An Initiation Codon Mutation in CD18 in Association with the Moderate Phenotype of Leukocyte Adhesion Deficiency*

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by mutations in the CD18 gene which codes for the Bz integrin subunit. We studied two patients, the first of which had a moderate LAD phenotype and expressed only 9% of CD11/CD18 on blood leukocytes. RNA from lymphoblasts was re-verse-transcribed, and the cDNA was amplified, cloned, and sequenced. An ATG to AAG alteration in the initiation codon was detected in 39 of 45 (87%) cDNA clones. This mutation was detected in the father, but not in the mother. The maternal defect was shown to be a frameshift mutation with the deletion of a single T in the aspartic acid codon at position 690 (GAT), 11 amino acids N-terminal to the beginning of the transmembrane domain. This mutation predicts a polypep-tide which would terminate without transmembrane or cytoplasmic domains. The frameshift mutation was also found in the second patient who had the severe phenotype of LAD ( ~ 1 % of CDll/CDlS), indicating that this allele does not encode a functional protein. The partial expression in the patient with a moderate phenotype must be derived from the initiation codon mutation and may be due to a low level of initiation of translation of the CD18 mRNA at the second codon

Leukocyte adhesion deficiency (LAD)' is an autosomal recessive disease caused by deficient expression of the p2 integrins (or CD11/CD18 glycoprotein complexes) on the surface of leukocytes. The characteristic features of LAD are delayed umbilical cord severance, poor wound healing, lack of pus formation, persistent leukocytosis, recurrent soft tissue and periodontal infections, and a high risk for developing recurrent life-threatening bacterial and fungal infections. Severe and moderate phenotypes of LAD have been described in which the severity of clinical infections or other complications is directly related to the degree of p2 integrin deficiency (1). Severe LAD patients are at high risk for systemic and * This work was supported in part by United States Public Health Service Grants AI 23521, AI 19031, and DE09079. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. often life-threatening infections in infancy as a consequence of an almost total deficiency of CDll/CD18 complex expression on the blood leukocytes. Somewhat higher levels of CD11/CD18 complex expression on leukocytes of moderate LAD patients (2.5-10% of normal levels) account for their less severe clinical presentation and frequent survival into adulthood.
The p2 integrins include LFA-1, Mac-1, and p150,95. They are heterodimeric glycoproteins composed of a common 95,000-dalton @ subunit encoded by the CD18 gene in a noncovalent association with one of three distinct 01 subunits encoded by the CDll gene family. LAD is caused by heterogeneous mutations in the common 0 subunit (CD18) of the leukocyte integrins. Although LAD is inherited as a single gene defect in the p subunit, there is a secondary absence of 01 subunits. Biosynthesis of 01 subunits in LAD cells is normal (2, 3), but they are not expressed on the cell surface because the p subunits are defective. A deficiency of CD11/CDl8 complexes on the surface of the LAD leukocytes accounts for a variety of functional impairments in uitro, including abnormalities of adhesion-dependent chemotaxis, aggregation, phagocytosis of iC3b-opsonized particles, complement or antibody-dependent cytotoxicity, and transendothelial migration (4, 5).
We have investigated the molecular nature of the p subunit defect in two LAD patients to further understand the basis for the two clinical phenotypes. We identified an initiation codon mutation and a frameshift mutation in a patient with a moderate phenotype. The frameshift mutation was also present in a patient with a severe phenotype.

