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Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19
A B W Boldt1, C Grisbach, R Steffensen
1Department of Medical Pathology, Hospital de Clínicas, Federal University of Paraná, Curitiba, Brazil.
Abstract:
Deficiency of mannan-binding lectin-associated serine protease 2 (MASP-2) has been associated with infections, whereas high levels appear to increase the risk of inflammatory disorders. Nevertheless, MASP2 haplotypes have been poorly investigated. To overcome haplotyping cost and time consumption, we developed multiplex polymerase chain reactions with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs), reducing the number of necessary reactions from 18 to 7. SNPs were distributed from the promoter to the last exon, and a single PCR-SSP was used for p.D120G. We evaluated the phylogenetic relationships and global distribution of 10 identified haplotypes in 338 Danish individuals with known MASP-2 and MAp19 levels and 309 South Brazilians. Four haplotypes were associated with reduced MASP-2 levels in plasma (lower than 200 ng/mL). Simultaneous association with the highest MASP-2 (over 600 ng/mL) and lowest MAp19 levels (lower than 200 ng/mL) was demonstrated with the intron 9 mutation (Kruskal-Wallis p < 0.0001). Cumulative genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both populations. Rapid and low-cost screening of patients with multiplex MASP2 PCR-SSP could be used to identify clinical conditions where MASP-2 (or MAp19) levels may be disease modifying, possibly improving disease outcome through early therapeutic and preventive measures.
Insights
Investigating mannan-binding lectin-associated serine protease 2 (MASP-2) haplotypes using multiplex PCR-SSP reveals associations with MASP-2 levels. This rapid screening method can identify individuals at risk for MASP-2 related infections or inflammatory disorders.
Area of Science:
- Immunogenetics
- Molecular Biology
- Human Genetics
Background:
- Mannan-binding lectin-associated serine protease 2 (MASP-2) deficiency is linked to infections, while high levels correlate with inflammatory disorders.
- MASP2 haplotypes, crucial for understanding MASP-2 regulation, have been underexplored due to complex and costly haplotyping methods.
Purpose of the Study:
- To develop a cost-effective and time-efficient multiplex PCR-SSP method for MASP2 haplotyping.
- To investigate the phylogenetic relationships and global distribution of identified MASP2 haplotypes.
- To associate specific MASP2 haplotypes with MASP-2 and MAp19 plasma levels.
Main Methods:
- Developed multiplex PCR with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs) in the MASP2 gene, reducing reactions from 18 to 7.
- Evaluated phylogenetic relationships and global distribution of 10 identified haplotypes in Danish and South Brazilian populations.
- Correlated identified haplotypes with MASP-2 and MAp19 plasma levels using statistical analysis.
Main Results:
- Identified 10 MASP2 haplotypes, with four associated with reduced plasma MASP-2 levels (<200 ng/mL).
- An intron 9 mutation was significantly associated with both high MASP-2 (>600 ng/mL) and low MAp19 (<200 ng/mL) levels (Kruskal-Wallis p < 0.0001).
- Genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both studied populations.
Conclusions:
- The developed multiplex MASP2 PCR-SSP is a rapid and low-cost screening tool.
- This method can identify individuals with specific MASP-2 or MAp19 levels relevant to disease.
- Early identification may facilitate therapeutic and preventive strategies for MASP-2 related conditions.
