DNAH17 is associated with asthenozoospermia and multiple morphological abnormalities of sperm flagella.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is one kind of severe asthenozoospermia, which is caused by dysplastic development of sperm flagella. In our study, we sought to investigate the novel gene mutations leading to severe asthenozoospermia and MMAF. METHODS AND MATERIALS: The patient's spermatozoa were tested by Papanicolaou staining and transmission electron microscopy. Whole exome sequencing was performed on the patient with severe asthenozoospermia and MMAF. Sanger sequencing verified the mutations in the family. The expression of DNAH17 was detected by immunofluorescence and Western blot. RESULTS: Spermatozoa sample from the patient showed severe asthenozoospermia and MMAF. We detected biallelic mutations (c.C4445T, p.A1482V and c.C6857T, and p.S2286L) in DNAH17 (MIM:610063). The protein expression of DNAH17 was almost undetectable in spermatozoa from the patient with the biallelic mutations. CONCLUSION: These results demonstrated that DNAH17 may be involved in severe asthenozoospermia and MMAF.

Biallelic mutations of CFAP74 may cause human primary ciliary dyskinesia and MMAF phenotype.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by recurrent respiratory infections, nasosinusitis, tympanitis, and/or male infertility, all of which can severely impair the patient's quality of life. Multiple morphological abnormalities of the sperm flagella (MMAF) is one type of severe teratozoospermia and results from a variety of flagellar defects. In this study, we conducted whole-exome sequencing to identify and evaluate the genetic lesions in two patients with potential PCD and MMAF. Biallelic mutations in exon 10, c.983G>A; p.(Gly328Asp), and exon 29, c.3532G>A; p.(Asp1178Asn), of the CFAP74 (NM_001304360) gene were identified in patient 1 (P1), and biallelic mutations in exon 7, c.652C>T; p.(Arg218Trp), and exon 35, c. 4331G>C; p.(Ser1444Thr), of the same gene were identified in patient 2 (P2). Bioinformatic analysis suggested that these variants may be disease causing. Immunofluorescence confirmed that CFAP74 was absent in these patients' sperm samples. Intracytoplasmic sperm injection (ICSI) was carried out for P1, and his wife became pregnant after embryo transfer and gave birth to a healthy baby. To the best of our knowledge, this study is the first to identify the importance of CFAP74 in potential PCD and MMAF, contributing to the genetic diagnosis of these disorders and helping to predict pregnancy outcomes relevant in in vitro fertilization.

Loss-of-function mutations in SPEF2 cause multiple morphological abnormalities of the sperm flagella (MMAF).

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a kind of severe teratozoospermia. Patients with the MMAF phenotype are infertile and present aberrant spermatozoa with absent, short, coiled, bent and/or irregular flagella. Mutations in several genes can explain approximately 30%-50% of MMAF cases and more genetic pathogenies need to be explored. SPEF2 was previously demonstrated to play an essential role in sperm tail development in mice and pig. Dysfunctional mutations in SPEF2 impair sperm motility and cause a short-tail phenotype in both animal models. OBJECTIVE: Based on 42 patients with severe infertility and MMAF phenotype, we explored the new genetic cause of human MMAF phenotype. METHODS AND RESULTS: By screening gene variants in 42 patients with MMAF using whole exome sequencing, we identified the c. 12delC, c. 1745-2A > G, c. 4102 G > T and c. 4323dupA mutations in the SPEF2 gene from two patients. Both of these mutations are rare and potentially deleterious. Transmission electron microscope (TEM) analysis showed a disrupted axonemal structure with mitochondrial sheath defects in the patients' spermatozoa. The SPEF2 protein level was significantly decreased in the spermatozoa of the patients revealed by Western blot (WB) and immunofluorescence (IF) analyses. CONCLUSION: Our experimental findings indicate that loss-of-function mutations in the SPEF2 gene can cause the MMAF phenotype in human.

DNAH2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella.

