Human Reproduction, Vol.34, No.6, pp. 1155–1164, 2019 Advance Access Publication on May 23, 2019 doi:10.1093/humrep/dez048 ORIGINAL ARTICLE Reproductive genetics ADCY10 frameshift variant leading to severe recessive asthenozoospermia Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 and segregating with absorptive hypercalciuria Arvand Akbari1,2, Giovanni Battista Pipitone3, Zahra Anvar4,5, Mojtaba Jaafarinia1,2, Maurizio Ferrari3,6,7, Paola Carrera3,6,*, and Mehdi Totonchi8,9,* 1Department of Biology, Faculty of Science, Fars Science and Research Branch, Islamic Azad University, Marvdasht, Iran 2Department of Biology, Faculty of Science, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran 3Laboratory of Clinical Molecular Biology and Cytogenetics, IRCCS San Raffaele Hospital, Milan, Italy 4Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 5Department of Obstetrics & Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 6Genomic Unit for the Diagnosis of Human Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Hospital, Milan, Italy 7Vita-Salute San Raffaele University, Milan, Italy 8Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 9Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran *Correspondence address. Genetics and Stem Cell departments, Royan Institute, Tehran, Iran. E-mail: [email protected]; Genomic Unit for the Diagnosis of Human Disorders, IRCCS San Raffaele Hospital, Milan, Italy. E-mail: [email protected] Submitted on January 19, 2019; resubmitted on February 18, 2019; editorial decision on March 13, 2019 STUDY QUESTION: Can whole exome sequencing (WES) reveal a novel pathogenic variant in asthenozoospermia in a multiplex family including multiple patients? SUMMARY ANSWER: Patients were discovered to be homozygous for a rare 2-bp deletion in the ADCY10 coding region (c.1205 1206del, rs779944215). WHAT IS KNOWN ALREADY: ADCY10 encodes for soluble adenylyl cyclase (sAC), which is the predominant adenylate cyclase in sperm. It is already established that proper sAC activity and a constant supply of cAMP are crucial to sperm motility regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility gene for dominant absorptive hypercalciuria (OMIM#143870); however, no ADCY10 variations have been confirmed to cause human asthenozoospermia to date. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a highly consanguineous pedigree of asthenozoospermia. The subject family was recruited in Iran in 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The two patients were diagnosed as asthenozoospermic through careful clinical investigations. Both patients, respective parents, and an unaffected brother were subjected to WES. The discovered variant was validated by Sanger sequencing and segregated with the phenotype. To confirm the pathogenicity of the variant, sperm samples from both patients, 10 normozoospermic men and 10 asthenozoospermic patients not representing the variation, were treated with a cAMP analogue dissolved in human tubal fluid medium, followed by computer-assisted sperm analysis and statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered homozygous variant occurs at 10 amino acids upstream of the ADCY10 nucleotide binding site leading to a premature termination (p.His402Argfs∗41). Treatment of the patients’ sperm samples with a cell-permeable cAMP analogue resulted in a significant increase in sperm motility, indicating the pathogenic role of the variant. Moreover, absorptive hypercalciuria, segregating within the family, was also associated with the same variant following a dominant inheritance. LIMITATIONS, REASONS FOR CAUTION: Though nonsense-mediated decay is highly likely to occur in the mutated transcripts, we were not able to confirm this due to low RNA levels in mature sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our finding enlarges the phenotypic spectrum associated with the ADCY10 gene, previously described as a susceptibility gene for dominant absorptive hypercalciuria. © The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected]. 1156 Akbari et al. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Royan Institute, Tehran, Iran, and San Raffaele Hospital, Milan, Italy. The authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A Key words: male infertility / asthenozoospermia / absorptive hypercalciuria / familial exome sequencing / ADCY10 . Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 . activation of PKA. Through its phosphorylation of downstream Introduction . proteins, it is assumed that PKA activity is critically essential for . The World Health Organization (WHO) has described infertility as a . motility, capacitation, and fertilization competence of spermatozoon . reproductive system disease defined by the failure to achieve a clinical . (Hess et al., 2005). pregnancy after 12 months or more of regular unprotected sexual . In a more severe form, asthenozoospermia may exist as mul- intercourse (Zegers-Hochschild et al., 2009). It affects 15% of couples . tiple morphological abnormalities of the sperm flagella (MMAF) worldwide and it is estimated that in 50% of cases, the cause is the male . representing absolute loss of sperm motility occasionally. On the factor (Nieschlag et al., 2010). However, a more recent study revealed . whole, asthenozoospermia may manifest itself as an isolated disorder, . that male factor contributes to 60–70% of cases in the Middle East, . accompanied by other sperm abnormalities or as part of a syndrome . which is higher than average (Agarwal et al., 2015). It has been reported . in primary ciliary dyskinesia (PCD) (Zuccarello et al., 2008). To this . that 30–50% of male infertility cases have an underlying genetic cause . point, the following genes have been reported to cause isolated (Zorrilla and Yatsenko, 2013). Despite considerable efforts toward . asthenozoospermia: KLHL10 (Yatsenko et al., 2006), PLA2G6 (Visser discovery of these genetic factors, 30–40% of such cases still remain . et al., 2011), SEPT12 (Kuo et al., 2012), SLC26A8 (Dirami et al., 2013), idiopathic (Nieschlag et al., 2010). GALNTL5 (Takasaki et al., 2014), NSUN7 (Khosronezhad et al., . Asthenozoospermia, which is the most prevalent infertility pheno- . 2015), CATSPER1–4 (Sun et al., 2017), and SPAG17 (Xu et al., 2018). type (Curi et al., 2003), is a condition in which less than 32% of total . Ray et al. (2017) have extensively reviewed the genes involved in spermatozoa are progressively motile (WHO, 2010). It might result . MMAF and PCD. from structural or metabolic defects. Normal flagellar ultrastructure is . Herein, we provide the first report of a homozygous frameshift of utmost importance and its malfunction and/or structural defects in . variation (c.1205 1206del; dbSNP rs779944215) in the exonic region . various protein components of the axoneme result in asthenozoosper- . of ADCY10 associated with recessive asthenozoospermia, in an . mia (Coutton et al., 2015). Metabolic defects can lead to deficient . inbred family. Noticeably, all relatives who were heterozygous or . energy metabolism, oxidative stress, or inhibition of different pathways . homozygous for the variant had a history of developing calcium kidney such as malfunction of ion channels involved in the regulation of sperm . stones, confirming the association with absorptive hypercalciuria motility. These pathways are interrelated and abnormalities usually . (OMIM#143870). This is an original finding showing the same have adverse effects on various sperm functions. pathogenic variant associated with distinct phenotypes, depending . Apart from being an energy source, ATP is also involved in the . on recessive or dominant inheritance. regulation of sperm motility where it is used as a precursor by adeny- . late cyclase enzymes to produce cAMP (Visconti, 2012). It has been . shown that oxidative phosphorylation process inhibition by a chemical . Materials and Methods . compound or by knocking out Gapdhs, which encodes for an integral . Human subject recruitment sperm-specific component of the glycolytic pathway, both lead to . sperm immobility (Miki et al., 2004; Goodson et al., 2012). A 34-year-old male from the Fars province, Iran, with a chief com- . cAMP/PKA and calcium pathways are the most important signaling . plaint of infertility was referred to the infertility clinic at Royan Insti- . pathways of sperm motility regulation (Turner, 2006). Soluble adenylyl . tute. His parents were first cousins and genetic counseling revealed . cyclase (sAC) is the predominant adenylate cyclase in the principal . one infertile first cousin once removed who had first-cousin par- . piece of the sperm tail (Buffone et al., 2014). This enzyme is activated . ents as well (Fig. 1). Considering the level of consanguinity in the 2+ − . by Ca and bicarbonate (HCO3 ) to turn ATP into cAMP, which . family, they were chosen as candidates