Genetic analysis of patients with leukocyte adhesion deficiency: Genomic sequencing reveals otherwise undetectable mutations

PII: S0301-472X(01)00782-20301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(01)00782-2
Genetic analysis of patients with leukocyte adhesion deficiency: Genomic sequencing reveals otherwise undetectable mutations
Dirk Roos
Central Laboratory Netherlands Blood Transfusion Service (CLB) and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
b
Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
c
d
e
MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
(Received 22 August 2001; revised 22 October 2001; accepted 25 October 2001)
Objective.

Methods.
We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns.
Results.

252Arg
593Cys
supported some LFA-1 and p150,95 formation in COS cells. The other three pa- tients were compound heterozygotes in which only one allele had previously been character- ized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide de- letion.
Conclusion.
Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection. © 2002
International Society for Experimental Hematology. Published by Elsevier Science Inc.


2
subunit (CD18), belong to the integrin su- perfamily of adhesion receptors involved in cell-cell and cell- matrix interactions [6]. The severity of the clinical symptoms in LAD generally correlates with the level of remaining leu- kocyte integrin expression, which is heterogeneous among LAD patients. The disease is categorized in a severe form with less than 1%, and a moderate form with 3–10%, of nor- mal CD11/CD18 glycoprotein expression [7].

subunit has been determined by molecular cloning [8,9], and its
INTG2
gene has been lo-
Offprint requests to: D. Roos, Ph.D., CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; E-mail: [email protected]
D. Roos et al./Experimental Hematology 30 (2002) 252–261
253

2
integrins [11–21]. In the present study, we have investigated the genetic ba-
sis of the leukocyte adhesion deficiency in seven patients with the severe phenotype and in one patient with the mod- erate phenotype of the disease. For this purpose, we have developed a mutation assay based on direct sequencing from genomic DNA. We report five novel defects, one of which involves a rare intronic branchpoint mutation. Our genomic DNA sequencing method opens the possibility of prenatal diagnosis of LAD at an early stage of pregnancy.
Materials and methods
Patient samples
Blood from patients, available family members, and healthy donors (laboratory personnel) was drawn in citrate or EDTA by vena punc- ture, with informed consent, and arrived in our laboratory within 24 hours. DNA from EBV-B lymphocyte cell lines was sent by Dr. P. Lachmann (MRC Immunopathology Unit, Cambridge, UK [LAD patients 6 and 8] and Dr. L.A. Boxer (Dept. of Pediatrics, University of Michigan, Ann Arbor, MI, USA [LAD patient 7]).
cDNA amplification and sequencing








C. The PCR products were electrophoresed on agarose gels to control the size of the amplified products. Nucleotide se- quence analysis of the PCR products was performed with the di- rect dideoxy-chain-termination method and cycle sequencing or automated sequencing.





Genomic DNA sequencing
Genomic DNA was purified from total leukocytes or EBV-B lym- phocyte cells. All 16 exons of
INTG2





M of each dNTP, 50 mM KCl, 1.5 mM MgCl
2

L glass capillaries (Idaho Tech- nology).








C, for 50 cycles in a 96-well polycarbonate plate in an Omni- gene Thermocycler (Hybaid, Teddington, Middlesex, UK) with simulated tube control and calibration factor 200. The sequencing samples were purified in Multiscreen plates (Millipore, Molsheim, France) according the DyeTerminator Removal protocol (http:// www.millipore.com/analytical) and were subsequently loaded onto an ABI 377XL automated DNA sequencer (PE Applied Biosystems).
Expression of CD11/CD18 antigens on transfected COS cells
To evaluate the ability of the two CD18 mutant proteins with amino- acid replacements found in patients 4 and 5 to form a stable complex with any of the three CD11 subunits, COS cells were cotransfected with cDNA coding for these proteins in the expression vector pcDNA3 [23]. The mAbs MHM23 (gift of Dr. McMichael, Institute of Molecular Medicine, Oxford, UK) and IB4 (gift of Dr. Wright, Southampton, UK), which are specific for CD11/CD18 het- erodimers, were used to detect the expression of the transfected pro- teins. The transfection efficiency was about 20%.
Results
From the genomic DNA of the patients and the available family members, each exon of
INTG2
was separately ampli- fied and sequenced. Table 2 summarizes the CD18 muta- tions found.
Patient 1

