CCL3L1 Human

What is CCL3L1 and where is it located in the human genome?

CCL3L1 (C-C motif chemokine ligand 3 like 1) is a gene located on chromosome 17q12 in humans. It belongs to a cluster of genes including CCL3, CCL3L2, and CCL3L3, with which it shares significant sequence homology. The gene encodes the LD78beta isoform of MIP-1alpha (Macrophage Inflammatory Protein-1alpha), a chemokine involved in immune responses .

The CCL3L1 gene is part of a complex structurally variable region. CCL3L1 and its neighboring gene CCL4L1 are tandemly repeated, with variable copy numbers across individuals and populations. The genomic sequence of CCL3L1 can be found in several reference assemblies, including NT_187661.1 (Chromosome 17 Reference GRCh38.p14) and NC_000017.10 (Chromosome 17 Reference GRCh37.p13 Primary Assembly) .

Importantly, CCL3L1 shares extremely high sequence homology with some of its paralogs—particularly CCL3L3, with which it appears to be identical in coding sequence. The homology between genes in this cluster ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions .

How does CCL3L1 copy number variation occur in humans?

CCL3L1 copy number variation represents a complex form of structural variation where individuals carry different numbers of the gene in their genome. The total diploid copy number of CCL3L1 (and the neighboring CCL4L1 gene) ranges from 0 to 6 copies in Europeans, with higher copy numbers observed in other populations—up to 10 in Tanzanian and 14 in Ethiopian populations .

This variation occurs through segmental duplications in the 17q12 region. The CCL3L1 gene and its neighboring paralog CCL4L1 are tandemly repeated, creating a structurally variable region. The mechanism behind this variation likely involves non-allelic homologous recombination between similar sequences during meiosis, leading to insertions or deletions of gene copies .

What is the relationship between CCL3L1 and its paralogs (CCL3, CCL3L2, CCL3L3)?

CCL3L1 shares substantial sequence homology with a cluster of paralogous genes located on chromosome 17q12: CCL3, CCL3L2, and CCL3L3. These genes arose through evolutionary duplication events and retain significant similarity in their sequences .

The homology relationships between these genes are as follows:

• The complete gene sequences share 50-99% homology

• The exonic regions specifically share 70-100% homology

• CCL3L1 and CCL3L3 appear to be identical in their coding sequences (100% homology)

This high sequence similarity presents significant challenges for researchers attempting to specifically measure CCL3L1, as many assays designed for CCL3L1 will also detect its paralogs. Pairwise and multiple alignments of these genes show that primers and probes used in several previously described assays align with overlapping sequences in at least two of the four genes .

While these genes share high sequence similarity, they have functional differences. For example, the MIP-1alpha isoform encoded by CCL3L1 (LD78beta) has approximately six-fold greater affinity for the CCR5 receptor than the alpha isoform encoded by CCL3 (LD78alpha) .

How does CCL3L1 copy number vary across different human populations?

CCL3L1 copy number shows substantial variation across different human populations, with a clear pattern of higher average copy numbers in African populations compared to European populations. This variation reflects human demographic history and possibly selection pressures related to the gene's immune functions .

Based on data from the 1000 Genomes Project, the distribution of CCL3L1 copy numbers across major population groups is as follows:

Europeans typically carry between 0 and 5 copies of CCL3L1, with an average of approximately 2 copies. In contrast, African populations show a higher range (1-9 copies) and a higher average (4.19 copies). East Asian, Admixed American, and South Asian populations show intermediate values .

This population variation is important to consider in study design, particularly for case-control studies investigating potential associations between CCL3L1 copy number and disease.

What is the functional relationship between CCL3L1 and CCR5?

CCL3L1 encodes the LD78beta isoform of MIP-1alpha, which functions as a natural ligand for the CCR5 receptor. This CCL3L1-CCR5 signaling axis plays an important role in various inflammatory responses, including macrophage function and T-cell-dependent immune responses .

The relationship between these two genes is particularly significant because:

• The MIP-1alpha isoform encoded by CCL3L1 (LD78beta) has approximately six-fold greater affinity for the CCR5 receptor than the alpha isoform encoded by CCL3 (LD78alpha) .

• CCR5 itself has an important variant—a 32bp exonic deletion allele (rs333, CCR5d32) with a minor allele frequency of between 5–15% in Europeans. This deletion creates a non-functional receptor .

• The interaction between MIP-1alpha and CCR5 controls the recruitment of immune cells to inflammatory foci, which is relevant to various disease processes .

