Journal of Clinical Immunology

, Volume 29, Issue 1, pp 117–122

The Clinical Spectrum of Leukocyte Adhesion Deficiency (LAD) III due to Defective CalDAG-GEF1

Authors

  • Sara S. Kilic
    • Department of Pediatric ImmunologyUludag University School of Medicine
    • Meyer Children’s HospitalRappaport Faculty of Medicine, Technion
Article

DOI: 10.1007/s10875-008-9226-z

Cite this article as:
Kilic, S.S. & Etzioni, A. J Clin Immunol (2009) 29: 117. doi:10.1007/s10875-008-9226-z

Abstract

Introduction

Leukocyte adhesion deficiency (LAD) type III is a rare syndrome characterized by severe recurrent infections, leukocytosis, and increased bleeding tendency. All integrins are normally expressed yet a defect in their activation leads to the observed clinical manifestations.

Materials and Methods

Less than 20 patients have been reported world wide and the primary genetic defect was identified in some of them. Here we describe the clinical features of patients in whom a mutation in the calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1 (CalDAG GEF1) was found and compare them to other cases of LAD III and to animal models harboring a mutation in the CalDAG GEF1 gene.

Discussion

The hallmarks of the syndrome are recurrent infections accompanied by severe bleeding episodes distinguished by osteopetrosis like bone abnormalities and neurodevelopmental defects.

Keywords

Leukocytesplateletsintegrinsadhesioninfectionsbleedingimmunodeficiency

More than 25 years ago a defect in the crucial adhesion cascade of leukocytes was described and designated as leukocyte adhesion deficiency (LAD), characterized by severe recurrent bacterial and fungal infections, delayed separation of the umbilical cord, defective wound healing without pus formation and markedly increased leukocyte count. LAD is an autosomal recessive genetic disorder caused by mutations in the ITGB2 gene which encodes for the β subunit of the integrin (CD18), leading to the inability of leukocytes to adhere to the blood vessel endothelium [1]. Several hundreds of patients have been described worldwide and in most of them no or markedly reduced expression of CD18 on leukocyte surface was noted. Rarely, mutations in the gene lead to a non-functional but expressed CD18 and these cases are referred to as LAD1/variant [2].

Ten years later a second syndrome, LAD II, has been described [3]. This rare condition (less than ten patients reported so far) is characterized by milder infectious episodes, severe psychomotor and growth retardation, the Bombay blood phenotype and marked leukocytosis. The syndrome is caused by a general defect in fucose metabolism due to mutations in the gene FUCT1, which encodes for the specific transporter of fucose from the cytoplasma to the Golgi apparatus where the incorporation of fucose to various glycoprotein occurs [4]. The defect leads to the absence of Sialyl Lewis X (CD15), the ligand for the selectin, essential for the first step in the adhesion cascade, the rolling phase [5].

Recently a third syndrome, LAD III, has been reported mainly in patients of Turkish origin [69]. They present with features of LAD I aggravated by severe bleeding tendency reminiscent of Glanzmann’s thrombasthenia. LAD III is caused by defects in the activation of β1, β2, and β3 integrins leading to both abnormal platelets aggregation and severe leukocyte adhesion dysfunction. In contrast to both LAD I and Glanzmann’s, integrins’ expression levels are normal. Due to the complexity of integrin signaling networks, the integrin activation defects associated with LAD III may be heterogenous and related to numerous genes, jointly required for normal intracellular integrin activation [10] (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10875-008-9226-z/MediaObjects/10875_2008_9226_Fig1_HTML.gif
Fig. 1

Inside–outside signaling of integrin activation

We previously described three LAD III patients singled by transcriptional and translational defects in calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1 (CalDAG-GEF1), an important Rap1 and integrin activator in hematopoietic cells [11]. In the current report the clinical characteristics of our patients are highlighted and compared with those reported in other, genetically distinct, LAD III cases and in recently published animal models with CalDAG-GEF1 deficiency.

