A familial analysis of two brothers with azoospermia caused by maternal 46,Y, t(X; 1) (q28; q21) chromosomal abnormality.

Chromosomal abnormality is a primary genetic factor that lead to azoospermia and male infertility. Here, we report the cases of two brothers with primary infertility, whose chromosomes displayed a balanced translocation, and their karyotypes were 46,Y, t(X; 1) (q28; q21). Both presented an azoospermia phenotype without abnormal clinical symptoms. Their mother's karyotype was 46,X, t(X; 1) (q28; q21), and their father's chromosome karyotype was 46,XY. No abnormal changes were noted in the copy number of chromosome fragments in the whole genome. This study is the first to report showing that 46,Y, t(X; 1) (q28; q21) chromosomal abnormalities are associated with azoospermia.

[Clinical and molecular genetic analysis of a patient with 3-M syndrome].

OBJECTIVE: To analyze the clinical features and molecular genetic etiology of a patient with 3-M (Miller McKusick Malvaux) syndrome from a consanguineous parentage family, and to explore the relationship between genotype and phenotype. METHODS: After the consent of the proband's guardian and the informed consent form was signed, DNA was extracted from peripheral blood samples of the proband and her parents for chromosome microarray analysis, medical exome sequencing and parental verification. RESULTS: A total of 247.1 Mb loss of heterozygosity was found in the proband with a CytoScan 750K array. Furthermore, a homozygous variant (c.458dupG) of the OBSL1 gene was found using high-throughput sequencing, which was inherited from her parents. Based on the criteria and guidelines of genetic variation of American College of Medical Genetics and Genomics, the variant is predicted to be pathogenic (PVS1+PM2+PP4), and only one case was reported previously. CONCLUSION: Spina bifida occulta and lower eyelid fat pad may be a special phenotype of c.458dupG variant of the OBSL1 gene. Our study may provide a useful reference for evaluating the relationship between genotype and phenotype of 3-M syndrome type 2.

[Genetic analysis of a Chinese pedigree affected with congenital split-hand/foot malformation].

OBJECTIVE: To analyze the molecular genetics of a Chinese pedigree with congenital hand foot cleft. METHODS: Single nucleotide polymorphism microarray (SNP array) was used to analyze the whole genome copy number variation. RESULTS: SNP array analysis showed that there was a 433 kb repeat in 10q24.31-10q24.32 region, which contained LBX1, BTRC, POLL, OPCD and FBXW4 genes. CONCLUSION: Microduplication of chromosome 10q24.31-10q24.32 may be the cause of congenital hand foot cleft in this pedigree.

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.

[Clinical and genetic analysis of a patient with 17-hydroxylase/17,20-lyase deficiency].

OBJECTIVE: To explore the clinical and genetic characteristics of a patient with 17-hydroxylase/17,20-lyase deficiency. METHODS: The patient was infertile without contraception. Laboratory examination showed her chromosomal karyotype to be 46, XX. DNA sequencing was performed to detect variants of CYP17A1 gene in the patient and her family members. RESULTS: Sanger sequencing revealed that the patient has carried homozygous variant c.1486C>T in the exon 8 of the CYP17A1 gene, which resulted in substitution of arginine by cysteine (p.Arg496Cys). Her family members were all heterozygotes for the same variant. CONCLUSION: Homozygous variant of the CYP17A1 gene c.1486C>T probably underlay the 17-hydroxylase deficiency in this patient. Above finding has enabled accurate genetic counseling and prenatal diagnosis for her family.

Biallelic mutations in PMFBP1 cause acephalic spermatozoa.

The majority of men with defects in spermatogenesis remain undiagnosed. Acephalic spermatozoa is one of the diseases causing primary infertility. However, the causes underlying over half of affected cases remain unclear. Here, we report by whole-exome sequencing the identification of homozygous and compound heterozygous truncating mutations in PMFBP1 of two unrelated individuals with acephalic spermatozoa. PMFBP1 was highly and specifically expressed in human and mouse testis. Furthermore, immunofluorescence staining in sperm from a normal control showed that PMFBP1 localizes to the head-flagella junction region, and the absence of PMFBP1 was confirmed in patients harboring PMFBP1 mutations. In addition, we generated Pmfbp1 knock-out (KO) mice, which we found recapitulate the acephalic sperm phenotype. Label-free quantitative proteomic analysis of testicular sperm from Pmfbp1 KO and control mice showed 124 and 35 proteins, respectively, increased or decreased in sperm from KO mice compared to that found in control mice. Gene ontology analysis indicates that the biological process of Golgi vesicle transport was the most highly enriched in differentially expressed proteins, indicating process defects related to Golgi complex function may disturb formation of the head-neck junction. Collectively, our data indicate that PMFBP1 is necessary for sperm morphology in both humans and mice, and that biallelic truncating mutations in PMFBP1 cause acephalic spermatozoa.

