Leukocyte adhesion deficiency type III in an infant presenting with intestinal perforation and low percentage of natural killer cells: first case report from Iran

Abstract

Background

Leukocyte adhesion deficiency III (LAD III) is a very rare autosomal recessive primary immunodeficiency characterized by recurrent infections without pus formation and bleeding syndrome of Glanzmann-type and life-threatening infections. The main etiology of this condition is variations in the FERMT3 gene, which encodes kindlin-3, an integrin-binding protein. We present a toddler with unique symptom of intestinal perforation followed by prolonged bleeding due to Glanzmann-like thrombasthenia who was diagnosed as LAD-III.

Case presentation

This report presents a toddler with leukocyte adhesion deficiency type III (LAD III), who was diagnosed because of protracted surgical wound and gastrointestinal bleeding following surgery for small bowel perforation at the age of 16 months. The patient’s history was positive for febrile episodes after vaccinations, recurrent pulmonary infections, frequent severe epistaxis and ecchymotic purpuric lesions since early infancy. The presence of severe bleeding symptoms encouraged us to consider LAD III. Accordingly, sanger sequencing was performed which identified that the patient was homozygote for mutation in exon 14 of FERMT3 gene, the gene encoding for kindlin-3. Our patient also showed low percentages of CD16 and CD56 on peripheral blood flow cytometry, an unheard finding in LAD type III.

Conclusions

LAD III should be considered in differential diagnosis of any child with recurrent infections, persistent leukocytosis, and bleeding disorders. This is the first case of LAD III presenting with intestinal perforation. The present case also showed low percentage of natural killer cells which should be followed in further studies.

Introduction

Leukocyte adhesion deficiency type III (LAD III), fascinatingly named as “integrin activation defect in hematopoietic lineage cells” by Etzioni is an extremely rare type of LAD represented with defective activation of all beta integrins on leukocytes, lymphocytes and platelets [1]. Till now, less than 40 patients with LAD III have been reported worldwide in the literature [2, 3].

LAD III, initially named LAD I/variant is an autosomal recessive disorder due to mutations in the FERMT3 gene, encoding for kindlin-3 protein which acts as an important activator of integrins [4]. As a result, integrins on platelets and leukocytes could not be activated due to integrin signaling dysfunction that leads to recurrent bacterial infections and bleeding tendency at an early age [5]. Since very few cases of LAD III have been reported in the literature, characterization of new patients will disclose more phenotypes of this rare disorder. We present a toddler with unique symptom of intestinal perforation followed by protracted bleeding after surgery, due to Glanzmann-like thrombasthenia who was diagnosed as LAD-III confirmed by sanger sequencing analysis associated with low percentages of CD16 and CD56 on peripheral blood flow cytometry, an unheard finding in LAD type III.

Case presentation

A 16-month-old boy with normal growth and development for age was referred from department of Pediatric immunology for investigation of abnormal bleeding tendency following the surgery for small bowel obstruction. He was born at full term as the first child of consanguineous parents. The pregnancy and delivery were carried out without any complications. Family history was negative for any specific recognized disorder. The patient’s symptoms had started within a few days after vaccination at 4 months of age, when he developed fever. Thereafter, he was recurrently admitted with respiratory tract infections, during which profound unexplained leukocytosis and neutrophilia were the only remarkable findings on several complete blood counts. The patient’s medical history was remarkable for recurrent severe episodes of epistaxis and ecchymotic lesions over the trunk and extremities since early infancy. The parents mentioned that the umbilical cord was separated between 7 and 10 days of age. At the age of 10 months, he presented with bilateral hydrocele for which he underwent hydrocelectomy followed by bilateral scrotal hematoma which was absorbed very slowly. At the age of 16 months, he developed symptoms of small bowel obstruction. Non-enhanced abdominal CT-scan showed a dilated proximal loop and a collapsed distal loop of small bowel. On laparotomy, he was diagnosed to have perforated small intestine. He had longstanding surgical wound associated with gastrointestinal bleeding following the surgery; albeit without pus, for which he received packed cell transfusions along with intravenous tranexamic acid and even recombinant activated factor VII (rFVIIa) to achieve hemostasis.

