Genome sequencing of Pakistani families with male infertility identifies deleterious genotypes in SPAG6, CCDC9, TKTL1, TUBA3C, and M1AP.

BACKGROUND: There are likely to be hundreds of monogenic forms of human male infertility. Whole genome sequencing (WGS) is the most efficient way to make progress in mapping the causative genetic variants, and ultimately improve clinical management of the disease in each patient. Recruitment of consanguineous families is an effective approach to ascertain the genetic forms of many diseases. OBJECTIVES: To apply WGS to large consanguineous families with likely hereditary male infertility and identify potential genetic cases. MATERIALS AND METHODS: We recruited seven large families with clinically diagnosed male infertility from rural Pakistan, including five with a history of consanguinity. We generated WGS data on 26 individuals (3-5 per family) and analyzed the resulting data with a computational pipeline to identify potentially causal single nucleotide variants, indels, and copy number variants. RESULTS: We identified plausible genetic causes in five of the seven families, including a homozygous 10 kb deletion of exon 2 in a well-established male infertility gene (M1AP), and biallelic missense substitutions (SPAG6, CCDC9, TUBA3C) and an in-frame hemizygous deletion (TKTL1) in genes with emerging relevance. DISCUSSION AND CONCLUSION: The rate of genetic findings using the current approach (71%) was much higher than what we recently achieved using whole-exome sequencing (WES) of unrelated singleton cases (20%). Furthermore, we identified a pathogenic single-exon deletion in M1AP that would be undetectable by WES. Screening more families with WGS, especially in underrepresented populations, will further reveal the types of variants underlying male infertility and accelerate the use of genetics in the patient management.

A heterozygous missense variant in DLX3 leads to uterine leiomyomas and pregnancy losses in a consanguineous Iranian family.

Uterine leiomyomas (ULs) are benign solid tumors arising from the uterine myometrium. They are the most common pelvic tumors among females of reproductive age. Despite the universal prevalence of ULs and its huge impact on women's lives, the exact etiology and pathophysiologic mechanisms have not been fully understood. Numerous studies indicate that genetic factors play a crucial role in ULs development. This study aims to identify the probable genetic causes of ULs in a consanguineous Iranian family. Whole-exome sequencing (WES) on five family members with ULs revealed a likely pathogenic missense variant encoding for Y88C in the transactivation (TA) domain of DLX3 gene (c.263A > G; p.Y88C). Sanger sequencing of a total of 9 affected and non-affected family members indicated a segregation with disease with autosomal dominant inheritance. Moreover, targeted Sanger sequencing on 32 additional non-related patients with ULs showed none was heterozygous for this variant. MutPred2 predicted the pathogenicity of candidate variant by both phosphorylation and sulfation loss as actionable hypotheses. Project HOPE revealed that the identified variant residue is smaller and more hydrophobic comparing to the wild-type residue. I-TASSER and UCSF Chimera were also used for modeling and visualizing the predicted variant, respectively. This WES analysis is the first to report a variant in DLX3 variation associated with ULs pathogenicity in Iranian population highlighting the effectiveness of WES as a strong diagnostic method. However, further functional studies on this variant are needed to confirm the potential pathogenicity of this mutation.

Gene expression profile of placentomes and clinical parameters in the cows with retained placenta.

BACKGROUND: Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development. METHODS: Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing. RESULTS: We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17ß (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups. CONCLUSION: Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor.

Copy Number Variation of Circulating Tumor DNA (ctDNA) Detected Using NIPT in Neoadjuvant Chemotherapy-Treated Ovarian Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) can be used to characterize and monitor cancers. Recently, non-invasive prenatal testing (NIPT) as a new next-generation sequencing (NGS)-based approach has been applied for detecting ctDNA. This study aimed to investigate the copy number variations (CNVs) utilizing the non-invasive prenatal testing in plasma ctDNA from ovarian cancer (OC) patients who were treated with neoadjuvant chemotherapy (NAC). The plasma samples of six patients, including stages II-IV, were collected during the pre- and post-NAC treatment that were divided into NAC-sensitive and NAC-resistant groups during the follow-up time. CNV analysis was performed using the NIPT via two methods "an open-source algorithm WISECONDORX and NextGENe software." Results of these methods were compared in pre- and post-NAC of OC patients. Finally, bioinformatics tools were used for data mining from The Cancer Genome Atlas (TCGA) to investigate CNVs in OC patients. WISECONDORX analysis indicated fewer CNV changes on chromosomes before treatment in the NAC-sensitive rather than NAC-resistant patients. NextGENe data indicated that CNVs are not only observed in the coding genes but also in non-coding genes. CNVs in six genes were identified, including HSF1, TMEM249, MROH1, GSTT2B, ABR, and NOMO2, only in NAC-resistant patients. The comparison of these six genes in NAC-resistant patients with The Cancer Genome Atlas data illustrated that the total alteration frequency is amplification, and the highest incidence of the CNVs (≥35% based on TCGA data) is found in MROH1, TMEM249, and HSF1 genes on the chromosome (Chr) 8. Based on TCGA data, survival analysis showed a significant reduction in the overall survival among chemotherapy-resistant patients as well as a high expression level of these three genes compared to that of sensitive samples (all, p < 0.0001). The continued Chr8 study using WISECONDORX revealed CNV modifications in NAC-resistant patients prior to NAC therapy, but no CNV changes were observed in NAC-sensitive individuals. Our findings showed that low coverage whole-genome sequencing analysis used for NIPT could identify CNVs in ctDNA of OC patients before and after chemotherapy. These CNVs are different in NAC-sensitive and -resistant patients highlighting the potential application of this approach in cancer patient management.

