Mapping Variants in Genes Causing Skeletal Disorders

Abstract

Human skeletal disorders represent a very rare heterogenous group of deformities affecting patterning, homeostasis, and development of bones resulting in irregular size and shape of the human skeleton. A genetic basis has been revealed in a wide range of such congenital disorders conveying the role of several molecular mechanisms in limb patterning and development. In the present study, twenty-two families affected with distinct skeletal disorders were investigated at clinical and molecular level. The study was performed in several phases, including visits to remote areas of Pakistan to construct the family pedigrees and collection of blood samples. This was followed by clinical and radiological assessment of each disorder at local government hospitals. Genetic analysis was carried out using techniques such as Exome sequencing, Sanger sequencing, Bioinformatics analysis, and in few cases protein modelling. In a couple of cases, functional gene testing of KIAA0825-mRNA for the missense variant [p.(Val324Phe)], identified in family B, was performed in X. tropicalis and mouse embryos. In ten families, linkage was established using polymorphic STS microsatellite markers/SNP microarray. Sanger sequencing was then used to search for the pathogenic variants. In twelve other families, whole exome sequencing [WES] was performed to hunt for the disease-causing variants. Sanger sequencing was used to examine the segregation of the selected variants in each family. The pathogenicity of identified pathogenic variants was validated through online in-silico approaches. Analysis of the genetic data revealed eleven novel and six previously reported disease causing variants in eighteen families, presented here. The novel variants were identified in KIAA0825 for post-axial polydactyly, in ZRS for preaxial polydactyly, in DLX5 for SHFM, in GLI3 for Greig-Cephalopolysyndactyly syndrome, in EVC2 for Ellis-Van Creveld syndrome, in HOXD13 for syndactyly, in BBS7 for Bardet Biedl syndrome, and in DYM for Dyggve-Melchior-Clausen syndrome. In seven families, previously reported nucleotide variants were identified in EVC2, FGF16, GLI3, MKKS, ESCO2, and chromosome 10 duplication. Due to time constraint, segregation of selected variants in three other families were not completed. However, initial analysis revealed first direct evidence of involvement of a couple of previously unknown genes in causing polydactyly. DRSML QAU Abstract Mapping Variants in Genes Causing Skeletal Disorders XXI In functional gene study, KIAA0825-mRNA was expressed both in X. tropicalis and mouse embryos. In X. tropicalis, in-situ hybridization revealed impaired mesoderm in homozygous mutant embryos. Mouse in-situ hybridization showed insignificant results of KIAA0825 in limbs at stage E10.5, E11.5 and E13.5. However, KIAA0825-mRNA expression was seen in the neural tube/notochord at E11.5. CRISPR-knock out for KIAA0825 was generated in X. tropicalis. However, heterozygotes did not show any limbs/skeletal phenotypes. Identification of novel disease-causing variants has not only expanded spectrum of the mutations in the causative genes, but it has also made genetic counselling easier in the families, presented here, and in those carrying similar skeletal deformities in Pakistani population. The research work, presented here, has been published in the following peer-reviewed international journals. 1. Abdullah, Shah PW, Nawaz S, Hussain S, Ullah A, Basit S, Ahmad W [2020]. A homozygous nonsense variant in DYM underlies Dyggve-Melchior-Clausen syndrome associated with ectodermal features. Molecular Biology Report 47[9]: 7083-7088. 2. Abdullah, Yousaf M, Azeem Z, Bilal M, Liaqat K, Hussain S, Ahmad F, Ghous T, Ullah A, Ahmad W [2019]. Variants in GLI3 cause Greig cephalopolysyndactyly syndrome. Genetic Testing and Molecular Biomarkers 23[10]: 744-750. 3. Khan H, Abdullah, Ahmed S, Nawaz S, Ahmad W, Rafiq MA [2021]. Greig Cephalopolysyndactyly Syndrome: Phenotypic variability associated with variants in two different domains of GLI3. Klinische Pädiatrie 233[02]: 53-58. 4. Abdullah, Miss E, Bilal M, Lakhani S, Ahmad W [2022]. Variants in KIAA0825 causing postaxial polydactyly is probably involved in SHH signalling pathway. [In Preparation] 5. Abdullah, Bilal M, Khan H, Lakhani S, Ahmad W, [2022]. A Heterozygous Frameshift Variant in DLX5 and 445kb-Microduplication Harboring BTRC Cause Split-hand/foot Malformation in two Different Families. [Submitted] 6. Abdullah, Khan H, Bilal M, Ahmad W, [2022]. Sequencing revealed a nonsense mutation in FGF16 and polyalanine extension in HOXD13 causing Syndactyly. [In Preparation]