Molecular characterization of leukocyte adhesion deficiency-I in Indian patients: Identification of 9 novel mutations

doi:10.1016/j.bcmd.2015.01.012

Blood Cells, Molecules, and Diseases

Volume 54, Issue 3, March 2015, Pages 217-223

https://doi.org/10.1016/j.bcmd.2015.01.012 Get rights and content

Abstract

Purpose

Leukocyte adhesion deficiency type-I (LAD-I) is caused by mutations in the ITGB2 gene, encoding the β₂-subunit of β₂-integrin (CD18) which leads to markedly reduced expression of CD18 on leukocytes resulting into recurrent life threatening infections. Here we aim to identify the molecular defects underlying LAD-I in Indian patients and correlate with the clinical presentation.

Methods

Blood was collected from 30 patients and their parents for absolute neutrophil count, expression of CD18 and CD11 by flow cytometry and DNA extraction. PCR and DNA sequencing of the ITGB2 gene was done for mutation characterization.

Results

Phenotypically, 22 patients were LAD-I⁰, 1 was LAD-I⁻ and 7 were LAD-I⁺ showing no expression and reduced expression of CD18 respectively. Nine novel mutations in 15 patients and 11 known mutations in 16 patients were detected. Prenatal diagnosis was performed for 5 families.

Conclusion

In this study 30 patients were phenotypically and genotypically evaluated for a less known disease LAD-I. Unavailability of curative options to majority of the patients and high cost of supportive care emphasize the need to increase awareness about a suspicious case so that timely management can be given to the patient and prenatal diagnosis can be offered to their families.

Introduction

Leukocyte trafficking mediated by adhesive interactions between myeloid leukocytes and inflamed endothelial cells (EC) is critical for defense against bacteria and wound healing. Primary immune deficiencies associated with defects of leukocyte trafficking are categorized under leukocyte adhesion deficiency (LAD). Three distinct forms of this group of disorders have been described: LAD- I, II and III (also described as LAD I variant). LAD-I is a rare form of autosomal recessive disorders caused by mutations of the gene ITGB2, encoding or the common β-chain of the β₂ integrin family, designated as CD18 [1], [2], [3], [4]. The β₂ integrins is a family of four heterodimeric proteins composed of α and β subunits. The four α subunits (CD11) coupled to common β₂ subunit forms four heterodimer proteins: LFA-I(α_Lβ₂ or CD11aCD18), MAC-1(α_Mβ₂ or CD11bCD18), p150,95 (α_Xβ₂ or CD11cCD18) and CR4 (α_Dβ₂ or CD11dCD18). β₂ subunit is a common protein and defect in its expression results in similar decrease in the expression of α subunit of integrins [3], [4], [5], [6]. They are expressed selectively by leukocytes and mediate leukocyte adhesion to the endothelium and their migration to sites of inflammation. LAD-I is the commonest form of LAD syndromes and till date more than 500 cases have been reported. Clinically, LAD-I is characterized by leukocytosis, recurrent infections, omphalitis with delayed cord separation, impaired pus formation and slow wound healing.

Study of CD18/CD11 expression pattern can help in differentiating various phenotypes of LAD-I such as LAD-I⁰ for < 5% expression, LAD-I⁻ for diminished expression i.e. between 5% and 20% and LAD-I⁺ for normally present but nonfunctional [7]. Generally, patients with LAD-I⁰ phenotype are more susceptible to severe infections leading to death in infancy whereas patients with LAD-I⁻ and LAD-I⁺ often survive till adulthood [7].

The defect in expression pattern is directly linked to the type of mutations in the ITGB2 gene. Majority of the point mutations are reported in the ~ 240 residue domain, which is highly conserved in all β₂ integrin subunits and coded by exons 5 to 9 of ITGB2 gene, whereas others are scattered throughout the gene [7], [8], [9]. So far, 76 different allelic mutations have been described [7]. All reported mutations are systematically characterized for the LAD-I patients in western countries and few of Asian countries [7], [8], [9], [10], [11]. Apart from few case reports on clinical manifestations [12], [13], [14], [15], [16], [17] there is paucity of data on the mutation pattern from India. Molecular characterization is required for confirmation of diagnosis in patients with LAD-I and also during prenatal diagnosis. Thus the aim of our study was to delineate the molecular defects in ITGB2 gene in LAD-I and correlate with the clinical and immunophenotypic manifestations in these patients.

