Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

Area of Science:

• Genetics
• Immunology
• Molecular Biology

Background:

• Copy number variations (CNVs) in CC chemokine ligand 3-like1 (CCL3L1) have been linked to HIV-1 susceptibility, but results are inconsistent.
• CCL3L1 is part of a gene cluster on chromosome 17q12, including CCL3, CCL3L2, and CCL3L3, involved in host defense and inflammation.
• Existing CNV assays for CCL3L1 have been developed, but their specificity is questionable due to gene homology.

Purpose of the Study:

• To evaluate the specificity of existing RT-PCR based CNV assays for CCL3L1.
• To investigate the homology between CCL3L1 and its related genes (CCL3, CCL3L2, CCL3L3).
• To understand the impact of assay specificity on the reported association between CCL3L1 CNVs and HIV-1 susceptibility.

Main Methods:

• Pairwise and multiple sequence alignments of CCL3L1, CCL3, CCL3L2, and CCL3L3 gene sequences using MEGA 4 and BioEdit.
• Alignment of sense primers, anti-sense primers, and probes from previously described CNV assays against the aligned chemokine gene sequences.
• Analysis of assay concordance and median copy number measurements in different ethnic groups.

Main Results:

• High homology (50-99% overall, 70-100% exonic) was found between CCL3L1, CCL3, CCL3L2, and CCL3L3, with CCL3L1 and CCL3L3 being identical.
• Primers and probes from existing assays showed overlapping matches with at least two of the four genes, indicating a lack of specificity for CCL3L1.
• Concordance between four different assays ranged from 0.44-0.83, revealing significant inconsistencies and discordant results.

Conclusions:

• Inconsistencies in HIV-1 association studies may be attributed to non-specific CNV assays yielding heterogeneous results.
• Separate determination of CNVs for CCL3, CCL3L2, and CCL3L3 is necessary.
• Future studies should differentiate between individual genes within the cluster to accurately assess their roles in disease pathogenesis.