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 (sAC), which is the predominant adenylate cyclase in . It is already established that proper sAC activity and a constant supply of cAMP are crucial to regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility 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 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 / 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 . 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 cases have an underlying genetic cause . . point, the following 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 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 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 for whole exome sequenc- . is consumed by PKA to phosphorylate serine/threonine residues of . ing (WES) in case the cause of infertility could not be determined . a number of downstream proteins. These proteins target a spe- . by routine clinical procedures. A written consent form was signed . cific subset of flagellar proteins for tyrosine phosphorylation including . by each participant after receiving information about the study pro- . . axonemal dynein, which is an essential step in the initiation of sperm . cedure and aims. This study was approved by the Royan Institute . motility (Turner, 2006). Interestingly, sAC knockout mice were infertile . ethics committee and conducted in accordance with the declaration . due to complete loss of progressive motility in (Esposito . of Helsinki. . et al., 2004). Furthermore, targeted deletion of the catalytic subunit . . of the sperm-specific isoform of PKA was also reported to cause . Conventional karyotyping . . male infertility in mice as a consequence of decreased sperm motility . Karyotype analysis was performed on trypsin-banded metaphase chro- . (Skålhegg et al., 2002). . mosomes. The analysis was performed by a standard protocol to . ADCY10 is the only human gene that encodes for sAC with a highly . generate a resolution of 550 bands per haploid set, from a single . biased expression in testis (www.ncbi.nlm.nih.gov/gene/55811). This . cell of the corresponding parent. Normally, 30 random metaphase . enzyme synthesizes cAMP, which is the required substrate for the . spreads per sample were targeted. Results were reported based on sAC deficiency leads to severe asthenozoospermia 1157