Section snippets

Patients

Thirty patients with LAD-I included in this study were evaluated over a period of 5 years. They were referred to our institute from different parts of India for the primary immunodeficiency workup based on their clinical manifestations. These patients were unrelated and belong to different communities. At initial diagnosis, detailed clinical history of patients was recorded. The study was approved by the Ethical Committee of National Institute of Immunohaematology. After an informed consent from

Clinical characteristics

Clinical characteristics of 30 LAD-I patients are depicted in Table 1. In 22 patients LAD-I⁰ phenotype was seen with median age at presentation being 10.5 days whereas LAD-I⁻ phenotype was seen in 1 patient diagnosed at the age of 4 months and LAD-I⁺ phenotype was seen in 7 patients diagnosed between 20 days and 16 years of age. In LAD-I⁰ phenotype clinical examination showed omphalitis as one of the most common feature with delayed cord separation usually after the 14th day of life. Recurrent

Discussion

This is a study of clinical and molecular features of 30 LAD-I patients from India diagnosed over a period of five years. LAD-I is a rare autosomal recessive disorder affecting about 1 per 10 million individuals with no described racial or ethnic predilection [24]. However due to high degree of consanguineous marriages, it is one of the commonest severe primary immunodeficiencies reported from India [25]. Consanguineous marriage was reported in 41.4% of the cases in the present study without

Conclusion

This is a study from India where 30 patients were clinically, immunophenotypically and genotypically evaluated for a disease like LAD-I which is not much reported from a vast country like India. However, with the improvement in the medical facilities more and more cases will be seen. There is a need to increase awareness among the primary health care providers and the pediatrician about a suspicious case so that timely management can be given to the patient and prenatal diagnosis can be offered

Contributor's statement

MM* and KG conceptualized the paper. KI, MG, SC, AM, MR and SM helped in laboratory assessment of the patients, flow cytometry and molecular characterization. MM*, KI and SC prepared the manuscript. MD, MM, SS, DS and AA helped in clinical diagnosis and management of the patients. Finally the manuscript has been approved by all.

(MM*—Manisha Madkaikar)

This work was supported by the Indian Council of Medical Research (61/1/2007/BMS).

Acknowledgment

Authors want to acknowledge Dr. Sheela Namboodari, Dr. D. Kaul from Sir Gangaram Hospital, New Delhi, Dr. J. Deepika from Kanchi Kamakotti Hospital, Cochin and Dr. Pritesh from Aditya Hospital, Hyderabad, for referring these patients to us for diagnosis.

