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Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain; however, the genetics of this heterogeneous and etiologically complex malformation is incompletely understood. Heterozygous mutations in SIX3, a transcription factor gene expressed in the anterior forebrain and eyes during early vertebrate development, have been frequently detected in human HPE cases. However, only a few mutations have been investigated with limited functional studies that would confirm a role in HPE pathogenesis. Here, we report the development of a set of robust and sensitive assays of human SIX3 function in zebrafish and apply these to the analysis of a total of 46 distinct mutations (19 previously published and 27 novel) located throughout the entire SIX3 gene. We can now confirm that 89% of these putative deleterious mutations are significant loss-of-function alleles. Since disease-associated single point mutations in the Groucho-binding eh1-like motif decreases the function in all assays, we can also confirm that this interaction is essential for human SIX3 co-repressor activity; we infer, in turn, that this function is important in HPE causation. We also unexpectedly detected truncated versions with partial function, yet missing a SIX3-encoded homeodomain. Our data indicate that SIX3 is a frequent target in the pathogenesis of HPE and demonstrate how this can inform the genetic counseling of families.

Background: Single median maxillary central incisor (SMMCI) is a rare anomaly that may occur alone or associated with other conditions, frequently as part of the holoprosencephaly (HPE) spectrum. However, it has been suggested that SMMCI alone, or associated with some midline defects, may be considered a different entity from HPE (OMIM: 147250). Families with SMMCI, without HPE cases, are difficult to counsel for the risk of HPE in future generations because the same midline defects described as part of the "SMMCI syndrome" can also be part of the HPE spectrum., Methods: We screened five cases of SMMCI for mutations in three HPE genes, SHH, TGIF, and SIX3., Results: A missense mutation c.686C>T was found in the gene SIX3 of one patient, which did not differ from the accepted 20% of known HPE gene mutations among all HPE cases. Our results and an extensive literature review of gene mutations in patients with SMMCI showed that 27/28 of them were in HPE genes: SHH (n = 21), SIX3 (n = 3), TGIF (n = 1), GLI2 (n = 1), and PTCH (n = 1), and only one in the SALL4 gene., Conclusions: The clinical findings in patients with SMMCI without HPE in families with mutations in HPE genes cannot be distinguished from the findings reported in the SMMCI syndrome. Therefore, persons with SMMCI and their relatives should be carefully investigated for related midline disorders, especially of the HPE spectrum, and all known HPE genes screened., (2007 Wiley-Liss, Inc.)

Here, we report six Brazilian patients with holoprosencephaly caused by SIX3 mutations. Missense mutations were more common than frameshift mutations. Comparison of patients with missense versus frameshift mutations was essentially unremarkable. Our cases suggest that SIX3 mutations result in a more severe phenotype than other gene mutations for holoprosencephaly. One patient had a double SIX3 mutation, which has not been reported previously. In our SIX3 mutations, three were transmitted by the paternal side, two were transmitted by the maternal side, and one was a de novo event. Mutations in normal parents with severe involvement of their offspring does not allow prediction of phenotypic severity, which makes genetic counseling difficult.