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Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain
Riitta Paakkanen1, Hanna Vauhkonen, Katja T Eronen
1Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland. riitta.paakkanen@helsinki.fi
Insights
A new real-time qPCR method accurately detects complement component 4 (C4) copy number variation and deficiencies, including the common C4A CT insertion mutation. This improves diagnosis of C4A deficiency, aiding disease association studies.
Area of Science:
- Immunogenetics
- Molecular Diagnostics
- Human Complement System
Background:
- Low protein levels and copy number variation (CNV) of complement component 4 (C4A and C4B) are linked to various diseases.
- Existing high-throughput methods for C4 CNV analysis often miss the common C4A CT insertion (CTins) mutation, a key cause of low C4A protein levels.
Purpose of the Study:
- To develop and validate a SYBR® Green labelled real-time quantitative polymerase chain reaction (qPCR) method for accurate C4 CNV and CTins detection.
- To assess the impact of CTins on C4A deficiency frequency and its association with diseases.
Main Methods:
- A novel real-time qPCR approach using a specific concentration range and SYBR® Green labelling was developed to detect C4 CNV and CTins.
- The method was validated across three sample sets and applied to over 1600 patient samples.
Main Results:
- The developed qPCR method accurately identified C4 CNV and CTins.
- CTins were responsible for C4A deficiency in over 70% of carriers, and its assessment prevented misclassification in 20% of patients with C4A deficiency.
- C4A deficiency was found to be more prevalent in patients compared to the healthy reference population (OR = 1.60, p = 0.039).
Conclusions:
- Real-time qPCR, particularly with SYBR® Green labelling and CTins detection, provides a straightforward way to analyze functional C4 gene copy numbers.
- Accurate determination of C4A deficiency frequency, by including CTins assessment, is crucial for understanding genotypic and phenotypic disease associations.
Abstract:
Low protein levels and copy number variation (CNV) of the fourth component of human complement (C4A and C4B) have been associated with various diseases. High-throughput methods for analysing C4 CNV are available, but they commonly do not detect the most common C4A mutation, a silencing CT insertion (CTins) leading to low protein levels. We developed a SYBR® Green labelled real-time quantitative polymerase chain reaction (qPCR) with a novel concentration range approach to address C4 CNV and deficiencies due to CTins. This method was validated in three sample sets and applied to over 1600 patient samples. CTins caused C4A deficiency in more than 70% (76/105) of the carriers. Twenty per cent (76/381) of patients with a C4A deficiency would have been erroneously recorded as having none, if the CTins had not been assessed. C4A deficiency was more common in patients than a healthy reference population, (OR = 1.60, 95%CI = 1.02-2.52, p = 0.039). The number of functional C4 genes can be straightforwardly analyzed by real-time qPCR, also with SYBR® Green labelling. Determination of CTins increases the frequency of C4A deficiency and thus helps to elucidate the genotypic versus phenotypic disease associations.