Materials and Methods

Case 1 and 2

Two sisters born to second degree consanguineous parents (Fig. 2) shared a similar clinical presentation consisting of severe bleeding tendency and recurrent severe infections from early infancy (described in [11]). The family originated in Eastern Turkey. The older sister was first hospitalized at 8 months of age with anemia, bleeding tendency, and otitis media. She required multiple erythrocyte and platelet transfusions due to bleeding mainly from the oral cavity. She suffered from recurrent infectious episodes (pneumonias, otitis media, and sepsis) since birth. She had many bruises on her body following an uneventful delivery. Her umbilical cord detached at 20 days. Physical examination revealed mild hepatosplenomegaly. Her laboratory investigation showed leukocytosis with neutrophilia and anemia (Table I). Bone marrow aspiration was normal. Bone X-ray of the extremities demonstrated increased mineral density, very similar to those seen in patients with osteopetrosis. In her follow-up period, she required erythrocyte transfusions every 3 weeks due to bleeding attacks. She died of massive pulmonary bleeding at 15 months of age. Her sister was admitted with similar complaints from early childhood including many pulmonary infections and bleeding episodes. Her laboratory investigation detected major bleeding diathesis and severe leukocytosis (Table I). Platelet aggregation triggered by G protein-coupled receptor (GPCR) agonists (ADP, epinephrine, arachidonic acid, and thrombin) was completely absent in patient-derived platelets. Still, ristocetin stimulation resulted in almost normal aggregation [11]. Her skeleton survey showed increased bone density (Fig. 3). Genetic analysis revealed that she was homozygous for a splice junction mutation in the CalDAG-GEF1 gene (Fig. 4). She required erythrocyte and platelet transfusions every 3 or 4 weeks for 2 years. During the last 2 years she was hospitalized several times with pneumonias. Recently she underwent bone marrow transplantation.
https://static-content.springer.com/image/art%3A10.1007%2Fs10875-008-9226-z/MediaObjects/10875_2008_9226_Fig2_HTML.gif
Fig. 2

Pedigrees of three families with LAD-III patients

https://static-content.springer.com/image/art%3A10.1007%2Fs10875-008-9226-z/MediaObjects/10875_2008_9226_Fig3_HTML.gif
Fig. 3

Bone X-Ray of lower extremities showing increased density

https://static-content.springer.com/image/art%3A10.1007%2Fs10875-008-9226-z/MediaObjects/10875_2008_9226_Fig4_HTML.gif
Fig. 4

Chromatograms showing the mutation in the three patients

Table I

Laboratory Features of LAD-III Patients

Clinical data

Family 1

Family 2

Family 3

Pt 1 YB

Pt 2 AB

Pt 3 SA

Pt 4 MK

Age at onset

1 day

1 month

1 day

1 week

Age at diagnosis

15 months

1.7 years

2 months

7 months

Bleeding disorder

Severe

Severe

Severe

Severe

Umbilical cord detachment (day)

20th

20th

26th

7th

Weight

50p

50p

50p

<3p

Growth

50p

50p

90p

<3p

Neurological defect

Motor retardation

Mild motor retardation

Convulsion

Seizures

 

 

Motor retardation

Developmental delay

 

 

 

Motor retardation

Hepatosplenomegaly

Yes

Yes

Yes

Yes

Recurrent severe infections

Yes

Yes

Yes

Yes

Increase bone density

Yes

Yes

Yes

Yes

Laboratory data (earliest recorded)

Hb (g/dl)

8.7

9.7

9.9

7.3

Plt (× 109/L)

191

281

115

244

WBC (×109/L)

50.4

Ne %

40

34.1

Ne %

54

38.9

Ne %

22

75.7

Ne%

44

Ly %

50

Ly %

30

Ly %

58

Ly%

46

Mo %

10

Mo %

12

Mo %

16

Mo%

10

HSC transplant

No

Pending

No

No

Platelet aggregation response to ADP, epinephrine, and collagen

ND

Decreased

Decreased

Decreased

Lymphocyte subsets %, (absolute mm3)

CD3

35 (8820)

37 (6813)

53 (4535)

51 (17.759)

CD19

36 (9072)

38 (6997)

16 (1369)

25 (8705)

CD4

23.5 (5796)

22 (4051)

29 (2481)

21.7 (7312)

CD8

9.7 (2444)

12 (2209)

22 (1882)

23.2 (8009)

CD16+Cd56

17.2 (4284)

6.5 (1104)

27 (2310)

10.3 (3482)

Neutrophil gate (%)

CD18

98.4

99.7

97.3

97.5

CD11a

99

99.8

99.5

99.9

Outcome

Died at 15 months

Alive

Died at 7 months

Died at 1.5 years

Case 3

A healthy appearing male infant was delivered at 38 weeks gestation by vaginal delivery. He was the second product of second degree-related parents (Fig. 2). His birth weight was 2,450 g. At delivery, mucosal and skin bleeding was observed. He received erythrocyte and platelet infusions. His clinical features are presented in Table I. Laboratory investigation showed leukocytosis and Glanzmann disease like thrombasthenia. As of 2 weeks of age, he was frequently hospitalized because of bacterial and fungal sepsis episodes and died at 7 months of age. Quantitative PCR analysis showed very low levels of CalDAG-GEF1.

