A variant in the sonic hedgehog regulatory sequence (ZRS) is associated with triphalangeal thumb and deregulates expression in the developing limb

Abstract

A locus for triphalangeal thumb, variably associated with pre-axial polydactyly, was previously identified in the zone of polarizing activity regulatory sequence (ZRS), a long range limb-specific enhancer of the Sonic Hedgehog (SHH) gene at human chromosome 7q36.3. Here, we demonstrate that a 295T>C variant in the human ZRS, previously thought to represent a neutral polymorphism, acts as a dominant allele with reduced penetrance. We found this variant in three independently ascertained probands from southern England with triphalangeal thumb, demonstrated significant linkage of the phenotype to the variant (LOD = 4.1), and identified a shared microsatellite haplotype around the ZRS, suggesting that the probands share a common ancestor. An individual homozygous for the 295C allele presented with isolated bilateral triphalangeal thumb resembling the heterozygous phenotype, suggesting that the variant is largely dominant to the wild-type allele. As a functional test of the pathogenicity of the 295C allele, we utilized a mutated ZRS construct to demonstrate that it can drive ectopic anterior expression of a reporter gene in the developing mouse forelimb. We conclude that the 295T>C variant is in fact pathogenic and, in southern England, appears to be the most common cause of triphalangeal thumb. Depending on the dispersal of the founding mutation, it may play a wider role in the aetiology of this disorder.

Preaxial polydactyly: interactions among ETV, TWIST1 and HAND2 control anterior-posterior patterning of the limb

“…The ZRS is necessary and sufficient to promote Shh expression specifically in the limb bud, but not elsewhere in the developing embryo. In addition, multiple point mutations scattered across the ZRS are individually linked to ectopic Shh expression in the anterior limb bud (Lettice et al, 2003;Maas and Fallon, 2005;Gurnett et al, 2007;Furniss et al, 2008), suggesting that the ZRS also mediates the repression that is essential for restricting Shh expression to the posterior limb bud. Although a number of transcription factors have been shown to regulate Shh expression in the limb bud (Buscher et al, 1997;Bourgeois et al, 1998;Qu et al, 1998;Zhang et al, 2009), few have been demonstrated to directly bind the ZRS (Capellini et al, 2006;Galli et al, 2010).…”

Enhancer-adoption as a mechanism of human developmental disease

“…These defects include preaxial polydactyly type 2 and type 3 (PPD 2/3, MIM#s 174500, 174600), triphalangial thumb polysyndactyly (TPTPS; MIM# 190605)), syndactyly type IV (SD4; MIM# 186200), and Werner's mesomelic syndrome (MIM# 188770). Well over 20 different point mutations in human, mouse, and the domestic cat are known to cause PPD [Albuisson et al, 2011;Dobbs et al, 2000;Farooq et al, 2010;Furniss et al, 2008;Gurnett et al, 2007;Lettice et al, 2003;Lettice et al, 2008;Sato et al, 2007;Wieczorek et al, 2010]. In addition, chromosomal rearrangements that result in genomic duplications of the ZRS are associated with TPTPS and SD4 [Klopocki et al, 2008;Sun et al, 2008].…”

Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb

“…Forelimb defects similar to those seen in Hox5 mutants have been identified in patients with HOS caused by Tbx5 mutations (32)(33)(34)(35)(36), and Hox genes have been reported to be capable of driving Tbx5 expression (46), OS caused by Sall4 mutations (29,30), TownesBrocks syndrome caused by Sall1 mutations (31,47) and SaethreChotzen syndrome caused by Twist1 mutations, which also show limb phenotypes in mutant mice (48)(49)(50). Mutations of both the human and mouse limb enhancer of Shh, ZRS (7,18,19,24), and human multiple congenital anomaly/mental retardation syndromes caused by mutations of Plzf, as well as loss-of-function mutations in Plzf in mice (37,39), lead to similar phenotypes. Based on these similarities, we investigated the expression of Tbx5, Sall1, Sall4, Twist1, and Plzf in our Hox5 mutants.…”

“…Some mutations in the Shh limb enhancer ZRS lead to loss of posterior digits reminiscent of loss of Shh function (3,4,8,17). In addition, many point mutations in the ZRS identified in spontaneous mouse mutants (Hx and M100081) (18), human patients (PPD2, Cuban mutation, Werner mesomelic syndrome, and others) (5)(6)(7)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), chickens (17), and cats (28) that lead to anteriorized and/or ectopic expression of Shh, indicating that the ZRS enhancer not only directs activation of Shh in the ZPA, but also is responsible for repression of Shh in the anterior limb.…”