Genetic skeletal disorders (GSDs) are a group of rare and diverse bone growth dis-orders that affect the development and homeostasis of bones, resulting in abnormal size and shape of the skeleton. These disorders, which can be syndromic or non-syndromic, are a significant public health concern, affecting approximately 1 in 4000 people worldwide and leading to high healthcare costs and reduced quality of life. Understanding the underlying pathophysiology and genetic defects responsible for these disorders is essential for providing appropriate clinical interventions for individuals with different types of GSDs.
The current investigation focused on clinical and molecular assessments of seven-teen families that were affected by different types of skeletal disorders. The study was carried out in several stages, starting with the construction of family pedigrees and collection of blood samples from remote regions of Pakistan. Next clinical and radiological evaluations of each disorder were conducted at local government hospi-tals. Genetic analysis was performed using various techniques, including exome sequencing, sanger sequencing, bioinformatics analysis, and in some cases, protein modelling. In a couple of cases, functional gene testing of AFF3 for the missense variant p. (Arg972Pro), identified in a family, was performed in Zebrafish model and LRP4 missense variant p.(Asp99His), identified in two families, was performed on cell lines to access Luciferase activity.
Linkage was confirmed in five families (G, H, O, P, Q) using polymorphic STS mi-crosatellite markers, followed by the search for pathogenic variants using sanger sequencing. Nine other families (C, D, E, F, I, J, K, L, M) underwent whole exome sequencing to identify disease-causing variants. In the latter case, Sanger sequenc-ing was utilized to test segregation of the selected variants in each family. Based on the reported information, candidate genes were sequenced in three families (A, B, N). The pathogenicity of the identified variants was validated through online in-silico tools.
The study provided first time evidence of involvement of four genes (AFF3, EF-CAB7, HOXD12, TRIM35) in developing specific skeletal disorders in humans. The variant identified in AFF3 caused isolated syndactyly, in EFCAB7 isolated postaxial polydactyly, in HOXD12 synpolydactyly and in TRIM35 preaxial polydactyly and thumb hypoplasia. Eight novel disease-causing variants were identified in seven previously reported genes. CHST3 gene found mutated in spondyloepiphyseal dys-plasia Omani type, GLI3 in Greig cephalopolysyndactyly syndrome, DYNC2H1 in short-rib thoracic dysplasia, IHH in brachydactyly, type A1, ROR2 in brachydac-tyly, type B1, KIAA0825 in postaxial polydactyly and in LRP4 with preaxial poly-dactyly in feet and mesoaxial polydactyly in hands in one family and Cenani-lenz syndactyly syndrome in two families. In addition, the present study revealed previ-ously reported pathogenic variants in three genes, including GJA1 in Syndactyly, type III, CCN6 in progressive pseudorheumatoid dysplasia and GLI3 in Greig ceph-alopolysyndactyly syndrome.
In conclusion, identification of new disease-causing genes/variants has not only broadened the range of mutations found in the genes responsible for the conditions, but it has also facilitated genetic counseling for families affected by skeletal de-formities in the Pakistani population, as well as those with similar conditions.
The research work, presented here, has been published in the following peer-reviewed international journals.
Khan H, Koh G, Chong AE, Zahid M, Hussain S, Ali H, Ahmad W, Xue S. A novel variant in AFF3 underlying isolated syndactyly. Clinical Genetics. 2023 Mar;103(3):341-5.
Khan H, Chong AE, Bilal M, Nawaz S, Abbasi S, Hussain A, Hussain S, Ullah I, Ali H, Xue S, Ahmad W. Novel variants in the LRP4 underlying Cenani-Lenz Syndactyly syndrome. Journal of Human Genetics. 2022 May;67(5):253-9.
Khan H, Ahmed S, Nawaz S, Ahmad W, Rafiq MA. Greig Cephalopolysyndactyly Syndrome: Phenotypic Variability Associated with Variants in Two Different Do-mains of GLI3. Klinische Pädiatrie. 2021 Mar;233(02):53-8.
Khan H, Ullah K, Jan A, Ali H, Ullah I, Ahmad W. A Variant in the LDL Recep-tor Related Protein Encoding Gene LRP4 Underlying Polydactyly and Phalangeal Synostosis in a Family of Pakistani Origin. Congenital Anomalies.
Bilal M, Khan H, Khan MJ, Haack TB, Buchert R, Liaqat K, Ullah K, Ahmed S, Bharadwaj T, Acharya A, Peralta S. Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly. European Journal of Human Genetics. 2023 Sep 8:1-5.
Khan H, Abdullah, Bilal M, Liaqat K, Bharadwaj T, Acharya A, Ali H, Ahmad W, Leal SM. Clinical and genetic characterization of a HOXD12 variant in synpolydac-tyly patients. [In preparation].
Abdullah, Miss E, Bilal M, Khan H, Liaqat K, Lakhani S, Bharadwaj T, Acharya A, Ali H, Schrauwen I, Leal SM, Ahmad W [2022]. Variants in KIAA0825 causing postaxial polydactyly is involved in SHH signaling pathway. [In Preparation].
Khan MJ, Khan H, Abdullah, Bilal M, Zahid M, Ahmed S, Nawaz S, Tanoli A, Ullah K, Ullah F, Ullah I, Ali H, Ahmad W [2022]. Clinical and Genetic Character-ization of Various Genes Involved in Short Stature. [In Preparation].