. . USA). Briefly, Nextera Rapid Capture Exome kit (Illumina, San Diego, . . California, USA) was used to construct the library and capture tar- . . ® . get regions followed by paired-end 150-bp sequencing via NextSeq . . 500/550 High Output Kit v2. Real-time image analysis and base calling . . were performed by sequence Real-time Analysis 2 (Illumina, San Diego, . . California, USA). Resulting BCL files were converted to FASTQ format . . and demultiplexed by bcl2fastq2 conversion software (Illumina, San . . Diego, California, USA). Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 ...... Exome data analysis . . FASTQ files were subjected to adapter trimming by PICARD . . . 4.0.2.1 (Broad Institute, Cambridge, MA, USA) and mapped against . . the reference genome (assembly GRCh38/hg38) by Burrows- . . Wheeler Aligner (BWA 0.7.17-r1188). Downstream analysis was . . performed using GATK 4.0.2.1 following GATK best practices . Figure 1 Pedigree of a multiplex consanguineous family . and variants were called by HaplotypeCaller. Resulting VCF files . with severe asthenozoospermia and calcium kidney stones. . . underwent preliminary and custom filtering steps and the final VCF A pair of first cousins in generations III (III-4 and III-5) and IV (IV-1 and . . file was subjected to annotation by ANNOVAR. Shortlisted high- IV-2) have produced two severely asthenozoospermic males in two . . impact variants were manually visualized by Interactive Genomics consecutive generations (black squares). Calcium kidney stones are . . Viewer 2.4.4 (Broad Institute, Cambridge, MA, USA). also prevalent in this family. Both infertile males and family members . . indicated with striped and checkered squares and circles have a history . . of calcium kidney stones. The proband (V-2) who is marked with a ‘P’, . Segregation analysis by Sanger sequencing . his father (IV-1), the other affected member (IV-4) and his mother (III- . . Specific primers flanking the homozygous frameshift deletion in 5) underwent whole exome sequencing (WES) after routine clinical . . the ADCY10 gene were designed by Primer3web 4.1.0 and used procedure failed to determine the cause of their infertility. Analysis . . to amplify the variation site by conventional polymerase chain of WES data revealed that V-2 and IV-4 were homozygous for a . .  frameshift deletion in ADCY10 (NM 018417:exon11:c.1205 1206del) . reaction (PCR). The sequences of primers were as follows: F: 5 - .   that encodes the soluble adenylate cyclase (sAC) which is a compo- . CCCTCATTCCATCAACATATAGCC-3 and R: 5 -TTTCCCAACCC .  nent of the cAMP/PKA pathway in the regulation of sperm motil- . AGACATTATTTCC-3 . Amplification was done using Taq DNA . ity. ADCY10 is also associated with hypercalciuria (MIM#143870), a . Polymerase Master Mix (Ampliqon, Odense, Denmark). Resulting PCR . common cause of calcium kidney stones with an autosomal domi- . products were purified using PureIT ExoZAP (Ampliqon, Odense, . nant inheritance pattern. Sanger Sequencing results of the living and . Denmark). Afterward, the purified PCR products were sequenced + + . available family members are shown on the pedigree with / , . by BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied + − − − . / and / indicating homozygous for the wild-type, heterozygous . . Biosystems, Foster City, CA, USA) followed by ethanol precipitation and homozygous for the mutated allele, respectively. IV-3 who is . . for purification. The products were then assayed by 3130xl Genetic homozygous for the wild type allele is not asthenozoospermic and has . . Analyzer (Applied Biosystems, Foster City, CA, USA). Final data were never developed kidney stones. The checkered members (III-2 and III- . . analyzed by Sequencing Analysis version 5.2 (Applied Biosystems, 4) are deceased and therefore not available for analysis. However, a . . Foster City, CA, USA) and FinchTV version 1.5.0 (Geospiza Inc.) history of calcium kidney stones was reported by family members. V- . . 1 and IV-7 were not available for analysis. . . . . Preparing sperm culture medium . . Human tubal fluid (HTF) medium was used for treatment of sperm the latest International System for Human Cytogenetic Nomenclature . . (McGowan-Jordan et al., 2016). . samples and was prepared using 0.5935 g of sodium chloride (NaCl), . . 0.035 g of potassium chloride (KCl), 0.0050 g of potassium dihydrogen . . phosphate (KH2PO4), 0.03 g of calcium chloride dihydrate (CaCl2.2H2O), Hormonal assay . . 0.0024 g of magnesium sulfate (MgSO4), 0.21 g of sodium bicarbonate Levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), . . (NaHCO ), 0.0036 g of sodium pyruvate (C H NaO ), 0.05 g of . 3 3 3 3 (PRL), and testosterone (T) were measured in all patients as . . D-glucose, 0.006 g of penicillin G sodium salt (C16H17N2NaO4S) part of routine hormonal assay for male infertility as recommended . . and streptomycin sulfate salt (C21H39N7O12. 1.5 H2SO4), 0.001 g by the Practice Committee of the American Society for Reproductive . . of phenol red (C19H14O5S), 0.2485 g of HEPES (C8H18N2O4S), Medicine (2015). . . 0.0021 g of EDTA ((HO2CCH2)2NCH2CH2N(CH2CO2H)2), and . . 0.54 g of sodium lactate (C H NaO ), all dissolved in 100 ml of . 3 5 3 DNA extraction and WES . incubated distilled water. The pH of the solution was set at 7.35–7.45 . . Blood samples from participants were drawn in EDTA-containing tubes . and slight deviations were corrected by NaOH or HCl (WHO, 2010). . and genomic DNA was isolated from blood by salting-out procedure . All mentioned components were procured from Sigma-Aldrich, St. . (Miller et al., 1988). WES was performed on III-5, IV-1, IV-3, IV-4, . Louis, Missouri, USA. This mixture was filtered through a syringe filter . ◦ and V-2 on NextSeq® 500 platform (Illumina, San Diego, California, . and stored at 4 C as HTF stock solution. 1158 Akbari et al.

