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Two Novel Frame Shift, Recurrent and De Novo Mutations in the ITGB2 (CD18) Gene Causing Leukocyte Adhesion Deficiency in a Highly Inbred North African Population.

Fathallah DM, Jamal T, Barbouche MR, Bejaoui M, Hariz MB, Dellagi K - J. Biomed. Biotechnol. (2001)

Bottom Line: We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus.Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect.In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
We have identified four different mutations causing leukocyte adhesion Deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum including de novo and recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

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Related in: MedlinePlus

Detection of mutations in Tunisian LAD patients CD18 andcDNA. The DNA sequences shown were performed on cloned PCRproducts obtained by RT/PCR of the patients PBMC total RNA. (a)Nucleotide sequence showing the deletion of a single G (1920delG)in the glycine codon at position 640 causing a frame shift introducing a stop codon 16 residues downstream. The mutated CD18 protein ends as following: GPFGFS RTAARRVRPCSCRTTP• (b) Detection of the G (1497delG) in the aspartic acid at position 500, causing a frame shift and a stop codon 28 residues downstream. The CD18 protein is truncated as following: SCRKFS TTTPSS AQGWRTVSAG SACATPATSPAS• (c) The C to T transition causing the substitution Arg 593 by Cyst detected in patient K is indicated by an asterisk. (d) Nucleotide sequence showing the missense mutation that affects residue 284 of the Cd18 in patients S and S changing a C into a G. FS = frame shift.
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Figure 3: Detection of mutations in Tunisian LAD patients CD18 andcDNA. The DNA sequences shown were performed on cloned PCRproducts obtained by RT/PCR of the patients PBMC total RNA. (a)Nucleotide sequence showing the deletion of a single G (1920delG)in the glycine codon at position 640 causing a frame shift introducing a stop codon 16 residues downstream. The mutated CD18 protein ends as following: GPFGFS RTAARRVRPCSCRTTP• (b) Detection of the G (1497delG) in the aspartic acid at position 500, causing a frame shift and a stop codon 28 residues downstream. The CD18 protein is truncated as following: SCRKFS TTTPSS AQGWRTVSAG SACATPATSPAS• (c) The C to T transition causing the substitution Arg 593 by Cyst detected in patient K is indicated by an asterisk. (d) Nucleotide sequence showing the missense mutation that affects residue 284 of the Cd18 in patients S and S changing a C into a G. FS = frame shift.

Mentions: Northern blot showed that all the patients exhibited a normal sized CD18 specific mRNA (data not shown). We used the RT/PCR strategy to generate CD18 specific cDNA fragments. We have first made a 1.6 kb fragment corresponding to most of the region coding for the extracellular domain of the CD18 subunit. The majority of the mutations reported todate mapped to this region of the molecule. Sequencing of severalindependently generated PCR products from each patient revealed(see Figure 3) that all the patients have a deletion ofthe G at position 1497 (1497delG) causing a frame shift and yielding a premature stop codon, 28 residues downstream of the deletion. Patient R was homozygous for this mutation. PCR products from both parents subcloned into the pCDNA3 vector exhibited clones missing the G at position 1497 as well as normal clones. This finding is in agreement with patient R's parents being first cousins. The second mutation, identified in patient M, was another single-bp deletion: 1920delG that results in a frame shift and the occurrence of a stop codon 16 residues down stream of thedeletion. The third mutation was a G to A transition affecting codon 284 (GGC/AGC) leading to the substitution of a conserved glycine by a serine (G284S). Patients S and I were compound heterozygous 1497delG/G284S. The G284S mutation was reported independently in at least one unrelated patient [16]. The fourth mutation was a single nucleotide transition in codon 593 (CGT/TGT) that leads to the replacement of the arginine normally encoded at this position by a cysteine (R593C). This mutation found in patient K, was reported as an inherited mutation in two other patients, one of whom being with certainty of different ethnic origin [16, 17]. Sequencing of multiple RT/PCR products generated from the patient's parents showed that no one of them carried this mutation. Patient K was a compound heterozygous 1497delG/R593C. The latter occurred on a different allele than the one carrying the 1497delG. It has previously been shown using CD18 transfection studies that the G284S and R593C mutations prevent β2 integrin heterodimer formation andtherefore account for LAD phenotype [16, 17]. The mutations were confirmed in all the individuals studied by amplification and sequencing from the genomic DNA of the corresponding coding exon: G284S (Exon 7) 1497delG (Exon 12), 1920delG, and R593C within Exon 13 and Exon 14,respectively.


