Biallelic mutations in spermatogenesis and centriole-associated 1 like(SPATC1L)cause acephalic spermatozoa syndrome and male infertility

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients,and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome.Spermatogenesis and centrioleassociated 1 like(SPATC1L)is indispensable for maintaining the integrity of sperm head-to-tail connections in mice,but its roles in human sperm and early embryonic development remain largely unknown.Herein,we conducted whole-exome sequencing(WES)of 22 infertile men with acephalic spermatozoa syndrome.An in silico analysis of the candidate variants was conducted,and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility.We identified biallelic mutations in SPATC1L(c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X)from a patient whose sperm displayed complete acephalia.Both SPATC1L variants are rare and deleterious.SPATC1L is mainly expressed at the head–tail junction of elongating spermatids.Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro.Moreover,none of the patient’s four attempts at intracytoplasmic sperm injection(ICSI)resulted in a transplantable embryo,which suggests that SPATC1L defects might affect early embryonic development.In conclusion,this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome.Furthermore,WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

Clustered regulatory interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 nuclease(Cas9),the third-generation genome editing tool,has been favored because of its high efficiency and clear system composition.In this technology,the introduced double-strand breaks(DSBs)are mainly repaired by non-homologous end joining(NHEJ)or homology-directed repair(HDR)pathways.The high-fidelity HDR pathway is used for genome modification,which can introduce artificially controllable insertions,deletions,or substitutions carried by the donor templates.Although high-level knock-out can be easily achieved by NHEJ,accurate HDR-mediated knock-in remains a technical challenge.In most circumstances,although both alleles are broken by endonucleases,only one can be repaired by HDR,and the other one is usually recombined by NHEJ.For gene function studies or disease model establishment,biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes.Thus,there is an urgent need for an efficient biallelic editing system.Here,we developed three pairs of integrated selection systems,where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker.Flanked by homologous arms containing the mutated sequences,the selection cassettes were integrated into the target site,mediated by CRISPR/Cas9-induced HDR.Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance.We tested this novel method on the amyloid precursor protein(APP)and presenilin 1(PSEN1)loci and demonstrated up to 82.0%biallelic editing efficiency after optimization.Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

Constitutional Mismatch Repair-Deficiency Syndrome Is a Rare Cause of Cancer Even in a Highly Consanguineous Population

Biallelic germline mutations in the mismatch repair genes, including MLH1, MSH2, MSH6 or PMS2, lead to a recessive constitutional mismatch repair-deficiency (CMMR-D) syndrome characterized by early onset malignancies in children and young adults. Because consanguinity unmasks autosomal recessive disorders, we hypothesized that the frequency of CMMR-D is inflated in the highly consanguineous population of Saudi Arabia. In this study, 371 pediatric and young adult?patient samples from Saudi Arabia that cover the tumor spectrum of CMMR-D syndrome were analyzed for biallelic germline mutations in the MLH1, MSH2, MSH6 and PMS2 with the use of direct genomic sequencing. However, none of the 371 patients involved in the study was found to have biallelic pathological mutations of MLH1, MSH2, MSH6 or PMS2. This result indicates that CMMR-D is exceptionally rare among pediatric cancer patients and adult early onset cancer patients, even in the highly consanguineous Saudi population. Our findings suggest that larger cohorts will be needed, particularly in outbred populations, to determine the frequency of CMMR-D and that routine screening for this syndrome among cancer patients is not warranted.