On the basis of this finding, in the second phase, we went on to sequence all coding exons of HPCA in the same 151 samples and also in a second cohort of 288 non-autosomal-dominant, young-onset (<40 years of age) subjects exhibiting dystonia that was either generalized or most prominent in the upper limbs or cervical or cranial region (i.e., we prioritized a distribution similar to the phenotype observed in other families). We found only one additional variant of any kind, and this was in the same sample that harbored the p.Thr71Asn substitution. The additional variant was a missense mutation (c.568G>C [p.Ala190Thr]) located toward the end of HPCA exon 4, which encodes the C terminus of the protein. The nucleotide involved is conserved (PhyloP = 2.015; PhastCons = 1), and the affected amino acid is conserved in most species, except the fly (D. melanogaster) and the Tasmanian devil (S. harrisii). However, only MutationTaster predicts it to be disease causing; SIFT, PROVEAN, and PolyPhen-2 predict that the substitution will be tolerated. The p.Ala190Thr substitution is not located in any EF-hand domain, and the mechanism by which it might impair protein function is less obvious. However, we note that it has previously been suggested that the C-terminal regions of other NCS proteins might be involved in fine-tuning their response or determining target specificity.20, 21