Identification of Heterozygous Mutation of FZD4 in Congenital Cavus Foot Deformity Patients
The proband’s mother (Figure 1. Ⅱ-2, Patient) was the second child born to healthy parents; there was no cavus foot deformity or other skeletal deformities among other five members. Upon physical examination of the proband. The 20X coverage for the RefSeq coding region was 98.23% (Suppl. Fig. 1). A novel missense mutation (exon2:c.1589G>A:p.G530E) in exon 2 of FZD4 (NM_012193) were detected in every cavus foot deformity patient but not in the healthy families (Figure 2A). One guanine ribonucleotide was altered to an adenine ribonucleotide in codon 1589, which caused a change in the reading frame from glycine (Gly) to glutamic acid (Glu). Sanger sequencing was also performed for this family, which confirmed the heterozygous mutation in the proband and her brother, was inherited by Patient Ⅱ-2, not in any of the healthy families (Fig. 2A). There was no evidence of Cavus Foot Deformity or of other skeletal diseases in the parents of Patient Ⅰ-2. Furthermore, mutations in FZD4 gene were further examined in twenty-three congenital CFD patients, FZD4 mutation (FZD4:NM_012193:exon1:c.205C>T:p.H69Y) in one congenital CFD patient was found (Fig. 2B). In addition, the loci FZD4:p.G530 and p.H69 (NP_036325.2) were highly conserved among a diverse range of species (Figure 2C). The mutations may result in the loss‑of‑function of FZD4. Both of the two mutations were located in the Topological Domain in FZD4, encoding a transmembrane region (26). In the 1,000 Genomes database (www.internationalgenome.org), Exome Sequencing Project 6500 database (evs.gs.washington.edu/EVS/), and gnomAD genome database (gnomad.broadinstitute.org), the allele frequencies of the c.1589G>A mutation were 0.000998403, 0, and 0.0000956, and c.205C>T mutation were 0.0002, 0.0002, and 0.0005, respectively (Table 1). The SIFT and MutationTaster predicted the mutations as likely to cause damage to the FZD4 protein function (Table 1). To the best of our knowledge, gene FZD4 have not been reported in cavus foot deformity previously. Thus, the present study identified FZD4 mutations as a new gene associated with the congenital cavus foot deformity.
Clinical Characteristic of Congenital Cavus Foot Deformity Patients
The feet in the proband is talipes cavus deformity, which is the most significant complaint, contributing to abnormal elevation of longitudinal arch. The deformity accompanies compound abnormality of the foot, which including adduction of the forefoot, plantar flexion of the midfoot and varus of the hindfoot. Along with the plantar flexion of the forefoot and midfoot is osseous uplift on the dorsal part of the midfoot. There is a subsequent thickening of the skin, with callous formation, under metatarsal heads, result from increasement of bearing under the forefoot. The myodynamia testing of the anterior tibial muscle tendon received a grade of 2/3, which result in weakness of foot dorsiflexion. Consequently, excessive dorsiflexion of toes appears in dorsal expansion position of ankle joint. However, myodynamia of the posterior tibial muscle, peroneus longus and brevis, flexor digitorum longus and flexor digitorum longus is achieved grade 5. Radiologically, from the lateral radiograph, the M’eary angle of right foot and left foot are 49° and 50° (greater than 10°) respectively, Hibbs angle of right foot and left foot are 35° and 37° (less than 150°) respectively, and calcaneal pitch angle (PTA) of right foot and left foot both are 19mm (greater than 30°).
According to the previous study, bilateral cavus involvement usually suggests an underlying nerve or muscle disease, and 80% of Charcot-Marie-Tooth (CMT) patients having pes cavovarus and 65% of patients with cavovarus having CMT (27). However, the familial cavus patients with this deformity do not have a neuromuscular problem or progressive weakness and muscle imbalance. And according to the clinical and radiographic results, there is no other abnormalities of the spinal cord, such as occult dysraphism, tethered cord, polio, myelodysplasia, and so on. Moreover, peripheral nerves diseases (motor and sensory neuropathies), for example, CMT disease, Dejerine-Sottas disease, or Refsum disease in patients in this study was not found.
Morphological, histological, and IHC analyses of the bone tissue in the cavus foot deformity patient and healthy controls
FZD4 is deficient in the patient's bone tissue. According to Western blot analysis and IHC analyses, the proband’s heel bone tissue (CFD) showed a significant deficiency expression of the FZD4 compared to the healthy controls (C1,C2) (Figure 3A). Additional bands below FZD4 was resulted from an important paralog of this gene is FZD10, which is just above 55kb. Micro-CT and X-ray analyses of the bone fragments revealed no significant difference among cavus foot deformity patient and healthy controls in parameters BMD, BV/TV, Tb.N, Tb.SP and Tb.Th (Suppl. Fig. 2). Probably because of the sample size is too small. The result of IHC assessment of bone fragments was in accordance with Western blot analysis, FZD4 shows lighter in the proband’s heel bone tissue (CFD) compared to the healthy controls (C1,C2) (Figure 3B).