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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 β subunit (CD18) of the β₂ 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 Le^a and Le^b blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed β 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 β integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.
Leukocyte adhesion deficiency type1 in Algeria
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Patients 3 to 11 belong to four unrelated families and have the same exonic mutation represented by c.562C > T, also in exon 6 which is the replacement of Arginine at position 188 by Stop codon. This mutation has been already reported in patients from different ethnic origin (Madkaikar et al., 2015). In the patient P12, we identified a novel homozygous nonsense mutation c.1401C > A.
Leukocyte adhesion deficiency type I (LAD-I) is an autosomal recessive disease characterized by impaired leukocyte diapedesis to sites of infection. The disorder is caused by mutations in the ITGB2 (integrin beta-2) gene. Herein, we report the first series of Algerian patients with LAD-I. In this study, we enrolled twelve patients. All suffered from the severe form of the disease, with no significant delay in umbilical cord separation, except for two patients, however omphalitis was present in most cases. The analysis of the spectrum of mutations showed three different homozygous mutations, two were known mutations (missense mutation: c.533C˃T and nonsense mutation: c.562C > T) and one was novel nonsense mutation (c.1401C > A). Thus, the three mutations affect CD18 and are responsible for the severe form of the disease.
β2 Integrin Gene (ITGB2) mutation spectra in Pakistani families with leukocyte adhesion deficiency type 1 (LAD1)
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It is expected because due to absence of adhesion molecules, neutrophils fail to migrate to site of infection (Wada et al., 2011). Similar conditions in LAD1 patients were reported previously in other populations (Tipu, 2017; Nigar et al., 2018; Bashir et al., 2018; Yassaee et al., 2016; Madkaikar et al., 2015). Due to timely referral of the patients by physicians for proper PID workup, diagnosis of the disorder was made between one to thirty months in our 12 patients.
Leukocyte adhesion deficiency I (LADI) is an autosomal recessive type of primary immunodeficiency characterized by occurrence of repeated bacterial infections, impaired pus formation and wound healing. Genetic variations in the β-2 integrin subunit encoding gene ITGB2 have been implicated in causing the disorder. In the present study, we have investigated twelve patients presenting LAD1 features. After collecting clinical and family history, flow cytometry was used to determine levels of CD18 in the patients. Clinical history revealed that umbilical cord separation occurred mostly after 19 days in the patients. Recurrent skin infections were found in seven patients. Eight patients had at least one elder sibling who died due to repeated infections. All patients had marked neutrophilia with only 0.77% of neutrophils expressing CD18. Total 12 patients suffering from LAD1 were Sanger sequenced for ITGB2 gene. Five variants, including a novel p.(Cys286Phe) and four previously reported [p.(Gly273Arg), p.(Asp128Tyr), p.(Cys62*), IVS7 + 1G > A] were identified in 8 cases, while no pathogenic variant was observed in remaining four cases. This study represents the first comprehensive clinical and genetic characterization of LAD1 in Pakistani population. This will facilitate diagnosis and genetic counselling of patients with immunodeficiency disorders in Pakistani population.
A novel mutation of the ITGB2 gene in a Chinese Zhuang minority patient with leukocyte adhesion deficiency type 1 and glucose-6-phosphate dehydrogenase deficiency
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To explore the clinical and molecular characteristics of a Chinese Zhuang minority patient with leukocyte adhesion deficiency type-1 (LAD-1) and glucose-6-phosphate dehydrogenase deficiency (G6PDD).
Routine clinical and physical examinations were performed, and patient data was collected and analyzed. Protein expression levels of Itgb2 and glucose-6-phosphate dehydrogenase (G6pd) proteins were assessed by flow cytometry and the glucose-6-phosphate (G6P) substrate method, respectively. Whole exome sequencing was performed to investigate genetic variations of the patient and his parents.
The patient had fester disease and delayed separation of the umbilical cord at birth. Staphylococcus was detected in the fluid secretion of the auditory meatus of the patient. He exhibited a recurrent cheek scab, swollen hand, and swollen gum. Hematological examination indicated dramatic elevation of leukocytes including lymphocytes, monocytes, neutrophils and eosinophils. A novel homozygous mutation was detected in the ITGB2 gene of the patient, which was determined to be a two nucleotide deletion at the site of c.1537–1538 (c.1537–1538delGT), causing a frameshift of 24 amino acids from p.513 and inducing a stop codon (p.V513Lfs*24). A base substitution mutation was identified at c.1466 (c.1466G>T) of G6PD on chromosome X of the patient, which resulted in an amino acid change from arginine to leucine at p.489 (p.R489L). The patient also showed deficient lymphocyte expression of CD18 (2.99%) and significant downregulation of the G6pd protein.
The patient was diagnosed with G6PDD and moderate LAD-1. The combination of LAD-1 and G6PDD in this case may have been due to the high incidence of genetic disease in this minority ethnic population. Analyzing existing LAD-1 and G6PDD cases from different populations can facilitate disease diagnosis and treatment. Particularly, reporting pathogenic mutations of LAD-1 and G6PDD will be crucial for genetic testing and prenatal diagnosis in an effort to decrease the incidence of these diseases.
Mutation spectra of the ITGB2 gene in Iranian families with leukocyte adhesion deficiency type 1
2016, Human Immunology
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Among the different diagnosis approaches, molecular analysis of the ITGB2 gene is very crucial for confirmation of diagnosis, carrier testing and treatment decisions, which can decrease the percentage of patients who suffer from LAD1. ITGB2 gene mutations are heterogeneous and majority of them are located in highly conserved domain, Von Willebrand Factor type A (VWFA), of β2 integrin subunit [4,5,24]. This highly conserved domain is coded by exons 5–9 of the gene [17] and required for the common enzyme activity.
Leukocyte adhesion deficiency type 1 (LAD1) is an autosomal recessive disorder clinically characterized by severe, recurrent bacterial infections, impaired pus formation and wound healing. It is caused by mutation in the ITGB2 gene, encoding the β₂ integrin subunit of the leukocyte adhesion cell molecule. This study aimed to identify disease causing mutations in 19 consanguineous families diagnosed with LAD1.
Blood samples were collected after informed and written consent was obtained. Genomic DNA was extracted from peripheral blood of patients and their parents. PCR amplification of the ITGB2 gene was done using specific primers followed by sequencing for mutation detection.
A total number of 14 alterations scattered throughout the ITGB2 gene were ascertained in which 10 mutations were previously reported, including c.329−6C>A, c.382G>T, c.715G>A, c.843delC, c.897+1G>A, c.1062A>T, c.1143delC, c.1877+2T>C, c.1907delA and c.2147G>C. Four novel likely pathogenic mutations consisting of c.576dupC (Asn193GlnfsX72), c.706G>A (Gly236Arg), c.897+1G>T and c.1030G>T (Glu344^∗), were identified. The majority of these mutations were located in exon six, suggesting this exon as a hotspot region probably.
This study emphasis on allelic heterogeneity of the ITGB2 gene in Iranian patients diagnosed with LAD1. Our results suggest that every population should develop a mutation database for rare genetic disorders to take advantage in genetic counseling clinic as well as genetic testing for rapid diagnostic purposes.
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View all citing articles on Scopus