Multiple morphological abnormalities of flagella (MMAF) is one kind of severe teratozoospermia. Gene mutations reported in previous works only revealed the pathogenesis of approximately half of the MMAF cases, and more genetic defects in MMAF need to be explored. In the present study, we performed a genetic analysis on Han Chinese men with MMAF using whole-exome sequencing. After filtering out the cases with known gene mutations, we identified five novel mutation sites in the DNAH2 gene in three cases from three families. These mutations were validated through Sanger sequencing and absent in all control individuals. In silico analysis revealed that these DNAH2 variations are deleterious. The spermatozoa with DNAH2 mutations showed severely disarranged axonemal structures with mitochondrial sheath defection. The DNAH2 protein level was significantly decreased and inner dynein arms were absent in the spermatozoa of patients. ICSI treatment was performed for two MMAF patients with DNAH2 mutations and the associated couples successfully achieved pregnancy, indicating good nuclear quality of the sperm from the DNAH2 mutant patients. Together, these data suggest that the DNAH2 mutation can cause severe sperm flagella defects that damage sperm motility. These results provide a novel genetic pathogeny for the human MMAF phenotype.

Digital PCR Detection of mtDNA/gDNA Ratio in Embryo Culture Medium for Prediction of Embryo Development Potential.

PURPOSE: The ratio of mitochondrial DNA to genomic DNA (mtDNA/gDNA) in embryo culture medium as a predictor of embryonic development is a new method of noninvasive embryo screening. However, current tests based on this concept have proven inconsistent. The aim of this study was to define the predictive value of the ratio of mtDNA/gDNA for embryonic developmental potential. MATERIALS AND METHODS: We used digital PCR to measure mtDNA/gDNA ratios in day 3 culture media of 223 embryos from 56 patients. We compared the relationship between the predictive value of mtDNA/gDNA ratio and each of embryo fragmentation, embryo morphological grade, and blastocyst formation. RESULTS: mtDNA/gDNA ratio decreased significantly with a decrease in embryo rating: 22.54 (44.66); 31.25 (36.97) and 46.33 (57.11); Grades A vs C, P = 0.006; B vs C, P = 0.015. mtDNA/gDNA ratio increased overall with an increase in embryo fragment content but did not differ significantly between high-, -medium, and poor-quality embryos. Interestingly, this trend differed from that of the unformed blastocysts. mtDNA/gDNA ratio of cleavage stage embryos forming blastocysts was lower (P=0.005). Trends of mtDNA/gDNA ratio differed according to inner cell mass (ICM) and trophectoderm (TE) levels, but not significantly. mtDNA/gDNA ratio in day 3 culture medium was not significantly improved over morphological scores. CONCLUSION: We hereby show the correlation of mtDNA/gDNA ratio in the culture medium of developing embryos. The correlation between the mtDNA/gDNA ratio and early embryonic development was controversial. Furthermore, an increase in mtDNA/gDNA ratio might indicate reduced development potential, but the difference remains insufficient for application as a clinical predictor.

EIF4G1 is a novel candidate gene associated with severe asthenozoospermia.

BACKGROUND: Asthenozoospermia (AZS), also known as asthenospermia, is characterized by reduced motility of ejaculated spermatozoa and is detected in more than 40% of infertile patients. Because the proportion of progressive spermatozoa in severe AZS is <1%, severe AZS is an urgent challenge in reproductive medicine. Several genes have been reported to be relevant to severe asthenospermia. However, these gene mutations are found only in sporadic cases and can explain only a small fraction of severe AZS, so additional genetic pathogenies need to be explored. METHODS AND RESULTS: By screening the variant genes in a patient with severe AZS using whole exome sequencing, we identified biallelic mutations c.2521C>T: p.(Pro841Ser) (NC_000003.11: g.184043412C>T) in exon13 and c.2957C>G: p.(Ala986Gly) (NC_000003.11: g.184045117C>G) in exon17 in the eukaryotic translation initiation factor 4 gamma 1 gene (EIF4G1, RefSeq: NM_004953.4, OMIM: 600495) of the patient. Both of the mutation sites are rare and potentially deleterious. Transmission electron microscopy analysis showed a disrupted axonemal structure with mitochondrial sheath defects. The EIF4G1 protein level was extremely low, and the mitochondrial marker cytochrome c oxidase subunit 4I1 (COXIV, OMIM: 123864) and mitochondrially encoded ATP synthase 6 (ATP6, OMIM: 516060) protein levels were also decreased in the patient's spermatozoa as revealed by WB and IF analysis. This infertility associated with this condition was overcome by intracytoplasmic sperm injections, as his wife became pregnant successfully. CONCLUSION: Our experimental findings indicate that the EIF4G1 gene is a novel candidate gene that may be relevant to severe AZS.