. . Density gradient centrifugation for sperm . Results . isolation . . . Clinical description of patients and family Sperm samples were subjected to a simple wash with HTF at 2500 rpm . . for 8 min (Beydola et al., 2014). Next, density gradient centrifugation . members . (DGC) was used to effectively remove seminal plasma and achieve . The proband V-2 who was a product of a consanguineous marriage . liquefaction by Allgrad® (Lifeglobal, CT, USA) according to manufac- . between first cousins (IV-1 and IV-2) was referred with a chief com- . turer’s instructions. . plaint of primary infertility. He reported having regular intercourse (2–3 . Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 . . times a week) during 12 years of marriage with no success in pregnancy Sperm sample treatment with cAMP . . while exhibiting normal erection and ejaculation. Physical examina- . analogue . tion by a urologist indicated normal development of male external . . genitalia, normal bilateral testis and testis size, and normally palpable Dibutyryl cAMP sodium salt (bucladesine sodium salt) was used as . . the cAMP analogue as it can easily permeate the cell through sim- . vas deferens with no varicocele. He had no history of exposure to . ple diffusion and activate cAMP-related pathways (Sigma-Aldrich, St. . chemotoxic materials or engagement in unhealthy activities affecting . Louis, Missouri, USA). Also, 0.00983 g of dibutyryl cAMP sodium salt . fertility, except for smoking. There was no history of systemic or . (MW = 491.37 g) was dissolved in 1 ml of HTF to prepare a stock . respiratory diseases. The noteworthy point in his medical record was . solution of 20 mM. . his and his parents’ history of kidney stones for which he regularly took . . medication. Further genetic counseling revealed one first cousin once HTF was mixed with human serum albumin (Vitrolife, Sweden) . ◦ . and incubated in a CO2 incubator for 3 h at 37 C prior to use. A . removed (IV-4) with infertility who was the son of consanguineous . simple wash was performed on sperm samples (Edwards, 1969, Early . parents as well. This cousin, his late father (III-4), his mother (III-5), and . Stages of Fertilization in vitro of Human Oocytes Matured in vitro). . two of his brothers (IV-5 and IV-6) also had a history of developing . Briefly, 2 ml of sperm sample was mixed with 2 ml of incubated HTF, . kidney stones. After being clinically examined, the cousin’s medical . pipetted gently and centrifuged at 2000 rpm for 15 min. The pellet was . history was obtained (Table I). Both patients (V-2; IV-4) underwent . . hormonal assay for LH, FSH, PRL, and T and all hormone levels were resuspended in 2 ml of HTF and distributed among treatment tubes. . . Then, 5 and 20 μl of dibutyryl cAMP sodium salt stock solution, equal . within the normal range (Table II). The patients were also tested by . to 0.05 mg and 0.2 mg, respectively, of the Bucladesine sodium salt . karyotyping and the results complied with apparent normal males . powder, was added to 500 μl of treatment media. . (46,XY). Routine showed the absence of spermatozoa . . with rapid progressive motility (Class A) in both patients and very . Sperm motility assessment . low percentages of spermatozoa with medium progressive motility . . The effect of treatment on sperm motility was assessed by Sperm . (Class B) (Table II). ® . Class Analyzer computer-assisted sperm analysis (CASA) system . . (MICROPTIC, Barcelona, Spain) with lower reference limits set to the . Detecting frameshift variation in ADCY10 . WHO guidelines. . WES was performed on III-5, IV-1, IV-3, IV-4, and V-2 and an average . . coverage of 150X was reached. Of the total reads, 91% passed the . Accession number . ≥ . quality filter (Q 30). Given the similarity in phenotypes of the two . The variant has been submitted to the ClinVar database under . patients and high level of consanguinity in the pedigree, we hypothe- . SCV000746242. . sized that the pathogenic variant was possibly homozygous and shared

Table I Medical history of the infertile patients.

