NR5A1/SF-1 Collaborates with Inhibin α and the Androgen Receptor.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that regulates steroidogenesis and reproductive development. NR5A1/SF-1 variants are associated with a broad spectrum of phenotypes across individuals with disorders of sex development (DSDs). Oligogenic inheritance has been suggested as an explanation. SF-1 interacts with numerous partners. Here, we investigated a constellation of gene variants identified in a 46,XY severely undervirilized individual carrying an ACMG-categorized 'pathogenic' NR5A1/SF-1 variant in comparison to the healthy carrier father. Candidate genes were revealed by whole exome sequencing, and pathogenicity was predicted by different in silico tools. We found variants in NR1H2 and INHA associated with steroidogenesis, sex development, and reproduction. The identified variants were tested in cell models. Novel SF-1 and NR1H2 binding sites in the AR and INHA gene promoters were found. Transactivation studies showed that wild-type NR5A1/SF-1 regulates INHA and AR gene expression, while the NR5A1/SF-1 variant had decreased transcriptional activity. NR1H2 was found to regulate AR gene transcription; however, the NR1H2 variant showed normal activity. This study expands the NR5A1/SF-1 network of interacting partners, while not solving the exact interplay of different variants that might be involved in revealing the observed DSD phenotype. It also illustrates that understanding complex genetics in DSDs is challenging.

Variety of genetic defects in GnRH and hypothalamic-pituitary signaling and development in normosmic patients with IHH.

Introduction: Normosmic isolated hypogonadotropic hypogonadism (nIHH) is a clinically and genetically heterogeneous disorder. Deleterious variants in over 50 genes have been implicated in the etiology of IHH, which also indicates a possible role of digenicity and oligogenicity. Both classes of genes controlling GnRH neuron migration/development and hypothalamic/pituitary signaling and development are strongly implicated in nIHH pathogenesis. The study aimed to investigate the genetic background of nIHH and further expand the genotype-phenotype correlation. Methods: A total of 67 patients with nIHH were enrolled in the study. NGS technology and a 38-gene panel were applied. Results: Causative defects regarded as at least one pathogenic/likely pathogenic (P/LP) variant were found in 23 patients (34%). For another 30 individuals, variants of unknown significance (VUS) or benign (B) were evidenced (45%). The most frequently mutated genes presenting P/LP alterations were GNRHR (n = 5), TACR3 (n = 3), and CHD7, FGFR1, NSMF, BMP4, and NROB1 (n = 2 each). Monogenic variants with solid clinical significance (P/LP) were observed in 15% of subjects, whereas oligogenic defects were detected in 19% of patients. Regarding recurrence, 17 novel pathogenic variants affecting 10 genes were identified for 17 patients. The most recurrent pathogenic change was GNRHR:p.Arg139His, detected in four unrelated subjects. Another interesting observation is that P/LP defects were found more often in genes related to hypothalamic-pituitary pathways than those related to GnRH. Conclusions: The growing importance of the neuroendocrine pathway and related genes is drawing increasing attention to nIHH. However, the underestimated potential of VUS variants in IHH etiology, particularly those presenting recurrence, should be further elucidated.

Digenic CHD7 and SMCHD1 inheritance Unveils phenotypic variability in a family mainly presenting with hypogonadotropic hypogonadism.

Objectives: CHARGE syndrome is a congenital hereditary condition involving multiple systems. Patients are easily misdiagnosed with idiopathic hypogonadotropic hypogonadism (IHH) due to the overlap of clinical manifestations. An accurate clinical diagnosis remains challenging when the predominant clinical manifestation resembles hypogonadotropic hypogonadism. Methods: This original research is conducted based on the genetic finding and analysis of clinical cases. Whole-exome sequencing (WES) and in-silico analyse were performed on two sisters to investigate the pathogenesis in this family. Homology modelling was conducted to evaluate structural changes in the variants. Results: WES and Sanger sequencing revealed two siblings carrying a nonsense mutation (NM_017780.4: c.115C > T) in exon 2 of CHD7 inherited from a mildly affected mother and a missense mutation (NM_015295.3: c.2582T > C) in exon 20 of SMCHD1 inherited from an asymptomatic father. The nonsense mutation in CHD7 was predicted to generate nonsense-mediated decay, whereas the missense mutation in SMCHD1 decreased protein stability. Conclusions: We identified digenic CHD7 and SMCHD1 mutations in IHH-associated diseases for the first time and verified the synergistic role of oligogenic inheritance. It was also determined that WES is an effective tool for distinguishing diseases with overlapping features and establishing a molecular diagnosis for cases with digenic or oligogenic hereditary disorders, which is beneficial for timely treatment, and family genetic counseling.