Exome sequencing identifies compound heterozygous KCTD7 mutations in a girl with progressivemyoclonus epilepsy.

Progressive myoclonic epilepsies (PME) are a clinically and genetically heterogeneous group of rare diseases characterized by myoclonic seizures, tonic-clonic seizures, and neurological deterioration. Here, we genetically analyzed a Chinese patient affected by infantile-onset progressive myoclonic epilepsy. We applied next-generation whole exome capture sequencing with Sanger direct sequencing to the proband and her unaffected parents. Two compound heterozygous mutations were identified in the KCTD7 gene. The first mutation [c. 434A > G(p.Q145R)] was inherited from her father, while the second [c.631C > T(p.R211X)] was inherited from her mother. The two were co-segregated with disease phenotype in the family. To our knowledge, this is the first report of KCTD7 mutations causing PME in the Chinese population, with c. 434A > G in particular being a novel mutation. Our findings supported the important role of KCTD7 in PME and broadened the gene's mutation spectrum. Thus, this study contributes to genetic diagnoses and counselling of families with PME.

Novel Mutations in CFAP44 and CFAP43 Cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF).

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease that causes primary infertility. However, the genetic causes for approximately half of MMAF cases are unknown. Whole exome sequencing analysis of the 27 patients with MMAF identified several CFAP44 mutations (3 homozygous: c.2935_2944del: p.D979*, c.T1769A: p.L590Q, c.2005_2006del: p.M669Vfs*13; and putative compound heterozygous: c.G3262A: p.G1088S and c.C1718A: p.P573H.) and CFAP43 acceptor splice-site deletion (c.3661-2A>-) mutations in 5 and 1 patients, respectively. Real-time quantitative polymerase chain reaction assays also demonstrated that CFAP44 expression was very weak in patient (P)1 and P3, and CFAP43 expression was lower in P6 than in the control. Immunofluorescence analysis of CFAP43 showed lower CFAP43 protein expression levels in P6 than in the normal control. This study demonstrated that biallelic mutations in CFAP44 and CFAP43 cause MMAF. These results provide researchers with a new insight to understand the genetic etiology of MMAF and to identify new loci for genetic counselling of MMAF.

Identification of a novel mutation in FGFR1 gene in patients with Kallmann syndrome by high throughput sequencing.

Kallmann syndrome (KS) is a rare clinical and genetic heterogeneity disease, which is familial or sporadic. KS is known to have three patterns of inheritance: X linked recessive inheritance, autosomal dominant inheritance and rare autosomal recessive inheritance. Here, we report a sibling pedigree with autosomal dominant inheritance of KS, and we identified a novel heterozygous frameshift mutation c.299_300insCCGCAGACTCCGGCCTCTATGC (p.C101Rfs*17) in FGFR1 gene using whole-exome sequencing (WES). The mutation and affection status were cosegregated. The mutation is not present in the dbSNP, 1000 Genome, ExAC, and gnomAD databases. The discovery of this new mutation in the FGFR1 gene enriches the spectrum of FGFR1 mutations in patients with KS. ABBREVIATIONS: FGFR1: fibroblast growth factor receptor 1; HH: hypogonadotropic hypogonadism; KS: Kallmann syndrome; MRI: magnetic resonance imaging; WES: whole-exome sequencing.

TDRD6 is associated with oligoasthenoteratozoospermia by sequencing the patient from a consanguineous family.

Oligoasthenoteratozoospermia (OAT) is characterized as low sperm count, decreased sperm motility and structural abnormalities of the sperm head in the same patient. However, very few studies reported the genetic alterations associated with OAT. Here we report a 38-year-old patient with OAT from a consanguineous family, with 2-6 million/mL sperm density, 2.1-3.8% normal sperm morphology and immotile sperm. Whole-exome sequencing (WES) identified homozygous variant c.1259A>G:p.Y420C in the TDRD6 gene. TDRD6 is a testis-specific expressed protein that was localized to the chromatoid bodies in germ cells and played an important role in the nonsense-mediated decay pathway. This rare variant co-segregated with the OAT phenotype in this family. Bioinformatic analysis also suggested the variant a pathogenic mutation. Two intracytoplasmic sperm injection (ICSI) cycles were carried out in the patient's wife, but she did not become pregnant after embryo transfer. So the mutations in TDRD6 may be associated with human male infertility and early embryonic lethality.

Use of targeted sequence capture and high-throughput sequencing identifies a novel PKD1 mutation involved in adult polycystic kidney disease.