Novel STAG3 variant associated with primary ovarian insufficiency and non-obstructive azoospermia in an Iranian consanguineous family.

Non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) present the most severe forms of male and female infertility. In the last decade, the increasing use of whole exome sequencing (WES) in genomics studies of these conditions has led to the introduction of a number of novel genes and variants especially in meiotic genes with restricted expression to gonads. In this study, exome sequencing of a consanguineous Iranian family with one POI and two NOA cases in three siblings showed that all three patients were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 (NM_001282717.1:c.1942G > A: p.Ala648Thr; NM_001282717.1:c.1951_1953del: p. Leu652del). Both variants occur within a short proximity of each other affecting the relatively conserved armadillo-type fold superfamily feature. STAG3 is a specific meiotic cohesin complex component that interacts with the α-kleisin subunit through this feature. Protein homology modeling indicated that the in-frame deletion destabilizes kleisin biding by STAG3. Although the missense variant did not seem to affect the binding significantly, protein homology modeling suggests that it further destabilizes kleisin binding when in double homozygous state with the deletion. Our findings are in line with several other studies having associated deleterious variants affecting this region with male and female infertility in humans and mouse models. This is the first report associating an in-frame STAG3 variant with NOA and POI in a single family. SUMMARY SENTENCE: A patient with primary ovarian failure and her two brothers with non-obstructive azoospermia were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 that potentially disrupt the protein's meiotic functions.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? SUMMARY ANSWER: Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. WHAT IS KNOWN ALREADY: MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. LARGE SCALE DATA: We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. LIMITATIONS, REASONS FOR CAUTION: Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. WIDER IMPLICATIONS OF THE FINDINGS: Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Correlations between Overexpression of SOX2OT Long Non-coding RNA and Susceptibility to Breast Cancer.

BACKGROUND AND OBJECTIVE: The SOX2OT lcnRNA has been recognized as a positive regulator in the transcription regulation of the SOX2 gene. Recent studies have approved the dysregulation of SOX2OT lncRNA expression patterns in some common cancer types, including esophageal, lung, and breast cancer. The objective of the present study was to investigate the correlation between overexpression of SOX2OT lcnRNA and susceptibility to breast cancer. METHODS: SOX2OT lncRNA expression profiling in 15 breast cancer and normal tumour-adjacent breast tissue samples was performed by using qRT-PCR. To evaluate the diagnostic potential of the SOX2OT lncRNA, we performed ROC curve analyses. RESULTS: The expression of SOX2OT lncRNA in patients suffering from breast cancer revealed a significant overexpression in comparison with the healthy group (P<0.001). Significantly, the elevated circulating SOX2OT lncRNA was found specific to breast cancer and could differentiate breast cancer from controls with 100% of both sensitivity and specificity. Based on the Kaplan- Meier analysis, there was no significant correlation between SOX2OT lcnRNA expression and overall survival. CONCLUSION: The results confirmed the association between breast cancer and higher SOX2OT lncRNA expression. According to the ROC curve results, SOX2OT lcnRNA could be a new measurable indicator of the breast cancer and a potential therapeutic target for breast cancer patients.

ADCY10 frameshift variant leading to severe recessive asthenozoospermia and segregating with absorptive hypercalciuria.

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. 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.

Rare case of an oligospermic male with 46,XX/46,XY tetragametic chimerism.

Chimerism, a rare human disorder, is assumed to be the result of an amalgamation of two separate zygotes in a single embryo. Studies have shown that the phenotypic spectrum of chimerism is variable and there is no definite genotype-phenotype correlation in patients with chimerism, therefore a majority of cases might remain undiagnosed. This study aims to investigate the possible mechanism of the chimerism in a 46,XX/46,XY infertile and phenotypically normal male, with 46,XX blood karyotype and normal spermatogenesis. We have used Interphase-FISH analysis to study the CEPX and CEPY regions on buccal and urine samples as well as molecular analysis of polymorphic short tandem repeats (STR) markers from 34 loci in order to discover the origin of 46,XX/46,XY. Analysis of X-linked and autosomal STR markers on blood, buccal tissue, urine, fibroblast and testis biopsy samples of the proband along with the blood sample of the patient's parents and siblings, showed divergent karyotypes in different tissues and tetragametic chimerism was diagnosed.

ARA lncRNA, is upregulated in liver and breast tumor tissues.

Important regulatory roles of long non-coding RNAs (lncRNAs) have been recently found, and reported as useful biomarkers in cancer. To identify a potential expression of the new discovered lncRNA (ARA), during promotes cell proliferation, apoptosis inhibit, migration and cell cycle arrest, we firstly evaluate its expression in two cancer tissues (breast cancer and liver cancer) and then compared its variability expression in tumor versus non-tumor samples. Expression profile of ARA lncRNA was evaluated using qRT-PCR in paired tumor and marginal non-tumor samples collected from patients who had been referred to the Shiraz General. After RNA extraction from tissue samples, cDNA synthesis and RT-qPCR method were performed according to the protocols. ARA lncRNA expression level was calculated using 2-ΔΔCt method. Principal-component analysis followed by receiver operating characteristic curve analyses was performed to evaluate the diagnostic potential of selected lncRNA. Our data revealed a significant upregulation (P < 0.001) of ARA in breast and liver tumor tissues, in comparison to same patients non-tumor marginal samples. Also, there was a significant difference between the expression of ARA lncRNA in breast cancer and liver cancer patients (P < 0.05). In conclusion, the results of our study suggest a possible role of ARA lncRNA in proliferation of breast and liver tissues, as well as its potential usefulness as a novel diagnostic biomarker for breast and liver tumors.