Developmental genetic analysis of post-axial longitudinal limb reduction defect (PALLRD) in Miller syndrome and nonclassical Miller syndrome
Aldridge, Kishan Victoria
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This project aimed to provide a greater understanding of limb development through the characterisation of Mendelian disorders. The more specific aim was to identify the developmental basis of the Post Axial Longitudinal Limb Reduction Deformity (PALLRD) seen in the autosomal recessive Miller syndrome caused by mutations in Dihydroorotate Dehydrogenase (DHODH). In addition whole exome sequence analysis was used to identify further causative variants in a group of individuals with Non Classical Miller syndrome. These individuals were negative for mutations in DHODH although they had a clinically overlapping PALLRD. A single novel variant was discovered in Fibroblast Growth Factor Receptor 1 gene (FGF1) in one individual in this cohort. Due to the known vital role of FGF signaling in limb bud development the functional significance of this variant was investigated further. In vitro data suggested that this variant has a dominant negative effect. Finally I compared the differential gene expression profile of embryonic mouse forelimb and hindlimb at a later stage of development. Digital Gene Expression Serial Analysis of Gene Expression (DGE-SAGE) produced gene-expression profiles of the forelimbs and hind limbs from 14.5 days post conception (d.p.c) murine embryos. This data included known differentially expressed genes as well as novel candidate genes that are putative regulators of limb growth. Whole mount In Situ Hybrisation (WISH) and Quantitative Real Time Polymerase Chain Reaction (qRTPCR) provided corroborating evidence for the differential expression of a subset of these genes between the forelimbs and hind limbs. This project suggests a role for DHODH in limb bud cell proliferation. It also demonstrates a novel potentially dominant negative mutation within FGFR1 in an individual with a limb deformity. Finally a subset of genes involved in regulating the differential growth between the forelimb and hindlimb were presented.