Homozygous mutations in CCDC34 cause male infertility with oligoasthenoteratozoospermia in humans and mice

BackgroundOligoasthenoteratozoospermia is a typical feature of sperm malformations leading to male infertility. Only a few genes have been clearly identified as pathogenic genes of oligoasthenoteratozoospermia.Methods and resultsHere, we identified a homozygous frameshift variant (c.731dup, p.Asn244Lysfs*3) in CCDC34, which is preferentially expressed in the human testis, using whole-exome sequencing in a cohort of 100 Chinese men with multiple morphological abnormalities of the sperm flagella (MMAF). In an additional cohort of 167 MMAF-affected men from North Africa, Iran and France, we identified a second subject harbouring a homozygous CCDC34 frameshift variant (c.799_817del, p.Glu267Lysfs*72). Both affected men presented a typical MMAF phenotype with an abnormally low sperm concentration (ie, oligoasthenoteratozoospermia). Transmission electron microscopy analysis of the sperm flagella affected by CCDC34 deficiency further revealed dramatic disorganisation of the axoneme. Immunofluorescence assays of the spermatozoa showed that CCDC34 deficiency resulted in almost absent staining of CCDC34 and intraflagellar transport-B complex-associated proteins (such as IFT20 and IFT52). Furthermore, we generated a mouse Ccdc34 frameshift mutant using CRISPR-Cas9 technology. Ccdc34-mutated (Ccdc34mut/mut) male mice were sterile and presented oligoasthenoteratozoospermia with typical MMAF anomalies. Intracytoplasmic sperm injection has good pregnancy outcomes in both humans and mice.ConclusionsOur findings support that CCDC34 is crucial to the formation of sperm flagella and that biallelic deleterious mutations in CCDC34/Ccdc34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.

Mutations of the Histone Linker H1-4: An Expanded Cohort and Functional Characterization of Frameshift Mutant H1.4 in Neurons

Background: Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, brittle bones, and dysmorphic facies. De novo heterozygous mutations in H1-4 (HIST1H1E) encoding the linker histone H1.4 are found in patients with RMNS; however, the underlying mechanisms causing the pronounced neurological manifestations are not understood. The majority of reported mutations in H1-4 are small insertions or deletions that create a shared frameshift, resulting in an H1.4 protein that is both truncated and possessing an abnormal C-terminal tail.Methods: Seven Rahman syndrome subjects with C-terminal frameshift mutations as well as three patients with heterozygous null mutations in H1-4 were described in detail. Lymphoblastoid cells from these patients were used to identify transcriptional abnormalities in RMNS. Wildtype or mutant frameshifted human H1.4 protein was exogenously expressed in primary rat hippocampal neurons, and neuronal structure and function were assessed using immunohistochemistry and multi-electrode array recordings. Results: Individuals with heterozygous null variants in the H1-4 gene lack several key RMNS phenotypes, supporting the hypothesis that RMNS is due to a gain-of-function of the frameshift mutant H1.4 protein. In cultured rat hippocampal neurons, H1.4 was localized to the nucleus, though the frameshift mutant H1.4 had a distinct subnuclear distribution and enlarged nuclei. Overexpression of frameshift mutant H1.4 had minimal effects on dendritic morphology; however, it significantly reduced neuronal firing rate relative to neurons overexpressing wildtype human H1.4. Limitations: Given the small number of RMNS cases worldwide, the true breadth of phenotypes remains unknown. Though our data do not show robust differential expression of any genes, larger cohorts for clinical and molecular studies will be needed to gain reliable data.Conclusions: These data are the first to characterize the consequence of frameshift mutant H1.4 in neurons. These data provide new insights into the breadth of phenotypes and causes of neurological dysfunction in RMNS and highlight the need for future studies on the function of histone H1.4 in neurons.

Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth

ArabidopsisAINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of milletSCR1,GNC, andAN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of theANT1ortholog in the C4 milletSetaria viridisby the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model forANT1regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles ofANT1in several developmental processes beyond its known roles in plant growth and floral organogenesis.

Study of the Retrotransposon-Derived Human PEG10 Protease

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. Previous works have demonstrated that a mutation in the coding sequence of this gene is lethal with regard to embryological age due to defects of placental development. In addition, PEG10 is implicated in several malignancies, such as pancreatic cancer and hepatocellular carcinoma. The PEG10 gene encodes two protein isoforms, which are translated by a typical retroviral frameshift mechanism. The Gag-like protein (RF1PEG10) is encoded by reading frame 1, whilst reading frames 1 and 2 accounts for the Gag-Pol-like polyprotein (RF1/RF2PEG10). The protease (PR) domain of RF2PEG10 contains an -Asp-Ser-Gly- sequence, which refers to the conservative -Asp-Ser/Thr-Gly- active-site motif of retroviral aspartic proteases. The function of the aspartic protease domain of RF2PEG10 remains unclear. In order to further investigate the function of the PEG10 protease (PRPEG10), a frameshift mutant was generated (fsRF1/RF2PEG10) for comparison with the RF1/RF2PEG10 form. To study the effects of PRPEG10 on cellular proliferation and viability, mammalian HEK293T and HaCaT cells were transfected with plasmids encoding for either the frameshift mutant (fsRF1/RF2PEG10) or a PR active-site (D370A) mutant fsRF1/RF2PEG10. Based on our findings, an fsRF1/RF2PEG10 overexpression resulted in an increased cellular proliferation, compared to the mutant form. Interestingly, transfection with fsRF1/RF2PEG10 had a detrimental effect on cell viability. We hypothesize that PRPEG10 may play a cardinal role in the function of this retroviral remnant, possibly implicated in cellular proliferation and the inhibition of apoptosis.

Functional Study of the Retrotransposon-Derived Human PEG10 Protease

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. The mRNA of PEG10 encodes two protein isoforms: the Gag-like protein (RF1PEG10) is coded by reading frame 1, while the Gag-Pol-like polyprotein (RF1/RF2PEG10) is coded by reading frames 1 and 2. The proteins are translated by a typical retroviral frameshift mechanism. The protease (PR) domain of RF2PEG10 contains an -Asp-Ser-Gly- sequence, which corresponds to the consensus -Asp-Ser/Thr-Gly- active-site motif of retroviral aspartic proteases. The function of the aspartic protease domain of RF2PEG10 remains unclear. To elucidate the function of PEG10 protease (PRPEG10), we designed a frameshift mutant (fsRF1/RF2PEG10) for comparison with the RF1/RF2PEG10 form. To study the effects of PRPEG10 on cellular proliferation and viability, mammalian HEK293T and HaCaT cells were transfected with plasmids coding for either RF1/RF2PEG10, the frameshift mutant (fsRF1/RF2PEG10), or a PR active-site (D370A) mutant fsRF1/RF2PEG10. Our results indicate that fsRF1/RF2PEG10 overexpression results in increased cellular proliferation. Remarkably, transfection with fsRF1/RF2PEG10 had a detrimental effect on cell viability. We hypothesize that PRPEG10 plays an important role in the function of this retroviral remnant, mediating the proliferation of cells and possibly implicating it in the inhibition of apoptosis.