{'fraction_non_alphanumeric': 0.029262186057428995, 'fraction_numerical': 0.015648227838197325, 'mean_word_length': 5.222979552093476, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Lack of support for an association between CLEC4M homozygosity and protection against SARS coronavirus infection

To the Editor: Chan et al.1 reported that individuals homozygous for a tandem repeat polymorphism (VNTR) in exon 4 of CLEC4M were protected against SARS coronavirus infection (odds ratio of 0.7), whereas heterozygotes were more susceptible to infection. This repeat region encodes the extracellular neck domain of the L-SIGN (‘liver/lymph node-specific ICAM-3 grabbing nonintegrin’) molecule, which is responsible for oligomerization into a functional tetramer. Functional studies by Chan et al. suggested that the protective effect was due to formation of a homotetramer of L-SIGN, with apparently higher affinity for viral ligands, in homozygous subjects. However, the authors also indicated that a similar protective effect was observed in cells that expressed L-SIGN with only two repeats (see Supplementary Fig. 6 in Chan et al.). This finding is not consistent with the hypothesis that formation of homotetrameric L-SIGN accounts for protection against trans infection, because L-SIGN with two repeats cannot form stable tetramers2,3, and monomeric receptors show much lower affinity and avidity for viral ligands3. Furthermore, the presence of a variety of alternatively spliced CLEC4M mRNAs, including isoforms with partial deletion in the neck region, suggests that the correlation between genotype and function may not be a simple one4. As about half of the Chinese population consists of heterozygotes, the results of Chan et al., if confirmed, bear important public health implications for SARS susceptibility. We tried to replicate these findings with another collection of 177 individuals with SARS. All affected individuals had a laboratory-confirmed diagnosis of SARS infection by either PCR tests for SARS coronavirus or serology. We studied three independent control samples of Hong Kong Chinese: (i) anonymous archival cord blood samples (n = 463), to determine population genotype frequencies; (ii) healthy elderly individuals aged >70 years (n = 163), to determine if age had any effect on genotype frequencies and (iii) a further sample collected from local university students (n = 248). A fourth population sample collected in Beijing (in northern China) was used to determine if there was any subpopulation structure (that is, variation of allele frequencies across different parts of China) for this polymorphism. We purified genomic DNA from whole-blood samples and performed PCR to genotype the VNTR in exon 4 of CLEC4M using the same protocol as described previously1. We confirmed genotype calls by duplicated assays. Genotype frequencies and homozygote proportions are shown in Table 1. The genotype distributions and homozygote proportions of the three groups of controls were not different from those of individuals with SARS (P = 0.72). Genotype frequencies of all samples,

except the group of university students (P = 0.028), were in Hardy-Weinberg equilibrium (by a Markov chain method in GENEPOP). Furthermore, we compared the genotypes among individuals with SARS with different prognoses. If L-SIGN homozygosity is a protective factor against infection, it may also be associated with better prognosis after acquiring the infection. Therefore, we also examined whether homozygotes had a better prognosis by classifying individuals with SARS who had an uneventful recovery versus those who had severe disease and were admitted to the intensive care unit for mechanical ventilation support (an approach similar to that reported previously 5 ). However, we did not detect any significant association (P = 0.9, Supplementary Table 1 online).
Sample size is the main limitation of both studies. However, these two samples already represent the few 'large' collections of individuals with SARS available for genetic study. To estimate the size of an overall effect, we performed a meta-analysis of the two data sets together by the Mantel-Haenszel test using control groups in Hardy-Weinberg equilibrium (two groups of controls in this study and random controls from Chan et al.; total n = 1,468; 462 affected individuals and 1,006 controls). The combined odds ratio was not significant (combined OR = 0.84; 95% confidence interval: 0.66-1.06, P = 0.14).
The difference in the results between the two studies was basically accounted for by a difference in the homozygote proportions in the controls (45.4% in this study versus 55.0% in Chan et al.), while the homozygote proportions among individuals with SARS are almost identical (46.9% here versus 46.3% in Chan et al.). The reason for the discrepancy in the homozygote proportions in the 'control' groups is not clear. However, a subpopulation difference in allelic and genotypic frequencies exists between northern and southern Chinese. The seven-repeat allele was more prevalent in the Beijing sample (0.7 in Beijing versus 0.64 in Hong Kong; P = 0.05), which also largely accounted for the higher proportion of homozygotes (55.7% in Beijing versus 46.0% in Hong Kong; P = 0.02). Unrecognized subpopulation structure may confound genetic association studies. Results in the study by Chan et al. suggested that this confounding factor might be present. There were three groups of controls, including two groups of hospital controls (health care workers who worked in SARS wards and affected individuals attending various outpatient clinics) and a group of blood donor controls. Interestingly, genotype distributions from both groups of hospitalbased controls deviated significantly (P < 0.0001) or marginally (P = 0.05) from Hardy-Weinberg equilibrium.
In addition, other yet-unknown mechanisms (such as alternative splicing of the neck region, which could interfere with formation of homotetramers among homozygotes) may account for the discrepancy between the two studies. Replication is an important approach to verify any significant genetic association findings 6,7 , and additional association studies are required to establish the putative protective effect of L-SIGN homozygosity against SARS or other infections.