IV-4 V-2 ...... 38 34 Age 10 y 12 y History of I infertility Once ectopic NO Abortion/pregnancy Smoker Smoker Smoking/drinking A first cousin once removed A first cousin once removed Family history Bilateral normal Bilateral normal Testes NO NO Varicocele Physical examination Palpable Palpable Vas deferens Asthenteratozoospermic Asthenozoospermic Semen analysis NO 7 y ago Varicocelectomy surgery NO NO History of systemic diseases NO NO Exposure to gonadotoxins and/or chemical toxins

Information obtained from genetic counseling, semen analysis, physical examination and historical investigation by urologist. sAC deficiency leads to severe asthenozoospermia 1159

Table II Semen analysis and hormonal assay results of the infertile.

IV-4 V-2 Normal range (WHO, 2010) ...... Volume (ml) 4.4 4.2 2–7 PH 7.8 7.8 >7.2 Sperm count (∗106/ml) 100 37 >15

Class A 0% 0% >32% Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 Class B 2% 3% Class C 39% 54% Class D 59% 43% <60% Normal morphology 2% 5% >4% Vitality test 95% 90% >58% Hormonal assay Test Result Normal range PRL (mlU/lit) 95.4 130.8 72–459 LH (mlU/ml) 8.32 5.63 1.0–10 FSH 2.4 10.6 1.5–12.4 (mlU/ml) T (ng/ml) 6.2 7.0 2.0–8.0

Class A: percentage of rapidly progressive sperm; Class B: percentage of medium progressive sperm; Class C: percentage of non-progressive sperm; Class D: percentage of immotile sperm. PRL, prolactin; LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone

