Introduction

Mannose-binding protein (MBP) is a member of the family of proteins known as collectins [1], characterized by the presence of carbohydrate recognition domains linked to collagenous regions. The protein binds mannose and N-acetyl-glucosamine-terminated glycoproteins [2, 3] and can activate the classical pathway of complement independent of antibody [4–6]. We have also shown that a common opsonic defect presenting as frequent unexplained infections in early life is associated with low serum levels of MBP [7]. Subsequently, we identified two frequent mutations in exon 1 encoding the N-terminal part of the collagenous domain [8, 9]. In Eurasian populations, a point mutation in codon 54 (GGC → GAC), when translated, substitutes aspartic acid (D) for glycine (G), and in Gambians, a comparable mutation in codon 57 (GGA → GAA) substitutes glutamic acid (E) for glycine. These mutations disrupt the fifth and sixth collagenous Gly-X-Y repeats by inserting a bulky dicarboxylic acid in place of the glycine residue. Since glycine residues occupy key axial positions in the structure of the collagen helix, we have predicted that these disruptions could lead to instabilities in the secondary structure, render the protein vulnerable to degradation and result in low serum concentrations [8, 9]. Recent X-ray crystallographic studies of a collagen-like peptide containing a single (Gly → Ala) substitution of the consensus sequence have confirmed a subtle alteration of the conformation, with local untwisting of the triple helix [10].

We found the codon 57 mutation to be remarkably common in the Gambian population with a gene frequency of 0.29 in adults and 0.23 in newborns [9]. Similarly, the frequency of the codon 54 mutation was also high, with a gene frequency of 0.16 in British Caucasians and 0.11 in Hong Kong Chinese [9]. The codon 54 mutation was also independently identified in both Danish [11] and Eskimo [12] populations with a frequency of 0.13. Subsequently, the codon 57 mutation was confirmed in Kenyans (gene frequency 0.23) and a rare third mutation was identified in codon 52 (resulting in an Arg → Cys replacement) at a frequency of 0.05 in both Danish and Kenyan populations [13].

In this report, we have extended the populations studied for MBP mutations to include (1) groups from the south Pacific, namely Papua New Guinea and Vanuatu (Melanesia) and (2) two further sub-Saharan populations, namely the Xhosa from South Africa and San Bushmen from Namibia. In addition to genotyping, serum protein MBP levels were determined for all groups except that from Papua New Guinea.

Materials and Methods

Subjects and Samples

Gambian and Chinese Population Groups. These adult population groups and the sampling methods used have been described previously [9]. Briefly, two Gambian cohorts were studied comprising 100 adult males and 99 newborns (40 male, 59 female). The Chinese population consisted of 123 adult donors (63 male, 60 female). In addition, for certain comparisons of MBP protein levels, unpublished data for samples of Chinese cord blood (n = 60) were also used.

Papua New Guinea. Blood samples were collected in a malaria case control study by Dr. David Laloo from individuals living in and around Port Moresby on the south coast of Papua New Guinea. The buffy coat was separated from each sample, frozen and transported to the UK on dry ice for subsequent DNA extraction. 50 of these samples (25 from individuals who presented with acute malaria and 25 from age-matched controls) were made available for the present study.

Vanuatu (Formerly the New Hebrides). As part of a large infant cohort study, cord blood was collected from consecutive births on the islands of Espiritu Santo and Maewo in the northern part of Vanuatu. After collection, cells and plasma were separated and frozen at −20°C and transported to the UK on dry ice, where DNA was extracted from the cell sample. For the present investigation, 112 of the babies (59 from Espiritu Santo and 53 from Maewo) who were unrelated and had been regularly followed in the cohort study were selected for analysis.

Xhosa. Samples of whole blood and serum (n = 55) were collected from blood transfusion donors in Cape Town. Separated serum and blood, collected into EDTA tubes, were frozen and shipped to London in dry ice, and stored at −70°C.

San Bushmen. Sekele San DNA (n = 59) with matching plasma was collected from unrelated subjects in Namibia, air freighted to London in dry ice and stored at −70°C until required.

Analysis of Mutations

The codon 54 and codon 57 mutations were analysed by a combination of techniques following PCR amplification as previously described [9]. The restriction enzymes BanI and MboII were used for primary ascertainment of the codon 54 and codon 57 genotypes, respectively. Selected samples were subjected to DNA sequencing using Sequenase (Cambridge Bioscience).