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.

Patients with multiple morphological abnormalities of the sperm flagella harbouring CFAP44 or CFAP43 mutations have a good pregnancy outcome following intracytoplasmic sperm injection.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a rare type of male infertility. Mutations in DNAH1, CFAP43 and CFAP44 are the main aetiology of the disorder. Previously, good intracytoplasmic sperm injection (ICSI) outcomes were reported for MMAF patients with DNAH1 mutations. However, the ICSI prognosis for MMAF patients with CFAP43 or CFAP44 mutations was not known. We designed a retrospective cohort study. Molecular genetic testing identified six MMAF patients with biallelic CFAP44 (CFAP44+ group) or CFAP43 mutations and 12 patients with homozygous or compound heterozygous DNAH1 mutations (DNAH1+ group). A control group consisted of age-matched, non-MMAF men. For MMAF patients carrying CFAP44 mutations, the recorded rates of fertilisation, transferable embryos, pregnancy and delivery after ICSI were 76.47%, 88.46%, 50.0% and 50.0% respectively. The fertilisation rate was significantly higher in the CFAP44+ group than in the DNAH1+ group (76.47% vs. 54.5%, p = 0.0196). There were no statistically significant differences in the rates of transferable embryos, implantation, clinical pregnancy and miscarriage between the CFAP44+ group and either the DNAH1+ group or the age-matched control group. Our results support a good ICSI prognosis for MMAF patients carrying CFAP44 or CFAP43 mutations.

[Analysis of a pedigree with partial trisomy 9 and partial monosomy 13 derived from a maternal balanced t(9;13) translocation].

OBJECTIVE: To determine the nature and origin of aberrant chromosomes in a child with multiple anomalies and psychomotor retardation. METHODS: Routine G-banding was carried out to analyze the karyotypes of the patient and his parents, and next generation sequencing for copy number variations (CNV-seq) was used for the fine mapping of the aberrant chromosomal regions. RESULTS: The proband and his uncle exhibited psychomotor retardation, craniofacial malformation, infantile external genitalia, and concealed penis. Cytogenetic analysis indicated that the child has a 46,XYqh+,+(9),t(9;13)(q13;q12),pat,-13 karyotype. His uncle was XYqh+,+(9),t(9;13)(q13;q12)mat,-13, his father was 46,XYqh+,t(9;13)(q13;q12)mat, his grandmother was 46,XX,t(9;13)(q13;q12), and his grandfather was 46,XYqh+. The result of CNV-seq assay for the child was 46,XY,+9p(pter-p13.2,-40 Mb×3). No deletion was detected. CONCLUSION: The partial trisomy 9 and partial monosomy 13 probably underlie the phenotypic abnormalities in the child. Combined chromosomal karyotyping and DNA sequencing can facilitate delineation of the nature and origin of the aberrant chromosomes.

A novel TEX11 mutation induces azoospermia: a case report of infertile brothers and literature review.

BACKGROUND: Testis-expressed gene 11 (TEX11) is an X-linked gene and essential for meiotic recombination and chromosomal synapsis. TEX11 deficiency causes meiotic arrest and male infertility, and many TEX11 mutations have been found in azoospermic and infertile men. CASE PRESENTATION: This study reported one novel TEX11 mutation (2653G → T, in exon 29, GenBank accession number, NM_031276) in two brothers with azoospermia. This mutation was firstly screened out by whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon 29. Surprisingly, the same exonic missense mutation (W856C) was observed in two brothers but not in their mother. Histological analysis of testicular biopsy from both brothers revealed meiotic arrest and no post-meiotic round spermatids and mature spermatozoa were observed in the seminiferous tubules. TEX11 expression was observed strongly in spermatogonia and weakly in spermatocytes, but not in Sertoli cells and interstitial cells. CONCLUSIONS: We identified one novel TEX11 mutation in two brothers and summarized the literature regarding TEX11 mutations and male infertility. This study and previous literature indicate that TEX11 mutations are closely associated with male infertility, especially azoospermia, although auxiliary clinical analyses are needed to figure out the causes of male infertility.