Polycystic kidney disease (PKD) is a common inherited disease that is characterized by a progressive development of renal cysts. Approximately 85% of PKD cases are due to mutations in the polycystin 1 (PKD1) gene. Here, we report a pedigree containing nine patients with autosomal dominant PKD (ADPKD). Using targeted exome sequencing of PKD1 and PKD2 genes, we identified a novel heterozygous frameshift mutation c.3976_3977insCT (p.F1326Sfs*21) in the PKD1 gene that segregated between affected and unaffected family members. This mutation is currently not present in the 1000 Genomes Project nor ExAC databases and is therefore a novel PKD1 mutation involved in ADPKD. These results provide a novel sequence variant for the genetic analysis of this disease.

HMGB1 Contributes to the Expression of P-Glycoprotein in Mouse Epileptic Brain through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products.

The objective of the present study was to investigate the role of high-mobility group box-1 (HMGB1) in the seizure-induced P-glycoprotein (P-gp) overexpression and the underlying mechanism. Kainic acid (KA)-induced mouse seizure model was used for in vivo experiments. Male C57BL/6 mice were divided into four groups: normal saline control (NS) group, KA-induced epileptic seizure (EP) group, and EP group pretreated with HMGB1 (EP+HMGB1 group) or BoxA (HMGB1 antagonist, EP+BoxA group). Compared to the NS group, increased levels of HMGB1 and P-gp in the brain were observed in the EP group. Injection of HMGB1 before the induction of KA further increased the expression of P-gp while pre-treatment with BoxA abolished this up-regulation. Next, the regulatory role of HMGB1 and its potential involved signal pathways were investigated in mouse microvascular endothelial bEnd.3 cells in vitro. Cells were treated with HMGB1, HMGB1 plus lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) [toll-like receptor 4 (TLR4) antagonist], HMGB1 plus FPS-ZM1 [receptor for advanced glycation end products (RAGE) inhibitor], HMGB1 plus SN50 [nuclear factor-kappa B (NF-κB) inhibitor], or vehicle. Treatment with HMGB1 increased the expression levels of P-gp, TLR4, RAGE and the activation of NF-κB in bEnd.3 cells. These effects were inhibited by the pre-treatment with either LPS-RS or FPS-ZM1, and were abolished by the pre-treatment of SN50 or a combination treatment of both LPS-RS and FPS-ZM1. Luciferase reporter assays showed that exogenous expression of NF-κB p65 increased the promoter activity of multidrug resistance 1a (P-gp-encoding gene) in endothelial cells. These data indicate that HMGB1 contributes to the overexpression of P-gp in mouse epileptic brain tissues via activation of TLR4/RAGE receptors and the downstream transcription factor NF-κB in brain microvascular endothelial cells.

Effects of brain IKKß gene silencing by small interfering RNA on P-glycoprotein expression and brain damage in the rat kainic acid-induced seizure model.

Multidrug resistance mediated by over-expression of P-glycoprotein (P-gp) in brain is an important mechanism accounting for the drug-therapy failure in epilepsy. Over-expression of P-gp in epilepsy rat brain may be regulated by inflammation and nuclear factor-kappa B (NF-κB) activation. Inhibitory κ B kinase subunit ß (IKKß) is an up-stream molecular controlling NF-κB activation. With the small interfering RNA (siRNA) technique and kainic acid (KA)-induced rat epileptic seizure model, the present study was aimed to further evaluate the role of NF-κB inhibition, via blocking IKKß gene transcription, in the epileptic brain P-gp over-expression, seizure susceptibility, and post-seizure brain damage. siRNA targeting IKKß was administered to rats via intracerebroventricular injection before seizure induction by KA microinjection; scrambled siRNA was used as control. Brain mRNA and protein levels of IKKß and P-gp were detected by RT-PCR and immunohistochemistry. NF-κB activity was measured by electrophoretic mobility shift assay. Latency to grade III or V seizure onset was recorded, brain damage was evaluated by neuronal cell counting and epileptiform activity was monitored by electroencephalography. IKKß siRNA pre-treatment inhibited NF-κB activation and abolished P-gp over-expression in KA-induced epileptic rat brain, accompanied by decreased seizure susceptibility. These findings suggested that epileptogenic-induced P-gp over-expression could be regulated by IKKß through the NF-κB pathway.