To the Editor:
In the January 2006 issue, Chan et al. 1 reported a significant association between severe acute respiratory syndrome (SARS) and a variable number of tandem repeats (VNTR) polymorphism in exon 4 of CLEC4M in a collection of individuals from Hong Kong. CLEC4M encodes L-SIGN ('liver/lymph node-specific ICAM-3 grabbing nonintegrin'), which serves as a receptor for many viruses, including SARS coronavirus (CoV) 2 . Individuals homozygous for CLEC4M tandem repeats were reported to be less susceptible to SARS CoV infection. The authors also showed that cells homozygous for CLEC4M repeats had a higher binding capacity for SARS CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, both genetic and functional studies suggested that homozygosity for CLEC4M was associated with protection against SARS CoV infection.
It is important to bear in mind that association studies require replication in independent populations 3 . We therefore attempted to replicate the findings of Chan et al. by genotyping the VNTR polymorphism in three additional collections of case-control samples from northern China: (i) the 'Beijing community population' , consisting of 339 individuals with SARS and 227 random controls recruited from the community 4 ; (ii) the 'Beijing health care worker (HCW) population' , consisting of 42 health care workers infected with SARS during the course of hospital duty and 40 health care workers who had worked in SARS wards but remained free of disease and were confirmed to be seronegative for SARS 5 and (iii) the 'Tianjin population' , consisting of 60 individuals with SARS and 129 disease-free controls (including 85 random controls and 44 health care workers) 6 . The three collections of case-control samples and their ascertainment criteria have been described in detail previously (Supplementary Methods online) 4-6 . All groups except the individuals with SARS from the Beijing community were in Hardy-Weinberg equilibrium. We found no significant differences in allele, genotype and homozygote or heterozygote frequencies between affected individuals and controls in the three populations ( Table 1 and  Supplementary Table 1 online). Early reports have shown that some nongenetic factors, such as comorbid conditions (including diabetes mellitus, hypertension, heart disease, tuberculosis, asthma and malignancy), are risk factors for the development of SARS 7,8 and may confound the contribution of genetic factors to this disorder. However, after stratification by comorbid conditions, the association remained nonsignificant in our Beijing community population ( Table 1). Of the 287 affected individuals without comorbid conditions, 19 were individuals with severe SARS who were identified by their admission to intensive care units or by their death, and the remaining 268 were individuals with mildly symptomatic SARS. To account for this, we assessed whether there was an association between homozygosity for CLEC4M tandem repeats and severity of SARS, but we found none ( Table 1). This result may be due to the limited number of individuals with severe SARS in the current study and will require confirmation in additional studies.
Collectively, the results in our three collections of case-control samples from northern China are not supportive of the findings of significant association between the VNTR polymorphism and SARS risk reported by Chan et al. 1  Additionally, just by using our Beijing community sample set, we have successfully confirmed the positive associations between the mannosebinding lectin polymorphisms and SARS risk 4 that were observed previously in a case-control sample from Hong Kong 10 . Furthermore, the consistency of the negative associations in our Beijing HCW population and our Tianjin population strengthens our results.
Second, there may be a small, populationspecific difference in the contribution of CLEC4M polymorphism to SARS susceptibility. This might occur if there were population differences in linkage disequilibrium pattern or allele frequencies of CLEC4M. Indeed, the homozygote or heterozygote frequencies in individuals with SARS in our Beijing community or Beijing HCW populations were significantly different from those reported for affected individuals in Hong Kong community or Hong Kong HCW populations (P = 0.0066 and P = 0.017, respectively; Supplementary Table 1). Additionally, the difference in allele frequency between Hong Kong outpatient controls and Tianjin random controls and the differences in homozygote or heterozygote frequencies between Hong Kong HCW controls and Tianjin HCW controls were also significant (P = 0.0064 and P = 0.017, respectively; Supplementary Table 1). Furthermore, there was also a significant difference in the genotype and homozygote or heterozygote frequency between the Chinese population and Europeans 1 .
Another possibility is that Chan et al. unwittingly neglected to account for the potential confounding factors that may distort the contribution of CLEC4M VNTR polymorphism to SARS susceptibility. Although Chan et al. took stringent precautions to stratify their samples by 'health care worker' , population samples may still differ by many other factors that depend on setting and context of recruitment, such as age of the subjects at SARS onset, sex of the subjects and any comorbid conditions. Unfortunately, Chan et al. do not provide data with regard to such important information on confounding factors. Last, the initial findings of Chan et al. may not represent real associations and might be false positives. In genetic association studies of common diseases, there is a very low prior probability of detecting a true association result when accounting for statistical adjustment for multiple comparisons. Indeed, the genetic associations presented by Chan et al. were marginally statistically significant (P = 0.027, 0.045 and 0.031, when comparing all SARS samples to random controls, community SARS to outpatient controls and HCW SARS to HCW controls, respectively). Thus, we did not find any significant differences in allele, genotype and homozygote or heterozygote frequencies between cases and controls in our three independent populations of northern Chinese. Although the biological plausibility of L-SIGN and the functional evidence of the VNTR polymorphism in the original report remain interesting, we urge that the association between CLEC4M polymorphism and SARS be investigated in other subpopulations of ethnic Chinese origin (for example, Taiwanese or Guangdong Chinese) or in those of different ancestry, such as Europeans.