune defects), lymphocytosis (defective adaptive immunity) and life-threatening mucocutaneous bleeding (impaired primary hemostasis) (14, 15, 29, 30).

Noticeable features of his phenotype (see reference 8, and Table 3) include: (1) Delayed umbilical cord separation for about 3 weeks (reference mean ± SD time = 6.61 ± 2.33 days; confidence of interval 95% = 6.16–7.05) (31); (2) Spontaneous petechiae in the neonatal period; (3) Severe epistaxis in early infancy (his first episode was at 8 mo of age, requiring transfusion); (4) Normal secondary hemostasis; (5) Normal platelet count; (6) Abnormal platelet function screen (PFA-100® test, >300 s for both channels); (7) Normal von Willebrand factor antigen and activity; (8) Impaired platelet aggregation with all agonists (ristocetin, thrombin, ADP, epinephrine, arachidonic acid, and collagen); (9) Normal expression of platelet receptors (e.g., CD41/CD61, or integrin α_IIbβ₃); (10) Normal expression of leukocyte adhesion molecules (e.g., CD18, or integrin β₂); (11) Absent hepatosplenomegaly and lymphadenopathy; (12) Prominent thymus on the chest radiograph; (13) Absence of fever; (14) Normal inflammatory biomarkers (CRP and ferritin); (15) Persistent marked leukocytosis/ granulocytosis (most notably, high monocyte and eosinophil counts); and (16) Persistent lymphocytosis (high T cell and B cell counts; with C19 B cell count higher than CD3 T cell count).

It is worth emphasizing that the recurrent severe episodes of epistaxis were the foremost clinical manifestation. This problem triggered the extensive hemostatic evaluation and genetic testing. In our community, diagnostic exome sequencing provides about 50% yield in detecting inborn errors metabolism (36). Thus, this test was requested for his investigation. It is also worth noting that his hemostatic findings suggested variants in FERMT3 (see below).

The differential diagnosis of leukocytosis includes infection, leukemoid reaction [leukocytosis with circulating immature white and red blood cells (“left shift”), mimicking chronic myelomonocytic leukemia (CMML) and chronic myelogenous leukemia (CML)], and lymphoproliferation (including leukemia) (37). The eosinophilia may mimic atopy. The large thymus may mimic T cell lymphoma. Nevertheless, persistent marked elevations of granulocytes and lymphocytes suggest a leukocyte adhesion defect (8).

The differential diagnosis of impaired primary hemostasis (summarized in Table 2) includes von Willebrand disease, acquired platelet dysfunction (e.g., use of non-steroidal anti-inflammatory drugs), inherited platelet dysfunction [e.g., Glanzmann thrombasthenia (GT), Bernard-Soulier syndrome (BSS), ADP receptor defect, and gray platelet syndrome], and vascular anomaly (e.g., Ehlers-Danlos syndrome) (37). It is important to note, Glanzmann thrombasthenia is characterized by a diminished platelet aggregation with ADP, epinephrine, collagen, thrombin, and arachidonic acid, with normal agglutination with ristocetin. Bernard-Soulier syndrome, on the other hand, is associated with normal platelet aggregation with all agonists, except ristocetin. Diminished platelet aggregation in response to ADP alone is characteristic of ADP receptor defect. In gray platelet syndrome, platelet aggregation is variable and can be normal; however, thrombocytopenia is typically present (38).

Defects in natural killer cell migration, adhesion and activation have been also reported in LAD3 (39). Thus, this entity represents a multifaceted inborn error of immunity. This consideration is especially important for countries which implement universal BCG vaccination at birth, such as India and United Arab Emirates. Furthermore, defects in bone-resorbing osteoclast adhesion have been reported in LAD3 (32, 40), justifying its inclusion in the differential diagnosis of impaired bone homeostasis (8).

The expediency of his abnormal “platelet function screen” is evident. This test assesses primary hemostasis when the platelet count is normal, and has replaced the old “bleeding time.” Thus, the platelet function screen test is essential in the current hemostatic evaluation (41).

Treatment of LAD3 is supportive and includes topical thrombin in conjunction with an anti-fibrinolytic agent, tranexamic acid or aminocaproic acid. The response to anti-fibrinolytic agents should be observed, and the dose should be adjusted as necessary. Platelet transfusion is indicated in severe bleeding, or it can be administered weekly as prophylaxis when necessary (8). Precautions with regard to circumcision and skin and mucosal (including dental) hygiene are necessary. Prompt antimicrobial therapy in case of infection is also vital, particularly as fever and inflammatory biomarkers may not be present or prominent (42). Prophylaxis for pneumocystis pneumonia (typically with the use of trimethoprim/sulfamethoxazole) is needed (see Table 3).

In one report, platelet and granulocyte functions (including leukocytosis) and radiographic signs of osteosclerosis (osteopetrosis) were normalized after allogeneic hematopoietic stem cell transplantation (8). Thus, this therapeutic intervention is successful, especially if applied in infancy (43–46). Of note, our patient had no radiographic signs of osteopetrosis, as shown in Figure 1. Genetic prevention remains a foreseen goal.

As noted above, he had received the live vaccine BCG (Bacillus Calmette–Guérin). Leucocyte adhesion deficiencies are included in the list of inborn errors of immunity (IEI) that describes complications to the BCG vaccination in India (47). In a retrospective study, inborn errors of immunity were identified in 52 of 90 (58%) individuals who had a localized or disseminated BCG complication; these disorders included LAD1 (47). Although he had no evidence of BCG or TB (tuberculosis) disease, these complications (which are generally severe in infants) should be considered, especially in high TB burden countries such as India. Therefore, LAD3 should be added to the list of “defects in intrinsic and innate immunity” where BCG should be avoided as a precaution, and a prophylactic treatment for BCG disease should be considered (48, 49). Moreover, it is prudent to change the time of BCG administration from birth to 1 year of age, when most IEI become more recognizable (50). It is well to reemphasize that a cautious consideration of BCG complications in infants is necessary, especially in regions with a high prevalence of IEI such as UAE.

It is important to realize that leucocyte adhesion deficiencies are rarely considered in clinical practices, and are infrequently reported in the literature. They go underdiagnosed, mainly due to the infrequent genetic testing (51).

Table 3 compares the clinical findings of the studied toddler with a few of the previously reported children. Typically, these individuals show symptoms and are diagnosed in early infancy. Bleeding and granulocytosis are consistent findings. Infections are frequent and span a wide-spectrum of pathogens (bacterial and fungal), including Pneumocystis jirovecii, Acinetobacter baumannii, and Salmonella species (Table 3).

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Tawam Human Research Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

A-KS, RV, AY, SA-H, and LK: conceived, designed, and structured the report. RV: performed the variant analysis. A-KS, AY, AAA, HA, and AA: analyzed and interpreted the clinical data. AE: analyzed and interpreted the pathologic data. A-KS, AY, RV, and LK: wrote the original draft. A-KS, RV, and AY: reviewed and edited the paper. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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