This ligand-receptor relationship has been the target of therapeutic development, with small molecule CCR5 antagonists like maraviroc (an approved antiretroviral drug) already in clinical use, and therapeutic monoclonal antibodies in development .

What are the most accurate methods for measuring CCL3L1 copy number variation?

Measuring CCL3L1 copy number variation accurately has been a significant challenge due to the multiallelic nature of the variation and the high sequence homology with its paralogs. Several methods have been developed, with varying degrees of accuracy :

• Quantitative PCR (qPCR): Early studies used qPCR assays, but these have limitations due to a low signal-to-noise ratio. These assays are prone to batch effects and can be inaccurate, particularly for distinguishing higher copy numbers .

• Paralogue Ratio Test (PRT): This is considered the gold standard approach for measurement of CCL3L1 copy number variation. The triplex PRT assay produces three independent estimates of copy number per test:

  • PRT is a comparative PCR method that amplifies a test and reference locus using the same pair of primers

  • This is followed by capillary electrophoresis and quantification of the two products

  • The triplex assay provides three measurements that can be averaged for increased accuracy

  • In studies, this approach shows consistent results in 95% of samples

• Next-Generation Sequencing (NGS): Whole genome sequencing data can be analyzed using tools like CNVrd2 to estimate copy number:

  • This approach analyzes sequence read depth across the CCL3L1 region

  • Segmentation scores obtained are clustered using a Gaussian mixture model

  • Prior information is used in a Bayesian model to infer integer copy numbers

  • While promising, this approach still requires validation against established methods

• PCR-based Assays with Specific Design Considerations: When designing PCR assays for CCL3L1, researchers must carefully check primer and probe specificity:

  • Previous studies have shown that many assays designed for CCL3L1 also detect its paralogs

  • Pairwise and multiple alignments of all four chemokine genes should be performed

  • Primers and probes must be checked against these alignments to ensure specificity

For large-scale studies, a combined approach is often best—using PRT as a gold standard and validating other high-throughput methods against it. In the UK Biobank study, there was strong concordance between PRT and sequencing-based methods, though with a joint error rate of approximately 5% for the range seen in Europeans (copy numbers 0 to 5) .

How does CCL3L1 copy number affect gene expression levels?

CCL3L1 copy number has a clear gene dosage effect on mRNA expression levels, demonstrating the functional importance of this copy number variation. Research has confirmed this relationship through multiple approaches :

• Direct correlation with mRNA levels: Studies comparing CCL3L1 copy numbers with transcript levels (measured by RNAseq in B-lymphoblastoid cell lines) showed a clear positive correlation:

  • There is a statistically significant positive correlation between CCL3L1 copy number and expression level (r² = 0.25, slope = 6.9, p < 2×10⁻¹⁶)

  • This indicates that approximately 25% of the variation in CCL3L1 expression can be explained by copy number variation

• Ratio of CCL3L1 to CCL3 expression: The relationship is even stronger when examining the ratio of CCL3L1 to CCL3 expression:

  • CCL3L1 copy number accounts for approximately 50% of the total variation in the CCL3L1:CCL3 mRNA ratio

  • This suggests that while CCL3L1 is expressed at lower levels than CCL3, its relative expression compared to CCL3 is strongly determined by copy number

• Functional implications: The gene dosage effect has potential functional significance because:

  • The MIP-1alpha isoform encoded by CCL3L1 (LD78beta) has much stronger affinity to the CCR5 receptor than the isoform encoded by CCL3 (LD78alpha)

  • Even though CCL3L1 is expressed at lower levels than CCL3, its higher receptor affinity may make even small changes in expression biologically relevant

What is the evidence for association between CCL3L1 copy number and respiratory diseases?

Despite the plausible biological role of CCL3L1 in respiratory function and inflammation, current evidence does not support a significant association between CCL3L1 copy number variation and respiratory diseases, particularly in terms of lung function :

• Biological plausibility: There are several reasons to suspect CCL3L1 might be involved in respiratory diseases:

  • In mice, MIP-1alpha is implicated in virus-mediated inflammation of the lung, pulmonary eosinophilia following paramyxovirus infection, and clearance of pulmonary infections

  • In humans, MIP-1alpha controls the recruitment of immune cells to inflammatory foci

  • Increased levels of MIP-1alpha mRNA are found in bronchial epithelial cells of COPD patients

  • Increased protein levels are found in the sputum of COPD patients, where increased macrophage and neutrophil infiltration in the lung is a key pathology

