Response: Adherence to the LAD variant form

The leukocyte adhesion deficiency type-1 (LAD-1)/variant syndrome was coined in 1997.¹  At the time, we designated the constellation of clinical symptoms and some laboratory findings a separate entity, and since then some 15 additional patients with this syndrome have been found. As described in our recent paper in Blood,²  the syndrome can be discriminated by increased levels of leukocytes (mostly granulocytes), the clinical presence of some degree of (hepato)splenomegaly, and overt bleeding tendency combined with a simple screening test for neutrophils.

Although all beta-integrins are normally expressed, the activation mechanism of beta-2 and beta-3 integrins is disturbed, with Rap1 a clear candidate for causative mutations. Whereas Rap1a is ubiquitously expressed, Rap1b is expressed in hematopoietic cells in particular. The reduced aggregation response in Rap1b^−/− mice was overcome at higher doses of collagen, which differs from the responses observed in the patients described, making us doubt whether Rap1 activation was really involved. This was subsequently shown by our in vitro experiments in a number of patients, with a normal Rap1 activation in neutrophils.² 

On the other hand, Alon et al initially described an Arab patient with defective Rap1 activation in an Epstein-Barr virus (EBV)–B-cell line upon stimulation with chemokines but with normal Rap1 activation when directly activated by phorbol ester.³  In 2 other patients the authors have now detected splice site mutations in CalDAG-GEF1.⁴  Although not determined, Rap1 activation is presumed to be again disturbed, in contrast to our own observations. Etzioni and Alon also refer to the paper of Crittenden et al, who showed platelet aggregation defects, and that of Bergmeier et al, who are now describing abnormal leukocyte mobilization in experimental inflammation models in CalDag-Gef1 knockout mice.^5,6  Thus, some of the patients with a clinical syndrome of a combined leukocyte and platelet adhesion defect may suffer from abnormalities in CalDAG-GEF1 regulation.

In our Turkish families, there are some (partly unpublished) results that are clearly different. First, we have found that none of the LAD-1/variant patients showed defective Rap1 activation.² 

Second, using both monoclonal antibodies as well as polyclonal antibodies (in fact, the same as used by Etzioni and Alon), we have detected normal levels of the CalDAG-GEF-1 protein with the expected size and no reduced or variant proteins of a smaller molecular weight in the lysates of EBV cell lines or neutrophils from the patients. This is in contrast with results obtained in the 2 patients described by Etzioni and Alon.

Third, we tested CalDAG-GEF1 as an obvious candidate gene in our patients about a year ago but could not detect any mutation in the gene encoding this key enzyme in Rap1 activation at the genomic or the cDNA level.

Finally, we have localized the defect in a different gene unrelated to Rap1 metabolism. In all our families there is a homozygous deletion currently under investigation. The small deletion we found in homozygous form in the patients and in heterozygous form in the parents was not present in more than 80 unrelated Turkish individuals tested thus far.

Together, this fits with the idea that the LAD-1/variant syndrome does not cluster with LAD-III.

Even though an update has been published on patient groupings (ESID Meeting, Budapest, Hungary, October 5-8, 2005), including the name “LAD-III” for all patients with beta-integrin activation defects, this is merely a reflection of the urge to make life easier for medical practice and clinical immunologists. At this moment, we believe that premature clustering of all these syndromes into one category would be a mistake, very much like the same misleading grouping nowadays recognized in another clinical defect known as common variable immunodeficiency (CVID).⁷ 

Therefore, we will refrain from the suggestion by Etzioni and Alon to cluster all beta-integrin activation defects until we fully understand the defect of our patients.