Human INHBB gene variant (c.1079T>C:p.Met360Thr) alters testis germ cell content, but does not impact fertility in mice

Testicular derived inhibin B (α/βB dimers) acts in an endocrine manner to suppress pituitary production of follicle stimulating hormone (FSH), by blocking the actions of activins (βA/B/βA/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (βB-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate Inhbb M364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male Inhbb M364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area, in Inhbb M364T/M364T males compared to wildtype males. Despite this testis phenotype, male Inhbb M364T/M364T mutant mice retained normal fertility. Serum hormone analyses however, indicated that the Inhbb M364T variant resulted in reduced circulating levels of activin B, but did not affect FSH production. We also examined the effect of this p.Met360Thr, and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man, on inhibin B and activin B in vitro biosynthesis. It was found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.

Successful Results of Intracytoplasmic Sperm Injection of a Chinese Patient With Multiple Morphological Abnormalities of Sperm Flagella Caused by a Novel Splicing Mutation in CFAP251

Asthenospermia is one of the most important causes of male infertility. Among asthenospermia, multiple morphological abnormalities of sperm flagella (MMAF) are relatively rare idiopathic conditions characterized by multiple defects in sperm flagella. Although many studies focusing on the genetic factors of MMAF have been conducted, its pathogenesis and treatment effect remain largely unknown. Here, we report a male patient from a nonconsanguineous Chinese family who exhibited a typical MMAF phenotype revealed by morphological analysis. We identified splicing mutations in CFAP251 (c.1192-3C>G), and the mutation was proven to cause exon skipping. In addition, western blotting and immunofluorescence analysis of the spermatozoa from the proband and a control subject revealed a significantly lower expression of CFAP251 protein due to pathogenic mutation. Interestingly, the patient’s mother was a heterozygous carrier for the mutation, but his father was not, and finally, the inheritance pattern was proven to be maternal uniparental disomy. We applied an intracytoplasmic sperm injection and achieved a successful pregnancy. Above all, our findings expand the spectrum of CFAP251 pathogenic mutations and provide more indications for clinical genetic counseling and assisted reproductive treatment for such patients.

Molecular and Cytogenetic Analysis of Romanian Patients with Differences in Sex Development

Differences in sex development (DSD) are often correlated with a genetic etiology. This study aimed to assess the etiology of DSD patients following a protocol of genetic testing. Materials and methods. This study prospectively investigated a total of 267 patients with DSD who presented to Clinical Emergency Hospital for Children Cluj-Napoca between January 2012 and December 2019. Each patient was clinically, biochemically, and morphologically evaluated. As a first intervention, the genetic test included karyotype + SRY testing. A high value of 17-hydroxyprogesterone was found in 39 patients, in whom strip assay analysis of the CYP21A2 gene was subsequently performed. A total of 35 patients were evaluated by chromosomal microarray technique, and 22 patients were evaluated by the NGS of a gene panel. Results. The karyotype analysis established the diagnosis in 15% of the patients, most of whom presented with sex chromosome abnormalities. Genetic testing of CYP21A2 established a confirmation of the diagnosis in 44% of patients tested. SNP array analysis was particularly useful in patients with syndromic DSD; 20% of patients tested presented with pathogenic CNVs or uniparental disomy. Gene panel sequencing established the diagnosis in 11 of the 22 tested patients (50%), and the androgen receptor gene was most often involved in these patients. The genes that presented as pathogenic or likely pathogenic variants or variants of uncertain significance were RSPO1, FGFR1, WT1, CHD7, AR, NIPBL, AMHR2, AR, EMX2, CYP17A1, NR0B1, GNRHR, GATA4, and ATM genes. Conclusion. An evaluation following a genetic testing protocol that included karyotype and SRY gene testing, CYP21A2 analysis, chromosomal analysis by microarray, and high-throughput sequencing were useful in establishing the diagnosis, with a spectrum of diagnostic yield depending on the technique (between 15 and 50%). Additionally, new genetic variants not previously described in DSD were observed.

Epigenotype, Genotype, and Phenotype Analysis of Taiwanese Patients with Silver–Russell Syndrome

Background: Silver–Russell syndrome (SRS) is a clinically and genetically heterogeneous disorder characterized by severe intrauterine growth retardation, poor postnatal growth, characteristic facial features, and body asymmetry. Hypomethylation of the imprinted genes of the chromosome 11p15.5 imprinting gene cluster and maternal uniparental disomy of chromosome 7 (mUPD7) are the major epigenetic disturbances. The aim of this study was to characterize the epigenotype, genotype, and phenotype of these patients in Taiwan. Methods: Two hundred and six subjects with clinically suspected SRS were referred for diagnostic testing, which was performed by profiling the methylation of H19-associated imprinting center (IC) 1 and the imprinted PEG1/MEST region using methylation-specific multiplex ligation-dependent probe amplification and high-resolution melting analysis with a methylation-specific polymerase chain reaction assay. We also applied a whole genome strategy to detect copy number changes and loss of heterozygosity. Clinical manifestations were recorded and analyzed according to the SRS scoring system proposed by Bartholdi et al. Results: Among the 206 referred subjects, 100 were classified as having a clinical diagnosis of SRS (score ≥ 8, maximum = 15) and 106 had an SRS score ≤ 7. Molecular lesions were detected in 45% (45/100) of the subjects with a clinical diagnosis of SRS, compared to 5% (5/106) of those with an SRS score ≤ 7. Thirty-seven subjects had IC1 hypomethylation, ten subjects had mUPD7, and three subjects had microdeletions. Several clinical features were found to be statistically different (p < 0.05) between the “IC1 hypomethylation” and “mUPD7” groups, including relative macrocephaly at birth (89% vs. 50%), triangular shaped face (89% vs. 50%), clinodactyly of the fifth finger (68% vs. 20%), and SRS score (11.4 ± 2.2 vs. 8.3 ± 2.5). Conclusions: The SRS score was positively correlated with the molecular diagnosis rate (p < 0.001). The SRS subjects with mUPD7 seemed to have fewer typical features and lower SRS scores than those with IC1 hypomethylation. Careful clinical observation and timely molecular confirmation are important to allow for an early diagnosis and multidisciplinary management of these patients.

