Identification and functional analysis of a rare variant of gene DHX37 in a patient with 46,XY disorders of sex development.

BACKGROUND: 46,XY sex reversal 11 (SRXY11) [OMIM#273250] is characterized by genital ambiguity that may range from mild male genital defects to gonadal sex reversal in severe cases. DHX37 is an RNA helicase that has recently been reported as a cause of SRXY11. So far, a total of 21 variants in DHX37 have been reported in 58 cases with 46,XY disorders of sex development (DSD). METHODS: Whole exome sequencing (WES) was conducted to screen for variations in patients with 46,XY DSD. The subcellular localization of mutant DHX37 proteins was detected by immunofluorescence. And the levels of mutant DHX37 proteins were detected via Western blotting. RESULTS: A novel pathogenic variant of DHX37 was identified in a patient with 46,XY DSD c.2012G > C (p.Arg671Thr). Bioinformatics analysis showed that the protein function of the variant was impaired. Compared with the structure of the wild-type DHX37 protein, the number of hydrogen bonds and interacting amino acids of the variant protein were changed to varying degrees. In vitro assays revealed that the variant had no significant effect on the intracellular localization of the protein but significantly reduced the expression level of the protein. CONCLUSIONS: Our finding further expands the spectrum of the DHX37 variant and could assist in the molecular diagnosis of 46,XY DSD patients.

DHX37 Is a Promising Prognostic Biomarker and a Therapeutic Target for Immunotherapy and Chemotherapy in HCC.

DHX37, a member of the DEAD/H-box RNA helicase family, has been implicated in various diseases, including tumors. However, the biological characteristics and prognostic significance of DHX37 in HCC remain unclear. In this study, we use R software 3.6.3 and multiple bioinformatics analysis tools, such as GDSC, HPA, STRING, TISCH, and TIMER2, to analyze the characterization and function of DHX37 in HCC. In addition, Western blot (WB) and immunohistochemistry (IHC) based on clinical samples validated some of the findings. DHX37 was more highly expressed in HCC samples compared to adjacent non-tumor tissues. Higher DHX37 expression is correlated with various clinicopathological characteristics in HCC, including AFP, adjacent hepatic tissue inflammation, histologic grade, T stage, and pathologic stage. Survival analysis revealed that the high DHX37 group had significantly shorter overall survival (OS), progress-free interval (PFI), and disease-specific survival (DSS) compared to the low DHX37 group. By analyzing the correlation between DHX37 and the IC50 of chemotherapeutic drugs, the results showed that DHX37 expression level was negatively correlated with the IC50 of 11 chemotherapeutic drugs. Further analysis indicated that DHX37 and its co-expressed genes may play important roles in activating the cell cycle, DNA repair, chemokine signaling pathways, and regulating the immune response, which leads to a poor prognosis in HCC. High expression of DHX37 is an independent risk factor for poor prognosis in HCC, and DHX37 is expected to be a potential target to inhibit tumor progression. Targeting DHX37 may enhance chemotherapeutic drug sensitivity and immunotherapeutic efficacy in HCC.

DHX37 and NR5A1 Variants Identified in Patients with 46,XY Partial Gonadal Dysgenesis.

The group of disorders known as 46,XY gonadal dysgenesis (GD) is characterized by anomalies in testis determination, including complete and partial GD (PGD) and testicular regression syndrome (TRS). Several genes are known to be involved in sex development pathways, however approximately 50% of all cases remain elusive. Recent studies have identified variants in DHX37, a gene encoding a putative RNA helicase essential in ribosome biogenesis and previously associated with neurodevelopmental disorders, as a cause of PGD and TRS. To investigate the potential role of DHX37 in disorders of sexual development (DSD), 25 individuals with 46,XY DSD were analyzed and putative pathogenic variants were found in four of them. WES analyses were performed on these patients. In DHX37, the variant p.(Arg308Gln), recurrent associated with DSD, was identified in one patient; the p.(Leu467Val), predicted to be deleterious, was found together with an NR5A1 loss-of-function variant in patient 2; and, the p.(Val999Met) was identified in two unrelated patients, one of whom (patient 3) also carried a pathogenic NR5A1 variant. For both patients carrying DHX37 and NR5A1 pathogenic variants, a digenic inheritance is suggested. Our findings support the importance of DHX37 variants as a cause of disorders of sex development, implying a role in testis development.

