Abstract

Introduction: CUL4B gene is ubiquitin ligase belongs to the Cullinring ubiquitin ligase family. In recent study we have identified a series of MTNR1B, SENP3, AKAP3 and PLOD3 genes and their functional interaction to the ligand binding capacity after prediction of 3D structure to the drug like methotrexate, which plays a significant role during spermatogenesis. Although, the role of Cul4B gene mutation and its impact to the ligand binding during development and differentiation of male gonad (testis) remains unexplored in fertility.

Objective: The present study has been designed to characterize the mutation of CUL4B gene sequence and correlate with functional aspect to the ligand binding site to understand the translational event of non-frame shift mutated (deleted) nucleotide sequence GGAGGA of DNA. The findings of mutations try to correlate with anatomical feature (testicular size) of the gonad and fertility.

Material and methods: Blood samples were collected from the cases of clinically diagnosed non obstructive azoospermia (NOA) with respective age matched controls. Study was carried out using whole genome sequencing (NGS) from Illumina (USA) to characterize the nature of mutation. The study was further extended to develop 3D structural analysis of the candidate gene CUL4B and protein (ligand) binding activity with drug like methotrexate using molecular docking (iTASSER) techniques for gene coded functional changes in testis.

Results: Findings of NGS DNA sequencing confirm the non - frame shift mutation in homozygous condition of CLU4B gene involving deletion at position 1761-1746 of GGAGGA nucleotide sequences. After decoding of mutation region (gene) confirm loss of non-essential amino acid glycine glycine resulting changes in physiological function during spermatogenesis in testis. The predicted 3D helical structure of CLU4B gene showing binding of amino acid residue to ligand (methotrexate) as model to evaluate the functional activity of the gene by using bioinformatics tools.

Conclusion: DNA sequencing analysis confirms the non-frame shift mutation of CUL4B gene in nonobstructive azoospermia male in homozygous condition. After mutation of GGAGGA sequence confirm either loss or deletion of non-essential amino acid residue (glycine-glycine) failed to bind to the ligand respective ligand (protein) resulting interference to the development of testis and infertility.

Keywords: CUL4B gene; Infertility; DNA sequencing; 3D protein structure