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Title: Identification and Elucidation of Molecular Basis of Inherited Bone Disorders in Pakistani Population
Authors: Ajmal, Muhammad
Keywords: Biological & Medical Sciences
Issue Date: 2018
Publisher: Quaid-i-Azam University, Islamabad.
Abstract: A large number of disease phenotypes of inherited bone disorders are reported among more than 6000 documented Mendelian disorders, wherein various genetic factors have been elaborated. Among multiple factors that contribute to the spread of genetic diseases, the consanguinity is the leading environmental factor creating the hype of genetic diseases especially in Pakistan. Lack of diseases knowledge, inappropriate diagnosis, unsuitable management, unavailability of supportive treatment and financial constraints all promote the dark side of genetic diseases especially inherited bone diseases. Further, the comprehension towards these diseases is limited due to inadequate genetic research work and research facilities. To understand the genetic insight into inherited bone disorders, six Pakistani families associated with diverse genetic bone diseases including; osteopetrosis, achondroplasia and multiple hereditary exostosis, were selected and enrolled in this study. The genetic basis of these bone diseases were elucidated using various molecular biology approaches from traditional linkage analysis to high-throughput sequencing methodologies. Various tools of emerging bioinformatics field were also implicated to forecast the effects of identified genetic variations on proteins involved in the development of disease phenotypes. The genetic elucidation of four autosomal recessive osteopetrosis families with classical phenotypic features including marble bones, macrocephaly, frontal bossing, exophthalmoses and visuals loss, craniofacial abnormalities and hepatosplenomegaly revealed novel homozygous substitution and deletion in TCIRG1 gene in 2 families while no pathological mutation was identified in other 2 families. The family 1OPT revealed novel donor splice site homozygous substitution mutation in intron 10 of TCIRG1 gene (c.1165+1G>A). The identified mutation resulted in aberrant splicing; therefore it is likely to be pathogenic. The family 2OPT showed a novel homozygous deletion c. 624delC in exon 6 of the TCIRG1 gene encodes a3 subunit of V-ATPase complex, which resulted in a frame shift producing a truncated protein of 208 aa instead of 830 aa. In silico studies have also revealed deleterious effects of frame shift mutation that involves a loss of part or the entire V-ATPase V0-complex domain. Since subunit a3 plays a key role in bone resorption process. The study confirms the role of this pathogenic mutation in Abstract Identification and Elucidation of Molecular Basis of Inherited Bone Disorders in Pakistani Population Page XVI infantile osteopetrosis. The TCIRG1 gene was screened in 2 more autosomal recessive osteopetrosis families (3OPT and 4OPT) and did not find any pathogenic genetic change in affected individuals, suggesting the involvement of some other gene(s) responsible for infantile malignant osteopetrosis. The identification of a novel deletion mutation not only supports the genetic heterogeneity of osteopetrosis but also highlights crucial role of V-ATPase a3 subunit of V0- complex domain in bone resorption process. Our findings may provide further insight in understanding the disease pathogenesis and help towards the development of targeted therapies. Similarly, the genetic screening of autosomal dominant achondroplasia family (1ACH) with short stature, rhizomelic abnormalities and prominent bowed legs exposed a frequently recurrent heterozygous c.1138 G>A (p.G380R) substitution in coding exon 8 of FGFR3 gene. Through sequencing, the identified genetic variation was confirmed in all affected subjects of the family while healthy individuals and controls were negative for this genetic change. Moreover, the findings were validated by PCR based RFLP analysis as c.1138 G>A substitution generated a unique recognition site for SfcI endonuclease. The substitution inserted adverse effects on FGFR3 protein stability, which was predicted by in silico studies of mutant FGFR3 protein. The hereditary multiple exostosis (HME), has an autosomal dominant mode of inheritance with almost asymptomatic phenotypes but expresses comprehensive clinical manifestation especially in male with more severity. Typical HME symptoms include; exostoses, restricted joint movement, knee deformities, short stature, affected limb growth, neuropathy, vascular compression and pain during developmental phase. The malignant transformation of asymptomatic osteochondroma into chondrosarcomas is the most dire and distressing condition. The 1HME family found to be associated with EXT1 gene at chromosome 8q24 locus. EXT1 gene screening in HME patients, showed an intermittently reported heterozygous deletion in exon 1 of EXT1 gene (c.247delC). The deletion not only replaces the arginine with glycine amino acid in the protein sequence at position 83 but also resulted in frame shift with formation of an early stop codon “TAG” at 135 codon position in protein sequence. Consequently, translation of premature truncated EXT1 protein, exostosin-1, with Abstract Identification and Elucidation of Molecular Basis of Inherited Bone Disorders in Pakistani Population Page XVII only 134 aa instead of 746 aa is the outcome. Further, an in silico exploration study has predicted the translated protein with 134 aa revealed harmful effects of deletion that involved in complete or partial decreased stability of protein. Perhaps, the mutated EXT1 gene led to impaired polymerase activity that may have caused the abnormal biosynthesis and expression of heparan sulfate, leading to disrupted signaling in endochondral growth plate. The elucidation of recurrent and novel genetic variations in the present study, will positively support in rapid, precise and accurate genetic screening as well as pre/ or post-natal molecular diagnosis of inherited bone disorders. Although, the identification of gene variants has provided significant insight into the disease pathogenesis; however, further hidden aspects of disorders, contributing to pathogenesis, can be unveiled by making use of recent advancements in the field of molecular biology. In this regard gene expression studies by using mammalian cell line or genetically modified animals can be of great importance in understanding the pathways involved in skeletal development and differentiation. Similarly, comparative study of the mutant and wild-type proteins characteristics such as stability, half-lives, cellular localization/ accumulation etc. can improve our understanding on disease pathogenesis. Findings of such type of studies may provide the basis of future treatment of these inherited diseases. At present, the findings of this study can be useful in providing genetic counseling and carrier screening to the affected families in order to minimize and/ or eradicate the genetic bone disorders in our population.
Gov't Doc #: 22099
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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