View full text

Molecular characterization of leukocyte adhesion deficiency-I in Indian patients: Identification of 9 novel mutations

Abstract

Purpose

Methods

Results

Conclusion

Introduction

Section snippets

Patients

Clinical characteristics

Discussion

Conclusion

Contributor's statement

(MM*—Manisha Madkaikar)

Acknowledgment

Exp. Hematol.

Blood Cells Mol. Dis.

Curr. Opin. Immunol.

J. Biol. Chem.

Blood

Biochem. Biophys. Res. Commun.

An inherited abnormality of neutrophil adhesion: its genetic transmission and its association with a missing protein

N. Engl. J. Med.

Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis

J. Exp. Med.

The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features

J. Infect. Dis.

Leukocyte adhesion deficiency and other disorders of leukocyte adherence and motility

Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response

Immunol. Rev.

Regional localization of CD18, the beta-subunit of the cell surface adhesion molecule LFA-1, on human chromosome 21 by in situ hybridization

Ann. Hum. Genet.

Hematologically important mutations: leukocyte adhesion deficiency (first update)

Blood Cells Mol. Dis.

Clinical and laboratory findings in Iranian patients with leukocyte adhesion deficiency (study of 15 cases)

J. Clin. Immunol.

A novel point mutation in CD18 causing leukocyte adhesion deficiency in a Chinese patient

Chin. Med. J.

Leucocyte adhesion defect-1

Indian Pediatr.

Amyloidosis in a child with leucocyte adhesion deficiency type-1: an unusual association

Indian J. Pediatr.

Clinical profile of leukocyte adhesion deficiency type 1

Indian Pediatr.

Leukocyte adhesion deficiency: a case report and review

J. Dent. Child. (Chic.)

Leukocyte adhesion defect type 1 presenting with recurrent pyoderma gangrenosum

Indian J. Dermatol.