TP53 mutations in cancer: Molecular features and therapeutic opportunities (Review).

The tumour suppressor factor p53 plays an essential role in regulating numerous cellular processes, including the cell cycle, DNA repair, apoptosis, autophagy, cell metabolism and immune response. TP53 is the most commonly mutated gene in human cancers. These mutations are primarily non­synonymous changes that produce mutant p53 proteins characterized by loss of function, a dominant negative effect on p53 tetramerisation and gain of function (GOF). GOF mutations not only disrupt the tumour­suppressive activities of p53 but also endow the mutant proteins with new oncogenic properties. Recent studies analysing different pathogenic features of mutant p53 in cancer­derived cell lines have demonstrated that restoring wild­type p53, rather than removing GOF mutations, reduces cancer cell growth. These findings suggest that therapeutic strategies for reactivating wild­type p53 function in cancer cells may bring a greater benefit than approaches halting mutant p53. This approach could involve the use of small molecules, gene therapy and other methods to re­establish wild­type p53 activity. This review describes the complexity of the biological activities of different p53 mutants and summarizes the current therapeutic approaches to restore p53 function.

ATXN2 loss of function results in glaucoma-related features supporting a role for Ataxin-2 in primary open-angle glaucoma (POAG) pathogenesis.

Glaucoma is a leading cause of irreversible blindness worldwide. The most common form, primary open-angle glaucoma (POAG), is a genetically complex trait with high heritability. Genome-wide association studies have identified significant POAG and IOP association of a genomic region on chromosome 12 that includes ATXN2 as well as 7 other genes. Association of protein disrupting ATXN2 variants in the NEIGHBORHOOD case-control cohort and the UK Biobank suggests that ATXN2 is a key gene in this locus. To investigate functional effects, we utilized a zebrafish (Danio rerio) CRISPR/Cas9 edited atxn2-knockdown line to show that loss of atxn2 results in reduced eye size, diminished retinal ganglion cells (RGC), increased intraocular pressure (IOP), and impaired visual function in zebrafish. Complementation assays supported functional effects for 14 POAG-associated human ATXN2 missense variants. These results suggest a loss-of-function mechanism underlying a potential role for ATXN2 in POAG pathogenesis.

Polymorphic pseudogenes in the human genome - a comprehensive assessment.

BACKGROUND: Over the past decade, variations of the coding portion of the human genome have become increasingly evident. In this study, we focus on polymorphic pseudogenes, a unique and relatively unexplored type of pseudogene whose inactivating mutations have not yet been fixed in the human genome at the global population level. Thus, polymorphic pseudogenes are characterized by the presence in the population of both coding alleles and non-coding alleles originating from Loss-of-Function (LoF) mutations. These alleles can be found both in heterozygosity and in homozygosity in different human populations and thus represent pseudogenes that have not yet been fixed in the population. RESULTS: A methodical cross-population analysis of 232 polymorphic pseudogenes, including 35 new examples, reveals that human olfactory signalling, drug metabolism and immunity are among the systems most impacted by the variable presence of LoF variants at high frequencies. Within this dataset, a total of 179 genes presented polymorphic LoF variants in all analysed populations. Transcriptome and proteome analysis confirmed that although these genes may harbour LoF alleles, when the coding allele is present, the gene remains active and can play a functional role in various metabolic pathways, including drug/xenobiotic metabolism and immunity. The observation that many polymorphic pseudogenes are members of multigene families argues that genetic redundancy may play a key role in compensating for the inactivation of one paralogue. CONCLUSIONS: The distribution, expression and integration of cellular/biological networks in relation to human polymorphic pseudogenes, provide novel insights into the architecture of the human genome and the dynamics of gene gain and loss with likely functional impact.

Specific analysis of SOD1 enzymatic activity in CSF from ALS patients with and without SOD1 mutations.