EXPERIMENTAL PROCEDURES
Patient Material-Epstein-Barr virus-transformed lymphoblasts were obtained from a male who was described in 1979 as the 9-yearold son of healthy unrelated parents (6). He had chronic otitis media, gingivitis, periodontitis, and severe skin lesions which contained Staphylococcus aureus and very few neutrophils even though the patient had chronic leukocytosis. Functional defects in neutrophils and the patient's clinical presentation were consistent with the subsequent diagnosis of LAD (patient 10 in Anderson and Springer (7)). The patient was classified as having the moderate form of the disease based on the severity of the clinical symptoms and the presence of approximately 9% of normal levels of CD11/CD18 complexes on the surface of f-Met-Leu-Phe-stimulated blood neutrophils as assessed by immunofluorescence flow cytometry (1). These findings were verified by immunoprecipitation of only trace amounts of a normal sized CD18 precursor protein from cell lysates and by diminished amounts of a normally sized mRNA by Northern blot analysis (patient ll in Kishimoto et ai. (2)). The severe patient is a previously unreported 5-year-old male whose clinical presentation and leukocyte studies are consistent with the severe phenotype.
Immunofluorescent Analysis-Indirect immunofluorescent analysis was performed on patient lymphoblasts with a FACScan (Becton, Dickinson & Co., Mountain View, CA) using the anti-CD18 antibody 714 CD18 Mutations 715 IB4 (generously provided by Samuel D. Wright, Rockefeller University) conjugated to biotin as described previously (8).
Isolation of RNA and the Amplification of CD18 cDNA for Cloning-Total RNA was isolated from lymphoblasts from the patients, their parents, and healthy controls (9).
A 30-pg aliquot of RNA was incubated with 4 pg of oligo(dT)12-1e (Pharmacia LKB Biotechnology Inc.) a t 55 "C in a total volume of 24 pl of H 2 0 for 3 min, and first strand cDNA was synthesized in a final volume of 40 p1 and contained the following: 0.1 M Tris-HCI (pH 7.5), 10 mM dithiothreitol, 1 mM each dNTP, 1.4 units of RNasin (Promega Biotec, Madison, WI), and 36 units of avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Petersburg, FL). After 45 min a t 42 "C, an additional 36 units of reverse transcriptase was added followed by an additional incubation of 45 min. The RNA was hydrolyzed by the addition of 60 pl of 0.7 M NaOH containing 40 mM EDTA and incubation a t 65 "C for 10 min. The cDNA product was precipitated with ethanol and resuspended in 100 pl of water prior to amplification.
The cDNA for CD18 was amplified using the polymerase chain reaction (PCR) with primers that flank the coding region of the molecule (Fig. 1). These primers included restriction sites a t their 5' end for easy cloning into an M13 vector. PCR reactions were in a volume of 50 p1 and contained the following: 6.7 mM MgC12, 67 mM Tris-HCI (pH 824, 10 mM &mercaptoethanol, 10% (v/v) dimethyl sulfoxide, 170 pg/ml bovine serum albumin, 1 mM each dNTP, 16 p1 of cDNA product, 0.56 mM each primer, and 5 units of Amplitaq" polymerase (Perkin-Elmer-Cetus). The PCR reactions were carried out for 25 cycles using a denaturation step a t 94 "C for 1 min, an annealing step a t 55 "C for 2 min, and an extension step a t 72 "C for 4 min. The amplified fragment was collected by ethanol precipitation, digested with XbaI and HindIII, isolated from low melting agarose, and directionally cloned into M13mp18 cut with the same enzymes. For PCR analysis of M13 phage clones, individual phage plaques were touched with a toothpick and the toothpick dipped into the reaction mix to provide template. T o assay for the presence of the initiation of codon mutation, a 205-bp fragment containing the initiation codon and a single AluI site was amplified directly from phage by PCR using primers 1 and 3 ( Fig. 1).
DNA Seqwncing"Ml3mplS clones were grown in Escherichia coli JM103. White plaques containing inserts were selected; singlestranded DNA was isolated and was sequenced using the dideoxynucleotide chain termination method (10) with Sequenase reagents (United States Biochemical Corp.). For the moderate patient, the entire coding region of the molecule was sequenced using specific oligonucleotides from the CD18 cDNA as primers.
Allele-specific Oligonucleotide Hybridization-A 499-bp segment of the CD18 sequence (base pairs 1930-2429, numbering according to Kishimoto et al. (11)) was amplified as described above using primers 2 and 4 ( Fig. 1). The cDNAs from the moderate patient and his parents were amplified to analyze the origin of the mutation at base 2142. PCR product (1 pl) was mixed with 1 p1 of 2.5% bromphenol blue, and 39 pl of 0.4 N NaOH containing 25 mM EDTA and spotted onto Zeta-Probe blotting membrane (Bio-Rad). Alternatively, 20 p1 of M13 phage supernatants from infected cultures were spotted directly on the membrane using the Minifold I1 slot-blot apparatus (Schleicher & Schuell). The DNA was immobilized by baking a t 80 "C in a vacuum oven for 2 h. Two 19-mer sense strand oligonucleotides were synthesized to match the normal gene sequence (CTATGTGGATGAGAGCCGA) or to match the frameshift mutant Primers 1 and 2 were used together for the amplification of fulllength CD18 cDNA. Primers 1 and 3 were used to amplify a 205 bp segment of the cDNA surrounding the initiation codon for analysis with AluI. Primers 2 and 4 were used to amplify a 499-bp region a t the 3' end of the coding region for hybridization analysis with AS0 probes. Synthetic DNA sequences (lowercase) were attached to some of the homologous sequence (uppercase) to create a restriction endonuclease recognition site in the amplified product for directional cloning. The underlined sequence is an XbaI site in primer 1 and is a HindIII site in primers 2 and 3.
sequence (CTATGTGGAGAGAGCCGAG). The oligonucleotides were end-labeled with [r-"'P]ATP using T4 polynucleotide kinase (12) and were purified with NENSORB'" 20 nucleic acid purification cartridges (Du Pont-New England Nuclear Research Products). The filters were hybridized as described elsewhere (13) at 55 "C in the presence of 10-fold molar excess of unlabeled competitor oligonucleotide (14). Filters were washed in 40 mM Na2HP0, (pH 7.2) with 1% sodium dodecyl sulfate at room temperature for 5 min and a t 42 "C for 15 min and exposed to X-Omat AR film (Eastman Kodak).