Case 4

A 6-month-old boy suffered from recurrent pneumonias and widespread petechiae on the skin since 1 week of age (reported in [11]). The patient presented with septicemia, oral candidiasis, failure to thrive, anemia, and marked leukocytosis (up to 80 × 109 WBC/I). He is the only child of healthy, consanguineous parents (Fig. 2). The family originated from Eastern Turkey and their medical history was unremarkable. A chest X-ray showed bilateral pneumonia and increased density was observed on his bone X-ray. Bone marrow aspiration demonstrated hypercellular bone marrow (Table I). Platelet aggregation response to platelet agonists was markedly reduced as in case 2. Several CalDAG-GEF1 transcripts were nearly completely absent from the blood. Quantitative PCR analysis showed very low levels, (>30-fold lower) of CalDAG-GEF1 compared to controls [11]. The patient was homozygous for markers localized at the CalDAG-GEF1 chromosomal locus11q13.1 and identical mutation as in family 1 was found [11] (Fig. 4). He was hospitalized many times for pneumonia, sepsis, or bleeding. At 15 months the patient was admitted with intestinal infection and was operated because of intussusception. His abdomen wall wound did not heal (Fig. 5) and he died after a long hospitalization due to pulmonary hemorrhage.
https://static-content.springer.com/image/art%3A10.1007%2Fs10875-008-9226-z/MediaObjects/10875_2008_9226_Fig5_HTML.jpg
Fig. 5

Unhealed abdominal operation wound

Discussion

Leukocyte arrest at target endothelial sites is nearly exclusively mediated by integrin receptors expressed on all circulating hematopoetic cells. These maintain their integrins (β1 and β2) in a generally non-adhesive state that can be rapidly activated subject to interactions with various agonists, predominantly chemoattractants and chemokines presented by the endothelium [10]. Likewise, platelets maintain their major fibrinogen receptor, the integrin αII3 in an inactive conformation, which is converted by chemokines which bind to GPCR [12]. The small GTPase Rap-1 has been implicated as the major intracellular activator of integrins both in leukocytes and platelets. A crucial molecule for Rap-1 activation is CalDAG-GEF1, which was found to be defective in our patients. It belongs to the CalDAG-GEF/Ras GRP family of intracellular signaling molecule with a guanine nucleotide exchange factor (GEP) domain that catalyzes the exchange of GTP for GDP bond to Rap1 [10].

The significance of vascular integrins has been demonstrated by numerous studies in murine knock out models as well as in rare human LAD syndromes.

In LAD I severe, life-threatening bacterial infections occur subject to mutations in the gene encoding β2 integrin. Another genetic disorder, called Glanzmann’s thrombasthenia is associated with mutations in the gene encoding the β3 integrin, resulting in dysfunctional platelet aggregation with severe bleeding tendency [13].

LAD III, which is due to abnormal activation of all integrins is characterized by both severe infections and recurrent bleeding episodes. We believe that on clinical ground alone one can accurately differentiate the three LAD syndromes (Table II).
Table II

Leukocyte Adhesion Deficiency Syndromes

 

LAD I

LAD II

LAD III

Clinical manifestation

Number of cases reported

Hundreds

Less than 10

10–20

Consanguinity rate

++

++

++

Recurrent severe infections

+++

+

+++

Leukocytosis

+++

+++

+++

Delayed separation of the umbilical cord

+++

++

Growth retardation

+++

+

Bleeding tendency

+++

Laboratory findings

CD18 expression

↓↓↓ or absent

N

N

SLeX expression

N

absent

N

Integrin defect in

β2

N

β1β2β3

Neutrophil rolling

N

↓↓↓

N

Neutrophil adherence

↓↓↓

↓↓↓

Platelets aggregation

N

N

↓↓↓

Primary genetic defect

ITGB2

FUCT1

Ca1DAGGEF1 4/13 of cases

We have previously shown that Rap1 activation is defective in some cases of LAD III [8] leading to integrin dysfunction. The high incidence of infections in this syndrome is mainly due to the leukocyte adhesion dysfunction. It has been also shown that defects in NADPH oxidase, observed in LAD III [11], may also contribute to susceptibility to infections.

