They found that a 98.6-kilobase duplication on chromosome 18 near the ALX4
gene, which plays an important role in mammalian eye development, was strongly associated with variation in blue eye color, primarily in Siberian Huskies but also in non-merle Australian Shepherds.
The association of SEPT9 with TAC1 methylation assay yielded a sensitivity of 73.1% and a specificity of 92.3%  while its association with TMEFF2 and ALX4
further increased both sensitivity (80.7%) and specificity (90.0%) .
This list includes ADHFE1, ALX4
, CNRIP1, EID3, ELMO1, ESR1, FBN1, HLTF, LAMM, NEUROG1, NGFR, RARB, RXRG, RYR2, SDC2, SEPT9, SFRP2, SOCS3, SOX17, THBD, TMEFF2, UCHL1, and VIM genes.
Lef1 works in association with Bmps to activate Alx4
to signal mesenchymal condensation and is required to activate Fgf4 expression for the Wnt cascade [Hudson et al., 1998; Nie et al., 2006b].
To achieve this combination of technical sensitivity and specificity, we optimized qPCR assays selected from the tissue studies that used the HM technology [TUSC3, tumor suppressor candidate 3; TMEFF2; ZDHHC22, zinc finger, DHHC-type containing 22; VTN; FOXL2, forkhead box L2; NGFR; ALX4
, aristaless-like homeobox 4; SIX6, SIX homeobox 6; EYA4; and SEPT9] (14).