Comprehensive molecular analysis identifies eight novel variants in XY females with disorders of sex development.

Disorders of sex development (DSD) are a group of clinical conditions with variable presentation and genetic background. Females with or without development of secondary sexual characters and presenting with primary amenorrhea (PA) and a 46,XY karyotype are one of the classified groups in DSD. In this study, we aimed to determine the genetic mutations in 25 females with PA and a 46,XY karyotype to show correlations with their phenotypes. Routine Sanger sequencing with candidate genes like SRY, AR, SRD5A2, and SF1, which are mainly responsible for 46,XY DSD in adolescent females, was performed. In a cohort of 25 patients of PA with 46,XY DSD, where routine Sanger sequencing failed to detect the mutations, next-generation sequencing of a targeted gene panel with 81 genes was used for the molecular diagnosis. The targeted sequencing identified a total of 21 mutations including 8 novel variants in 20 out of 25 patients with DSD. The most frequently identified mutations in our series were in AR (36%), followed by SRD5A2 (20%), SF1 (12%), DHX37 (4%), HSD17B3 (4%), and DMRT2 (4%). We could not find any mutation in the DSD-related genes in five (20%) patients due to complex molecular mechanisms in 46,XY DSD, highlighting the possibility of new DSD genes which are yet to be discovered in these disorders. In conclusion, genetic testing, including cytogenetics and molecular genetics, is important for the diagnosis and management of 46,XY DSD cases.

DHX37 and 46,XY DSD: A New Ribosomopathy?

Recently, a series of recurrent missense variants in the RNA-helicase DHX37 have been reported associated with either 46,XY gonadal dysgenesis, 46,XY testicular regression syndrome (TRS), or anorchia. All affected children have non-syndromic forms of disorders/differences of sex development (DSD). These variants, which involve highly conserved amino acids within known functional domains of the protein, are predicted by in silico tools to have a deleterious effect on helicase function. DHX37 is required for ribosome biogenesis in eukaryotes, and how these variants cause DSD is unclear. The relationship between DHX37 and human congenital disorders is complex as compound heterozygous as well as de novo heterozygous missense variants in DHX37 are also associated with a complex congenital developmental syndrome (NEDBAVC, neurodevelopmental disorder with brain anomalies and with or without vertebral or cardiac anomalies; OMIM 618731), consisting of microcephaly, global developmental delay, seizures, facial dysmorphia, and kidney and cardiac anomalies. Here, we will give a brief overview of ribosome biogenesis and the role of DHX37 in this process. We will discuss variants in DHX37, their contribution to human disease in the general context of human ribosomopathies, and the possible disease mechanisms that may be involved.

Contribution of Clinical and Genetic Approaches for Diagnosing 209 Index Cases With 46,XY Differences of Sex Development.