Recently Kuijpers et al. [14] described nine patients with LAD III (referred to them as LAD I/variant). All of them originated from the Anatolia area in Turkey, while our patients are from Eastern Turkey. While recurrent infections, bleeding tendency, leukocytosis, and defective integrins activation were reported for these patients, their clinical course seems to be milder and the majority of patients remained alive even without bone marrow transplantation and no mutations in CalDAG-GEF1 were found [14]. Thus, as previously proposed [9] different defects in the intracellular activation of both platelets and leukocytes integrins can lead to LAD III syndromes with differences in the clinical presentation [15].

CalDAG-GEF1 appears to be specifically expressed within the hematopoietic system as well as in neurons, especially in the stratum of the basal ganglia [16]. In addition to the hematopoietic dysfunctions, other non-hematological features mark our patients.

Although the development milestones were found to be retarded in all patients they all showed normal cognitive and social skills. Two of them had epileptic activity and required antiepileptic treatment. The epileptic activity was not correlated to brain compression due to bleeding or subdural effusion. CalDAG-GEF1 protein levels in patients’ nerves were never tested and we cannot thus establish whether the expression level of the GEF in the nervous system is equally reduced as in hematopoietic cells or to a lesser degree subject to differences in mRNA processing and translation between nerve and blood cells. Whether reduced CalDAG-GEF1 levels in patient neutrons could affect neuron function remains to be determined. Future studies are needed to address multiple potential roles of CalDAG-GEF1 as a Rap-1 effector and integrator of integrin activation events in neurons.

Notably, all four patients had increased bone density on X-ray similar to that seen in patients with osteopetrosis. Mutations in several genes, including the gene encoding receptor activator of nuclear factor-kb ligand (RANKL), a crucial osteoclast growth factor involved in osteoclast differentiation and function, were identified in patients with osteopetrosis. Osteoclasts are hematopeitic derived cells of myeloid origin [17]. It has been shown that once RANKL starts osteoclast differentiation the αvβ3 integrin on osteoclasts mediates key matrix interactions essential for osteoclast maturation [18]. The large similarity between the platelet integrin GpIIb3 and the osteoclast αvβ3 suggests that activation of the alfavbeta3 integrin is both Rap-1 and CalDAG-GEF1 dependent and is therefore defective in the LAD III cases identified by us. Loss of osteoclast-matrix adhesion may impair osteoclast bone resorbing activities and explain the increased bone density measured in our LAD III cases.

Recently, several animal models of LAD III have been reported. Knock out CalDAG-GEFI deficient mice exhibit the same platelet and neutrophil adhesion defect [19]. Still no spontaneous bleeding or apparent infections in these mice were observed [19]. It should be noted that the mice were kept in a restricted pathogen-free lining space. Furthermore no abnormalities in bone density on X-ray were observed in the CalDAG-GEF1 knock out mice (Graybiel AM., personal communication).

Mutations in CalDAG-GEF1 gene were also reported in calf and dogs [20, 21]. Interestingly, in these two animal models, only platelet aggregation defect was found while leukocyte count was normal and no neutrophil adhesion defect was observed (Boudreaux MK., personal communication). As in our patients, the main cause of death was due to bleeding episodes. It appears that CalDAG GEF1 is indispensable for platelets aggregation in all species, while several other Rap-1 pathways may substitute for loss of CalDAG-GEF1 in leukocytes in different species. Interestingly, murine lymphocytes lack CalDAG-GEF1 in contrast to human lymphocytes [10] and thus, the dependence on this GEF for normal leukocyte integrin activation may be particularly high in humans.

Acknowledgement

We would like to thank Prof. Alon for critically reviewing the paper and to Prof. Rechavi’s group for helping in the genetic analysis of the families.

Recently we found that these patients have also mutation in Kindlin 3 (Blood Accepted).

Copyright information

© Springer Science+Business Media, LLC 2008