RESULTS
Fluorescence-activated Cell Sorting of the Patients' Lymphoblasts-Biotin-conjugated anti-CD18 antibody and phycoerythrin-streptavidin were used to stain lymphoblasts (8). Comparisons of relative fluorescence intensities in a cell sorter revealed CD18 levels 9% of normal in the moderate patient and 1% of normal in the severe patient (Fig. 2).
The Paternal Mutation of the Moderate Patient is in the Initiation Codon of CDI8-Full-length cDNA was amplified from normal individuals and from the two patients, and the PCR products were of the expected size. The products were cloned into M13mp18, and several clones from the patients and from the controls were sequenced. Sequence analysis revealed an ATG to AAG change in the initiation codon in each of four clones from the moderate patient (Fig. 3). This mutation changes a methionine codon a t position 1 to a lysine codon and is designated M1K. This change in the DNA sequence would disrupt the initiation of translation.
This single base change of a T to an A created an additional AluI site in the cDNA of the moderate patient. It was possible to screen all of the phage clones from this patient for the presence of the mutation by amplifying a small region surrounding the initiation codon. The PCR products were digested with AluI and analyzed on a polyacrylamide gel to test

CD18 Mutations
the amplified product with AZuI, the two bands unique to the altered initiation codon (93 and 70 bp) appeared with the amplified cDNA from the moderate patient and his father, but not with that from the mother or the control (Fig. 4).

The Maternal Mutation in the Moderate Patient Is a Single Base Deletion i n Codon 690"
Four of the M13 clones from the moderate patient were chosen for further analysis because they did not contain the paternal mutation as evidenced by the absence of the additional AluI site. These clones which should contain the putative maternal mutation were sequenced and were shown to contain a normal initiation codon. However, a single base deletion in the coding region of the molecule, 11 amino acids N-terminal to the start of the transmembrane domain was detected in three of four maternal clones sequenced. The T in the GAT codon of the aspartic acid a t position 690 (D690) at base 2142 is present in the normal control and in the paternal clones but is deleted in the maternal clones (Fig. 5). This deletion creates a frameshift mutation which is expected to terminate the protein without proper transmembrane or cytoplasmic domains.
For family studies, allele-specific oligonucleotides (ASOs) of 19 bp complementary to the mutant or the wild type sequence a t base 2142 were synthesized and end-labeled with [-p"P]ATP. The cDNAs from the moderate patient and his parents were amplified and transferred to a nylon membrane for analysis with ASOs (Fig. 6). Hybridization detected the frameshift mutation in the moderate patient and his mother, but not in the father. One of the phage clones from the patient contained neither the paternal or the maternal mutation. This single clone is thought to have arisen as the result of a DNA repair event when heteroduplex molecules formed in the PCR reaction were introduced into bacteria (15, 16).

The Severe Patient Also Has the Mutation in Codon 690-
The D690 frameshift mutation was also found in four of eight cDNA clones from the severe patient (data not shown). Sequencing of cDNA clones from the parents revealed that this   (PT) and for a normal control ( N L ) .
The asterisk indicates the deleted base. The other mutant allele in the severe patient has not been identified.