Codon 52 genotyping and independent confirmation of codon 54 and codon 57 genotype assignments were obtained by oligonucleotide-specific hybridization using a modification of the procedure of Wordsworth et al. [14]. For this, aliquots of the final amplification mixture were rendered single stranded prior to transfer to nitrocellulose (Hybond N, 0.45 urn, Amersham). The PCR product (15 was mixed with denaturing buffer (86 µl Tris 10 mM, EDTA 0.5 mM, pH 7.5; 8 µl NaOH 6 M, 6 µl EDTA 0.5 M, pH 7.5) in microtitre plates on ice for 10 min, neutralized with 110 µl of 2 M ammonium acetate and then transferred to nitrocellulose filters using a Bio-Rad manifold. The filters were prewetted in water and each well washed with 200 µl 1 M ammonium acetate before and after transfer.

Filters were washed briefly in 6 × SSC and exposed face down to UV for 3 min. Duplicate filters were incubated for 10 min at 31°C in 1.8 × SSC, 0.2% (wt/vol) Ficoll, 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) BSA, 0.5% (wt/vol) SDS and yeast tRNA (400 µg/ml) and then hybridized with 32P-end-labelled oligonucleotide probes (activity 1 × 108 counts/min/mg) in 1.8 × SSC for approximately 8 h at 31 °C. The following probes were used:

Table 1
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The filters were washed for 10 min in 6 × SSC before exposing to film for 1 h at −70°C to establish a baseline prior to higher-stringency washes used to remove any probe not completely matched to the product sequence. The wash temperature was based on the theoretical melting temperature of the oligonucleotide probe. After the high-temperature washes, the filters were exposed to film as appropriate for the signal strength. Filters were stripped using a solution of NaOH 50 mM, Tris 2 mM pH 7.5, NaCl 0.5 M, followed by 3 × 5 min washes in 6 × SSC, checked by autoradiography and then hybridized with the next probe.

Nomenclature

The following nomenclature will be used throughout this paper for genotypic variants: WT = an individual with the wild-type MBP gene sequence; WT/G54D = an individual heterozygous for the codon 54 mutation; G54D/G54D = an individual homozygous for the codon 54 mutation; WT/G57E = an individual heterozygous for the codon 57 mutation; G57E/G57E = an individual homozygous for the codon 57 mutation, and WT/R52C = an individual heterozygous for the codon 52 mutation.

Assays for MBP

Serum levels of MBP were determined using a modification of the symmetrical ELISA procedure as previously described [9]. The commercially available murine monoclonal antibody clone 131-1 (Cymbus Bioscience, Southampton, UK) was used as the capture reagent and a biotinylated preparation of the same antibody was used as detector.

Statistical Analysis

Serum MBP data for various population groups were compared at the 5% level using the non-parametric Mann-Whitney rank-sum test to investigate the null hypothesis that the population medians are equal.

Results

MBP Genotypes of South-West Pacific Populations

Vanuatu

Populations from the islands of Maewo and Santo were analysed for the presence of the three known MBP mutations. No individuals were found with either the codon 52 or codon 57 mutations and only 3 individuals out of a total population of 112 were found to be heterozygous for the codon 54 mutation. These data are shown in table 1 and compared with previously published frequency data for a Chinese population.

Table 1 Frequency analysis for the MBP codon 54 mutation in populations from the south-west Pacific and south-east Asia
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Serum levels of MBP were determined in both populations and the medians and ranges for these populations are compared with our previous data for Chinese cord samples in table 2.

Table 2 Cord blood MBP levels in populations from Vanuatu compared with Hong Kong Chinese
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As previously shown in the Chinese population, WT/G54D (heterozygous) individuals had serum levels of MBP which were significantly lower than those of the corresponding wild-type individuals (p > 0.0001 for all comparisons).

Papua New Guinea

Genotyping data only were available for the Papua New Guinea population and these are included in table 1. Individuals typing as WT/G54D were essentially evenly divided between the controls (3/25) and the malaria-infected (4/24) subgroups. Again, no individual typed positive for either the codon 52 or codon 57 mutations.

MBP Genotype of Sub-Saharan African Populations

MBP genotyping results for the Xhosa (South African) and San Bushmen populations are shown in table 3. No individual in either population typed positive for the codon 52 mutation.

Table 3 Frequency analysis for MBP codon 54 and codon 57 mutations in sub-Saharan African populations
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The Xhosa population was similar to the Gambian with a codon 57 gene frequency of 0.27 and no evidence for the codon 54 mutation. In contrast, the San Bushmen had a much lower frequenc of the codon 57 mutation and four individuals were found to be heterozygous for the codon 54 mutation. The latter results were confirmed by sequencing.

The serum MBP levels of both the Xhosa and San Bushmen are compared with our previously published Gambian data in table 4.

Table 4 Serum MBP levels in three sub-Saharan African populations tabulated with respect to genotype
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The serum levels of MBP in the wild-type San Bushmen were significantly lower than those of wild-type Xhosa (p = 0.0024) but did not differ significantly from the wild-type Gambians.

WT/G57E individuals of all three population groups had serum MBP levels which were significantly lower than the corresponding wild-type individuals (p < 0.0001 for all comparisons).

