A novel pathogenic variant of IHH for Brachydactyly type A1


 Brachydactyly type A1 (BDA-1, MIM 112500) is a genetically heterogeneous autosomal-dominant disorder mainly characterized by shortening or missing of the middle phalanges. Brachydactyly type A1 (BDA-1) is caused by heterozygous pathogenic variants in a specific region of the N-terminal active fragment of Indian Hedgehog (IHH). In this study, we reported a Chinese family with 8 members affected with Brachydactyly type A1. After performing whole-exome sequencing in the proband, we identified a novel heterozygous missense variant c.299A>G (p.D100G) at the mutational hotspot of IHH gene. The variant co-segregated with BDA-1 in the pedigree, showed 100% penetrance for phalange phenotype with variable expressivity. This finding expanded the Brachydactyly type A1-related mutational spectrum of IHH gene.

Autonomous Region. The pedigree is shown in Figure 1.

Genetic analysis
Whole-exome sequencing and Sanger sequencing Whole exome sequencing using the genomic DNA of proband IV-1 (Figure 1) was performed. Agilent SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA) was used for target capture. The library was sequenced on Hiseq2500 platform (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. A custom pipeline mainly built on the Genome Analysis Toolkit (GATK) was used for sequence data analysis and annotation. Identification of causal variant was aided by the TGex software (LifeMap Sciences,USA). The candidate IHH variant was validated by Sanger sequencing and its pathogenicity classified following ACMG/AMP guidelines [6].

Clinical phenotype
We ascertained a five-generation pedigree with BDA1.Twenty two family members, including 8 affected members and 14 unaffected family members, were participated in this study ( Figure 1). The proband (Ⅳ-2) was a 30-year-old women, who presented with mild disproportionate short stature with a height SDS of -2.4SD. Her hand radiographs showed varying degrees of shortening of the middle phalanx of the second to fifth fingers, and the middle phalanges in digit five was fused to the terminal phalange as only one interdigital joint was visible. She also showed bilateral shortening of metacarpals bone 3-5. Her foot radiographs showed shortening of all digits, the middle phalanges of third to fifth toe were revealed that the novel missense variant had deleterious effects. According to the ACMG standards and guidelines for the interpretation of sequence variants [6], the novel variant is pathogenic.

Discussion
Brachydactyly type A1 is characterized by hypoplasia/aplasia of the middle phalanges of digits 2-5. Indian hedgehog (IHH) was the first identified gene to be associated with BDA1 [7].The IHH gene, which encodes a member of the Hedgehog family of signaling proteins, is known for its role in endochondral ossification: regulate the balance between growth and ossification of the developing bones [5]. The IHH protein operates through a feedback control mechanism. IHH binds to the patched (PTC) receptor, which functions in association with smoothened (SMO), to activate the GLI complex of transcription factors [8][9][10]. From there, these transcription factors continue to signal and regulate downstream genes affecting patterning.
IHH mutations affect Hh signaling at multiple levels, which impair chondrocyte maturation and proliferation, resulting in failure of osteoblast development in endochondral bones [7]. So far, about 14 IHH pathogenic variants have been reported to be association with BD IHH is expressed in the prehypertrophic chondrocytes of cartilage, and regulates growth of bones by coordinating chondrocyte proliferation and differentiation [18]. Thus in addition to affect phalange bone growth, disregulation of IHH signaling could also affect long bone and stature. Biallelic IHH pathogenic variants cause acrocapitofemoral dysplasia acrocapitofemoral dysplasia (MIM 607778), a disorder characterized by severe disproportionate short stature, BDA1 and cone-shaped epiphyses in hands and hips [19].
Short stature is often a component of the Brachydactyly, but it is less frequent among DBA1 individuals. So far, short stature had only been observed in DBA1 individuals with pathogenic variants affecting Asp100 residue of IHH gene [3, [20][21]. Not all DBA1 individuals with these pathogenic variants exhibited short stature, but they were shorter than non-carrier family members [3, [19][20][21], suggesting variants at this mutational hotspot can affect the growth to various degree but with reduced penetrance for short stature. In this Chinese family with a novel variant at residue 100, short stature was not 100% penetrant: the proband (Ⅳ-2), proband's uncle (III-4) and cousin (Ⅳ-5) had short stature, whereas her father (III-2) and uncle (III-5)'s height had normal stature. Other factors are believed to be involved in affecting the final stature of an individual with IHH pathogenic variants. Such pedigrees provided opportunities for identify co-determinants for human heights. Gabriela et al. observed that in Brazilian and Spanish populations heterozygous deleterious IHH variants are more frequent in short stature cohort (1.6%) than in general population (0.017% in gnomAD; P<0.001) [22], supporting the notion that reduced IHH signaling may be responsible for a reduced growth of the long bones and short stature [3,7].The role of IHH variants in non-syndromic short stature also needs further study.
In conclusion, a novel missense variant (c.299 A>G) affecting the mutational hotspot (residue 100) of IHH resulting in brachydactyly type A1 was identified in a Chinese family.
Sufficient evidence support the pathogenicity of this novel variant. High penetrance for the phalange phenotype and variable expressivity were observed in this family. Short stature was only observed in a subset of affected family members. The findings of this report will further help our understanding the phenotype-genotype correlations of IHH pathogenic variants and related disorders including brachydactyly type A1.