DISCUSSION
Two patients, one with moderate phenotype and the other severe, were studied and found to be compound heterozygotes. Although unrelated, they share the same frameshift mutation just prior to the transmembrane domain. The moderate patient has also inherited from his father a mutation in the CD18 initiation codon. Both mutations would be expected to impair the synthesis or function of the protein.
The frameshift mutation would be expected to produce a truncated / 3 subunit that is 56 amino acids smaller than a normal molecule, thus lacking a transmembrane and a cytoplasmic domain. Although the formation of cup heterodimers occurs before the complex is transported to the cell surface (17)(18)(19), and the cytoplasmic domain and transmembrane regions are not required for heterodimer assembly (20,21), it has been shown that regions of the cytoplasmic domain of CD18 are important in the regulation of the adhesiveness of LFA-1 to ICAM-1 in a COS cell expression system (21). However, immunofluorescent analysis showed no significant CD18 on the cell surface in the severe patient, indicating that both alleles present in this patient do not produce a significant amount of mature CD18.
In contrast, the moderate patient's lymphoblasts express CD18 on the cell surface at a level of 9% of normal. The increased CD18 expression in the moderate patient compared to the severe patient must be the result of the mutant CD18 initiation codon. Amplification of cDNA from the moderate patient indicated that the mRNA with the frameshift mutation was less abundant, presumably due to decreased stability as has been seen for some nonsense and frameshift mutations (22,23).
We hypothesize that the moderate phenotype is due to a low level of initiation of translation of the mRNA from the paternal allele, particularly at the second codon (CUG). The current "scanning" model of initiation of translation proposes that the 40 S ribosomal subunit binds to the upstream region of an mRNA transcript near the m7G cap structure and proceeds toward the 3' end of the transcript, searching for the first AUG triplet that will initiate translation. However, it is also important that the initiation codon be in a good context for efficient initiation to proceed. In a survey of 699 eukaryotic genes, a consensus sequence of GCCG C C A / & C m G was found by Kozak (24,25). Furthermore, the G a t position +4 and the purine at position -3 were shown to be especially important in defining a good context.
Mutations in an initiation codon have been shown to play  a role in disease in the following instances: the a1 or a 2 globin gene causing hemoglobin H disease (26)(27)(28), the @ globin gene causing @-thalassemia (29), the phenylalanine hydroxylase gene causing phenylketonuria (30), the a subunit of the stimulatory G protein of adenylate cyclase causing Albright hereditary osteodystrophy (31), the apolipoprotein C-I1 gene causing type I hyperlipoproteinemia (32), and the ornithine aminotransferase gene causing gyrate atrophy of the retina (33).
In the case of @-thalassemia, phenylketonuria, hyperlipoproteinemia, and gyrate atrophy of the retina, the phenotype is consistent with relatively complete protein deficiency. In the case of the a globin genes and the a subunit of stimulatory G protein, it is presumed that the mutant allele is inactivated, but the data did not address the possibility of low levels of initiation of translation at an alternative codon.
There are now many well characterized examples of mRNAs whose translation is initiated a t non-AUG triplets (34-42). Specific precedent for the use of CUG as an initiation codon exists for the high molecular weight forms of human fibroblast growth factor (40, 41), for the int-2 gene in mouse (37), for the c-myc gene in humans (38), and for the ltk receptor tyrosine kinase gene in the mouse (42). Fig. 7 details nucleotide similarity to the consensus sequence for the normal CD18 gene and for the paternal allele with the mutated initiation codon. The second codon in the CD18 gene is CUG and occurs in a context which would be reasonably favorable for a low level of initiation. The G at position +4 and the purine in the -3 position are both in favorable alignment. The next AUG codon in the mRNA occurs at base 73, but is not in frame. Subsequent in frame AUG codons occur distal to the leader peptide, but they would not allow proper targeting of the protein to the endoplasmic reticulum. The possibility of initiation at the second CUG codon in this case is particularly attractive because (i) there was previous evidence that the detectable protein was of normal size; (ii) based on amplification of cDNA, the mRNA from the paternal allele appears more abundant than that from the maternal allele; (iii) the context of the CUG codon is particularly favorable for initiation as shown in Fig. 7; and (iv) initiation at this CUG codon would yield an almost perfectly normal protein.
It is estimated that a non-AUG codon in a good context will function a t a level of approximately 3-5% of an AUG codon in the same context (35).
The molecular basis of the LAD defect has been described for only a few patients (43)(44)(45) (Table I). Homozygosity for a splicing mutation which caused skipping of a 90-bp exon resulted in a moderate phenotype due to correct splicing of a small fraction of the mutant transcripts (43). One patient with a moderate phenotype was heterozygous for a missense mutation substituting leucine for proline at position 149 (L149P), but the mutation on the other chromosome was not identified (45). One patient with a severe phenotype carried a missense mutation of arginine for glycine at position 169 (G169R). This patient was thought to be homozygous for this mutation or to carry an allele which did not produce a stable mRNA on the other chromosome. Both the L149P and G169R mutations were shown to cause defective association of CD18 with C D l l a subunit in a COS cell expression system. The mutated leucine and glycine are conserved in all known integrin fl subunits. One compound heterozygote patient with a moderate phenotype carried two missense mutations, cysteine for arginine at position 593 (R593C) and threonine for lysine at position 196 (K196T) (44). It was uncertain whether one or both alleles contributed to the moderate phenotype. With the two mutations reported here, there are now four missense mutations, one splicing mutation, one initiation codon mutation, and one frameshift mutation identified as causing leukocyte adhesion deficiency (Table I). This report also represents the first incidence of a common allele among unrelated LAD kindreds. Using ASOs, it will be possible to determine the frequency of this severe allele in the population of identified LAD patients.