Notable Carrier Risks for Individuals Having Two Copies of SMN1 in Spinal Muscular Atrophy Families with 2-copy Alleles: Estimation Based on Chinese Meta-analysis Data.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease mainly caused by homozygous deletion of SMN1. The 2-copy SMN1 allele may present in the families of SMA patients with homozygous deletion of SMN1, one of whose parents has two SMN1 copies. In such families, individuals having two SMN1 copies still have a chance to be "2 + 0" carriers. In this study, the risks for the parents, fetuses and other siblings having two SMN1 copies to be "2 + 0" carriers were estimated based on Chinese meta-analysis data and turned out to be rather striking. Our findings would help to optimize genetic counseling regarding spinal muscular atrophy.

Three novel mutations of STK11 gene in Chinese patients with Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder characterized by gastrointestinal (GI) hamartomatous polyps, mucocutaneous hyperpigmentation, and an increased risk of cancer. Mutations in the serine-threonine kinase 11 gene (SKT11) are the major cause of PJS. CASE PRESENTATION: Blood samples were collected from six PJS families including eight patients. Mutation screening of STK11 gene was performed in these six families by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) assay. Three novel mutations (c.721G > C, c.645_726del82, and del(exon2-5)) and three recurrent mutations (c.752G > A, c.545 T > C and del(exon1)) in STK11 were detected in six Chinese PJS families. Genotype-phenotype correlations suggested that truncating mutations trend to result in severe complications. CONCLUSION: These findings broaden the mutation spectrum of the STK11 gene and would help clinicians and genetic counselors provide better clinical surveillance for PJS patients, especially for ones carrying truncating mutation.

Chromosomal analysis of blastocysts from balanced chromosomal rearrangement carriers.

Balanced chromosomal rearrangements (CRs) are among the most common genetic abnormalities in humans. In the present study, we have investigated the degree of consistency between the chromosomal composition of the blastocyst inner cell mass (ICM) and trophectoderm (TE) in carriers with balanced CR, which has not been previously addressed. As a secondary aim, we have also evaluated the validity of cleavage-stage preimplantation genetic diagnosis (PGD) based on fluorescence in situ hybridization (FISH) of blastocysts from CR carriers. Blastocyst ICM and TE were screened for chromosomal aneuploidy and imbalance of CR-associated chromosomes based on whole-genome copy number variation analysis by low-coverage next-generation sequencing (NGS) following single-cell whole-genome amplification by multiple annealing and looping-based amplification cycling. The NGS results were analyzed without knowledge of cleavage-stage FISH results. NGS results for blastocyst ICM and TE from CR carriers were 86.49% (32/37) consistent. Of the 1702 (37 × 46) chromosomes examined, 99.47% (1693/1702) showed consistency. However, only 40.0% (18/45) of all embryos had consistent results for chromosomes involved in CR, as determined by blastocyst NGS and cleavage-stage FISH. Of the 85 CR-affected chromosomes analyzed by FISH, 37.65% (32/85) were incongruous with NGS results, with 87.5% (28/32) showing imbalanced composition by FISH but balanced composition by NGS. These results indicate that chromosomal composition of blastocyst ICM and TE in balanced CR carriers is highly consistent, and that PGD based on cleavage-stage FISH is inaccurate; therefore, using blastocyst TE biopsies for NGS-based PGD is recommended for identifying chromosomal imbalance in embryos from balanced CR carriers.

De novo exonic deletion of KDM6A in a Chinese girl with Kabuki syndrome: A case report and brief literature review.

Kabuki syndrome (KS) is a rare condition with multiple congenital anomalies and mental retardation. Exonic deletions, disrupting the lysine (K)-specific demethylase 6A (KDM6A) gene have been demonstrated as rare cause of KS. Here, we report a de novo 227-kb deletion in chromosome Xp11.3 of a 7-year-old Chinese girl with KS. Besides the symptoms of KS, the patient also presented with skin allergic manifestations, which were considered to be a new, rare feature of the phenotypic spectrum. The deletion includes the upstream region and exons 1-2 of KDM6A and potentially causes haploinsuffiency of the gene. We also discuss the mutation spectrum of KDM6A and clinical variability of patients with KDM6A deletion through a literature review. © 2016 Wiley Periodicals, Inc.

Morphometric analysis and developmental comparison of embryos from carriers with balanced chromosomal rearrangements in preimplantation genetic diagnosis cycles.

