Leukocyte adhesion deficiency type 1 (OMIM # 116920) (LAD1) is a rare (1 in every 10,000 live births) autosomal recessive syndrome resulting from mutations in the gene encoding CD18 protein (the integrin beta 2 chain, ITGB2).
ITGB2 has 16 exons located on 21q22.3. This gene is involved in adhesion and transmigration of human leukocytes in vivo, and thus patients with LAD1 have absent or severely reduced expression of ITGB2 chain on the surface of their leukocytes resulting in impaired accumulation of myeloid leukocytes at extravascular sites. The product of this gene belongs to the integrin beta chain family of proteins. A given chain may combine with multiple partners, ...view middle of the document...
Early diagnosis of LAD1 is vital to the success of treatment and prevention of aggressive infections. The main aims of this study are uncovering variations using sequence analysis of the ITGB2 gene in Iranian patients with LADl. The results of this study can be used in prenatal diagnosis in subsequent pregnancies and genetic counseling for LAD1 patient.
Materials and Methods
This study included 19 patients (10 boys and nine girls, ages between 60 days to 12 years) with initial diagnoses of LAD1 from consanguineous families. They were referred to the Children's Medical Center and Masih Daneshvari hospital having high levels of white blood cells in complete blood counts (CBC) (Table 1).
We obtained Blood samples from19 affected LAD1 and their parents after informed and written consent. Genomic DNA was prepared from whole blood by salting out method. Thirteen primer pairs were used to amplify 16 exons and exon–intron binderies of an ITGB2 gene under conditions available upon request. Amplifications of 50 µl Reactions were performed on a thermal cycler. PCR products were evaluated on 2% agarose gel, and purified PCR products were sequenced using the ABI 3130 DNA Analyzer (Applied Biosystems, Foster City, CA). Sequence analysis was performed using Chromas 2.4 and Lasergene (SeqMan ProV.1.7.0).
Clinical and Laboratory Findings
The most common clinical signs were infection, inflammation of the umbilicus and delayed umbilical cord separation (usually after three weeks) in these LAD1 patients. CBC in these patients indicated neutrophil dysfunction and an increased number of blood cells. In most cases, omphalitis and Skin ulcers symptoms were seen in the early stages of the disease (Table 1).
Mutation analysis carried out on ITGB2 gene sequences of 19 patients. After identification of mutation in an exon, sequencing was not performed to the remaining exons. DNA sequence analysis of ITGB2 gene in Iranian families, demonstrating an autosomal recessive form of LAD1, identified different types of mutations, including, four insertion/deletion and ten missense/nonsense mutations. Among the 19 families analyzed, ten different types of mutations previously reported were identified, including c.329-6C>A, c.382G>T, c.715G>A, c.843delC, c.897+1G>A, c.1062A>T, c.1143delC, c.1877+2T>C, c.1907delA and c.2147G>C. Sequence analysis of the ITGB2 gene revealed four novel mutations including; c.576dupC (Asn193GlnfsX72), c.706G>A (Gly236Arg), c.897+1G>T and c.1030G>T (Glu344*) (Table 2. Figure 1.). All variations were confirmed in parents as criers.
Figure 1. Sequence analysis of four novel variations in the gene ITGB2; Arrows indicates the location of mutations. A1, B1, C1 and D1 represent normal nucleotide sequences in the affected individuals. A2 (c.576dupC) and B2 (c.706G>A) indicate heterozygous variation in P6. C2 (c.897+1G>A, in P7 and P15) and C3 (c.897+1G>T, in P10) show splicing variant identified in intron 7. D2...