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Holoprosencephaly (HPE) is the most common malformation of the human forebrain, and may be due to cytogenetic anomalies, teratogens, occur in the context of a syndrome, or be due to mutations in single genes associated with non-syndromic HPE. Mutations in ZIC2, a transcription factor located on chromosome 13q32, are the second-most common cause of non-syndromic, non-chromosomal HPE. Blood samples from over 1000 individuals with HPE-spectrum disorders and their relatives were analyzed for sequence variations in ZIC2. We examined clinical details and included all other known previously published and unpublished cases of mutations in ZIC2 through a literature search and collaboration with other centers. We find mutations in ZIC2 in 8% of probands with HPE, and describe 153 individuals from 116 unrelated kindreds, including 137 patients with molecularly-determined mutations in ZIC2 and 16 patients with deletions of the ZIC2 locus. Unlike HPE due to mutations in other genes, the vast majority of cases are sporadic and the proportional distribution of HPE types differs significantly from previously published analyses of non-chromosomal non-syndromic HPE. Furthermore, we describe a novel facial phenotype in patients with mutations in ZIC2 which includes bitemporal narrowing, upsplanting palpebral fissures, a short nose with anteverted nares, and a broad and well-demarcated philtrum, and large ears. This phenotype is distinct from the standard facial dysmorphisms associated with non-chromosomal, non-syndromic HPE. Our findings show that HPE due to mutations in ZIC2 is distinct from that due to mutations in other genes. This may shed light on the mechanisms that contribute to the formation of the face and the forebrain and may help direct genetic counseling and diagnostic strategies.

Background Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. Objective To characterise genetic and clinical findings in patients with SIX3 mutations. Methods Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. Results In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. Conclusions Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.