Human X-linked CUL4B Gene Mutation Mediated Changes in Function during Development of Testis after Prediction of 3D Structure

Human Gene Mutation Mediated Abstract Introduction: CUL4B gene is ubiquitin 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. Present study explore the association of the E3 ubiquitin ligase CUL4B gene and deletion (loss) of GGA nucleotide sequences followed by change of translation event i.e. loss of non-essential amino acid (glycine)

family have two members, CUL4A and CUL4B play a crucial role for the survival of both male and female germ cells.
CUL4B is expressed in testis during spermatogenesis and mostly in post-meiotic spermatids stage and hardly expressed in spermatocytes [4,5]. CUL4B gene expression has distinct functions during development of gonad, but still to date, the crucial function(s) of CUL4B gene has not been clear in association with infertility. The rationale behind this study to recognize the impact of non-frame shift gene mutation of CUL4B and their interaction to ligand (protein) binding after prediction of 3D structure using known MTX drug as a model of infertility using molecular docking techniques. The present study might be helpful to explore the mechanism of abnormal differentiation of male gonad (testis) after gene protein drug interaction if mother exposed antenatally with anticancer molecules which might have interfare to the spectrum of cellular events of spermatogenesis and fertility.

Material and Methods
In the present study, clinically diagnosed cases of male infertility referred from OPD of AIIMS, Patna (Bihar, India) for Genetic investigations. The study was approved by Institutional Ethical Committee (IEC) and blood samples were collected by written informed consent from guardian. These cases of infertility are mainly categorized in three different groups i.e. non-obstructive azoospermia, obstructive azoospermia and oligozoospermia on the basis semen analysis by WHO [6] where the sperm count >20×106/ ml, progressive motility>50% and normal morphology >30% and established fertility (with one or more children) were included as controls. All patients were initially evaluated by clinician and conventional medical diagnostic including patient's background and physical examination analyses were performed. None of them had any history of childhood disease, environmental exposure, radiation exposure or prescription drug usage that could account for their infertility. The median age of patients included in the study was 35.4 years.

Isolation and characterization of mutation using whole exome sequencing (WGS)
Genomic DNA was isolated from clinically diagnosed cases of non-obstructive azoospermia and blood sample (1.0 ml) were collected after written informed consent and according to procedure of DNA isolation kit (Promega USA). Quantitative analysis of the DNA (ng/ul) was measured by Nanodrop spectrophotometer (Thermo. USA) and mutations of microdeletion of Y-chromosome regions (AZF a, b, c) were confirmed using PCR procedure before performed the sample for sequencing. After confirmation of mutation, genomic DNA was purified before initiation of sequencing,

Homology modelling by I-TASSER
The most common server is a hierarchical approach of using I-TASSER (Iterative Threading Assembly Refinement) based on algorithms for the prediction of protein structure and function.
It first identifies structural templates from the PDB by multiple Molecular docking is an essential technique for prediction of protein and ligand interaction which help to determine accuracy of complex geometry based on free binding energy and charges using auto dock tools software. Auto Dock parameter help to arrange distancedependent dielectric functions were used for the calculation of van der Waals and the electrostatic forces for stable binding [9]. The non-polar hydrogen atoms were merged and rotate during docking calculations with drug (MTX) ligand protein model.

Result
Cytogenetic location of chromosome-X (Homo sapiens annotation of cytogenetic GRCh38.p12) (https://www.ncbi.nlm. nih.gov/genome/tools/gdp) shown in Figure 1A, chromosomal loci of CUL4B gene mapped on Xq24 region. The DNA sequencing data reveals that CUL4B is gene mutation occurs in homozygous condition with deletion of GGA GGA nucleotide sequences at position 1741-1744, which encodes a protein consist 913 amino acids, although, the total length of nucleotide consist of 51241bp is depicted in Figure 1B.   (Table 1A). Thus, free binding energies suggested a better interaction between drug (MTX) with the mutated CLU4B protein as compared to the normal structure.   (A) Binding energies ranging from -1.33kcal/mol to -0.12kcal/mol in the case of normal CUL4B with MTX.
(B) Binding energies ranging from -2.52kcal.mol to 0.76kcal/mol in the cases of mutated CUL4B with MTX. The ranks are shown 1-10 on the basis of the best fit protein ligand structure in the case of normal protein.

Discussion
In of infertility. Interestingly, the non-coding regions were found to be useful for clinical diagnosis of unexplained cause of male infertility [10]. Furthermore, environmental factors including arsenic exposure are also responsible for reproductive dysfunction affecting male fertility [11]. During spermatogenesis, CUL4B is expressed in spermatogonia, differentiating secondary spermatocytes and spermatids during recombination and crossing over events. In human CUL4B deficiency causes development of hypogonadism in males [12]. Earlier, studies by our group identified the involvement of several genes regulating spermatogenesis including X linked ARSD (Arylsulfatase D) gene polymorphism [13][14][15][16]. In addition to these, several genetic polymorphisms have been demonstrated to be significantly associated risk factor with male infertility [17]. By In the present study, genetic factors for male infertility become prime important to understand the interaction between candidate gene CUL4B and their translation event (loss of glycine). Thus, the molecular modelling help to explain the physiological function based on ligand bindings sites to the target proteins (mutated).
Thus, authors conclude that, present study suggests that CUL4B gene mutation is responsible not only for the size of testis but may interfare to the events of spermatogenesis, if foetus exposed antenatally to teratogen like methotrexate. Hence, such molecules