10
10
/L), and pro- foundly defective chemotactic activity. The CD11b expres- sion on the leukocytes was 2.5% of the normal value. A sis- ter who also had LAD, with a similar clinical phenotype, died of recurrent infections at the age of 11 months.
Sequencing the genomic DNA of patient 1 showed that all
INTG2
exons were normal except exon 2. The PCR with primers around exon 2 yielded a nonspecific product. The PCR product spanning exon 1–5 of the cDNA was about 60 bp smaller than the normal size (Fig. 1A). Nucleotide se- quence analysis revealed that exon 2 (61 bp) was absent. The cDNA products of the parents contained both the nor- mal and the exon-2–deleted species. Analysis of the genomic sequences immediately flanking exon 2 of the parents re-
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D. Roos et al./Experimental Hematology 30 (2002) 252–261
vealed no abnormalities in either the donor or the acceptor splice sites of exon 2. Thus, the absence of exon 2 in the cDNA of patient 1 was probably not due to abnormal mRNA splicing. Therefore, we PCR amplified a larger region of ge- nomic DNA, from about 1000 nucleotides upstream of exon 2 to the beginning of intron 3. With control DNA, we ob- tained a product of about 1.6 kb, whereas with the patient’s DNA this was only about 0.8 kb (Fig. 1B). DNA from both
parents yielded the 1.6-kb as well as the 0.8-kb product. Se- quencing revealed a deletion of 833 nucleotides inclusive of exon 2 in the 0.8-kb product (Fig. 1C). Analysis of the par- ents’ genomic DNA confirmed their heterozygous status for this deletion. The deletion results in a shift in the reading frame and the loss of the initiating ATG start codon. A pep- tide of 16 amino acids may be produced when the biosynthe- sis starts at an ATG start site in exon 3 (Fig. 1D).
Table 1.
Exon 1: AL1A: sense (

















