Neurochemical studies indicate an association between SIDS and medullary 5-HT abnormalities in approximately 70% of cases, mandating the elucidation of potential abnormalities in the genetic program of 5-HT neuronal development in this disorder (23
). For this reason, we found it imperative to replicate the important study of Rand et al in which a rare insertion variant in the FEV gene was reported exclusively in SIDS cases of African-American descent (22
). African-American infants are more than twice as likely than non-Hispanic white infants to die of SIDS (33
); the SIDS rate for African-American infants is 1.15/1000 compared to 0.49/1000 in U.S Caucasian populations (1
). The documentation of a SIDS-related gene in African-Americans, e.g., the FEV insertion polymorphism, would be a major step towards explaining at the biologic level, the increased risk for SIDS in African-Americans. In contrast to the previous report (22
), however we observed FEV
c.128-(191_192)dupA in both SIDS and controls from all ethnic groups, not just SIDS cases of African-American descent.
Approximately one-third of African-American control individuals were heterozygous for c.128-(191_192)dupA demonstrating that this variant is in fact a common polymorphism among African-Americans. Furthermore, we found no evidence for a preferential association of this change with SIDS in the African-American population. We also observed a novel variant c.128-(301_306)delG in eight of the 99 Coriell African-American controls, as well as in two autopsied infant controls. This single base intronic deletion is in perfect linkage disequilibrium with c.128-(191_192)dupA, implying that it arose on an ancestral chromosome carrying the insertion polymorphism. As with the previously identified variant, c.128-(301_306)delG was also not associated with SIDS in African-Americans, but its clinical significance is unknown as we have limited power to investigate the influence of this second polymorphism on SIDS phenotype given the low incidence of the polymorphism in cases and controls and our limited sample size.
It is important to consider possible differences between the current study and that by Rand et al (22
) that may account for our inability to replicate the association of c.128-(191_192)dupA with SIDS in African-Americans. Clearly, these two studies report very different allele frequencies of the insertion polymorphism in the two sets of African-American populations; we identified it in 32 of 99 (32%) African-American control individuals while Rand et al. found it in only six of 98 (6%) total (zero of 49 non-SIDS controls). The difference between the two sets of controls is too large to be attributed to statistical sampling errors alone (p<1.36 × 10−6
comparing the two sets of controls). Possible explanations include technical differences in the assay and/or inaccuracies in base calling, inaccurate or inconsistent designations of racial affinity, and/or difference in the cases and controls used. Rand et al. investigated the entire FEV
gene; exons, intron-exon boundaries, and promoter sequence. We sought to replicate only their positive findings, thus we assayed a single amplicon containing the reported insertion polymorphism. However, except for a slight difference in the reverse primer used in our study compared to Rand et al., the experimental conditions were essentially the same, and were therefore unlikely to account for any discrepancy between the findings of the two studies. Both groups utilized direct DNA sequencing on both strands of genomic PCR products and we believe the likelihood of extensive miss typing due to technical artifact should be quite low given the robust nature of the technologies employed. The origin of African-American cases and controls in the study by Rand et al. (22
) is unknown, but, fortuitously, the Coriell Human Variation Panel of self-identified African-Americans is well-characterized genetically. Of 89 specimens reported in detail, the average contribution of African ancestry was 79% with 21% Caucasian contribution (34
). However, it is notable that the estimated genome-wide proportion of European ancestry among these specimens varied widely, from 1% to 62%. Thus studies of admixed populations, such as African-Americans, require large sample sizes to avoid obtaining a non-representative sample. Consequently, one contributing reason for differences between the two studies may have arisen from the possibility that different, non-representative samples of the population were studied by the two groups.
Although the overall sample size (78 SIDS cases and 355 controls) in our study was larger than the report by Rand et al. (96 SIDS cases and 96 controls of which about half were African-American), only seven African-American SIDS cases were available in our database. Thus, our power to detect association within the African-American subset was significantly lower than that of Rand et al. Nevertheless, our observation that roughly one-third of self- identified African-Americans are heterozygous for c.128-(191_192)dupA unequivocally demonstrates that this is a common polymorphism among African Americans and cannot be interpreted as a rare causative mutation. Regarding SIDS phenotype definition, it is possible that the difference in the classification of infant deaths accounted in small part for the different results, given that some cases (dying while awake) were potentially labeled as SIDS by Rand et al. but not in our study. Rand et al. classified cases according to the 1991 NICHD definition (35
); in our study, we classified cases according to the 2004 schema of Krous et al. that, unlike the NICHD definition, requires sudden death to be linked to a sleep period.
Of interest, we found a significant association for the insertion polymorphism within the SIDS cases in a Hispanic subset of our database. We caution, however, that the label Hispanic, like “African-American” applies to a heterogeneous group, often of mixed ancestry. Therefore, it is possible that the association signal was due to admixture, in particular with African-American ancestry, among SIDS cases. The Coriell HD100Mex panel is derived exclusively from the Mexican American community of Los Angeles, and may contain a smaller African contribution than the broader Hispanic population of SIDS cases and controls collected by the San Diego Medical Examiner's office. Furthermore, the association in our study was significant only before multiple testing correction and no other test showed a significant association for c.128-(191_192)dupA or c.128-(301_306)delG whether testing within groups or the entire sample. Therefore, unless and until this result can be replicated and confirmed in an independent data set, we conclude that there is little evidence to support an association of either polymorphism with SIDS in the existing datasets.
In conclusion, we were not able to replicate an association between the previously reported insertion mutation in the FEV
gene and SIDS in African-American infants. We caution, however, that our failure to confirm this finding in an independent SIDS dataset does not negate the potential significance of the FEV
gene to SIDS, particularly to its relationship with medullary 5-HT abnormalities in affected cases. Further work in SIDS is needed to explore the known functional regions of the FEV
gene, as abnormalities here could still lead to aberrant functioning of 5-HT neurons. Also, the interactions between the FEV
transcription factor and other transcription factors related to the 5-HT neuronal development cascade, e.g., lmx1b (16
), GATA2 (15
), need to be considered (36
). We still believe a full genetic screen of the FEV
gene is needed to gain complete insight into the molecular pathogenesis of the potential association between this important gene, SIDS, and medullary 5-HT abnormalities.