He underwent a thorough immunological assessment for evaluation of recurrent pulmonary infections and persistent leukocytosis. Preliminary tests of immune function including specific antibodies to vaccines (tetanus, Haemophilus influenzae B, and pneumococcus), immunoelectrophoresis and lymphocyte transformation test (LTT) were performed which were within normal limits. T-cell receptor excision circle (TREC) assay indicated normal results in the patient. Peripheral blood flow cytometry showed normal total lymphocyte counts, normal subsets of T (CD3, CD4, and CD8) and B lymphocytes (CD19 and CD20) and CD4/CD8 ratio of 1.6; however, very low percentages of CD16 and CD56 were observed (Table 1). Flow cytometry with anti-CD11/CD18 monoclonal antibodies displayed normal expression (more than 97%) of CD11a, CD11b, CD11C and CD18 integrins. To investigate for bleeding disorders, coagulation screening tests including PT, INR, and aPTT were within reference intervals except for a prolonged bleeding time (12 min).

Table 1 Peripheral blood flowcytometry of the patient with LAD type III

Table 2 Platelet aggregation results of the patient with LAD-III

In view of severe bleeding tendency in the patient and prolonged bleeding time, platelet light transmission aggregometry (LTA) was performed by Lummi-aggregometer (Chronolog Ltd. USA) which showed significant abnormal results. Aggregation of platelets in response to all primary platelet agonists were impaired (Table 2). In LTA, a reversible response to ristocetin 1.5 mg/dL was found, however, it showed diminished one-wave response to different dilutions of ADP, collagen, epinephrin and arachidonic acid. ATP release test also showed diminished response to ADP (Figure 1).Since, the expression of platelet integrins 𝛼IIb (CD41a) and 𝛽3 (CD61) was observed to be normal by more than 90%, a Glanzmann-like disease was taken into consideration. Considering the recurrent pulmonary infections, marked leukocytosis, bleeding tendency with Glanzmann-like pattern on LTA and normal expression of platelet integrins αIIbβ3, the patient was determined to undergo genetic study with a high index of suspicion for LAD-III. PCR amplification followed by sanger sequencing was performed for the entire coding and splicing regions of the FERMT3 (NM_031471.6) gene (c.1717 C > T) on Ch 11 which detected a pathogenic variant (p.Arg573*) on exon 14 of FERMT3 gene in the homozygous state confirming the diagnosis of LAD-III (figure 2). The sequencing data of the patient can be reached at: https://ngdc.cncb.ac.cn/gsa-human, with the accession ID of HRA010821 and Project ID: PRJCA037528.

The patient was started on prophylactic antibiotics and received platelet transfusions along with rFVIIa to control gastrointestinal bleeding and oozing of the blood from the surgical wound. The patient is persistently symptomatic awaiting to receive a matched hematopoietic stem cell transplantation.

Fig. 1

Lumiaggregometry of the patient showing diminished one-wave response to different dilutions of ADP, collagen, epinephrin and arachidonic acid. ATP release test shows diminished response to ADP

Fig. 2

Sanger sequencing for the entire coding and splicing regions of the FERMT3 gene (c.1717 C > T) on Ch 11. A homozygous pathogenic variant (p.Arg573*) was detected on exon 14 of FERMT3 gene

Discussion

LAD syndromes comprise disorders in which a particular defect in adhesion molecules occurs. LAD I is caused by genetic defects resulting in lack of expression of β subunit of the integrins (CD18), while LAD II is due to absence of fucosylated carbohydrate ligands for selectins [1]. In addition, there is a kind of LAD I in which CD18 is normally or partially expressed, but is dysfunctional [6]. LAD III was first reported in 1997 by Kuijpers et al. in a Turkish child of consanguineous parents who presented with a mild LAD I phenotype and profound leukocytosis who later on expressed symptoms of increased bleeding tendency. β2 expression was reported normal in that case; however, a defect in adhesion was noted. Since no mutation was found in the coding gene for the β subunit of CD18, they designated their case as LAD I/variant [7]. In LAD III which is a combination of LAD I and Glanzmann-like disorder, the defective protein kindlin-3 due to FERMT3 mutations contributes to the failure of activation of β1 and β2 integrins on the surface of granulocytes and lymphocytes, and β3 integrin (αIIbβ3) on the surface of platelets [8]. In fact, the very rare patients with LAD III have normal expression of CD18, CD15a and even all integrins on leukocytes and platelets. As creatively described by Etzioni, LAD III is a hematopoietic lineage restricted inside-out signaling defect [1]. Although LAD III is an extremely rare syndrome, literature review shows more cases from middle east countries with Turkish, Maltese, Pakistani and Indian origins which could be explained by high rate of consanguinity in these countries [5]. The last case report is a two-year-old male infant from Saudi Arabia with a novel variant of FERMT3 Gene [9]. The presented case displayed petechial ecchymoses and recurrent severe epistaxis since early infancy, and scrotal hematoma and protracted surgical wound bleeding following surgery for intestinal perforation. Interestingly, the bleeding tendency in patients with LAD III seems to be more severe than in patients with Glanzmann thrombasthenia [2]. However, classic aggregometry does not discriminate between these two conditions. In a comparative study employing a new flow cytometry-based aggregation assay, platelets in Glanzmann (and not in LAD-III) bound collagen and formed small functional aggregates (collagen-induced aggregates). These aggregates required functional GPIa/IIa (integrin α2β1), which patients with LAD III did not have it, explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective [10].

