Deletion of 2 amino acids in IHH in a Japanese family with brachydactyly type A1

Background Brachydactyly type A1 (BDA1) is an autosomal dominant disorder characterized by uniform shortening of the middle phalanges in all digits. It is associated with variants in the Indian Hedgehog (IHH) gene, which plays a key role in endochondral ossification. To date, heterozygous pathogenic IHH variants involving several codons, which are restricted to a specific region of the N-terminal active fragment of IHH, have been reported. The purpose of this study was to identify the pathogenic variant in a Japanese family with BDA1 and to evaluate its pathogenesis with regard to previous reports. Methods The proband, a 9-year-old boy, his siblings, and his father had shortened digits and a short stature of variable severity. Based on physical examinations, radiographic findings and family history, they were diagnosed with BDA1. This family is the first case of an isolated malformation in Japan. Sanger sequencing of IHH was performed on these individuals and on the proband’s unaffected mother. The significance of the variants was assessed using three-dimensional analysis methods. Results Sanger sequencing showed a novel IHH heterozygous variant, NM_002181.4:c.544_549delTCAAAG(p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del].. These two residues are located outside the cluster region considered a hotspot of pathogenic variants. Three-dimensional modelling showed that S182 and K183 are located on the same surface as other residues associated with BDA1. Analysis of residue interactions across the interface between IHH and its interacting receptor protein revealed the presence of hydrogen bonds between them. Conclusions We report a novel variant, NM_002181.4:c.544_549delTCAAAG (p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del] in a Japanese family with BDA1. Indeed, neither variations in codons 182 or 183 nor with such two-amino-acid deletions in IHH have been reported previously. Although these two residues are located outside the cluster region considered a hotspot of pathogenic variants, we speculate that this variant causes BDA1 through impaired interactions between IHH and target receptor proteins in the same manner as other pathogenic variants located in the cluster region. This report expands the genetic spectrum of BDA1.

(IHH) gene [1]. As a central signalling molecule mediating skeletal development, IHH plays an important role in modulating skeletal condensation, the growth and differentiation of chondrocytes, joint development and bone formation [2]. To date, more than 10 pathogenic variants in IHH have been identified in individuals with BDA1, and these variants cluster in the central region of the N-terminal active fragment [3]. Previous research has demonstrated that these residues constitute an interface that binds receptor proteins in a calcium-dependent manner, and the disruption of these calcium-dependent interactions by the variants causes BDA1. This is interpreted as a common mechanism of the BDA1-causing variants [4]. Most IHH variants have been characterized as missense [1,3,[5][6][7][8][9][10][11][12], except for one insertion [13] and one deletion [14].
Here, we report a five-generation Japanese family with BDA1. Although the novel two-amino-acid deletions of codons 182 and 183 revealed by Sanger sequencing were located outside the cluster regions of pathogenic variants reported previously, which are considered to constitute an interface that binds receptor proteins [3], a threedimensional analysis of IHH demonstrated that Ser182 and Lys183 are placed on the same surface as other previously reported mutations. We studied the pathogenesis of this variant through genetic and structural analyses.

Subjects
The proband (Fig. 1, V-5) is a 9-year-old boy who was born to non-consanguineous parents. He was referred to Kadono-Sanjo Children's Clinic for a congenital hand malformation, with shortening of all digits of the hands and a short stature. His siblings (V-4, 6), cousin (V-2), aunt (IV-4), father (IV-5) and grandmother (III-6) also showed shortened digits and a short stature of variable severity. We diagnosed this family with BDA1 based on physical examinations, radiographic findings and family history. Written informed consent was obtained from all participants in compliance with the Ethics Review Board at Kyoto University (Approval No. G1228, Approval date Feb. 17,2020). The pedigree is shown in Fig. 1.

Genetic analysis
Genomic DNA was extracted from the peripheral blood of the proband (V-5), his siblings (V-4, 6), his parents (IV-5, 6), his aunt (IV-4) and his cousins (V-2, 3). All three exons and intron-exon boundaries of IHH were amplified by PCR and subjected to Sanger sequencing. For exon 1, different primers for PCR and sequencing were used due to difficulties in primer design, whereas for exons 2 and 3, the same forward primer was used for PCR and sequencing (Table 1).
Three-dimensional structures and the spatial relationship between the variants in this case and the cluster regions of pathogenic variants reported previously were predicted using PyMOL (https:// pymol. org/2/). Moreover, residue interactions across the interface between IHH and receptor proteins were predicted using PDBsum (https:// www. unipr ot. org/ unipr ot/ Q14623).

Clinical phenotype
There was no history of other medical conditions, including nystagmus, musculoskeletal abnormalities, a developmental delay or scoliosis. On examination, both hands of the proband showed shortening of all digits, and the distal interphalangeal creases in the third to fifth digits were hardly identifiable. Radial clinodactyly of the fourth digit was observed (Fig. 2a). There were no dysmorphic findings other than those in the hands. Radiographs of the proband's hands revealed shortening of the middle phalanges of the second to fifth digits and fusion of the middle and terminal phalanges (Fig. 2c).

Discussion
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 [1]. The Hedgehog (Hh) family of secreted proteins regulates various developmental processes, maintains adult tissue homeostasis, and functions as a morphogen gradient [15,16].
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 [6]. 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. [3] 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. [11] reported a Swedish family with a novel Arg158Cys mutation, showing that Byrnes et al. 's [3] 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. [4], showed that SHH-CDO interactions require Fig. 5 Comparison of the amino acid sequences of human, mouse and chicken Hedgehog proteins. The S812 and K183 residues of IHH are evolutionarily conserved, as are the other BDA1-associated amino acid residues 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. [29] 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_549delTCA AAG (p.Ser182Lys183del) [NC_000002.12:g.219057461_219 057466del, 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) [13] and another with one amino acid deletion (p.Glu95del) [14], 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. [4] 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 BDA1related 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) [30].
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 [31]. A short stature of variable severity was observed among the Japanese family. Yang et al. [1] 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].

Conclusions
We report a novel in-frame deletion of IHH NM_002181.4:c.544_549delTCA AAG (p.Ser182Lys183del) [NC_000002.12:g.219057461_21905 7466del] in a Japanese family with BDA1. Although the two deleted amino acid residues are located outside of the cluster region that is considered a hotspot of pathogenic variants, these two residues constitute the interface of interactions between IHH and CDO, Ptc and Hip, as do the other BDA1-associated residues. Future functional assays and additional experiments will be required to confirm the pathogenicity of this variant. Nevertheless, we are certain that our current findings will expand the genetic spectrum of BDA1.