AL9B: antisense (Intron 9, 88 to 62) 5-TCCTGCTGACCTGCAGCAGGAGCTGAC-3
Exon 10: AL10A: sense (Intron 9, 63 to 42) 5-GGCCAGGCAGGGCCAGGACAAC-3
AL10B: antisense (Intron 10, 73 to 52) 5-GACCCTGGCTCCTTCTGGCTGT-3
Exon 11: AL11A: sense (Intron 10, 36 to 21) 5-GAGGGTCCCCACCCTTGAGCCCAGC-3
AL11B: antisense (Intron 11, 74 to 57) 5-CCTTCACCTGCCGACAC-3
Exon 12: AL12A: sense (Intron 11, 82 to 58) 5-AAGGGGGCCTGGAGGACAGGGCGGCT-3
AL12B: antisense (Intron 12, 59 to 41) 5-CCCAACACCAAGTCTGTGC-3
Exon 13: AL13A: sense (Intron 12, 86 to 60) 5-CCGGAGGGAGCAACCGCAGTGGGGTTT-3
AL13B: antisense (Intron 13, 36 to 19) 5-ATCCCTGCCCGCCCTGCC-3
Exon 14: AL14A: sense (Intron 13, 58 to 32) 5-CCCCGTTGCCGGAGCCCCGCACACCCT-3
AL14B: antisense (Intron 14, 51 to 29) 5-CCCGCAGCCGGAGCTCTCTGGAGCC-3
Exon 15: AL15A: sense (Intron 14, 79 to 60) 5-GGGTGACCTCGGACCTGTGG-3
AL15B: antisense (Intron 15, 74 to 50) 5-CCCGCAGCAGGAGGTCGCATAGTG-3
Exon 16: AL16A: sense (Intron 15, 73 to 49) 5-GTGTCCTCAGGGAGAAAGATTCTGC-3
AL16B: antisense (Exon 16, 2595 to 2573) 5-GACTGTGCCAGTCAGAGTGGAGC-3
Oligonucleotides used for PCR of intron 1 to exon 3 in family 1: sense (intron 1, 964 to 949) 5-CGAGGGCCTGTGAGTGACCACTGAG-3 antisense primer AL3B (intron 3, see above)
D. Roos et al./Experimental Hematology 30 (2002) 252–261 255
Patient 2 Patient 2 (female) had the severe form of LAD, with a total absence of leukocyte cell adhesion molecules and undetect- able mRNA for the CD18 subunit [24]. This patient suffered from impaired wound healing, necrosis, and staphylococcal infections until she died from ileum perforation at the age of 7 months. The only mutation found in the genomic DNA of patient 2 was an apparently homozygous T→G substitution in intron 5 at position 12 upstream of exon 6 (Fig. 2A). To investigate whether this mutation might affect the correct mRNA splicing, we analyzed the cDNA PCR product that includes exons 5, 6, and 7. This fragment was about 150 bp smaller than expected (not shown). Nucleotide sequencing revealed that the first 149 nucleotides of exon 6 were ab- sent, predicting a frameshift with 46 irrelevant amino acids after Ile166 and a premature stop codon at position 214 (Fig. 2B). The mother of patient 2 was found to be a carrier of the T→G mutation in intron 5 (not shown), but no abnor- malities were detected in the DNA from the father (Fig. 2A). The mutation was not found in the DNA from 30 con- trol donors.
The T→G mutation creates a target site for the restriction enzyme BstNI, which recognizes the 5-CCAGG-3 se- quence (mutant) but not the 5-CCATG-3 sequence (nor- mal). Allele-specific restriction enzyme analysis (ASRA) was performed on genomic DNA. The 367-bp PCR product obtained with primers AL6A and AL6B from DNA of an unrelated donor was digested with BstNI in three fragments of 234 bp, 80 bp, and 53 bp (Fig. 2C, lane 1).[FIG 2] In the sample from the patient, the 234-bp fragment was further digested into two fragments of 202 bp and 32 bp (Fig. 2C, lane 4). Of the parents, only the mother was found to be a carrier of the mutant allele, as deduced from the heterozy- gous restriction pattern (Fig. 2C, lane 3).
The apparent homozygosity of the patient for the T→G mutation at the genomic DNA level, and the absence of the mutation in the DNA of the father, prompted us to perform Southern blotting experiments to investigate a possible
large deletion in the paternal CD18 allele of the patient. Ge- nomic DNA was digested with either BamHI or EcoRI re- striction enzymes and probed with labeled full-length CD18 cDNA. RFLP patterns similar to those published previously [25] were obtained, with no significant differences between the patient, both parents, and a control (not shown). Pater- nity investigations showed a 99.7% probability of the pa- tient being the child of her legal parents.
We then investigated a number of polymorphisms in INTG2 in this family. Figure 2D shows that on at least two sites (intron 8 and exon 10) the patient had apparently not inherited the paternal markers. Therefore, the possibility of uniparental maternal disomy must be considered.
Patient 3 This patient (female) is the first child of consanguineous Swiss parents. She was diagnosed as severe LAD [no ex- pression of CD 11a,b,c or CD18 on the leukocytes, in- creased numbers of circulating leukocytes (40–60 109/L), late separation of the umbilical cord, omphalitis with S. au- reus infection]. She succumbed to liver failure after hap- loidentical bone marrow transplantation. A two-year-younger brother also proved to be an LAD patient. He was success- fully transplanted with parental, haploidentical, peripheral stem cells.