CONTEXT: Massively parallel sequencing (MPS) technologies have emerged as a first-tier approach for diagnosing several pediatric genetic syndromes. However, MPS has not been systematically integrated into the diagnostic workflow along with clinical/biochemical data for diagnosing 46,XY differences of sex development (DSD). OBJECTIVE: To analyze the contribution of phenotypic classification either alone or in association with genetic evaluations, mainly MPS, for diagnosing a large cohort of 46,XY DSD patients. DESIGN/PATIENTS: 209 nonsyndromic 46,XY DSD index cases from a Brazilian DSD center were included. Patients were initially classified into 3 subgroups according to clinical and biochemical data: gonadal dysgenesis (GD), disorders of androgen secretion/action, and DSD of unknown etiology. Molecular genetic studies were performed by Sanger sequencing and/or MPS. RESULTS: Clinical/biochemical classification into either GD or disorders of hormone secretion/action was obtained in 68.4% of the index cases. Among these, a molecular diagnosis was obtained in 36% and 96.5%, respectively. For the remainder 31.6% classified as DSD of clinically unknown etiology, a molecular diagnosis was achieved in 31.8%. Overall, the molecular diagnosis was achieved in 59.3% of the cohort. The combination of clinical/biochemical and molecular approaches diagnosed 78.9% of the patients. Clinical/biochemical classification matched with the genetic diagnosis in all except 1 case. DHX37 and NR5A1 variants were the most frequent genetic causes among patients with GD and DSD of clinical unknown etiology, respectively. CONCLUSIONS: The combination of clinical/biochemical with genetic approaches significantly improved the diagnosis of 46,XY DSD. MPS potentially decreases the complexity of the diagnostic workup as a first-line approach for diagnosing 46,XY DSD.

DHX37 gene heterozygous variant—a frequent cause of embryonic testicular regression syndrome / 中华内分泌代谢杂志

Objective:To report embryonic testicular regression syndrome(ETRS) caused by DHX37 heterozygous variant for the first time in China and summarize the clinical manifestations of ETRS as to improve the understanding of doctors for this disease.Methods:The clinical data and whole exome sequencing results of five cases of ETRS from Shenzhen Children′s Hospital were collected. The reported cases of DHX37 heterozygous variant were reviewed.Results:Five patients with ETRS visited the doctors at the age of 2 months to 5 years and 5 months. Three patients raised as males came to hospital due to virilition and 2 female patients visited a doctor due to clitoral hypertrophy. No uterus was detected by ultrasound in all patients. The gonadal pathologies from 4 cases displayed no testicular tissue or gonadal dysgenesis, complicated with gonadoblastoma in one case. The genetic testing revealed that the heterozygous variant(c.923G>A, p. R308Q) in DHX37 was found in 2 cases, without variant in other 3 cases. According to the review, ETRS and 46, XY gonadal dysgenesis due to DHX37 herozygous variant was firstly reported in 2019. A total of 40 cases, including 21 cases of ETRS, presented with the virilition or female phenotype, with the disappearance of testicular tissue as the main pathologies. There is no report in China.Conclusion:The article summarized the clinical manifestations and whole exome sequencing results of 5 patients with ETRS, among which two cases were caused by DHX37 variants and one was complicated with gonadoblastoma.

Clinical and genetic analysis of 46, XY partial gonadal dysgenesis caused by DHX37 mutation: One case report and literature review / 中华内分泌代谢杂志

One case of 46, XY partial gonadal dysgenesis due to a congenital defect of DEAH-box RNA helicase 37(DHX37) was reported. The clinical and genetic data of a boy who was admitted to the Department of Endocrinology and Metabolism, the First Affiliated Hospital of Henan University of Science and Technology due to ambiguous external genitalia in September 2020 were collected and analyzed. This 3-month-old male patient showed a micropenis, bilateral cryptorchidism, 46, XY karyotype, a decrease in testosterone, anti-Müllerian hormone, inhibin B, an increase in follicle stimulating hormone. Testis biopsy indicated gonadal dysgenesis. The proband harbored a de novo heterozygous mutation in the DHX37 gene c. 923G>A(p.Arg308Gln). DHX37 variants need to be considered for 46, XY gonadal dysgenesis.

A case of 46, XY disorders of sexual development caused by the heterozygous mutation of DHX37 gene / 中华实用儿科临床杂志

A clinical data, laboratory examination, genetic test results, diagnose and treatment of a patient with 46, XY disorders of sexual development (46, XY DSD) from a family of the Asp-Glu-Ala-His-box helicase 37 ( DHX37) gene mutation were retrospectively analyzed.The child was admitted in the Department of Genetics, Endocrinology and Metabolism, Jiangxi Children′s Hospital in June 2021.The DHX37 gene mutation was confirmed as a new pathogenic gene leading to 46, XY DSD in 2019.It is featured as autosomal dominant inheritance with incomplete externality.Its clinical manifestations are abnormal external genitalia, testicular degeneration insufficiency syndrome and gonadal insufficiency.This patient is the first 46, XY DSD case caused by the heterozygous variation of DHX37 gene c. 2020C>T (p.R674W) in China.This study can provide new ideas for diagnosis and treatment of 46, XY DSD children and reliable genetic evidence for family reproduction.