In terms of immunodeficiency, patients with LAD III experience milder symptoms in comparison to LAD I and their infections are not as severe as of LAD I [11]. There is a report of a 15-month-old infant who was admitted with intestinal infection and underwent surgery because of intussusception. His abdomen wall wound did not heal and the infant died due to pulmonary hemorrhage [12]. We assume that the occurrence of intestinal perforation in our presented case might also have been due to septicemia that resulted in a non-healing surgical wound with protracted bleeding following laparotomy.

Another unrevealed rare manifestation of LAD III among the clinical spectrum of the disease is osteopetrosis which is related to the crucial role of Kindlin-3 in osteoclast-mediated bone resorption. In the absence of Kindlin-3, osteoclasts have impaired adhesion to the bone surface due to the defect in the formation of integrin-dependent adhesion molecules. A detailed search in the literature identified four cases of LAD III in infants of Turkish origin who had increased bone density on x-ray similar to that in patients with osteopetrosis [12]. All four patients also had some degrees of motor retardation and hepatosplenomegaly. Our case had normal developmental milestones. It can be hypothesized that differences in disease severity among LAD-III patients may be due to their genetic backgrounds and there may be small amounts of mutated protein in vivo depending on the nature of the Kindlin-3 mutation.

We detected low percentages of natural killer cells on peripheral blood flow cytometry that was reproducible on two occasions. Natural killer cell migration, adhesion and activation have been reported to be impaired in LAD III [8]. This finding should be considered in countries such as our country (Iran) which BCG vaccination is recommended at birth, and this overemphasizes the importance of neonatal screening programs for inborn errors of immunity. kindlin-3 lowers the threshold for NK cell activation and loss of kindlin-3 has a pronounced effect on NK cell–mediated cytotoxicity.

NK cell phenotype is characterized in a female baby with LAD-III deficiency who was homozygote for a stop codon mutation in kindlin-3. There was no kindlin-3 detected in her blood. The investigators verified that percentages of the NK cells of the LAD-III patient were similar to that of healthy controls. They also observed that NK cell cytotoxicity of LAD-III patients was impaired when involving CD16 [13]. We strongly suggest that NK cell enumeration and also function should be followed in future studies in patients with LAD III.

However, hematopoietic stem cell transplantation is potentially curative in LADs, but it has limitations because of donor availability, graft-versus-host disease, and graft failure. Gene therapy using autologous CD34 + cells transduced with a lentiviral vector carrying integrin beta-2 theoretically could restore CD18 expression and has been used in clinical trials in patients with LAD-I [14]. There are very few preclinical studies about safety and efficacy of lentiviral-mediated gene therapy for patients with LAD-I. Cristina Mesa-Nunez et al. first demonstrated that gene therapy effectively corrected the phenotype of mice with severe LAD-I [15]. There is not any report of employing gene therapy in patients with LAD-III in the literature so far.

Conclusion

LAD III is an extremely rare primary immunodeficiency syndrome which should be considered in differential diagnosis of any child with recurrent infections, persistent leukocytosis, and bleeding disorders. It can be anticipated that more unknown clinical phenotypes will be discovered in the future depending on the nature of the Kindlin-3 mutations. Considering low percentages of natural killer cell markers in this patient, further quantitative and functional studies on natural killer cell subsets are strongly suggested in patients with LAD type III.