Nuclear Receptor Gene Variants Underlying Disorders/Differences of Sex Development through Abnormal Testicular Development.

Gonadal development is the first step in human reproduction. Aberrant gonadal development during the fetal period is a major cause of disorders/differences of sex development (DSD). To date, pathogenic variants of three nuclear receptor genes (NR5A1, NR0B1, and NR2F2) have been reported to cause DSD via atypical testicular development. In this review article, we describe the clinical significance of the NR5A1 variants as the cause of DSD and introduce novel findings from recent studies. NR5A1 variants are associated with 46,XY DSD and 46,XX testicular/ovotesticular DSD. Notably, both 46,XX DSD and 46,XY DSD caused by the NR5A1 variants show remarkable phenotypic variability, to which digenic/oligogenic inheritances potentially contribute. Additionally, we discuss the roles of NR0B1 and NR2F2 in the etiology of DSD. NR0B1 acts as an anti-testicular gene. Duplications containing NR0B1 result in 46,XY DSD, whereas deletions encompassing NR0B1 can underlie 46,XX testicular/ovotesticular DSD. NR2F2 has recently been reported as a causative gene for 46,XX testicular/ovotesticular DSD and possibly for 46,XY DSD, although the role of NR2F2 in gonadal development is unclear. The knowledge about these three nuclear receptors provides novel insights into the molecular networks involved in the gonadal development in human fetuses.

Genetic spectrum of Kallmann syndrome: Single-center experience and systematic review.

OBJECTIVE: To study phenotype-genotype data of Asian-Indian Kallmann syndrome (KS) from our center and systematically review the studies analyzing multiple congenital hypogonadotropic hypogonadism (CHH) genes in KS cohorts using next-generation sequencing. DESIGN, PATIENTS, MEASUREMENT: Five hundred twenty-two KS probands (our center n = 78, published studies n = 444) were included in this systematic review. Molecular diagnosis was considered if the likely pathogenic/pathogenic variant in known CHH gene/s was reported in the appropriate allelic state. Varsome prediction tool (following American College of Medical Genetics standards) was used to analyze the variants. RESULT: For our center, the molecular diagnosis was seen in 20.5% of probands and was seen more often with severe than partial reproductive phenotype (28.3% vs. 4%, p = .0013). Our center data adds eight novel variants. The molecular diagnosis was seen in 31% as per the systematic review and analysis. It ranged from 16.6% to 72.2% at different centers. The affected genes were FGFR1 (9.8%), ANOS1 (7.5%), PROKR2 (6.1%), CHD7 (5.4%), oligogenic (2.1%), and others <1% each (FGF8, SOX10, PROK2, SEMA3A, IL17RD, and GNRHR). FGFR1 and ANOS1 were the commonly affected genes globally, whereas PROKR2 was commonest in studies from China and CHD7 from Japan, South Korea and Poland. CONCLUSION(S): This systematic review highlights that the genetic yield is 31% in KS probands, with distinct regional variations. The association of severe reproductive phenotype with the higher genetic yield needs further validation.