A case of 46, XY disorders of sexual development caused by the heterozygous mutation of DHX37 gene / 中华实用儿科临床杂志

A clinical data, laboratory examination, genetic test results, diagnose and treatment of a patient with 46, XY disorders of sexual development (46, XY DSD) from a family of the Asp-Glu-Ala-His-box helicase 37 ( DHX37) gene mutation were retrospectively analyzed.The child was admitted in the Department of Genetics, Endocrinology and Metabolism, Jiangxi Children′s Hospital in June 2021.The DHX37 gene mutation was confirmed as a new pathogenic gene leading to 46, XY DSD in 2019.It is featured as autosomal dominant inheritance with incomplete externality.Its clinical manifestations are abnormal external genitalia, testicular degeneration insufficiency syndrome and gonadal insufficiency.This patient is the first 46, XY DSD case caused by the heterozygous variation of DHX37 gene c. 2020C>T (p.R674W) in China.This study can provide new ideas for diagnosis and treatment of 46, XY DSD children and reliable genetic evidence for family reproduction.

Expanding DSD Phenotypes Associated with Variants in the DEAH-Box RNA Helicase DHX37.

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown.

Using CRISPR to enhance T cell effector function for therapeutic applications.

T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.

Pathogenic variants in the DEAH-box RNA helicase DHX37 are a frequent cause of 46,XY gonadal dysgenesis and 46,XY testicular regression syndrome.

PURPOSE: XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. METHODS: We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. RESULTS: Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. CONCLUSION: DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

Next-Generation Sequencing Reveals Novel Genetic Variants (SRY, DMRT1, NR5A1, DHH, DHX37) in Adults With 46,XY DSD.

CONTEXT: The genetic basis of human sex development is slowly being elucidated, and >40 different genetic causes of differences (or disorders) of sex development (DSDs) have now been reported. However, reaching a specific diagnosis using traditional approaches can be difficult, especially in adults where limited biochemical data may be available. OBJECTIVE: We used a targeted next-generation sequencing approach to analyze known and candidate genes for DSDs in individuals with no specific molecular diagnosis. PARTICIPANTS AND DESIGN: We studied 52 adult 46,XY women attending a single-center adult service, who were part of a larger cohort of 400 individuals. Classic conditions such as17ß-hydroxysteroid dehydrogenase deficiency type 3, 5α-reductase deficiency type 2, and androgen insensitivity syndrome were excluded. The study cohort had broad working diagnoses of complete gonadal dysgenesis (CGD) (n = 27) and partially virilized 46,XY DSD (pvDSD) (n = 25), a group that included partial gonadal dysgenesis and those with a broad "partial androgen insensitivity syndrome" label. Targeted sequencing of 180 genes was undertaken. RESULTS: Overall, a likely genetic cause was found in 16 of 52 (30.8%) individuals (22.2% CGD, 40.0% pvDSD). Pathogenic variants were found in sex-determining region Y (SRY; n = 3), doublesex and mab-3-related transcription factor 1 (DMRT1; n = 1), NR5A1/steroidogenic factor-1 (SF-1) (n = 1), and desert hedgehog (DHH; n = 1) in the CGD group, and in NR5A1 (n = 5), DHH (n = 1), and DEAH-box helicase 37 (DHX37; n = 4) in the pvDSD group. CONCLUSIONS: Reaching a specific diagnosis can have clinical implications and provides insight into the role of these proteins in sex development. Next-generation sequencing approaches are invaluable, especially in adult populations or where diagnostic biochemistry is not possible.

Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells.

CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.