2792 C. Lopez Rodriguez, A. Nueda, B. Grospierre et al. Eur. J. Immunol. 1993. 23: 2792-2798 z Cristina Lopez Rodriguezoo, Arsenio NuedaoA, Barbara GrospierreA, Francisco Shchez-Madrid., Alain Fischer., Timothy A. Springer. and Angel L. CorbiO Unidad de Biologia Molecularo and Secci6n de Inmunologia., Hospital de La Princesa, Madrid, INSERM zyxwvut U132, Hospital Necker Enfants Malades'., Paris and the Center for Blood Research, Harvard Medical School., Boston Characterization of two new CD18 alleles causing severe leukocyte adhesion deficiency* Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by heterogeneous mutations within the gene encoding the common zy fJ subunit (CD18) of the three leukocyte integrins LFA-1 (CDlldCDlS), Mac-1 (CDllb/CD18), and p150,95 (CDlldCD18). Based on the level of expression of CD18 on patient leukocytes, two phenotypes of LAD have been defined (severe and moderate) which correlate with the severity of the disease. We have investigated the molecular basis of the disease in two unrelated severe patients (HS and ZJO). Both patients share a complete absence of CD18 protein precursor and cell surface expression, but they differ in the level of CD18 mRNA, which is normal in HS and undetectable by Northern blot in ZJO. Determination of the primary structure of the patient HS CD18 mRNA revealed a 10-base pair deletion between nucleotides 190-200 (CD18 exon 3), which eliminates residues 41-43 and causes a frameshift into a premature termination codon 17 base pairs downstream from the deleted region. The 10-base pair frameshift deletion maps to a region of the CD18 gene where aberrant mRNA processing has been detected in HS and two other unrelated LAD patients. In the ZJO patient, amplification of lymphoblast CD18 mRNA demonstrated the presence of a non-sense mutation in the third nucleotide of the triplet encoding Cys534 (TGC zyx + TGA),within exon 12. Both genetic abnormalities were also detected at the genomic level, and affect the restriction pattern of their corresponding genes, thus enabling the detection of the mutant alleles among healthy heterozygous alleles in family studies. The identification of two new LAD CD18 alleles, either carrying a non-sense mutation (ZJO) or a partial gene deletion (HS), further illustrates the heterogeneity of the genetic alterations in LAD. 1 Introduction Cellular adhesion plays an essential role in the onset and regulation of immune and inflammatory processes. Most leukocyte adhesive functions are absolutely dependent on the expression and function of the leukocyte-specific heterodimers LFA-1 (CDllaKDlS), Mac-1 (CDllb/ CDlS), and p150,95 (CDllc/CD18), which comprise the p2 integrin subfamily [l]. Their essential role in leu- kocyte binding to and migration through the endothelial layer in inflamed tissues is illustrated by the existence of an immunodeficiency disorder termed leukocyte adhesion deficiency (LAD). LAD is an autosomal recessive disease caused by the deficient expression of the three p2 integrins on the cell surface of leukocytes [l, 21. LAD patients exhibit delayed umbilical cord separation, impaired pus [I 118701 * This work was supported by CICYT SAL89-0883, FIS 93/0134 and CAM 212/92 (to A.L.C.), NIH grant CA31798 (toT.A.S.), and Fundacion Ram6n Areecs. Recipient of a predoctoral fellowship from the Comunidad Autonoma de Madrid. A Recipient of a predoctoral fellowship from the Ministerio de Educaci6n. Correspondence: Angel L. Corbi, Unidad de Biologia Molecular (Planta 9). Hospital de la Princesa. Diego de Leon 62, E-28006 Madrid, Spain (Fax: 34 13 09 24 96) Abbreviation: LAD: Leukocyte adhesion deficiency Key words: Adhesion deficiency / 02 integrin / CD18 formation, and recurrent bacterial and fungal infections of soft tissues, as a consequence of the lack of neutrophil migration into sites of inflammation [1-41. Two clinical phenotypes of LAD (severe and moderate or partial), which normally correlate with the severity of the disease, have been defined according to the level of p2 integrin expression on patient leukocytes [3]. LAD is originated by heterogeneous mutations within the common 62 (CD18) subunit gene zyxw [S, 61, which is located on chromosome 21q22 [7].The diversity of the alterations has led to the definition of several types of LAD (I-V), whose classification is based on the size and levels of the CD18 subunit precursor, the CD18 messenger RNA, and the resulting phenotype [S]. Analysis of severe and moderate LAD CD18 alleles has identified aberrant splicing events [8-11], mis-sense muta- tions [9-151, and a one bp deletion [ 141within the structural region of the CD18 gene. The molecular analysis of LAD patients has provided insight into the structure-function relationships in p subunit integrins and may identify specific regions within the p2 integrin gene which are more prone to naturally ocurring genetic alterations. In the present report we describe the identification of two new types of genetic abnormalities leading to severe LAD, namely a 10-bp frameshift deletion (patient HS) and a non-sense mutation (patient ZJO). Interestingly, both LAD alleles alter the restriction pattern of the CD18 gene, facilitating their detection among healthy carriers. Our results expand the repertoire of genetic abnormalities causing LAD and further indicate that the heterogeneity of the molecular basis of LAD is comparable to that of other genetic diseases such as thalassemia and hemophilia. oO14-2980/~~/1111-2792$1O. zyxwvuts 00 + .25/0 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1993