• Large-scale studies find no association: Despite this biological plausibility, a large study of approximately 5,000 individuals from UK Biobank found:

  • No evidence for association of CCL3L1 copy number with lung function

  • No evidence for association of CCR5d32 genotype with lung function

  • No evidence for association of combinations of genotypes at the two loci with lung function

• Study design considerations: The UK Biobank study specifically examined:

  • Forced Expired Volume in 1 second (FEV1) as a binary trait

  • Individuals selected from the extremes of the lung function distribution to increase power

  • A sample size that, while modest by GWAS standards, is large for studies involving labor-intensive PRT assays

• Implications for drug development: These findings suggest that repositioning CCR5 antagonists (like maraviroc) is unlikely to be successful for the treatment of airflow obstruction. Although the MIP-1alpha-CCR5 signaling axis can be disrupted by artificial CCR5 antagonists, there is no evidence that this axis has a functional effect on lung function .

How do CCL3L1 and CCR5 variations interact to potentially influence disease susceptibility?

While direct evidence for interaction effects between CCL3L1 copy number and CCR5 variants on disease susceptibility is limited in the search results, we can draw insights about how these variations might interact based on their biological relationship :

• Functional relationship: CCL3L1 encodes a ligand (LD78beta isoform of MIP-1alpha) that has approximately six-fold greater affinity for the CCR5 receptor than the alpha isoform encoded by CCL3. Meanwhile, the CCR5d32 variant creates a non-functional receptor .

• Potential interaction mechanisms:

  • In individuals with high CCL3L1 copy numbers, there may be increased activation of CCR5-mediated signaling due to higher expression of the high-affinity ligand

  • In individuals carrying the CCR5d32 variant, this signaling would be reduced due to fewer functional receptors

  • Combinations of these genotypes could potentially have interactive effects on immune function and disease susceptibility

• Study findings: The UK Biobank study specifically examined combinations of genotypes at the two loci and found no evidence for association with lung function. This suggests that, at least for respiratory function, there is no significant interaction effect .

• Drug target implications: Understanding the interaction between these genetic variants is relevant for drug development:

  • CCR5 antagonists already exist (e.g., maraviroc)

  • Therapeutic monoclonal antibodies targeting this pathway are in development

  • Drug targets with genetic evidence supporting their involvement in disease are generally more likely to be successful in clinical development

  • The lack of genetic association with lung function suggests limited potential for CCR5 antagonists in treating respiratory conditions

Further research is needed to explore potential interaction effects in other disease contexts, particularly in conditions where either CCL3L1 or CCR5 variations have shown individual associations.

What challenges exist in studying CCL3L1 genomic variation and how can they be addressed?

Studying CCL3L1 genomic variation presents several significant challenges that researchers must address to obtain reliable results :

• Measurement accuracy challenges:

  • Early studies used qPCR assays with low signal-to-noise ratios, leading to potential measurement errors

  • The multiallelic nature of CCL3L1 copy number variation makes accurate determination difficult

  • There is a need for technical replicates and multiple measurement approaches to increase confidence in copy number calls

• Paralog homology issues:

  • CCL3L1 shares extremely high sequence homology with its paralogs (CCL3, CCL3L2, and CCL3L3)

  • CCL3L1 and CCL3L3 appear to be identical in their coding sequences

  • This high sequence similarity complicates the design of specific assays

• Assay specificity problems:

  • Many previously utilized RT-PCR based CNV assays are not specific for only CCL3L1

  • Primers and probes often align with overlapping sequences in at least two of the four paralogous genes

  • This leads to discordant calls and inconsistencies between different assays (concordance between assays ranges from 0.44-0.83)

• Population stratification concerns:

  • CCL3L1 copy number varies significantly across human populations

  • Europeans typically carry 0-5 copies while other populations can have up to 14 copies

  • This population variation can confound association studies if not properly controlled

These challenges can be addressed through several methodological approaches:

• Use of gold standard measurement techniques:

  • The triplex Paralogue Ratio Test (PRT) is considered the gold standard for CCL3L1 copy number measurement

  • This method provides three independent estimates that increase accuracy when averaged

• Multiple measurement validation:

  • Using multiple independent measurement approaches and comparing results

  • Validating high-throughput methods against established standards

• Careful primer and probe design:

  • Performing pairwise and multiple alignments of all four chemokine genes

  • Checking primer and probe specificity against these alignments

  • Testing assays on samples with known copy numbers