Correlation Analysis of Genotypes and Phenotypes in Chinese Male Pediatric Patients With Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) can be divided into Kallmann syndrome (KS) and normosmic HH (nHH). The clinical and genetic characteristics of CHH have been studied in adults, but less in pre-adults. The medical records of patients with CHH in our gonad disease database from 2008 to 2020 were evaluated. In total, 125 patients aged 0 to 18 years were enrolled in our study. KS patients had a higher incidence of micropenis compared with nHH (86.2% vs. 65.8%, p=0.009), and 7 patients (5.6%) had hypospadias. Among the 39 patients with traceable family history, delayed puberty, KS/nHH, and olfactory abnormalities accounted for 56.4%, 17.9%, and 15.4%, respectively. In total, 65 patients completed the hCG prolongation test after undergoing the standard hCG test, and the testosterone levels of 24 patients (22.9%) were still lower than 100 ng/dL. In 77 patients, 25 CHH-related genes were identified, including digenic and trigenic mutations in 23 and 3 patients, respectively. The proportion of oligogenic mutations was significantly higher than that in our previous study (27.7% vs. 9.8%). The most common pathogenic genes were FGFR1, PROKR2, CHD7 and ANOS1. The incidence rate of the genes named above was 21.3%, 18.1%, 12.8% and 11.7%, respectively; all were higher than those in adults (<10%). Most mutations in CHH probands were private, except for W178S in PROKR2, V560I in ANOS1, H63D in HS6ST1, and P191L and S671L in IL17RD. By analyzing family history and genes, we found that both PROKR2 and KISS1R may also be shared between constitutional delay of growth and puberty (CDGP) and CHH. L173R of PROKR2 accounts for 40% of the CHH population in Europe and the United States; W178S of PROKR2 accounts for 58.8% of Chinese CHH patients. Micropenis and cryptorchidism are important cues for CHH in children. They are more common in pediatric patients than in adult patients. It is not rare of Leydig cell dysfunction (dual CHH), neither of oligogenic mutations diagnosed CHH in children. Both PROKR2 and KISS1R maybe the potential shared pathogenic genes of CDGP and CHH, and W178S in PROKR2 may be a founder mutation in Chinese CHH patients.

New findings in oligogenic inheritance of congenital hypogonadotropic hypogonadism.

Introduction: Congenital hypogonadotropic hypogonadism results from a dysfunction of the hypothalamic-pituitary-gonadal axis, which is essential for the development and function of the reproductive system. It may be associated with anosmia, referred to as Kallmann syndrome, or a normal sense of smell. Numerous studies have proven that hypogonadotropic hypogonadism is not simply a monogenic Mendelian disease, but that more than one gene may be involved in its pathogenesis in a single patient. The oligogenic complex architecture underlying the disease is still largely unknown. Material and methods: Targeted next-generation sequencing (NGS) was used to screen for DNA variants in a cohort of 47 patients with congenital hypogonadotropic hypogonadism. The NGS panel consists of over 50 well-known and candidate genes, associated with hypogonadotropic state. Results: Here we report the identification of new oligogenic variants in SPRY4/SEMA3A, SRA1/SEMA7A, CHD7/SEMA7A, CCDC141/POLR3B/POLR3B, and PROKR2/SPRY4/NSMF. These genes are known to contribute to the phenotype of hypogonadotropic hypogonadism, yet our results point to potential new "partners" underlying digenic and trigenic patterns. Conclusions: The finding supports the importance of oligogenic inheritance and demonstrates the complexity of genetic architecture in hypogonadotropic hypogonadism. It also underlines the necessity for developing fine-tuned guidelines to provide a tool for adequate and precise sequence variant classification in non-Mendelian conditions.

Next-Generation Sequencing Analysis Reveals Frequent Familial Origin and Oligogenism in Congenital Hypothyroidism With Dyshormonogenesis.

Context: Congenital hypothyroidism (CH) is related to dyshormonogenesis in 15% to 40% of the world population and associated with homozygous or heterozygous variants in the main genes of the hormone synthesis pathway. Emerging diagnostic tools, such as next-generation sequencing (NGS), have been used to efficiently explore panels of genes and identify complex mechanisms of pathogenesis. Objective: We explored 19 candidate genes known to be causative for permanent or transient CH to evaluate the role of complex gene variations in CH phenotype. Patients Design and Setting: Using the NGS approach, we studied 65 newborns with thyroid dyshormonogenesis (TDH). New variants were assessed in silico for pathogenicity. Results: Among the 65 infants, 56.9% presented a variant in one or more genes of the thyroid hormone synthesis axis. We identified homozygous or compound heterozygous variants in the TG, DUOX2, TPO, or SLC5A5 genes in 10 infants and heterozygous variants in DUOX2, TG, TPO, and TSHR in 19 others. In seven cases, a heterozygous variant in the TG gene was the unique anomaly detected, but related to disturbed hormonal balance. Oligogenic variants were found in eight infants associated with severe CH and goiter in five of them. Conclusion: The systematic exploration of genes involved in thyroid hormone synthesis by NGS in TDH showed high diagnostic relevance. Oligogenic inheritance could be related to phenotypic heterogeneity and a high frequency of goiter.

