Blended phenotypes resulting from the contribution of two or more genetic variants to the disease of a patient pose a significant diagnostic challenge. Correlating between the phenotypes and the genotypes of the affected patients is difficult in these cases, especially in the absence of a large family segregating the condition. We report a child born to consanguineous Syrian parents with a complex phenotype including a skeletal dysplasia, characterized by a small thorax and phalangeal shortening, as well as cardiac anomalies and sensorineural hearing loss. Molecular analysis identified the presence of three potentially disease-causing genetic variants. These include homozygous variants in the IHH and TTC12B genes, known to be associated with acrocapitofemoral dysplasia (OMIM# 607778) and a form of short-rib thoracic dysplasia (OMIM# 613819), respectively, in addition to a heterozygous variant in the COL11A1 gene, associated with dominant forms of skeletal dysplasia, such as Marshall (OMIM# 154780) and Stickler (OMIM# 608481) syndromes, and hearing loss (OMIM# 618533). We propose that the complex phenotype observed in the patient results from the contribution of l three of these variants. This case highlights some of the challenges encountered in the genetic counseling of families with rare genetic conditions.

Brachydactyly A1 (BDA1) is a rare disorder characterized by the disproportionate shortening of fingers and/or toes with or without symphalangism. Mutations in Indian hedgehog signaling molecule (IHH), which impair the effect of functional IHH protein derived from its precursor IHH, are commonly identified in patients with BDA1 or acrocapitofemoral dysplasia (ACFD). The ultrasound phenotype of fetuses with IHH mutations has rarely been described. To better understand the consequences of IHH mutation, we analyzed the characteristics of a Chinese fetus with BDA1 caused by a novel heterozygous IHH mutation. Clinical data and genomic DNA were collected from the proband and family members. Whole-exome sequencing (WES) was performed to identify potential causative mutations. Sequence analysis was performed to investigate the conservation of the affected leucine residue in IHH. Protein 3D modeling was performed to predict the effects of the mutation on protein structure. In vitro overexpression transfection experiments in human embryonic kidney 293T (HEK293T) cell lines were performed to evaluate the pathogenicity of the identified mutation. The fetal proband carried a novel heterozygous mutation in IHH (NM_002181.4: c.331_333delCTG, NP_002172.2: p.Leu111del) inherited from the father; this mutation manifested as shortening of the limbs, with more severe shortening observed in the proximal extremities than in the distal extremities, as evidenced by ultrasound. The Leu111 residue is highly conserved among vertebrates, and deletion of this residue destabilizes the protein structure. Western blotting analysis of HEK293T cells in overexpression transfection experiments revealed that the Leu111del mutation led to an increase in the level of the IHH precursor and a reduction in the level of functional IHH protein compared with those in HEK293T cells expressing wild-type IHH, indicating that this mutation might cause IHH protein dysmaturity. The novel heterozygous mutation c.331_333delCTG (p.Leu111del) in the IHH gene is the likely cause of BDA1 in this Chinese fetus. This mutation causes IHH protein maturation failure. These findings contribute to our understanding of the molecular pathogenesis of BDA1 and the clinical identification of fetal BDA1.

Acrocapitofemoral dysplasia (ACFD) is a rare autosomal recessive disorder, characterized by postnatal onset of disproportionate short stature with short limbs, brachydactyly, cone-shaped epiphysis, narrow thorax, and relatively large head. To date, only three homozygous missense mutations have been reported in the signaling amino terminal domain (201-308 amino acids) of the IHH gene in three ACFD families from Belgian, Dutch, and Turkish ethnicities. In the present study, we have investigated two patients in a Pakistani family affected with ACFD. Whole exome sequencing (WES) followed by Sanger sequencing was carried out for mutational screening. The variant was further validated by in silico modeling and molecular dynamics simulation analysis. Data analysis revealed a novel homozygous missense variant [c.518C>A; p.(Ala173Asp)] in exon 2 of the IHH (NM_002181.4) gene. The variant segregated within the family and was not observed in unaffected ethnically matched controls. In silico modeling and dynamic simulation analysis revealed that the variant disturbed the core structure of the domain and destabilized the loop region and the region surrounding the variant. This study reports the first case of ACFD from Pakistan and identifies the fourth novel missense variant in the IHH gene that led to the broadening of the phenotypic and genotypic spectrum of ACFD.

Inactivation mutations in the Indian hedgehog (Ihh) gene in humans cause numerous skeletal chondrodysplasias, including acrocapitofemoral dysplasia, brachydactyly type A1, and human short stature. The lack of an appropriate human-relevant model to accurately represent these chondrodysplasias has hampered the identification of clinically effective treatments. Here, we established a mouse model of human skeletal dysplasia induced by Ihh gene mutations via ablation of Ihh in Aggrecan-positive (Acan+) cells using Aggrecan (Acan)-creERT transgenic mice. Smoothen agonist (SAG) promoted Hh activity and rescued chondrocyte proliferation and differentiation by stimulating smoothened trafficking to the cilium in Ihh-silenced cells. SAG treatment corrected mouse stature and significantly decreased mortality without evidence of toxicity. Moreover, Ihh ablation in Acan+ cells produced enchondroma-like tissues near the growth plates that were significantly reduced by SAG treatment. These results demonstrated that SAG effectively treats skeletal dysplasia caused by Ihh gene mutations in a mouse model, suggesting that SAG may represent a potential drug for the treatment of these diseases and/or enchondromas.

Acrocapitofemoral dysplasia (ACFD) is a rare autosomal recessive skeletal dysplasia characterized by short stature with short limb dwarfism, brachydactyly, and a narrow thorax. Major radiographic features are egg-shaped capital femoral epiphyses with a short femoral neck and cone-shaped epiphyses, mainly in the hands and hips. To date, only four child patients from two families have been reported. We describe two adult patients with ACFD with a novel homozygous c.478C>T (p.Arg160Cys) mutation in IHH in the third family of the literature. The reported cases showed a middle phalanges which fused with distal phalanges in the fifth toes, the typical configuration of metacarpals, radial angulation and extremely short femoral neck. These findings could help the diagnosis of ACFD in adult patients. We hope that this new family will be a helpful guide for predicting and managing the prognosis of diagnosed children.