To the Editor: We appreciate the comments by Heller and Bolz1 in their letter “The Challenge of Defining Pathogenicity: The Example of AHI1” and welcome discussion on the pathogenicity of the variants in AHI1. Our study was a large-scale screening investigation of patients with inherited retinal dystrophy.2 We considered the two variants as damaging for the following reasons. (i) The first variant, E1086G, was reported as a known disease mutation in a previous study3 and in the RetinoGenetics database4 as well as the Human Gene Mutation Database (CM080033), the major database for human mutation repositories.5 We also reexamined the pathogenicity using multiple other databases.2 Our analyses indicate that this variant is predicted to be damaging by MutationTaster6 (score 0.999) and PolyPhen-2 (ref. 7) (score 0.994). In addition, the minor allele frequency for this variant is less than 0.01 in all four previously described databases.2 (ii) Similarly, the second variant, Y218C, is predicted to be damaging by PolyPhen-2 (score 0.985) and Sorting Intolerant From Tolerant (SIFT) (score 0.002). The minor allele frequency for Y218C is less than 0.005 in all examined databases. Segregation testing also demonstrated that these two variants are inherited as a paternal allele and a maternal allele, respectively. (iii) The purpose of this study was to elucidate the mutational spectrum and genotype–phenotype correlations of inherited retinal dystrophy. AHI1 was included in our sequencing panel because many previous studies have reported the genetic defects in AHI1 as a cause for Joubert syndrome type 3, a syndromic retinal dystrophy.8,9,10,11,12 Our study suggests that AHI1 is a candidate gene for nonsyndromic retinitis pigmentosa. However, considering that this is the first study identifying AHI1 mutations in patients with nonsyndromic retinitis pigmentosa, further studies are required to confirm our finding.
The crux of the doubt expressed by Heller and Bolz1 about the pathogenicity of these two variants is based on (i) the high frequency of the variants in the ExAC database (Cambridge, MA; http://exac.broadinstitute.org, as of December 2014) and (ii) the SH3 domain of the AHI1 protein at which E1086G is located not being essential for AHI1 function.13 First, the frequency of Y218C in the ExAC database is 5.6E−4 (64/114,524) and that of E1086G is 3.8E−3 (457/119,154), neither of which is considered high frequency in an autosomal-recessive pattern of inheritance. Of note, 12 individuals in ExAC are homozygous for E1086G. However, because the ExAC database was originally released in October 2014 and we submitted the manuscript in June 2014, we were unable to include this information in our report. Second, Heller and colleagues concluded in their study that the SH3 domain is not essential for AHI1 function based primarily on the finding that two homozygous truncating mutations, Arg1066* and Trp1088Leufs*16, are nonpenetrant in a zebrafish model. This conclusion is slightly overstated because (i) previous studies have reported a functional role of the SH3 domain,14,15 and the frameshift Trp1088Leufs*16 mutation in the SH3 domain was identified in patients with Joubert syndrome;8 (ii) the evidence from zebrafish is limited in Heller and colleagues’ study13 because the total number of fish examined is not given, creating a lack of statistical data, and the eyeball size seems to be decreased by e23i23MO injection; and (iii) previous studies have demonstrated that different mutations in the same gene may lead to drastically different retinal phenotypes in mice.16 We believe that Heller and colleagues’ viewpoint is extremely meaningful in this field; however, the exact role of the AHI1 protein domains in a mouse model remains unclear and warrants further investigation.
In brief, we agree with Heller and Bolz1 that the pathogenicity of E1086G should be further examined because of the homozygosity reported in the ExAC database (as of January 2015). It is important to note that a rapid expansion of exome resources in recent years has increased the amount of information regarding the pathogenicity of variants, which might lead to inconsistent results when different database are analyzed. For the purpose of serving as a valuable reference for genetic disease studies and precisely defining the pathogenicity of variants, a comprehensive and integrative database is necessary.
Disclosure
The authors declare no conflict of interest.
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Jin, ZB., Huang, XF. Response to Heller and Bolz. Genet Med 17, 508–509 (2015). https://doi.org/10.1038/gim.2015.48
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DOI: https://doi.org/10.1038/gim.2015.48