Genomic DNA sequencing of patient 3 revealed a novel homozygous C199T mutation in INTG2 exon 4, changing codon CAG for Gln67 into the stop codon TAG. Both par- ents were found to be heterozygotes for this mutation (not shown).
Patient 4 Patient 4 (female) was the second child of healthy Turkish parents, who are second-degree relatives with an older, healthy daughter. Patient 4 suffered from recurrent infec- tions and was treated with antibiotics. She died a few weeks after the diagnosis. Her lymphocytes and granulocytes showed no expression of CD18 and CD11b, consistent with
Table 2. Summary of the CD18 mutations identified in the LAD alleles
Patients Nucleotide change Amino acid change Phenotype
1 Deletion of 0.8 kb, including exon 2. Homozygous. Incorporation of 16 aberrant amino acids, premature stop Severe 2 nt500–646 in cDNA (partial exon 6 skipping) due to T→G mutation
at position 12 in intron 5. Maternal disomy? Incorpration of 48 aberrant amino acids, premature stop Severe
3 C199T (CAG→TAG) Homozygous Gln67stop Severe 4 T754C (TGG→CGG) Homozygous Trp252Arg Severe 5 C1777T (CGT→TGT) Homozygous? Arg593Cys Moderate 6 1. A→C at position 14 in intron 6, activation of cryptic splice site;
also C1756T (CGG→TGG) 1. Incorporation of 4 extra amino acids; also Arg586Trp Severe
2. Deletion TC 66,67 2. Incorporation of 35 aberrant amino acids, premature stop 7 1. G1021C (GCC→CCC) 1. Ala341Pro Severe
2. C1602A (TGC→TGA) 2. Cys534stop 8 1. T446C (CTA→CCA) 1. Leu149Pro Severe
2. deletion T2070 2. Incorporation of 24 aberrant amino acids, premature stop
256 D. Roos et al./Experimental Hematology 30 (2002) 252–261
Figure 1. Results obtained with the DNA from patient 1. (A) Size of PCR-amplified cDNA (sense primer in exon 1, antisense primer in exon 5). Lane 1, con- trol donor; lane 2, father; lane 3, mother; lane 4, patient; lane M, size markers (100 bp difference). (B) Size of PCR-amplified genomic DNA (sense primer in intron 1, antisense primer in intron 3). Lane 1, control donor; lane 2, father; lane 3, mother; lane 4, patient; lane M, size markers (upper 3, 200 bp difference; lower 5, 100 bp difference). (C) Sequence of PCR-amplified genomic DNA around position 721 in intron 1 and around position 51 in intron 2. Arrows indi- cate deletion boundaries in the patient. (D) Schematic representation of genomic DNA sequence, cDNA sequence, and predicted amino-acid sequence. Upper- case letters indicate coding sequences and lowercase letters indicate intron sequences. Exon sequences in genomic DNA are shown within boxes. Arrowheads indicate exon boundaries in cDNA. Thick lines indicate size difference in genomic DNA between control and patient.
D. Roos et al./Experimental Hematology 30 (2002) 252–261 257
Figure 2. Results obtained with the genomic DNA from family 2. (A) Sequence of PCR-amplified genomic DNA around position 12 in intron 5 of a con- trol donor, the patient, and her father. (B) Schematic representation of genomic DNA sequence, cDNA sequence, and predicted amino-acid sequence. Sym- bols as in Figure 1B. Arrow indicates T→G mutation at position 12 in intron 5 in the patient. Half circles indicate the normal outsplicing of intron 5 from wild-type mRNA (top) and the abnormal skipping of part of exon 6 from the mutated mRNA, due to activation of a cryptic splice site in exon 6 (bottom). (C) PCR-ASRA of genomic DNA. A PCR product of 367 nucleotides was digested with BstNI into fragments of 234 bp, 80 bp, and 53 bp. In the patient’s PCR sample, the 234-bp fragment was further digested into fragments of 202 bp and 32 bp. Lane M, 100-bp size markers; lane 1, control; lane 2, father; lane 3, mother; lane 4, patient. (D) Polymorphisms within INTG2. Single letters indicate homozygosity, double letters indicate heterozygosity. The sequence at posi- tion 46 in intron 8 in the mother could not be determined due to lack of material.
258 D. Roos et al./Experimental Hematology 30 (2002) 252–261
a severe type of LAD. A T754C transition was found in exon 7 of INTG2 of the patient, predicting a Trp252Arg substitution. Both parents were heterozygotes for the T754C mutation (not shown). Analysis performed on 30 unrelated control donors showed the normal T754 sequence. CD18 carrying the Trp252Arg mutation failed to support surface expression of LFA-1, Mac-1, and p150,95 on COS cell transfectants (Fig. 3).
