Here we report the largest single kindred with the most confirmed cases of a molecularly-defined SIX3 mutation. There is evidence that nine individuals in four generations carry the mutation, as shown by either direct sequencing or the presence of the SIX3 mutation-specific haplotype. At least an additional 4 individuals had clinical signs of HPE spectrum, and 2 individuals were presumed carriers, yielding presumptive positive findings in 15 individuals among 5 generations. It is possible that fewer individuals had the mutation. For example, individual II.5 might carry the same haplotype as those with the mutation, but not the mutation itself, and individual III.27 could have HPE due to a reason other than the mutation in SIX3. While a common mutation in at least these 15 individuals is the simplest explanation, it cannot be assumed that Occam’s razor provides the only explanation.
Study of this kindred recalls lessons learned in previous studies of HPE and other complex traits. First, this pedigree demonstrates the difficulties in the use of linkage analysis as a tool to discover disease-causing genes. Phenocopies are one complicating factor. Here, initial linkage analysis including the apparent phenocopy (IV.20) and her parent (III.11) did not demonstrate any meaningful linkage to the SIX3 locus. Even when direct mutation testing demonstrated that the phenocopy did not possess the mutation, repeat linkage analysis did not achieve statistical significance at the SIX3 locus.
Second, this pedigree demonstrates well the incomplete penetrance and variable expressivity seen in other kindreds with HPE. As described according to the “multiple hit” theory [Ming and Muenke, 2002
] other genetic and/or environmental factors likely affect phenotypic severity. In fact, the phenocopy described here (IV.20) may have been affected by these “modifying” factors, but may not herself have possessed the mutation in SIX3
. Despite the recent elucidation of the potential for direct Six3-Shh
genetic interactions in animals, there are no known cases of HPE in humans due to simultaneous mutations in SIX3
. Perhaps exonic coding mutations in SHH
are not found simultaneously with similar changes in SIX3
, but SHH
regulatory changes not routinely tested in CLIA laboratories may co-occur.
Third, this kindred illustrates the challenges of genetic counseling in cases such as these. Despite the fact that this sequence change was shown to be a significant loss-of-function mutation, the presence of the mutation in SIX3 was seen in both unaffected individuals and in individuals with severe HPE. In these circumstances, genetics professionals must be aware of this range of possibilities, and should attempt to incorporate this understanding both in the interpretation of test results and in making prognoses.
Finally, clinical examination of individuals with the SIX3 mutation who were considered to possess the HPE microform often had a sharply angular nose (described as “knifelike” by one clinician) without true hypotelorism or microcephaly. The correlation of this phenotype with the presence of the mutation was more obvious in retrospect, and highlights the fact that genetic disorders may manifest in ways not exactly as traditionally described. Along these lines, 15 years passed between the initial genetic consultation on the proposita and the elucidation of the molecular cause. This extended diagnostic period demonstrates the importance of perseverance despite initially negative studies, including applying new technology and testing newly discovered genes.