CETN1 variations cause idiopathic male infertility. D. V. S. SUDHAKAR1, A. KHATTRI1, R. PHANINDRANATH1, A. K. SHARMA1, J. RESHMA DEVI1, M. DEENADAYAL2, N. J. GUPTA3, S. PRASAD4, S. YOGENDRA1, K. THANGARAJ1 1) Centre for Cellular and Molecular Biology, Hyderabad, India; 2) Infertility Institute & Research Centre, Secunderabad, India; 3) Institute of Reproductive Medicine, Salt Lake, Kolkata, India; 4) Sridevi Nursing Home, Warasiguda, Hyderabad, India.

   Centrins are calmodulin-like, EF-hand containing calcium-binding proteins that are found in all eukaryotic cells from yeast to mammals. Centrins are encoded by 3 homologous genes (CETN1, CETN2, CETN3) in humans. The expression of centrin-1 (CETN1) is testis-specific, spermatogenic cell-specific and developmental stage-related. Our previous studies on several Y-chromosome, autosomal and mitochondrial genes revealed 25% of genetic causes responsible for male infertility. Therefore, our aim is to identify additional genetic factors that are associated with male infertility. We collected testicular biopsy from 6 obstructive and 5 Non-obstructive Azoospermic (NOA) men, isolated mRNA and performed gene expression analysis using Affymetrix array (2.0). We found several genes were differentially expressed, of which CETN1 was one among the highly down-regulated genes in NOA individuals. Moreover, recent studies have shown that Cetn1 (-/-) male mice were infertile. Hence, we selected this gene for further studies. We have sequenced the coding region of CETN1 in ethnically matched 656 infertile and 347 fertile Indian men and found five nucleotide substitutions in the entire CETN1 gene; of which two were 5UTR variants (rs_367716858, novel), one each was synonymous (rs114739741), non-synonymous (rs61734344) and 3UTR variant (rs568365). Non-synonymous nucleotide substitution (rs61734344) was found to be strongly associated with male infertility (PCorr<0.0005), replacing Methionine with Threonine (p.Met72Thr) in highly conserved region. Functional characterization of the above non-synonymous mutation (p.Met72Thr) was carried out using (overexpressed) wild type and mutant proteins. Biophysical studies revealed that the mutant protein (p.Met72Thr) is less structured and has considerable differences in the ion binding thermodynamics, compared to wild type protein. Since the mutant proteins spectroscopic and thermodynamic properties are hampered, we predicted that the mutation could probably lead to the compromised physiological functions of Centrin1, which is a calcium sensor. In addition to the substitutions, we found a novel seven base pair indel (g.581202_209) in the 3UTR region, which is also associated (PCorr=0.033) with male infertility. This is the first study on CETN1 gene mutations and their association with human male infertility.

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