Kallmann syndrome and idiopathic hypogonadotropic hypogonadism: The role of semaphorin signaling on GnRH neurons.

Idiopathic hypogonadotropic hypogonadism and Kallmann syndrome are rare genetic disorders characterized by isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) and delayed or absent puberty. Defective GnRH neuron migration during development or secretion of mature GnRH neurons secondary to molecular defects in several key developmental and neuroendocrine pathways are thought to be the primary causes of these disorders. Recent studies have highlighted the importance of semaphorins and their receptors in this system, by showing that these molecules play distinct roles during the development and plasticity of these neurons. Accordingly, mutations in the semaphoring-signaling pathway genes have been found in patients affected by IGD, underlying the importance of semaphorin-mediated signaling pathways in the neuroendocrine axis that control reproduction.

Oligogenicity, C9orf72 expansion, and variant severity in ALS.

"Oligogenic inheritance" is used to describe cases where more than one rare pathogenic variant is observed in the same individual. While multiple variants can alter disease presentation, the necessity of multiple variants to instigate pathogenesis has not been addressed in amyotrophic lateral sclerosis (ALS). We sequenced ALS-associated genes in C9orf72-expansion-positive and negative ALS patients, alongside unaffected controls, to test the importance of oligogenicity and variant deleteriousness in ALS. We found that all groups had similar numbers of rare variants, but that variant severity was significantly higher in C9orf72-negative ALS cases, suggesting sufficiency of C9orf72 expansion to cause ALS alone.

Mutation spectrum analysis of 29 causative genes in 43 Chinese patients with congenital hypothyroidism.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder with a genetic origin. The purpose of the present study was to analyze the mutation spectrum of CH patients in China. A targeted next­generation sequencing panel covering all exons of 29 CH­related causative genes was used in 43 Han Chinese patients with CH [11 dysgenesis and 32 glands in situ (GIS)]. The functional impact and pathogenicity of detected variants were analyzed using a comprehensive bioinformatics approach and co­segregation studies. A total of 47 rare non­polymorphic variants in 9 target genes associated with thyroid hormone synthesis (DUOX2, DUOXA2, TPO, TG, SLC26A4 and SLC5A5), thyroid stimulating hormone resistance (TSHR) and central hypothyroidism (PROP1 and TRHR) were identified in 31 patients (31/43, 72%). Of these variants, 8 were novel, including 3 in DUOX2, 2 in TPO, 3 in TSHR and 1 in SLC5A5. Variants were mostly affected by DUOX2, TG, TPO and TSHR. Approximately 44% of the patients (19/43) carried DUOX2 variants. The mutation detection rates in patients with GIS were higher compared with patients with dysgenesis [25/32 (78%) vs. 6/11 (54%)]. Oligogenic mutations were detected in 25.6% of the total cases and 35% of the mutated cases. Genetic basis was ascertained in 13 patients, reaching a diagnosis detection rate of 30%. In conclusion, genetic defects in dyshormonogenesis, mainly in DUOX2, were the main genetic cause of CH in the Chinese population. Oligogenicity is highly involved in CH pathogenesis and may thus be an important factor in common phenotypic variability observed in patients with CH.

Oligogenic Origin of Differences of Sex Development in Humans.

Sex development is a very complex biological event that requires the concerted collaboration of a large network of genes in a spatial and temporal correct fashion. In the past, much has been learned about human sex development from monogenic disorders/differences of sex development (DSD), but the broad spectrum of phenotypes in numerous DSD individuals remains a conundrum. Currently, the genetic cause of less than 50% of DSD individuals has been solved and oligogenic disease has been proposed. In recent years, multiple genetic hits have been found in individuals with DSD thanks to high throughput sequencing. Our group has been searching for additional genetic hits explaining the phenotypic variability over the past years in two cohorts of patients: 46,XY DSD patients carriers of NR5A1 variants and 46,XY DSD and 46,XX DSD with MAMLD1 variants. In both cohorts, our results suggest that the broad phenotypes may be explained by oligogenic origin, in which multiple hits may contribute to a DSD phenotype, unique to each individual. A search for an underlying network of the identified genes also revealed that a considerable number of these genes showed interactions, suggesting that genetic variations in these genes may affect sex development in concert.