Discussion

The mutations in codons 54 and 57 of the human MBP gene have previously been found at high frequencies in several population groups [9, 11–13], whereas the codon 52 mutation appears to be much less frequent. In a study of British Caucasians, Gambians and Chinese the codon 52 mutation was present at frequencies of 0.02, 0.01 and 0.01 respectively [own unpubl. obs.] and Madsen et al. [13] have reported frequencies for the same mutation of 0.05, 0.05 and 0 in Danish, Kenyan and Eskimo populations, respectively. In contrast, the frequency of the codon 54 mutation in four distinct populations ranged from 0.11 to 0.17 whereas the frequency of the codon 57 mutation ranged from 0.23 to 0.29 in three cohorts from east and west Africa. The presence of two independent but similar mutations in African and non-African populations led us to speculate that there might be some biological advantage associated with the heterozygous state [9]. We proposed that in such individuals there might be a reduced capacity for complement activation and immunopathologically mediated host damage. Another possibility, proposed by Garred et al. [12], envisages a reduced level of infectivity in MBP-deficient individuals for certain intracellular parasites and viruses which become coated with C3b opsonin and then gain access to phagocytic cells through C3 receptors. These two hypotheses are not necessarily mutually exclusive and both mechanisms may have been contributory factors at different times or in differing environments.

The serum MBP measurements performed in this study have again confirmed that the presence of either the codon 54 or codon 57 structural gene mutation usually has a profound effect on the circulating protein level. In common with previous studies, broadly distributed levels of MBP were observed in both wild-type and heterozygous individuals and are presumed to reflect polymorphisms in the promoter region of the MBP gene [15]. This may explain why the four San Bushmen genotyped as WT/G54D had MBP levels well within the wild-type range.

The populations investigated in this report are of particular interest since they include the first examples of groups having relatively low frequencies of the codon 54 and codon 57 MBP gene mutations. In the case of the populations from the south-west Pacific, we detected the codon 54 mutation previously identified in Hong Kong Chinese [9] but in a total population of 161 neither the codon 52 nor the codon 57 mutation was detected.

We have previously speculated that the codon 54 mutation in the MBP gene is likely to have arisen before the separation of the ancestral Chinese and European populations some 40,000 years ago [9]. On the basis of observations on classical genetic markers, it has been claimed that an earlier split (approximately 50,000–60,000 BP) of the ancestral non-African population separated Australians, New Guineans and south-east Asians from north Eurasians [16, 17]. A possible explanation of our findings for both the Papua New Guinea and Vanuatu samples is that they reflect this divergence before the codon 54 mutation occurred. The low frequency observed may simply reflect a more recent admixture of the codon 54 mutation. If there is a heterozygous advantage associated with the MBP structural gene mutations it seems unlikely that a previously high frequency of the mutant allele would decline to the present low levels and this has not occurred in the Greenland Eskimo who are presumed to have migrated out of Asia with the codon 54 mutation some 18,000 years BP.

This is the first report of a sub-Saharan African population which does not exhibit a high frequency of the codon 57 mutation. If the high frequencies observed elsewhere are indeed the result of positive selection, then it is not unreasonable to surmise that the San are different because the selection pressures acting on hunter-gatherers with respect to their exposure to bacterial infections and intracellular parasites such as mycobacteria and Leishmania are very much less than on settled agropastoralists living in close contact with each other.

The finding of the codon 54 mutation in 4/58 San is also of considerable interest. The origins of the Khoisan peoples, including the San and the !Kung, remain an extremely controversial issue. Analysis of mitochondrial DNA samples from the !Kung suggests a direct descent from primitive human ancestors [18]. In contrast, an analysis of gene frequencies by Cavalli-Sforza et al. [19] indicates that the Khoisan are the result of a relatively early admixture between Africans and Asians. If the latter hypothesis proves to be correct, the codon 54 mutation in the San has, like the codon 57 mutation, also failed to achieve a high frequency.

Although further studies are required to address the issues raised by the unique patterns in the San, the world distribution and frequencies of the two major mutations remain essentially distinct and suggest independent origins since the migration of Homo sapiens out of Africa. These distributions and possible migration routes are summarized schematically in figure 1. This does not include data for the much rarer codon 52 mutation which has previously been found at low frequencies in both African and non-African populations. More extensive studies in larger populations are needed before conclusions can be drawn concerning this mutation.

Fig. 1
figure 1

A Frequencies of the codon 54 and codon 57 mutations of MBP in various populations studied to date. [The data summarized are taken from ref. 9, 13 and the present study]. B Hypothetical dispersion routes for the codon 54 (——) and codon 57 (—.—.—) mutations of human MBP superimposed on a theoretical dispersion of human populations [16, 17]. The earliest migration into Papua New Guinea and Australia (.....) may have predated the codon 54 mutation.

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