. among the affected. Thus, an autosomal recessive inheritance mode . kidney stones in this pedigree followed a dominant inheritance pattern . was assumed. Subsequently, we started the analysis by investigating . (Fig. 1). . . variants that were homozygous in the proband V-2 and heterozygous in . The candidate variation for asthenozoospermia, a two . his father (IV-1); 16 900 variants fitted into this category. Variants with . deletion causing a frameshift producing a putative stop codon 41 . minor allele frequency >0.01 in ExAC and gnomAD were excluded. . codons after His402, is located 10 amino acids upstream of the . The remaining variants were categorized based on ACMG guideline . nucleotide binding site of the enzyme (Fig. 2a). This juncture is highly . for the interpretation of sequence variants (Richards et al., 2015). We . conserved across mammals and the discovered truncating variant . excluded synonymous Single Nucleotide Variation (SNVs), intronic and . can theoretically render the expression of the allele completely null . . intergenic variants that resulted in a list of 114 remaining variants. . (Fig. 2b). Both the affected subjects were confirmed to be homozygous . ∗ The effect of non-synonymous SNVs of this list was predicted using . for ADCY10:NM 018417:c.1205 1206del:p.(His402Argfs 41) by . various prediction tools such as SIFT, PolyPhen2, FATHMM, CADD, . Sanger sequencing (Fig. 2c). All of the family members genotyped . DANN, MetaLR, MetaSVM, Mutation taster, Mutation assessor, and . displayed a perfect correspondence with phenotype; only the . PROVEAN. For assessing conservation of the variation sites, GERP . homozygous mutated subjects segregated with the asthenozoospermia . ++ . , PhyLoP, and PhastCons were used. Variants with predicted high . phenotype while the heterozygous and the wild-type subjects were not . impacts that were mapped to genes involved in spermatogenesis, . associated with the affected phenotype. . sperm motility, maturation, and function or had restricted expression . . to testis were prioritized for segregation analysis with the infertility . . phenotype in the pedigree. The best candidate was the homozygous . Sperm motility rescue with cAMP analogue . frameshift variant c.1205 1206del, p.(His402Argfs∗41) in ADCY10 that . . Sperm samples were collected from 10 normozoospermic men, 10 . was reported in ExAC only once in a heterozygous state (dbSNP: . asthenozoospermic men, and the two patients. Sanger sequencing . rs779944215). . confirmed that all sample donors except for the two patients were . . homozygous for the wild-type allele at the site of ADCY10 frameshift . . . variation. After DGC and a simple wash, 0.5 ml of sperm sample was . . mixed with 0.5 ml of HTF in two three-microtube sets. Treatment was Variant confirmation and segregation . . performed with 0 (non-treated), 0.05, and 0.2 mg of dibutyryl cAMP . analysis . sodium salt, meaning that respectively 0, 5, and 20 μl of the analogue . ADCY10 has been linked with hypercalciuria (MIM#143870), which is a . was added from the 20-mM stock solution to the final medium volume . known cause of calcium kidney stones (Reed et al., 2002). Interestingly, . of 1 ml. Samples were incubated at 37◦CinaCO incubator at intervals . 2 . the proband (V-2) had kidney stones and was under medical care since . of 30 and 60 min. . stone removal (Fig. S1). Both of the proband’s parents (IV-1 and IV-2) . The percentage of spermatozoa with rapid progressive motility . and three first cousins once removed including the infertile one (IV-4, . (Class A), medium progressive motility (Class B) (World Health . IV-5, and IV-6) had kidney stones. We observed that the presence of . Organization, 1999), progressive motility (A + B) (WHO, 2010), and 1160 Akbari et al. ∗ ∗ ∗ ∗ ∗ ∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ (vs. non-treated) Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 m/s) p value μ ...... ents underwent treatment with cAMP analogue. Percentages of sperm with creases in sperm parameters after 30 and 60 minutes of treatment. The only exception ). Normozoospermic and asthenozoospermic patients showed a significant increase in the d samples and treated samples with 0.05 mg and 0.2 mg of cAMP analogue in each group via ∗∗ 0.01 < P , ∗ 0.05 < P ...... 1.28 10.46 11.73 8.11 0.08437 0.876.978.480.443.56 3.21 13.15 14.65 3.11 9.25 4.08 20.12 23.14 3.56 12.81 8.67 16.73 16.87 8.55 16.08 0.00928 _ 0.01089 0.007611 _ 5.76 6.59 12.35 14.29 0.01494 5.055.24 6.17 6.66 11.22 11.90 13.89 14.49 0.009852 _ 0.117.143.13 1.98 9.8 9.39 2.09 16.94 12.53 7.16 24.83 16.30 0.01012 _ 0.009449 1.68 7.62 9.30 9.20 0.02765 8.267.080.318.28 9.70 8.10 3.38 19.67 17.96 15.18 3.69 27.95 21.04 17.92 7.68 19.62 0.6144 0.9618 0.01135 _ 24.22 24.11 48.33 21.07 0.00961 10.26 7.85 18.11 21.76 _ 21.0622.53 22.36 22.54 43.42 45.07 18.52 20.77 0.02496 _ 18.1317.7216.46 23.15 20.58 22.35 41.28 38.30 38.81 15.69 17.01 15.42 _ 0.8349 0.9627 Class A (%) Class B (%) PR (%) VSL ( 0 0 0 0 0 0 0 0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 cAMP value of 0.08437. analogue (mg) P values less than 0.05 were considered statistically significant ( P ...... 60 60 30 60 30 60 (min) Results of sperm samples treatment with cAMP analogue. -test with the alternative hypothesis set to ‘less’. t V-2 30 IV-4 30 Groups Interval (Average of 10) (Average of 10) Normozoospermic mentioned four parameters after 30was minutes V2, of at treatment 0.2 but mg no of significant analogue increase after was 60 detected minutes, after which returned 60 a minutes. Both patients showed significant in Table III Four groups including 10 asthenozoospermic and 10 normozoospermic samples (who were confirmed as homozygous for the wild type allele) and the two pati progressive motility, sperms with class Aindependent motility, sperm with class B motility and straight line velocity (VSL) was compared between the non-treate Asthenozoospermic ...... sAC deficiency leads to severe asthenozoospermia 1161 Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019