The morphological parameters of embryos from 22 carriers with balanced chromosomal rearrangements (CRs) were quantified and evaluated to determine their possible link to chromosomal composition. The morphometric characteristics of 168 embryos diagnosed by fluorescence in situ hybridisation were measured using an imaging tool and then analysed retrospectively. The mean zygotic diameter of normal-balanced embryos was significantly smaller compared with that of abnormal embryos (P=0.015). In addition, the reduction in total cytoplasmic volume for Day-3 embryos was significantly lower in normal or balanced embryos than in abnormal embryos (P=0.027). Moreover, the pronuclear volumes of embryos that failed to reach the blastocyst stage were significantly smaller compared with those of blastocysts (P=0.016). These findings indicate that morphometric characteristics are correlated with developmental outcomes as well as with chromosomal composition in embryos from balanced CR carriers. However, an effective indicator of developmental outcomes may not accurately reflect chromosomal composition. Combining morphometric and traditional qualitative assessment may increase the precision and standardisation of embryo evaluation as well as contributing to improved efficiency of preimplantation genetic diagnosis by selecting embryos with high developmental potential and preferentially testing embryos predicted to have a low risk of chromosomal imbalance.

[Familial fragile X syndrome: A pedigree analysis].

OBJECTIVE: To investigate the clinical (including reproductive) manifestations and genetic characteristics of familial fragile X syndrome (FXS). METHODS: We collected the clinical data about a case of familial FXS by inquiry, testicular ultrasonography, semen analysis, determination of sex hormone levels, and examinations of the peripheral blood karyotype and Y chromosome microdeletions. Using Southern blot hybridization, we measured the size of the CGG triple repeat sequence of the fragile X mental retardation-1 (FMR1) gene and determined its mutation type of the pedigree members with a genetic map of the FXS pedigree. RESULTS: Among the 34 members of 4 generations in the pedigree, 3 males and 1 female (11.76%) carried full mutation and 9 females (26.47%) premutation of the FMR1 gene. Two of the males with full FMR1 mutation, including the proband showed a larger testis volume (>30 ml) and a higher sperm concentration (>250 ×106/ml), with a mean sperm motility of 50.5%, a mean morphologically normal sperm rate of 17.5%, an average sperm nuclear DNA fragmentation index (DFI) of 18.5%, a low level of testosterone, normal karyotype in the peripheral blood, and integrity of the azoospermia factor (AZF) region in the Y chromosome. One of the second-generation females carrying FMR1 premutation was diagnosed with premature ovarian failure and another 3 with uterine myoma. CONCLUSIONS: Some of the FXS males in the pedigree may present macroorchidism and polyzoospermia but with normal semen parameters. In the intergenerational transmission, premutation might extend to full mutation, with even higher risks of transmission and extension of mutation in males, especially in those with >80 CGG triple repeat sequences. Therefore, it is recommended that the couples wishing for childbearing receive genetic testing, clinical guidance, and genetic counseling before pregnancy and, if necessary, prenatal diagnosis and preimplantation genetic diagnosis.

[Analysis of the NDP gene in a Chinese family with X-linked recessive Norrie disease].

OBJECTIVE: The purpose of the current research was to investigate the NDP (Norrie disease protein) gene in one Chinese family with Norrie disease (ND) and to characterize the related clinical features. METHODS: Clinical data of the proband and his family members were collected. Complete ophthalmic examinations were carried out on the proband. Genomic DNA was extracted from peripheral blood leukocytes of 35 family members. Molecular analysis of the NDP gene was performed by polymerase chain reaction and direct sequencing of all exons and flanking regions. RESULTS: A hemizygous NDP missense mutation c.362G > A (p.Arg121Gln) in exon 3 was identified in the affected members, but not in any of the unaffected family individuals. CONCLUSIONS: The missense mutation c.362G > A in NDP is responsible for the Norrie disease in this family. This discovery will help provide the family members with accurate and reliable genetic counseling and prenatal diagnosis.

[Analysis of FGFR2 gene mutations in two Chinese families with Crouzon syndrome].

OBJECTIVE: To detect potential mutations of fibroblast growth factor receptor 2 gene (FGFR2) in two Chinese families with Crouzon syndrome. METHODS: Genomic DNA was extracted from peripheral blood leukocytes of 20 members from two affected families. All of the 18 exons of the FGFR2 gene were amplified with polymerase chain reaction and sequenced after purification. RESULTS: A missense mutation c.868T>C (p.W290R) in exon 8 of the FGFR2 gene was found solely in 2 affected members from family 1. Another missense mutation c.833G>T (p.C278F) in exon 8 was found solely in 5 affected members of family 2. CONCLUSION: The missense mutations of the FGFR2 gene are responsible for the Crouzon syndrome in the two families. The c.868T>C missense mutation is reported for the first time in Chinese population.