Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility.

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

KMT2D loss drives aggressive tumor phenotypes in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most lethal skin cancer. Due to ultraviolet light-induced damage, cSCCs have a high mutation rate, but some genes are more frequently mutated in aggressive cSCCs. Lysine-specific histone methyltransferase 2D (KMT2D) has a two-fold higher mutation frequency in metastatic cSCCs relative to primary non-metastatic associated cSCCs. The role of KMT2D in more aggressive phenotypes in cSCC is uncharacterized. Studies of other tumor types suggest that KMT2D acts to suppress tumor development. To determine whether KMT2D loss has an impact on tumor characteristics, we disrupted KMT2D in a cSCC cell line using CRISPR-cas9 and performed phenotypic analyses. KMT2D loss modestly increased cell proliferation and colony formation (1.4- and 1.6-fold respectively). Cells lacking KMT2D showed increased rates of migration and faster cell cycle progression. In xenograft models, tumors with KMT2D loss showed slight increases in mitotic indices. Collectively, these findings suggest that KMT2D loss-of-function mutations may promote more aggressive and invasive behaviors in cSCC, suggesting that KMT2D-related pathways could be targets for cancer therapies. Future studies to determine the downstream genes and mechanism of phenotypic effect are needed.

Differences in somatic TP53 mutation type in breast tumors by race and receptor status.

PURPOSE: Somatic driver mutations in TP53 are associated with triple-negative breast cancer (TNBC) and poorer outcomes. Breast cancers in women of African ancestry (AA) are more likely to be TNBC and have somatic TP53 mutations than cancers in non-Hispanic White (NHW) women. Missense driver mutations in TP53 have varied functional impact including loss-of-function (LOF) or gain-of-function (GOF) activity, and dominant negative (DNE) effects. We aimed to determine if there were differences in somatic TP53 mutation types by patient ancestry or TNBC status. METHODS: We identified breast cancer datasets with somatic TP53 mutation data, ancestry, age, and hormone receptor status. Mutations were classified for functional impact using published data and type of mutation. We assessed differences using Fisher's exact test. RESULTS: From 96 breast cancer studies, we identified 2964 women with somatic TP53 mutations: 715 (24.1%) Asian, 258 (8.7%) AA, 1931 (65.2%) NHW, and 60 (2%) Latina. The distribution of TP53 mutation type was similar by ancestry. However, 35.8% of tumors from NHW individuals had GOF mutations compared to 29% from AA individuals (p = 0.04). Mutations with DNE activity were positively associated with TNBC (OR 1.37, p = 0.03) and estrogen receptor (ER) negative status (OR 1.38; p = 0.005). CONCLUSIONS: Somatic TP53 mutation types did not differ by ancestry overall, but GOF mutations were more common in NHW women than AA women. ER-negative and TNBC tumors are less likely to have DNE+ TP53 mutations which could reflect biological processes. Larger cohorts and functional studies are needed to further elucidate these findings.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers-Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis.

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

X-linked ADGRG2 mutation and obstructive azoospermia in a large Pakistani family.

We performed whole exome sequencing to identify an unknown genetic cause of azoospermia and male infertility in a large Pakistani family. Three infertile males were subjected to semen analysis, hormone testing, testicular histology, ultrasonography, karyotyping, Y-chromosome microdeletion and CFTR testing. The clinical testing suggested a diagnosis of obstructive azoospermia (OA). To identify the cause, we performed whole exome sequencing (WES) for 2 infertile brothers and 2 fertile family members. For segregation analysis and variant confirmation, we performed Sanger sequencing. WES data analysis of the family revealed segregated variants in 3 candidate genes. We considered novel nonsense variant c.2440C > T(p.Arg814*) in X-linked gene ADGRG2 as biologically most plausible. It is predicted to truncate the protein by 204 amino acids (aa) at a key transmembrane domain. Adgrg2-knockout male mice show sperm loss due to obstructive fluid stasis, while ADGRG2 mutations cause OA in the infertile male patients. Our analysis of testicular histology reveals secondary severe reduction of spermatogenesis, consistent with human and knockout mouse phenotypes. The ADGRG2 nonsense mutation is absent in the largest population databases, ExAC and gnomAD. Analysis of the novel nonsense mutation in extended family members confirmed co-segregation of the mutation with OA in all affected males. The likely pathogenic nature of the mutation is supported by its truncation effect on the transmembrane domain and distinctive ultrasound results. The study demonstrates effectiveness of WES in discovering a genetic cause of azoospermia.

The -251 A/T Polymorphism in the IL8 Promoter is a Risk Factor for Acute Pancreatitis.

OBJECTIVES: Inflammation in the setting of acute pancreatitis (AP) is partially driven by pathogen recognition receptors that recognize damage-associated molecular patterns. Interleukin (IL)-8 is a chemotactic factor produced by pathogen recognition receptor-expressing cells. A single-nucleotide polymorphism in IL8 promoter region (-251 A/T) has been implicated in inflammatory diseases. We examined whether this IL8 polymorphism confers susceptibility to AP. METHODS: Patients with AP (n = 357) were prospectively recruited. Clinical data and blood were collected in subjects and controls (n = 347). Severity was defined following the Revised Atlanta Classification. Genotypes were assessed by quantitative polymerase chain reaction using TaqMan probes. RESULTS: Patients and controls had similar demographics and had no difference in Hardy-Weinberg (patients, P = 0.29; controls, P = 0.66). Twenty-five percent of patients developed severe AP. Compared with controls, the A/A genotype was more common in AP (P = 0.041; odds ratio, 1.42; 95% confidence interval, 1-1.99). Obese patients with the A/A genotype were more likely to develop mild AP (P = 0.047). CONCLUSIONS: The -251 polymorphism confers susceptibility to AP and disease severity in obese patients. However, its effect is moderate. One potential mechanism for this susceptibility is via increased IL8 production by innate cells, with subsequent enhanced neutrophil influx and pancreatic injury.

Familial deletion of the HOXA gene cluster associated with Hand-Foot-Genital syndrome and phenotypic variability.

Hand-Foot-Genital syndrome is a rare autosomal dominant condition characterized by distal limb anomalies and urogenital malformations. This disorder is associated with loss-of-function mutations in the HOXA13 gene. HOXA13 plays an important role in the development of distal limbs and lower genitourinary tract of the fetus. We report a novel familial 589 kb deletion in the 7p15.2 region identified in a male toddler and his mother. The proband had severe penoscrotal hypospadias, mild skeletal anomalies of the hands and feet, cardiac, renal, and gastrointestinal anomalies. His mother had a bicornuate uterus, cervical incompetence, and minor anomalies of her hands and feet. This family was found to have the smallest reported deletion of 7p15.2 to date, and presented with features typical of Hand-Foot-Genital syndrome in the mother, but much more severe phenotype in her son. This deletion included the entire HOXA cluster in addition to the SKAP2 and EVX1 genes. An RT-PCR analysis was performed to determine the expression of the HOXA genes in the proband and to explore a parent-of-origin effect. Our expression studies did not support the hypothesis of an imprinted status of the HOXA2, HOXA3, HOXA5, and HOXA11 genes in peripheral blood. To our knowledge, this is the first familial 7p15.2 deletion. This family raises possibility for sexual dimorphism as a mechanism for phenotypic variability in patients with the HOXA gene cluster deletions. © 2016 Wiley Periodicals, Inc.