Results from this study highlight a number of important findings regarding VACTERL association, which remains a poorly-understood condition. First, while previous studies have highlighted controversies regarding the inclusion of certain features, our results show that it may be hasty to ignore specific findings. Each component feature was observed in over half of the patients in our cohort, except for limb anomalies, which was seen in just under half. The fact that our cohort was overall well-characterized lends credence to the results here, as it is less likely that these patients had alternate diagnoses. However, it is very challenging to compare the results of our study with those of previous cohorts of patients diagnosed with VATER/VACTERL association. Methods of ascertainment, inclusion, and even criteria for diagnosis differed, and extraction of exact data from previous publications was difficult.
This study shows how the application of various types of statistical analyses can be used to dissect a complex condition. When looking at pairs of any two component features in isolation, we did not observe statistically significant association. However, when we considered the co-occurrence of features using clustering analysis techniques, we do observe trends which suggest the grouping of certain features in our cohort. two The application of two types of cluster analysis show that vertebral, cardiac, and renal anomalies tend to occur together when the cohort is considered as a whole. However, with LCCA, some clusters of patients (such as clusters 1, 2, and 4) do demonstrate this grouping of findings, but other subgroups do not. This demonstrates the power of LCCA to separate cohorts of patients into subpopulations, thus demonstrating heterogeneity that cruder analyses based on entire cohorts will miss.
The application of LCCA to complex diseases may result in better phenotypic classification of the patients according to different underlying biological factors [Acosta et al., 2008
]. These factors may include multiple interacting molecular and environmental insults occurring in a tightly ordered spatial and chronological manner during embryonic development. LCCA may thus be a valuable tool for investigating pleiotropy and variable expressivity in disorders such as VACTERL association.
Previous work has suggested that patients with VATER association might be subdivided into “upper” and “lower” groups, with patients with cardiac malformations belonging to the upper group, and patients with renal defects belonging to the lower group [Källén et al., 2001
]. Our LCCA analysis suggests other, more specific types of subgroups. First, there seem to be large and distinct subgroups (clusters 1, 2, and 3, which together comprise 51 of the 60 patients analyzed by LCCA) segregating upper vs. lower intestinal tract malformations. This makes intuitive sense, both in terms of spatiotemporal developmental separation of the upper and lower gastroinestinal tract and because different genes affect the development of the upper and lower intestines. More generally, these data suggest the possibility of exploring candidate genes specific for each cluster based on the component findings in that cluster and the known expression patterns of the genes. Data from this type of LCCA could also be helpful in attempting to unravel the causes of VACTERL. For example, with a larger sample size, it would theoretically be possible to conduct association-type analyses based on the clusters defined by LCCA. That is, distinct clusters could be compared with one another as “cases” and “controls”.
Second, patients who belong to a relatively small subgroup (cluster 4, which includes only seven patients) appear to have most or all features of VACTERL association. There may certainly be ascertainment bias in which more severely affected individuals are more likely to seek a study such as the one described here. However, this group also points to the possibility of eventual genotype-phenotype correlations: perhaps certain genetic changes tend to result in the presence of a distinct pattern of anomalies, as has been elegantly described in the case of mutations due to GLI3
[Johnston et al., 2005
The diversity of possible alternative diagnoses among patients excluded from the final statistical analysis (see the Methods section for specific details) emphasizes the importance of a thorough clinical evaluation by a geneticist familiar with VACTERL association and related disorders. Certain conditions that have features overlapping those of VACTERL association, such as CHARGE syndrome, 22q11.2 syndrome, deletion 22q11.2, and Baller-Gerold syndrome, already have genetic testing available. Results from this testing can be helpful for assigning a diagnosis and to identify associated medical problems that should be evaluated, for establishing prognosis, and for genetic counseling. Among the conditions that are part of differential diagnosis, Fanconi anemia is particularly noteworthy, as patients with this disorder have a high risk of hematologic abnormalities including bone marrow failure, myelodysplastic syndrome, and leukemia. The availability of chromosomal breakage assays as a sensitive test for Fanconi anemia makes it especially important not to miss this critical diagnosis [reviewed in Tamary and Alter, 2007
While some manifestations of VACTERL association may be obvious either prenatally or immediately after delivery, others can be more subtle, and may be ascertained only many years later. We advise clinicians encountering patients with features of VACTERL association to carefully evaluate for the presence of each of the component features, as well as for other features that may be associated with overlapping conditions. In our experience with our NIH study on VACTERL association, we have often identified the presence of subtle VACTERL anomalies that were not previously known. While the majority of these findings are likely to have no clinical significance (such as the presence of very subtle limb anomalies), others, such as mild structural renal or cardiac anomalies, warrant further clinical monitoring. Finally, just as it is important to perform laboratory-based testing to rule out Fanconi anemia, it is also important to assess for the presence of hydrocephalus (found in VACTERL-H, or VACTERL with hydrocephalus), as this condition may warrant immediate medical intervention.
In summary, it is interesting to ponder explanations for the presence of the distinct groups identified in our analysis, but replication of these results involving larger number of patients is critical to establish the validity of our findings. As our continued work into genetic and environmental causes of this condition proceeds it will be additionally revealing to reexamine our results in retrospect to see if there is indeed a plausible explanation for segregation into subgroups. While our analysis shows some trends which are worth pursuing, new advances in diagnostic genetic techniques, such as extremely high-density microarray platforms and the increasing availability of high-throughput sequencing (including genomic sequencing), will likely result in a causal explanation for many previously poorly-understood conditions. It will be fascinating to reanalyze this cohort as such explanations become available.