Coexistence of variants in TBK1 and in other ALS-related genes elucidates an oligogenic model of pathogenesis in sporadic ALS.

Variants in tank-binding kinase 1 (TBK1) are responsible for a significant proportion of amyotrophic lateral sclerosis (ALS) cases. In the present study, we analyzed variants in TBK1 extracted by targeted sequencing of 32 genes in a group of 406 Italian patients with ALS. We identified 7 different TBK1 variants in 7 sporadic cases, resulting in a frequency of 1.7%. Three patients had missense variants (p.R357Q, p.R358H, and p.R724C), one patient had a small deletion (p.E618del), and 3 had truncating variants (p.Y482*, p.R229*, and p.N681*). Notably, we found that 4 patients had an additional variant in ALS-related genes: 2 in OPTN and 2 in the 3'UTR region of FUS. By studying an independent group of 7 TBK1-mutated patients previously reported, we found another variant in the 3'UTR region of FUS in one patient. The presence of a second variant in TBK1 variant carriers is an interesting finding that needs to be investigated in larger cohorts of patients. These findings suggest that TBK1 belongs to the category of genes conferring a significantly increased risk but not sufficient to cause disease.

Evidence for a Common Genetic Origin of Classic and Milder Adult-Onset Forms of Isolated Hypogonadotropic Hypogonadism.

Multiple metabolic and inflammatory mechanisms are considered the determinants of acquired functional isolated hypogonadotropic hypogonadism (IHH) in males, whereas classic IHH is a rare congenital condition with a strong genetic background. Since we recently uncovered a frequent familiarity for classic IHH among patients with mild adult-onset hypogonadism (AO-IHH), here we performed a genetic characterization by next generation sequencing of 160 males with classic or "functional" forms. The prevalence of rare variants in 28 candidate genes was significantly higher than in controls in all IHH patients, independently of the age of IHH onset, degree of hypogonadism or presence of obesity. In fact, it did not differ among patients with classic or milder forms of IHH, however particular genes appear to be more specifically associated with one or the other category of IHH. ROC curves showed that Total Testosterone <6.05 nmol/L and an age of onset <41 years are sensitive cutoffs to identify patients with significantly higher chances of harboring rare IHH gene variants. In conclusion, rare IHH genes variants can frequently predispose to AO-IHH with acquired mild hormonal deficiencies. The identification of a genetic predisposition can improve the familial and individual management of AO-IHH and explain the heritability of congenital IHH.

Integrating clinical and genetic approaches in the diagnosis of 46,XY disorders of sex development.

46,XY differences and/or disorders of sex development (DSD) are clinically and genetically heterogeneous conditions. Although complete androgen insensitivity syndrome has a strong genotype-phenotype correlation, the other types of 46,XY DSD are less well defined, and thus, the precise diagnosis is challenging. This study focused on comparing the relationship between clinical assessment and genetic findings in a cohort of well-phenotyped patients with 46,XY DSD. The study was an analysis of clinical investigations followed by genetic testing performed on 35 patients presenting to a single center. The clinical assessment included external masculinization score (EMS), endocrine profiling and radiological evaluation. Array-comparative genomic hybridization (array-CGH) and sequencing of DSD-related genes were performed. Using an integrated approach, reaching the definitive diagnosis was possible in 12 children. The correlation between clinical and genetic findings was higher in patients with a more severe phenotype (median EMS 2.5 vs 6; P = 0.04). However, in 13 children, at least one variant of uncertain significance was identified, and most times this variant did not correspond to the original clinical diagnosis. In three patients, the genetic studies guided further clinical assessment which resulted in a reclassification of initial clinical diagnosis. Furthermore, we identified eight patients harboring variants in more than one DSD genes, which was not seen in controls (2.5%; P = 0.0003). In summary, taking into account potential challenges in reaching the definitive diagnosis in 46,XY DSD, only integrated approach seems to be the best routine practice.