Figure 2 Site of the variation within the C2 domain of the sAC. a) ADCY10 is located on reverse strand so 5 to 3 is shown from right to left. The C1 and C2 catalytic domains are encoded by exons 2 to 7 and 9 to 12 respectively. A GT deletion in exon 11 causes a frameshift at His402 which is located ten residues upstream of Asn412 of the NXXXR catalytic motif. The catalytic site of the enzyme is located at the interface of the C1/C2 dimer. This variation potentially causes a disruption in exon 12 and prevents C2 from forming a dimer with C1 rendering the enzyme dysfunctional. b) The amino acid sequence of the interval between the His402 and the NXXR catalytic motif is highly conserved across mammals. The last sequence belongs to Atlantic Salmon. Indeed, the whole sequences of C1 and C2 domains are highly conserved across the species. The Leu413 and Ile413 residues are both conserved and effective within the catalytic motif although they might cause some differences in the efficiency ofthe catalytic activity. c) Sanger Sequencing confirmed the variation in patients and close family members (Homo: Homozygous; Hetero: Heterozygous; WT: Wild-Type; MU: Mutant). 1162 Akbari et al.

. straight line velocity (VSL) parameters was compared statistically . into a hydrophobic cleft formed by the side chains of Ala97, Phe296, . before and after treatment by paired t-test with the alternative . Leu345, Phe336, and Val411 (Saalau-Bethell et al., 2014). Adenine is . . hypothesis set to ‘less’, i.e. a decrease is expected in the second data . coordinated by the carboxyl oxygen of Val406 to be recognized by the . group in comparison with the non-treated group. A P value <0.05 was . two ATP recognizing residues in Lys334 and Thr405 through hydrogen . considered statistically significant. . bonds between the adenine ring N1 and Lys334 and between the . In the normozoospermic group, treatment with 0.05 and 0.2 mg of . 6-amino group and Thr405 (Tesmer et al., 1997; Kamenetsky et al., . cAMP analogue for 30 min resulted in a significant increase in mea- . 2006; Kleinboelting et al., 2014). Further along the cleft, the highly .

