Case Report: A Case of Leukocyte Adhesion Deficiency, Type III Presenting With Impaired Platelet Function, Lymphocytosis and Granulocytosis

Fermitin family homolog 3 (FERMT3), alternatively kindlin-3 (KIND3), is an integrin binding protein (of 667 residues) encoded by the FERMT3 gene. The molecule is essential for activating integrin αIIbβ3 (the fibrinogen receptor) on platelets and for the integrin-mediated hematopoietic cell (including platelets, T lymphocytes, B lymphocytes, and granulocytes) adhesion. Its defects are associated with impaired primary hemostasis, described as “Glanzmann's thrombasthenia (MIM#273800)-like bleeding problem.” The defects are also associated with infections, designated as “LAD1 (leukocyte adhesion deficiency, type I; MIM#116920)-like immune deficiency.” The entity that joins the impaired primary hemostasis with the leukocyte malfunction has been termed “leukocyte adhesion deficiency, type III” (LAD3, autosomal recessive, MIM#612840), representing a defective activation of the integrins β1, β2, and β3 on leukocytes and platelets. Here, we report a male toddler with novel compound heterozygous variants, NM_178443.2(FERMT3):c.1800G>A, p.Trp600* (a non-sense variant) and NM_178443.2(FERMT3):c.2001del p.*668Glufs*106 (a non-stop variant). His umbilical cord separated at about 3 weeks of age. A skin rash (mainly petechiae and purpura) and recurrent episodes of severe epistaxis required blood transfusions in early infancy. His hemostatic work-up was remarkable for a normal platelet count, but abnormal platelet function screen with markedly prolonged collagen-epinephrine and collagen-ADP closure times. The impaired platelet function was associated with reduced platelet aggregation with all agonists. The expression of platelet receptors was normal. Other remarkable findings were persistent lymphocytosis and granulocytosis, representing defects in diapedesis due to the integrin dysfunction. The natural history of his condition, structure and sequence analysis of the variations, and comparison with other LAD3 cases reported in the literature are presented.

Fermitin family homolog 3 (FERMT3), alternatively kindlin-3 (KIND3), is an integrin binding protein (of 667 residues) encoded by the FERMT3 gene. The molecule is essential for activating integrin α IIb β 3 (the fibrinogen receptor) on platelets and for the integrin-mediated hematopoietic cell (including platelets, T lymphocytes, B lymphocytes, and granulocytes) adhesion. Its defects are associated with impaired primary hemostasis, described as "Glanzmann's thrombasthenia (MIM#273800)-like bleeding problem." The defects are also associated with infections, designated as "LAD1 (leukocyte adhesion deficiency, type I; MIM#116920)-like immune deficiency." The entity that joins the impaired primary hemostasis with the leukocyte malfunction has been termed "leukocyte adhesion deficiency, type III" (LAD3, autosomal recessive, MIM#612840), representing a defective activation of the integrins β 1 , β 2 , and β3 on leukocytes and platelets. Here, we report a male toddler with novel compound heterozygous variants, NM_178443.2(FERMT3):c.1800G>A, p.Trp600 * (a non-sense variant) and NM_178443.2(FERMT3):c.2001del p. * 668Glufs * 106 (a non-stop variant). His umbilical cord separated at about 3 weeks of age. A skin rash (mainly petechiae and purpura) and recurrent episodes of severe epistaxis required blood transfusions in early infancy. His hemostatic work-up was remarkable for a normal platelet count, but abnormal platelet function screen with markedly prolonged collagen-epinephrine and collagen-ADP closure times. The impaired platelet function was associated with reduced platelet aggregation with all agonists. The expression of platelet receptors was normal. Other remarkable findings were persistent lymphocytosis and granulocytosis, representing defects in diapedesis due to the integrin dysfunction. The natural history of his condition, structure and sequence analysis of the variations, and comparison with other LAD3 cases reported in the literature are presented.
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.

CASE REPORT
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, Hemostatic evaluation was also requested because of the severe epistaxis. His platelet counts and secondary hemostasis FIGURE 1 | Findings on the skin and chest radiographs. Healed BCG site and dry skin with bruises and scars are evident. Prominent thymus (arrowed) at 8 mo of age is evident. Please also note the normal skeletal radiographs. [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 R PFA Collagen/EPI and Collagen/ADP Test Cartridges, Siemens Healthcare Diagnostics GmbH; Marburg, Germany) showed prolonged collagenepinephrine 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 R 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 IIb β 3 ( Table 1). Spontaneous platelet aggregation, a pathologic finding, was not present.
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) (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.

I. Basic hemostatic work-up
Platelet count Platelet function screen von Willebrand factor (vWF) antigen and activity aPTT, PT/INR, thrombin time, and fibrinogen II. Platelet aggregation studies (for an abnormal platelet function screen with normal vWF) Abnormal agglutination with "ristocetin" only → BSS Abnormal aggregation with "thrombin, ADP, collagen, arachidonic acid and epinephrine" → GT Abnormal aggregation with "ADP" only → ADP defect Abnormal aggregation with all agonists → LAD3
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).
Frontiers in Pediatrics | www.frontiersin.org 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 antifibrinolytic 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)(44)(45)(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).

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

ETHICS STATEMENT
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.