Patient 5 Patient 5 (male) was of Gypsy origin. He suffered from se- vere recurrent bacterial infections. Two siblings died of bac- terial infections. Despite the severity of the LAD in this pa- tient (and his family), his granulocytes, monocytes, and lymphocytes were found to express low but detectable amounts of LFA-1, Mac-1, and p150,95 (2–10% of normal) [26]. Patient 5 died in a car accident at the age of 30 years.
Nucleotide sequence analysis of the genomic DNA from patient 5 showed an apparently homozygous C to T transi- tion at position 1777 of the coding sequence of INTG2, pre- dicting an Arg593Cys substitution in exon 13. Family mem- bers were not available for analysis. CD18 with the Arg593Cys
substitution appeared to allow CD11a/CD18 expression on transfected COS cells (Fig. 3). However, it was less effec- tive in supporting the expression of CD11c/CD18 and CD11b/CD18.
Patients 6, 7, and 8 Patients 6 (female) [18,21,27] and 8 (male) [12,14,27] have been described before; they suffer(ed) from recurrent bacterial infections, diminished neutrophil mobility in vivo, and total ab- sence of 2 integrins on the surface of their leukocytes. Patient 8 died at the age of 22 years from the outgrowth of a skin ulcer, which developed into septicemia. Patient 7 (female) [23] had about 1% of CD11b/CD18 expression on her neutrophils, leu- kocyte levels of 12 109/L (in periods without infection) to 40 109/L (during infectious periods), and recurrent mucositis and cutaneous infections with either Pseudomonas or Staphy- lococcus aureus. She developed severe graft-vs-host disease after a match-related bone marrow transplantation and died at the age of 12 years.
Patients 6, 7, and 8 had been analyzed for mutations in CD18 cDNA previously [14,18,21,23]. In each of the three cases, only a single mutated cDNA species had been found (entry “1” of the respective patients in Table 2). However, analysis of their genomic DNA had revealed the mutated as well as the normal sequences, suggesting that the mRNA of the other allele was expressed, if at all, at undetectable lev- els. With our genomic DNA sequencing method, the previ- ously published mutations were confirmed. In addition, a two-bp deletion (TC66,67) was found in exon 3 of INTG2 in patient 6, inducing a frameshift and a premature stop codon at codon 57; a C1602A missense mutation in exon 12 of pa- tient 7, changing codon TGC for Cys534 into the stop codon TGA; and a T2070 deletion in exon 15 of patient 8, induc- ing a frameshift and a premature termination of protein syn- thesis in codon 714.
Discussion We have characterized eight mutations in the CD18 gene of patients with LAD, of which five have not been reported previously (Fig. 4). The novel missense mutation Trp252Arg, identified in patient 4 with the severe type of LAD, is located in a region of CD18 that is highly conserved in all other hu- man integrin subunits and critical for the maintenance of the heterodimer. This Trp is also conserved in the sub- units of Drosophila, urchin, crayfish, worm, coral, and sponge [28]. The mutation leads to a deficiency of the CD18 and CD11b molecules on the leukocytes of the patient, as well as on cotransfected COS cells (Fig. 3), and thus suggests a role of this region in complexation of the CD18 subunit with CD11a, b, and c subunits. Indeed, other mutations have been described in this region that affect the complex forma- tion, maturation, and membrane expression [13,21,23,29,30]. Alternatively, this region may be of crucial importance for the intrinsic stability of the CD18 protein.
Figure 3. Expression of LFA-1, Mac-1, and p150,95 on CD11/CD18 trans- fected COS cells. Column 1: Transfection with wild-type CD18, alone or cotransfected with wild-type CD11a, CD11b, or CD11c. Detection with McAbs MHM23 or IB4. Control McAb is OX33. Column 2: Transfection with CD18252Arg (W252R, patient 4), alone or cotransfected with wild-type CD11a, CD11b, or CD11c. Column 3: Transfection with CD18593Cys (R593C, patient 5), alone or cotransfected with wild-type CD11a, CD11b, or CD11c.
D. Roos et al./Experimental Hematology 30 (2002) 252–261 259
The Arg593Cys substitution identified in the CD18 gene of patient 5 was previously reported in two other, unrelated patients with the moderate phenotype of LAD [13]. Both patients were compound heterozygotes for Arg593Cys with another mutation, i.e., Lys196Thr and Gly284Ser, respec- tively. We presume that patient 5 is homozygous for the Arg593Cys mutation, but we cannot exclude that he carries a (partial) deletion of INTG2 on one allele. Arginine-593 is located in a major cysteine-rich region at the C-terminal side of the extracellular domain of…