. Downloaded from https://academic.oup.com/humrep/article-abstract/34/6/1155/5492390 by Promedica Health System user on 20 July 2019 sured parameters with P values of 0.02496 and 0.00961, respectively. . conserved Asn412 and Arg416 of the NXXXR cyclase catalytic motif . However, in the 60-min interval, no significant increase was detected. . sit on opposite sides of the ribose ring and the α-phosphate. Both of . The asthenozoospermic group followed the same pattern, meaning . these residues are assumed to be essential for the stabilization of the . that treatment with both amounts of the cAMP analogue for 30 min . transition state (Yan et al., 1997). Accordingly, a frameshift variation at . showed a significant increase in sperm parameters with P values of . His402 may completely render the enzyme inactive as it affects various . 0.009852 and 0.01494 for 0.05 and 0.2 mg, respectively, while no . . functionally indispensable residues. . significant increase was observed after treatment for 60 min. However, . In 2004, Esposito et al. produced a knockout mouse model of sAC . for the patients, treatment with both volumes of analogue at both . by homologous recombination. The generated male mice were viable . intervals yielded significant increases. The only exception was treat- . and developed normal testes and but were sterile due to . ment of the V-2 sample with 0.2 mg of analogue at 60-min interval . . drastically reduced total and almost non-existent progressive motility (Table III). Graphical comparison of the four measured parameters . . of spermatozoa. Subsequently, the sAC-deficient spermatozoa were . in all groups (Supp. Figs 2–5) along with video files of non-treated . treated with cAMP-acetoxymethyl, which resulted in restoration of . (Supp. Movies 1, 2, 5, and 6) and treated (Supp. Movies 3, 4, 7, . progressive motility (Esposito et al., 2004). Based on these findings, . and 8) V-2 and IV-4 sperm samples are available in supplemental . Visser et al. shortlisted ADCY10 as candidates for screening in astheno- . files. . . zoospermic men. They reported two ADCY10 missense variations . . (c.1020-40A > C and c.3542A > G) that were detected in patients . . only. However, both variations were heterozygous and the functional Discussion . . assay performed to test the pathogenic capacity of c.3542A > G . sAC is the predominant adenylate cyclase in sperm (Hess et al., 2005). . . did not confirm any significant alteration in the enzyme activity . It is encoded solely by the ADCY10 gene. The longest transcript, cor- . (Visser et al., 2011). . responding to NM 018417, has 33 exons and translates into a 1610- . In the present study, the patients’ sperm samples displayed complete . amino-acid long polypeptide with a mass of ∼187 kDa. The C1 domain . absence of rapid and a conspicuous reduction of medium progressive . spans six exons in exons 2 to 7 and the C2 domain is encoded by four . motility, features that were similar to the phenotype observed in the . exons in exons 9 to 12 (Chen et al., 2014)(Fig. 2a). The discovered . . sAC-knockout mice (Esposito et al., 2004). Treatment of the patients’ . frameshift candidate pathogenic variation for asthenozoospermia is . sperm samples with a cAMP analogue was performed to confirm the . described as ADCY10:NM 018417:exon11:c.1205 1206del:p.H402fs . variant’s pathogenicity. Class A, class B, progressive motility, and VSL . in which a GT deletion causes a frameshift at the His402 residue, . of sperm in both patients were measured and compared with those . altering the translation downstream of the deletion and causing a . . of their own non-treated samples at two intervals of 30 and 60 min. premature termination after 41 residues in the new reading frame. . . The results showed a statistically significant increase in all measured . Since this region is highly conserved in mammals, we hypothesized that . parameters. Barring the normozoospermic and asthenozoospermic . this variation severely affects the function of the protein as it disrupts . samples that did not represent the variant in question, the patients’ . the C2 domain (Fig. 2a), particularly because the variation occurs 10 . samples treatment results were in agreement with the findings of . residues upstream of the NXXXR catalytic motif residing two highly . . Esposito et al. (2004), indicating a restoration of sperm motility thus conserved residues in Asn412 and Arg416 (Fig. 2b). Alternatively, it . . confirming deficiency of the sAC, as the cause of the phenotype in the . may be that the mutant transcript is subjected to nonsense-mediated . studied family (Table III and Figs S2–5). . mRNA decay, leading to a low/very low concentration of transcript. . On another note, OMIM listed ADCY10 as a disease gene for . Mammalian ACs possess two structurally similar catalytic domains . . familial idiopathic hypercalciuria, which is a common cause of kidney (C1 and C2) located on one polypeptide chain. The active enzyme . . stones (MIM#143870) (Reed et al., 2002). Interestingly, in the present . includes a dimer formed by either homo- or heterodimerization of . pedigree, both patients and all other relatives who were heterozygous . these catalytic sites and the catalytic unit of the enzyme is located at . for the frameshift variant had a history of calcium kidney stones, . the interface of the dimer (Kamenetsky et al., 2006). As C1 and C2 . confirmed by medical radiography of kidneys (Fig. S1). Only IV-3 who is . contribute different residues to the catalytic centers, the two resulting . . homozygous for the wild-type allele never experienced kidney stones. active sites are not identical and only one of them possesses all the . . On this account, the association between absorptive hypercalciuria and . features required for catalysis. The other one, which is degenerate, . mutations in ADCY10 was confirmed. Importantly, our finding enlarges . is the designated binding site for enzyme specific activators, that is . the phenotypic spectrum associated with this gene, as also causative of . forskolin for tmACs and bicarbonate for sAC. Hence, although C1 . a recessive form of asthenozoospermia. . . and C2 have the same overall structures, they serve different purposes . . (Tesmer et al., 1997; Zhang et al., 1997; Kamenetsky et al., 2006). The . . Supplementary data crystal structure of the human sAC in complex with calcium ions and . . an ATP analogue confirms a single ATP binding site. ATP is inserted . Supplementary data are available at Human Reproduction online. sAC deficiency leads to severe asthenozoospermia 1163

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