Two Novel Frame Shift, Recurrent and De Novo Mutations in the ITGB2 (CD18) Gene Causing Leukocyte Adhesion Deficiency in a Highly Inbred North African Population.

Fathallah DM, Jamal T, Barbouche MR, Bejaoui M, Hariz MB, Dellagi K - J. Biomed. Biotechnol. (2001)

Detection of mutations in Tunisian LAD patients CD18 andcDNA. The DNA sequences shown were performed on cloned PCRproducts obtained by RT/PCR of the patients PBMC total RNA. (a)Nucleotide sequence showing the deletion of a single G (1920delG)in the glycine codon at position 640 causing a frame shift introducing a stop codon 16 residues downstream. The mutated CD18 protein ends as following: GPFGFS RTAARRVRPCSCRTTP• (b) Detection of the G (1497delG) in the aspartic acid at position 500, causing a frame shift and a stop codon 28 residues downstream. The CD18 protein is truncated as following: SCRKFS TTTPSS AQGWRTVSAG SACATPATSPAS• (c) The C to T transition causing the substitution Arg 593 by Cyst detected in patient K is indicated by an asterisk. (d) Nucleotide sequence showing the missense mutation that affects residue 284 of the Cd18 in patients S and S changing a C into a G. FS = frame shift.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC129056&req=5

Figure 3: Detection of mutations in Tunisian LAD patients CD18 andcDNA. The DNA sequences shown were performed on cloned PCRproducts obtained by RT/PCR of the patients PBMC total RNA. (a)Nucleotide sequence showing the deletion of a single G (1920delG)in the glycine codon at position 640 causing a frame shift introducing a stop codon 16 residues downstream. The mutated CD18 protein ends as following: GPFGFS RTAARRVRPCSCRTTP• (b) Detection of the G (1497delG) in the aspartic acid at position 500, causing a frame shift and a stop codon 28 residues downstream. The CD18 protein is truncated as following: SCRKFS TTTPSS AQGWRTVSAG SACATPATSPAS• (c) The C to T transition causing the substitution Arg 593 by Cyst detected in patient K is indicated by an asterisk. (d) Nucleotide sequence showing the missense mutation that affects residue 284 of the Cd18 in patients S and S changing a C into a G. FS = frame shift.
Mentions: Northern blot showed that all the patients exhibited a normal sized CD18 specific mRNA (data not shown). We used the RT/PCR strategy to generate CD18 specific cDNA fragments. We have first made a 1.6 kb fragment corresponding to most of the region coding for the extracellular domain of the CD18 subunit. The majority of the mutations reported todate mapped to this region of the molecule. Sequencing of severalindependently generated PCR products from each patient revealed(see Figure 3) that all the patients have a deletion ofthe G at position 1497 (1497delG) causing a frame shift and yielding a premature stop codon, 28 residues downstream of the deletion. Patient R was homozygous for this mutation. PCR products from both parents subcloned into the pCDNA3 vector exhibited clones missing the G at position 1497 as well as normal clones. This finding is in agreement with patient R's parents being first cousins. The second mutation, identified in patient M, was another single-bp deletion: 1920delG that results in a frame shift and the occurrence of a stop codon 16 residues down stream of thedeletion. The third mutation was a G to A transition affecting codon 284 (GGC/AGC) leading to the substitution of a conserved glycine by a serine (G284S). Patients S and I were compound heterozygous 1497delG/G284S. The G284S mutation was reported independently in at least one unrelated patient [16]. The fourth mutation was a single nucleotide transition in codon 593 (CGT/TGT) that leads to the replacement of the arginine normally encoded at this position by a cysteine (R593C). This mutation found in patient K, was reported as an inherited mutation in two other patients, one of whom being with certainty of different ethnic origin [16, 17]. Sequencing of multiple RT/PCR products generated from the patient's parents showed that no one of them carried this mutation. Patient K was a compound heterozygous 1497delG/R593C. The latter occurred on a different allele than the one carrying the 1497delG. It has previously been shown using CD18 transfection studies that the G284S and R593C mutations prevent β2 integrin heterodimer formation andtherefore account for LAD phenotype [16, 17]. The mutations were confirmed in all the individuals studied by amplification and sequencing from the genomic DNA of the corresponding coding exon: G284S (Exon 7) 1497delG (Exon 12), 1920delG, and R593C within Exon 13 and Exon 14,respectively.

Bottom Line: We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus.Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect.In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
We have identified four different mutations causing leukocyte adhesion Deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum including de novo and recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

No MeSH data available.


Related in: MedlinePlus