We report a novel heterozygous variant in the IHH gene in a Japanese family with BDA1. BDA1 is inherited in an autosomal dominant manner and is characterized by hypoplasia or aplasia of the middle phalanges of digits 2–5. Approximately half of the analysed BDA1 cases are due to mutations in IHH . The Hedgehog (Hh) family of secreted proteins regulates various developmental processes, maintains adult tissue homeostasis, and functions as a morphogen gradient [15, 16].
Hh signalling is mediated by its N-terminal domain (HhN), and reception of the HhN signal is modulated by several cell surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), cell adhesion molecule-related, down- regulated by oncogenes (CDO), Hedgehog-interacting protein (Hip), and growth-arrest-specific 1 (Gas1) [2, 17,18,19,20,21,22]. In mammals, there are three homologues of the Hh family, Sonic (SHH), Indian (IHH), and Desert (DHH), each of which has tissue-specific functions. In limb development, SHH acts early, regulating patterning and growth . Mutations in SHH are known to cause holoprosencephaly in humans [2, 24]. IHH, which is produced by prehypertrophic chondrocytes during endochondral ossification, acts later and is not believed to affect patterning but to regulate endochondral bone formation by controlling chondrogenic differentiation and proliferation [25,26,27,28].
IHH was shown to be a disease locus for BDA1, with IHH mutations c.G283A (p.E95K), c. C300A (p.D100E), and c.G391A (p.E131K) being initially identified in three Chinese BDA1 families . Since the first report of these three variants, additional variants causing BDA1 have been identified. Reviewing published cases in addition to novel mutations identified by them, Byrnes et al.  concluded that all BDA1 variants involving codons 95, 100, 128, 130, 131 and 154 are limited to a 59-amino acid region of the N-terminal active fragment (IHH-N) that spans codons 95–154 Soon after this review, Stattin et al.  reported a Swedish family with a novel Arg158Cys mutation, showing that Byrnes et al.’s  proposal is not always the case. Since then, some novel BDA1 associated variants have been reported in various populations [7, 12, 13]. However, to date, variants other than those involving codons 95, 100, 128, 130, 131, 154, and 158 have never been identified. Using the X-ray crystal structure, McLellan et al. , showed that SHH-CDO interactions require calcium and that the CDO binding interface on SHH is conserved in nearly all Hh proteins. They further showed that this interface is a hotspot for mediating interactions between SHH and CDO, Ptc, Hip and Gas1, and mutations causing BDA1 including the one described by Sattin et al. map to this calcium-binding site and disrupt interactions with these partners. Kavran et al.  determined the crystal structures of IHH-N both alone and complexed with CDO, which allowed the direct visualization and interpretation of BDA1-causing mutations. They grouped BDA1-associated variants into two categories. The first group affects the calcium-binding region of IHH and includes E95K, E95G, D100N, D100E, and E131K. The second category of variants, with R128N, T154I, and T130N, interrupts hydrogen bond networks formed between IHH and CDO.
Here, we identified a novel in-frame deletion in IHH, designated NM_002181.4:c.544_549delTCAAAG(p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del, in a Japanese family with BDA1. This variant is distinct from previously identified variants in the following respects. First, codons 182 and 183 are somewhat distant from the limited 59-amino-acid region of IHH of pathogenic variants reported previously. Second, with the exceptions of a family with one amino acid insertion (p.Glu95_Asn96insLys)  and another with one amino acid deletion (p.Glu95del) , all the BDA1-causing IHH variants are missense mutations, and patients with two amino acid deletions have never been described. Although the variant we report here has such distinct features, we consider it to be pathogenic for the following reasons. First, this variant is absent from the population database. Second, this variant is in-frame two amino acid deletion that causes a protein length change in a well-conserved, non-repetitive region. Third, three-dimensional modelling showed that S182 and K183 are located on the same surface and bind receptor proteins as do other residues associated with BDA1 and that S182 and K183 on IHH interact with Glu922 and Glu897 on CDO via hydrogen bonding. This is consistent with the proposal of McLellan et al.  who reported that in the murine SHH-CDO complex, the murine SHH interface encompasses a region involving K88, E90, R124, H134, R154, R156, S178, and K179, with the S178 and K179 residues being equivalent to S182 and K183 in human IHH, respectively. Overall, we considered that residues K182 and S183 construct important elements of the interface that interacts with receptor proteins and that their deletion impairs the interactions between IHH and CDO, Ptc and Hip, thereby causing BDA1. As our study shows that S182 and K183 are involved in hydrogen bond networks, this would classify S182 and K183 into the latter group of variants proposed by Kavran et al. Our findings also predict the possibility of other BDA1-related variants being located on the interface between IHH and receptor proteins. According to the ACMG/AMP standards and guidelines for the interpretation of sequence variants, the novel variant is likely pathogenic (PM1, PM2, PM4, PP1, and PP4) .
Clinical phenotypes, with respect to digit malformation, showed no remarkable heterogeneity in the affected family members. On examination, all digits of the hands were short, and the distal interphalangeal creases in the third to fifth digits were barely identifiable. Radial clinodactyly of the fourth digit was observed in all affected individuals. Radiographically, the middle phalanges of all digits of the hands were present but uniformly shortened, especially in the second to fifth digits, and the middle phalanges were fused to terminal phalanges in the fifth digits. With the exception that proximal phalanges and metacarpals showed no remarkable findings, these phenotypes are consistent with the description characterized by Fitch and judged as mild . A short stature of variable severity was observed among the Japanese family. Yang et al.  summarized several case reports of BDA1 and concluded that short stature was present only with IHH variants at Asp100. Affected siblings (V-4, 6) fell under the category of normal stature, although they were relatively short in height. However, given the severe short stature of the proband, his father, aunt, and his affected cousin, in contrast to the normal stature of unaffected cousins V-1 and 3 (HSDSs -1.0 and -0.6, respectively), the variant p.Ser182Lys183del could induce short stature with variable expressivity. This supports the notion that reduced IHH signalling may be responsible for reduced growth of long bones and result in short stature [6, 10, 32].