In order to derive insight into the important functions and domains of the ZIC2 protein inferred from our mutational analysis, we have compiled all 83 of the known variants detected exclusively among HPE cases and not in controls into . Several important findings emerge from this compilation (broken down for convenience by mutation type in ). Regardless of mutation type these categories of variation have either been directly shown to reduce biological activity (Brown et al., 2005
) or would be predicted to lose activity by analysis of the effects on key structural motifs of virtually translated protein products.
A schematic representation of all of the mutations affecting the ZIC2 coding region sorted by their position and category
First, except for the poly-Alanine tract expansion/contraction cases, that continue to represent a prevalent mutation type (, see below), we observe that most mutations are detected in only a single family (i.e. family-specific alterations) and at least 50% can be demonstrated to be de novo
within the HPE proband's DNA (Dubourg, this study). There is a strikingly similar molecular pathology elucidated for the zinc-finger mutations in the X-linked ZIC3
gene and their proven effects on diminished DNA binding and reporter gene activation (Ware et al., 2004
; Zhu et al., 2008
). In this respect, heterozygous ZIC2
mutations differ from the hemizygocity of the X-linked ZIC3
gene or the homozygous hypomorph or null models of HPE in mice (Nagai et al., 2000
; Warr et al., 2008
Second, our findings clearly illustrate that there is a common feature of predicted loss-of-function attributable to virtually all of the HPE-specific variants, especially evident in our continued detection of a high frequency of frameshifts and truncations (>55%) also noted in the original studies (Brown et al. 1998
; Dubourg 2004
). None of the truncation mutations when hypothetically translated are consistent with a complete set of zinc-fingers; this is also true of the majority of the predicted frameshift variants (compare ). However, a significant number of frameshifts () or insertion/deletions () occur in the extreme COOH terminus of the protein indicating that simple variations affecting the zinc-finger domain are not the only mechanism affecting ZIC2 function and confirming previous interpretations (Brown et al. 2005
Thirdly, almost all of the mis-sense mutations occur in residues known to be either extensively conserved among the zinc-finger regions of all Zic-related genes (; see also Aruga et al., 2006
) or intrinsic to their ability to fold and interact with DNA. Specifically, each of the five zinc fingers is defined by a Cystidine2-Histidine2 (C2H2 motif) comprising four bonds that coordinate with a zinc-atom into a unique three-dimensional configuration (Redemann et al., 1988
). We observe that 5 of these 20 key residues are either directly mutated through codon changes or displaced by insertions (). In addition, we demonstrate that by compiling additional cases there are now numerous examples of codons or genetic elements important to the function of the protein that are targets of independent mutational damaging events in different families. For example, p.286His is not only an invariant residue within Zinc-finger #1 (ZF #1 in , and ) but it is also found mutated to different codons in three different HPE subjects, strengthening the interpretation that the zinc-fingers and their integrity are key to the normal function of the ZIC2 protein.
Detailed structure of the zinc-finger region
Of the four mis-sense alterations detected outside of the zinc-finger domain there is evidence for apparent clustering. The c.107A>C Gln36Pro variant has previously been shown to retain 170% activity in cell-based luciferase assays (Brown et al., 2005
). Interestingly, we now report that the adjacent residue c.109G>A Asp37Asn is altered in a novel HPE proband potentially supporting a role for this region of the NH2-terminus in ZIC2 function. Similarly, c.454_455delGAinsTT Asp152Phe has been shown to deleteriously affect function, while the nearby mutation c.382G>A Asp128Asn is presently untested. It is also worth noting that although the ZOC (Z
pa [Odd-paired] C
onserved) domain is highly conserved among ZIC proteins (Aruga et al., 2006
) it has not been implicated in our mutational analysis thus far.
Defects in splicing are predicted to follow from independent mutations in both donor and both acceptor sites (); the common consequence of these changes is to deprive the transcript from the ability to encode an intact set of zinc-fingers. Note that three apparently unrelated families have the same alteration in the splice acceptor site of the first intervening sequence (c.1076-1G>A or IVS1-1G>A). Even in-frame insertions tend to occur in key structural elements, such as ZF #3 and ZF #5 predicting altered DNA binding or its absence as the most common component of the molecular pathology.
One of the principal conclusions of the most rigorous functional analysis to date (Brown et al., 2005
) was that beyond the obvious importance of the zinc-finger domain (see above) there remained a significant fraction of all cases demonstrating length variations of either the poly-Histidine tract or especially the poly-Alanine tract (Brown et al. 2001
). While the former His length variation is currently postulated to be a simple population variant (stretches of 8-12 residues have been observed in both patients and controls) there is overwhelming evidence that an expansion of this poly-Alanine tract to 25 affects both DNA binding and reporter gene activation. We now show that length reductions to 5 or 10 aminoacids are also detected. While these two length reduction variations will require additional study, it is intriguing that the spacing between the zinc-fingers and the poly-Alanine tract is proven to be important for ZIC2 function, since the in-frame deletion of 12 aminoacids, variant #74 c.1330_1365del36, between the end of ZF #5 and the beginning of the poly-Alanine tract clearly reduces biological activity (Brown et al., 1998
Consistent with the fact that ZIC2 was initially described as a key gene within a minimal critical region of common HPE-associated cytogenetic re-arrangements, it follows that copy-number variations of the gene should be readily detectable and produce similar biological effects as a more typical loss-of-function allele through crucial coding region alterations. This is indeed the case, since the U. Rennes has identified four independent cases of ZIC2 loss through microdeletion (Bendavid and David, in preparation), the Maastricht team has detected one ZIC2 microdeletion (Paulussen et al., submitted), and our NIH studies have also identified one additional case (~1.25% of tested probands, Lacbawan and Muenke, in preparation). As new knowledge is gained relating to non-coding regulatory elements essential for ZIC2 expression there are likely to be additional mutational targets identified.