Genetic analysis of CHARGE syndrome identifies overlapping molecular biology.

PURPOSE: CHARGE syndrome is an autosomal-dominant, multiple congenital anomaly condition characterized by vision and hearing loss, congenital heart disease, and malformations of craniofacial and other structures. Pathogenic variants in CHD7, encoding adenosine triphosphate-dependent chromodomain helicase DNA binding protein 7, are present in the majority of affected individuals. However, no causal variant can be found in 5-30% (depending on the cohort) of individuals with a clinical diagnosis of CHARGE syndrome. METHODS: We performed whole-exome sequencing (WES) on 28 families from which at least one individual presented with features highly suggestive of CHARGE syndrome. RESULTS: Pathogenic variants in CHD7 were present in 15 of 28 individuals (53.6%), whereas 4 (14.3%) individuals had pathogenic variants in other genes (RERE, KMT2D, EP300, or PUF60). A variant of uncertain clinical significance in KDM6A was identified in one (3.5%) individual. The remaining eight (28.6%) individuals were not found to have pathogenic variants by WES. CONCLUSION: These results demonstrate that the phenotypic features of CHARGE syndrome overlap with multiple other rare single-gene syndromes. Additionally, they implicate a shared molecular pathology that disrupts epigenetic regulation of multiple-organ development.

Evaluating Mendelian nephrotic syndrome genes for evidence for risk alleles or oligogenicity that explain heritability.

BACKGROUND: More than 30 genes can harbor rare exonic variants sufficient to cause nephrotic syndrome (NS), and the number of genes implicated in monogenic NS continues to grow. However, outside the first year of life, the majority of affected patients, particularly in ancestrally mixed populations, do not have a known monogenic form of NS. Even in those children classified with a monogenic form of NS, there is phenotypic heterogeneity. Thus, we have only discovered a fraction of the heritability of NS-the underlying genetic factors contributing to phenotypic variation. Part of the "missing heritability" for NS has been posited to be explained by patients harboring coding variants across one or more previously implicated NS genes, insufficient to cause NS in a classical Mendelian manner, but that nonetheless have a sufficient impact on protein function to cause disease. However, systematic evaluation in patients with NS for rare or low-frequency risk alleles within single genes, or in combination across genes ("oligogenicity"), has not been reported. To determine whether, compared with a reference population, patients with NS have either a significantly increased burden of protein-altering variants ("risk-alleles"), or a unique combination of them ("oligogenicity"), in a set of 21 genes implicated in Mendelian forms of NS. METHODS: In 303 patients with NS enrolled in the Nephrotic Syndrome Study Network (NEPTUNE), we performed targeted amplification paired with next-generation sequencing of 21 genes implicated in monogenic NS. We created a high-quality variant call set and compared it with a variant call set of the same genes in a reference population composed of 2,535 individuals from phase 3 of the 1000 Genomes Project. We created both a "stringent" and a "relaxed" pathogenicity-filtering pipeline, applied them to both cohorts, and computed the burden of variants in the entire gene set per cohort, the burden of variants in the entire gene set per individual, the burden of variants within a single gene per cohort, and unique combinations of variants across two or more genes per cohort. RESULTS: With few exceptions when using the relaxed filter, and which are likely the result of confounding by population stratification, NS patients did not have a significantly increased burden of variants in Mendelian NS genes in comparison to a reference cohort, nor was there any evidence for oligogenicity. This was true when using both the relaxed and the stringent variant pathogenicity filter. CONCLUSION: In our study, there were no significant differences in the burden or particular combinations of low-frequency or rare protein-altering variants in a previously implicated Mendelian NS genes cohort between North American patients with NS and a reference population. Studies in larger independent cohorts or meta-analyses are needed to assess the generalizability of our discoveries and also address whether there is in fact small but significant enrichment of risk alleles or oligogenicity in NS cases that was undetectable with this current sample size. It is still possible that rare protein-altering variants in these genes, insufficient to cause Mendelian disease, still contribute to NS as risk alleles and/or via oligogenicity. However, we suggest that more accurate bioinformatic analyses and the incorporation of functional assays would be necessary to identify bona fide instances of this form of genetic architecture as a contributor to the heritability of NS.