Genetic Analyses Of Polydactyly And Syndactyly

Objective:We collected and classified 150 patients with limb malformations according to their clinical and radiological manifestations, to summarize the incidence of each type of malformations, and to identify new malformations among subjects. We have discovered a patient with a “doublefoot” malformation, blood samples from the proband and his parents were collected, exome sequencing was preceded in the pedigree with the “double foot” malformation, multi-step-screening was made for sequencing data annotation to identify the mutations that accounted for the doublefoot malformation.Methods:First, we collected clinical and radiological data for 150 patients with limb malformations. We have classified the patients according to the following criteria:(1)Polydactyly was classified by the location of the extra digits: preaxial, central,postaxial. Using Zuidam’s nomenclature: we followed the Rotterdam classification which consists of eight types, the type IV was subdivided into five types. Postaxial polydactyly(PAP) was classified into type A and type B regarding the maturation of the tissue, type A has a fully developed extra digit, while type B has the supernumerary digit that incompletely developed and consists of a boneless soft tissue rudiment.(2) The current classification scheme of syndactyly is a modified Temtamy & Mckusick system that incorporated the new clinical, genetic and molecular developments in this field. In the system, syndactyly was classified into type I-IX, and type I consists of four types(type Ia-type Id).(3) Bell introduced a classification of brachydactyly based on anatomic backgrounds, in which there are five types(type A-E), while type A was divided into subtypes A1-A6. Secondly, since the anomalies can occur either as an isolated malformation or as a part of complex congenial malformation syndromes, we evaluated the patients and considered the syndromes according to the congenital defects of other organs and systems. Lastly,we sorted out phenotypes that have not been reported.After the classification, we found a patient with “doublefoot” malformation,without other detectable birth defects. The doublefoot malformation is only reported in the mouse model, the pathogenesis of human doublefoot malformation is not clear.Peripheral blood samples were collected from the proband and his parents. DNA samples were extracted, exome sequencing were performed for the pedigree. Exome sequencing results were filtered against databases of HAPMAP8, db SNP130,and1000 Genome Project, common polymorphisms which had been reported were filtered, non-synonymous single nucleotide variants(SNVs), indels, copy number variants(CNVs) from the proband and his parents were compared to identify candidate genes, followed by Sanger sequencing to verify exome results.Results:1. Polydactyly: polydactyly were found in 121 cases, including 40 postaxial polydactyly(PAP)cases,78 preaxial polydactyly(PPD) cases, 4 central polydactyly cases, including 2 cases co-exist with PAP. One patient has the “doublefoot”malformation. Eighty five(85) cases have hand polydactylies, including 6 PAPs and79 PPDs; Sixty five(65) cases have foot polydactylies, including 56 PAPs, 6 PPDs,and 4 central polydactyles.2. All patients with PAP were classified as type A. PPD: type II: 22 cases, type III:2 cases, type IV: 36 cases, type V: 2 cases, type VI: 6 cases, and 11 cases cannot be classified due to incomplete data.3. Syndactyly(SD): 31 cases, type SD-I: 11 cases, type SD-Ic: 7 cases, type SD-Id:4 cases; type II(SPD): 6 cases; type SD-III: 4 cases; type SD-V: 1 case;type VI:3cases. 6 patients cannot be classified into the present classification, 1 patient is 3/4toes syndactyly. 2 patients are 1/2toes syndactyly and 3 cases are 2/3/4 fingers syndactyly.4. Brachydactyly(BD): 5 cases, and 4 cases also had with polydactylies or syndactylies. One patient is type BDA3, 4 cases could not be included into the classification.5. We have discovered some special phenotypes in subjects:(1) Doublefoot malformation mirror-image duplication of left foot;(2) Type SD-V with wide binocular distance, bulging eyes, and wide forehead;(3) Type II PPD with dextrocardia, congenital isolated kidney, and absence of left kidney;(4) Fusion of 2/3/4 fingers and aplasia of middle phalanx of 2-4 fingers, congenital heart disease;(5)Postaxial polydactyly of feet with congenital heart disease;(6) Type IV PPD, absence of thenar, and cleft lip;(7) Shortening of right hand and upper limb, fusion of 2-5fingers, collapsing of right thorax.6. Exome sequencing: After non-synonymous single nucleotide variants(SNVs),Indels, CNVs from the patient and his parents were compared, we found that the proband had 80 missense mutations and 35 indels different from his parents. Among them, two genes associated with limbs development: HOXD13 and ROR2.7. Sanger sequencing: We detected the same mutation of HOXD13 which resulted in Gly11 Ala in the patient and his mother; his father did not carry the mutation.Conclusion:1. Polydactyly is the most common limb malformation, hand preaxial polydactyly and foot postaxial polydactyly are the most common forms of polydactylies,meso-axial polydactylies are rare. Type IV occurs is most frequent preaxial polydactylies, while type II is the second most common type. Syndactyly is heterogeneous and type SD-I is one of the most common types. Brachydactyly often coexists with other limb malformations, such as polydactyly or syndactyly.2. One patient has the "doublefoot" malformation, and one patient may has Poland syndrome.3. We detected a missense HOXD13 mutation(Gly11Ala) in the doublefoot patient.His mother also had the same HOXD13 mutation, however, she did not exhibit the phenotype.