Background & Summary

Human mannose-binding lectin (MBL) is a C-type serum lectin produced in the liver that plays a crucial role in innate immunity1. MBL may mediate phagocytosis by binding to specific carbohydrate moieties on various pathogens, thereby utilising identified phagocyte receptors2. Additionally, MBL employs MBL-associated serine proteases (MASP)-1 and −2 to activate the MBL pathway of complement3. The MBL protein is encoded by the polymorphic MBL gene, which consists of four exons interrupted by three introns and is located on chromosome 10 (10q11.2-q21)4.

Single nucleotide polymorphisms (SNPs) at specific nucleotide positions have been identified: −550 (G/C or H/L), −221 (C/G or X/Y), +4 (C/T or P/Q), +223 (C/T or A/D), +230 (G/A or A/B) and +239 (G/A or A/C) within the promoter/5′ untranslated and exon 1 regions5. Functional characterisation of SNPs in the promoter region, altering MBL transcription, underscores the significance of these genetic differences for MBL circulating levels and expression6. In the exon 1 region, the wild-type (normal) allele is denoted as A, while the B (codon 52), C (codon 54) and D (codon 57) allelic variants are collectively referred to as O. Changes in amino acid resulting from exon 1 variations are believed to influence the functional properties of the MBL protein collagenous region7.

Theoretically, 64 unique haplotypes could be generated by combining those six polymorphisms. However, only eight common haplotypes are typically observed in population studies due to significant linkage disequilibrium (LD) between the promoter and exon 1 SNPs5,6. Different haplotypes have been associated with high MBL levels, such as HYPA, LYQA, and LYPA; an intermediate MBL level represented by LXPA; and haplotypes linked to low MBL levels, including HYPD, LYPB, LYQC, and LYPD8,9.

Within the promoter and exon 1 regions, at least 30 SNPs and six deletion sites have been previously identified10. However, SNPs at positions −550, −221, +4, +223, +230, and +239 are commonly investigated and recognised as common point mutation secretors. These SNPs are frequently studied to characterise MBL variants and determine serum MBL protein levels. This strategy can be implemented using either serum level measurements or genotyping method such as polymerase chain reaction-sequence-specific amplification (PCR-SSP), -sequence-based typing (PCR-SBT), -restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Previous studies have shown that these MBL SNPs are associated with autoimmune diseases such as rheumatoid arthritis (RA)11,12, Sjögren syndrome (SS)13,14 and systemic lupus erythematosus (SLE)15,16; as well as infectious diseases including pulmonary tuberculosis (TB)17,18, acute respiratory infection (ARI)19,20 and vulvovaginal candidiasis (VVC)21,22.

Despite a wide coverage of studies and a flux of research findings, studies on the MBL gene in Malaysia are uncommon. Here we preliminary described the human MBL sequencing datasets, which include six common point mutation secretors and five additional polymorphic sites in the promoter region among Kelantan individuals (Table 1, Fig. 1). A diagram illustrating the workflow in this study is presented in Fig. 2. Genomic DNA was extracted from blood samples and subjected to direct PCR and Sanger sequencing. The resulting 886 bp sequence products were assembled using molecular genetic software to identify MBL haplotypes, which were later used to construct an evolutionary tree. This preliminary study provides baseline information for future research on the association between MBL gene polymorphisms and targeted diseases. Additionally, the generated data will be representative of the Malaysian population and may encourage broader population studies in the future.

Table 1 The profile of MBL polymorphic sites at chromosome 10q11.2.
Fig. 1
figure 1

Location of SNPs’ point mutation on the promoter and exon 1 of MBL gene. Noted that the figure is not drawn to scale.

Fig. 2
figure 2

Workflow of research study. QC- Quality control, NCBI- National Centre for Biotechnology Information.


Ethical statement

This study was approved by the Human Research Ethics Committee of Universiti Sains Malaysia (USM/JEPeM/19090533) and was performed in accordance with the guidelines set forth by the National Blood Centre, Ministry of Health Malaysia. Signed informed consent and demographic background information were obtained from each participant who agreed to take part in the study, and the data will be published without revealing their identities.

Sample collection and DNA extraction

Genomic samples were collected from 30 unrelated individuals from Kelantan. Approximately, 10 cc of blood was drawn from the peripheral vein and stored in the ethylenediaminetetraacetic acid (EDTA) tube. The total DNA was extracted using the gSYNCTM DNA Extraction Kit (Geneaid, Taiwan), following protocol provided by the manufacturer.

MBL genotyping

The isolated genomic DNA was amplified using a set of forward reverse primers specific for MBL (Table 2)19. The PCR amplification was carried out using Veriti TM 96-well fast thermal cycler. Subsequently, the amplified PCR products were purified using the GeneJET PCR Purification Kit (Thermo Scientific, United States). DNA sequencing was performed using ABI 3100 DNA Sequencer at First Base Laboratories Sdn Bhd (Malaysia).

Table 2 Properties of MBL primers used.

Sequencing analysis

The raw sequences were visualised and analysed using SnapGene version 6.1 (Fig. 3). Subsequently, all sequences were aligned with the Homo sapiens MBL2 RefSeqGene (LRG_154) of chromosome 10, utilising Sequencher version 5.4.6. The identification of MBL haplotypes was based on the allelic mutations (Table 1)5. Additionally, a Neighbor-joining tree23 was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) version 1124, with Pan troglodytes (chimpanzee) sequences (AY970679 and AY970685)25 as an outgroup (Fig. 4).

Fig. 3
figure 3

Map of 884 bp MBL sequence.

Fig. 4
figure 4

Neighbor-joining (NJ) tree of MBL in this study. The evolutionary distances were computed using Kimura 2-parameter method.

Data Records

The sequencing data are accessible in the National Centre for Biotechnology Information (NCBI), assigned with the reference numbers ON619541-ON619546 (Table 3)26,27,28,29,30,31. These sequencing information details pertain to a subset of individuals.

Table 3 List of registered MBL haplotypes in the NCBI website.

Technical Validation

The MBL-PCR products underwent assessment to confirm the absence of contamination through 2% gel electrophoresis. Subsequently, the raw sequences were aligned with RefSeqGene (NG_008196), and each sequence was manually inspected to ensure the absence of three stop codons: UAA, UAG and UGA. The evolutionary history was inferred using the NJ method23. The percentage of replicate trees (>50%) in which the associated taxa clustered together in the bootstrap test (1,000 replicates) I indicated next to the branches32.