Sains Malaysiana 50(11)(2021): 3303-3311

http://doi.org/10.17576/jsm-2021-5011-14

Downregulation of WNT8A, LRP5, LRP6 and FGF8 in Malay Non-Syndromic Cleft Lip and/or Palate Patients

(Pengawalaturan Rendah WNT8A, LRP5, LRP6 dan FGF8 pada Pesakit Melayu Sumbing Bibir Bukan Sindrom dengan atau Tanpa Sumbing Lelangit)

NURUL SYAZANA MOHAMAD SHAH¹*, WAN AZMAN WAN SULAIMAN¹, SARINA SULONG² & AHMAD SUKARI HALIM³

¹Reconstructive Science Unit, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

²Human Genome Centre, School of Medical Sciences, Health Campus, Universiti Sains Malaysia

16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

³Director Office, Hospital Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

Received: 23 September 2020/Accepted: 1 March 2021

ABSTRACT

Non-syndromic cleft lip and/or palate (NSCL/P) is a one of the most common birth defects occurs as a result of multi-factorial determinants such as genetic and environmental factors. Genetic factor has been studying widely across different population in identifying genes causing cleft defects. This study aims to validate the role of fibroblast growth factors (FGFs) and signalling molecules Wingless-type (Wnt) to the occurrence of cleft lip and/or palate among Malay population. Tissue skin was obtained from consented NSCL/P patients who underwent the cleft lip repair operation at the upper lip skin area and non NSCL/P individual was obtained from patient having traumatic injury at the lip area as a control. Expression pattern of FGF8, FGF10, Wnt8a, and LRP5/6 were tested and validated using Western Blot (WB) and quantitative Reverse Transcriptase-PCR (qRT-PCR). The fold change difference of FGF8 (0.614 ± 0.1012-fold), FGF10 (0.7188 ± 0.1017-fold) and Wnt8a protein (0.9051 ± 0.0142-fold) was downregulated by 1-fold in cleft lip (CL) tissues compared to the normal meanwhile LRP5/6 protein (1.2201 ± 0.1404-fold) was found upregulated by 1-fold. Validation testing confirmed that expression of FGF8 (p=0.014), Wnt8a (0.0762 ± 0.0227), LRP5 (0.3577 ± 0.1362) and LRP6 (0.3093 ± 0.2541) were significantly reduced in CL tissues compared to normal. This is the first study identified defective regulation of Wnt8a, LRP5, LRP6 and FGF8 in NSCL/P among Malays. These novel findings clearly explained the important role of growth factors FGF and Wnt signalling pathway in lip and palate formation during craniofacial development.

Keywords: Craniofacial; gene expression; non-syndromic cleft; protein

ABSTRAK

Sumbing bibir bukan sindrom dengan atau tanpa sumbing lelangit adalah salah satu kecacatan kelahiran yang berlaku disebabkan oleh pelbagai faktor seperti faktor genetik dan persekitaran. Kajian ke atas faktor genetik giat dijalankan terhadap pelbagai populasi berbeza dalam mengenal pasti gen-gen penyebab kecacatan sumbing. Kajian ini bertujuan untuk memvalidasi peranan faktor pertumbuhan ‘Fibroblast growth factor’ (FGF) dan signal molekul ‘Wingless-type’ (Wnt) terhadap pembentukan sumbing bibir dengan atau tanpa lelangit dalam kalangan populasi Melayu. Tisu kulit diperoleh dengan kebenaran daripada pesakit sumbing yang mendapat rawatan pembedahan sumbing bibir dan individu bukan sumbing daripada pesakit yang mengalami kecederaan pada bahagian bibir sebagai kawalan. Pengekspresan FGF8, FGF10, Wnt8a dan LRP5/6 diuji dan divalidasi menggunakan teknik ‘Western Blot’ (WB) dan kuantitatif transkripsi berbalik-PCR’ (qRT-PCR). Protein-protein FGF8 (0.614 ± 0.1012-fold), FGF10 (0.7188 ± 0.1017-fold) dan Wnt8a (0.9051 ± 0.0142-fold) pada tisu sumbing menunjukkan penurunan sebanyak sekali ganda berbanding normal manakala protein LRP5/6 (1.2201 ± 0.1404- fold) menunjukkan peningkatan sekali ganda. Ujian validasi mengesahkan penurunan ekspresi FGF8 (p=0.014), Wnt8a (0.0762 ± 0.0227), LRP5 (0.3577 ± 0.1362) dan LRP6 (0.3093 ± 0.2541) secara signifikan pada tisu sumbing berbanding normal. Kajian ini adalah yang pertama mengenal pasti kecacatan regulasi Wnt8a, LRP5, LRP6 dan FGF8 pada NSCL/P dalam kalangan Melayu. Penemuan novel ini menjelaskan secara terperinci kepentingan peranan faktor pertumbuhan FGF dan laluan signal Wnt dalam pembentukan bibir dan lelangit ketika pembentukan kraniofasial.

Kata kunci: Ekspresi gen; kraniofasial; protein; sumbing bukan sindrom

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*Corresponding author; email: syazanashah@usm.my