Prenatal diagnosis of complete paternal uniparental isodisomy for chromosome 3: a case report

Background Uniparental disomy (UPD) is defined as an inheritance of two chromosomes from only one of the parents with no representative copy from the other. Paternal-origin UPD of chromosome 3 is a very rare condition, with only five cases of paternal UPD(3) reported. Case presentation Here, we report a prenatal case that is only the second confirmed paternal UPD(3) reported with no apparent disease phenotype. The fetus had a normal karyotype and normal ultrasound features throughout gestation. Copy neutral regions of homozygosity on chromosome 3 were identified by single nucleotide polymorphism (SNP) array. Subsequent SNP array data of parent–child trios showed that the fetus carried complete paternal uniparental isodisomy (isoUPD) of chromosome 3. The parents decided to continue with the pregnancy after genetic counseling, and the neonate had normal physical findings at birth and showed normal development after 1.5 years. Conclusions These findings provided further evidence to confirm that there were no important imprinted genes on paternal chromosome 3 that caused serious diseases and a reference for the prenatal diagnosis and genetic counseling of UPD(3) in the future.

Complete uniparental disomy of chromosome 1

Complete uniparental disomy of chromosome 1 (UPD1) is an uncommon genetic finding about which a specific phenotype has not yet been established. We present a boy who has complete paternal UPD1 and isolated developmental delay. We suggest that there is no clear phenotype of UPD1.

Clinical Utility of Noninvasive Prenatal Screening for Rare Chromosome Abnormalities in Singleton Pregnancies

Objective: To systematically investigate the clinical utility of noninvasive prenatal screening (NIPS) commercially used for the common fetal aneuploidies as a prenatal screening tool for rare chromosome abnormalities (RCAs). Design: Prospective study. Setting: Hospital-based. Population or Sample: 528 gravidas with positive NIPS results for RCAs. Methods: Gravidas with positive NIPS results for RCAs subsequently underwent amniocentesis for single nucleotide polymorphism array (SNP-array) were recruit. The degrees of concordance between NIPS and SNP-array were classified into full concordance, partial concordance, discordance related and discordance. Main Outcome Measures: The positive predictive values (PPVs) for rare aneuploidies and segmental imbalances, while incidental findings for regions of homozygosity/uniparental disomy (ROH/UPD), were used to evaluate the performance of NIPS. Results: Of the 528 gravidas with positive NIPS results, 29.2% were confirmed with positive prenatal SNP-array results (154/528). The PPVs for rare aneuploidies and segmental imbalances were 6.1% (7/115) and 21.1% (87/413), respectively. ROH/UPDs, as incidental findings, have been identified in 9.5% (50/528) of gravidas with positive NIPS results. The PPV for clinical significant findings was 8.9% (47/528), including 7 cases with mosaic rare aneuploidies, 35 with pathogenic/likely pathogenic copy number variants, and 5 with imprinting disorders. Conclusions: NIPS commercially used for the common fetal aneuploidies yielded low PPV for rare aneuploidies, moderate PPV for segmental imbalances, and incidental findings for ROH/UPD. For the low PPV for clinical significant findings, NIPS has limited clinical utility for RCAs. Prenatal SNP-array should be regarded as the first-tier test for positive NIPS, particularly for those involved imprinted chromosomes.

Molecular Changes in Prader-Willi Syndrome Neurons Reveals Clues About Increased Autism Susceptibility

Background: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hormonal dysregulation, obesity, intellectual disability, and behavioral problems. Most PWS cases are caused by paternal interstitial deletions of 15q11.2-q13.1, while a smaller number of cases are caused by chromosome 15 maternal uniparental disomy (PW-UPD). Children with PW-UPD are at higher risk for developing autism spectrum disorder (ASD) than the neurotypical population. In this study, we used expression analysis of PW-UPD neurons to try to identify the molecular cause for increased autism risk.Methods: Dental pulp stem cells (DPSC) from neurotypical control and PWS subjects were differentiated to neurons for mRNA sequencing. Significantly differentially expressed transcripts among all groups were identified. Downstream protein analysis including immunocytochemistry and immunoblots were performed to confirm the transcript level data and pathway enrichment findings.Results: We identified 9 transcripts outside of the PWS critical region (15q11.2-q13.1) that may contribute to core PWS phenotypes. Moreover, we discovered a global reduction in mitochondrial transcripts in the PW-UPD + ASD group. We also found decreased mitochondrial abundance along with mitochondrial aggregates in the cell body and neural projections of +ASD neurons.Conclusion: The 9 transcripts we identified common to all PWS subtypes may reveal PWS specific defects during neurodevelopment. Importantly, we found a global reduction in mitochondrial transcripts in PW-UPD + ASD neurons versus control and other PWS subtypes. We then confirmed mitochondrial defects in neurons from individuals with PWS at the cellular level. Quantification of this phenotype supports our hypothesis that the increased incidence of ASD in PW-UPD subjects may arise from mitochondrial defects in developing neurons.