Mutations in superoxide dismutase-1 (SOD1) are a cause of hereditary amyotrophic lateral sclerosis (ALS) through a gain of function mechanism involving unfolded mutant SOD1. Intrathecal gene therapy using the antisense-oligo-nucleotide drug tofersen to reduce SOD1 expression delays disease progression and has recently been approved in the United States and the European Union. However, the discovery of children homozygous for inactivating SOD1 mutations developing the SOD1 Deficiency Syndrome (ISODDES) with injury to the motor system suggests that a too low SOD1 antioxidant activity may be deleterious in humans. Measuring SOD1 activity in cerebrospinal fluid (CSF) in tofersen-treated patients is recommended but difficult due to low concentration and the presence of the isoenzyme SOD3. We here present a sensitive method to assess SOD1 activity by removing SOD3 from CSF samples using highly specific immobilized antibodies and subsequent measurement of the SOD activity. We validated the method on 171 CSF samples from ALS patients with and without mutations and controls and used paired erythrocyte samples for comparison. We found that ALS patients with wildtype SOD1, the SOD1 activity in CSF was equal to controls, but patients with mutant SOD1 show lower activity in CSF, even for patients with mutants previously reported to have full activity in erythrocytes. Activity variation in CSF was large among patients carrying the same SOD1 mutation and larger than in erythrocytes and in post-mortem nervous tissue. Additionally, we identified a discrepancy between the SOD1 activity and protein level measured with ELISA in both CSF and erythrocytes. Since antibodies used for SOD1 ELISA-quantification are raised against the natively folded wildtype SOD1, the concentration of mutant SOD1s may be underestimated. Analysis of SOD1 enzymatic activity in CSF is therefore a more reliable way to monitor the effect of SOD1-lowering drugs.

Long-term prognosis of patients with an SCN5A loss-of-function variant and progressive cardiac conduction disorder or Brugada syndrome.

BACKGROUND: The long-term prognosis of patients with a loss-of-function variant in the cardiac sodium channel gene SCN5A is unknown. OBJECTIVE: This study aimed to evaluate the long-term arrhythmic risk in patients with an SCN5A loss-of-function variant to identify predictors of arrhythmic events. METHODS: Probands and family members with (likely-)pathogenic SCN5A loss-of-function variants were retrospectively included. Clinical and ECG data at baseline and last follow-up were collected. Patients with a history of cardiac arrest, sustained ventricular tachycardia, symptomatic or documented atrial tachy- or bradyarrhythmia, or arrhythmogenic syncope, were categorized as symptomatic. Arrhythmic events at follow-up were defined as sudden death, aborted cardiac arrest, documented ventricular fibrillation and/or sustained ventricular tachycardia. RESULTS: We included 615 patients (349 males, 242 probands, 157 spontaneous type-1 Brugada-ECG and 111 symptomatic at baseline). During 9.5 (5.0-14.3) years follow-up, arrhythmic events occurred in 41 patients (6.7%), equating an overall event rate of 0.7%/year; 2.0%/year in symptomatic and 0.3%/year in asymptomatic patients. In the overall study population, symptoms at baseline, male sex, and QRS prolongation were identified as independent predictors of arrhythmic events. In asymptomatic patients, also male sex and QRS prolongation were identified as predictors. Asymptomatic women with QRS <100ms did not experience arrhythmic events at follow-up. CONCLUSION: Key predictors of arrhythmic risk in patients with an SCN5A loss-of-function variant, regardless of a Brugada syndrome diagnosis, are symptoms at baseline, male sex, and prolonged QRS. Our findings may enable more tailored management strategies in patients with an SCN5A loss-of-function variant based on their individual risk profiles.

Protein structural context of cancer mutations reveals molecular mechanisms and candidate driver genes.

Advances in protein structure determination and modeling allow us to study the structural context of human genetic variants on an unprecedented scale. Here, we analyze millions of cancer-associated missense mutations based on their structural locations and predicted perturbative effects. By considering the collective properties of mutations at the level of individual proteins, we identify distinct patterns associated with tumor suppressors and oncogenes. Tumor suppressors are enriched in structurally damaging mutations, consistent with loss-of-function mechanisms, while oncogene mutations tend to be structurally mild, reflecting selection for gain-of-function driver mutations and against loss-of-function mutations. Although oncogenes are difficult to distinguish from genes with no role in cancer using only structural damage, we find that the three-dimensional clustering of mutations is highly predictive. These observations allow us to identify candidate driver genes and speculate about their molecular roles, which we expect will have general utility in the analysis of cancer sequencing data.

A partial loss-of-function variant in STAT6 protects against T2 asthma.

BACKGROUND: Signal Transducer and Activator of Transcription 6 (STAT6) is central to Type 2 (T2) inflammation and common non-coding variants at the STAT6 locus associate with various T2 inflammatory traits, including diseases, and its pathway is widely targeted in asthma treatment. OBJECTIVE: To test the association of a rare missense variant in STAT6, p.L406P, with T2 inflammatory traits, including the risk of asthma and allergic diseases, and to characterize its functional consequences in cell culture. METHODS: We tested association of p.L406P with plasma protein levels, white blood cell counts and the risk of asthma and allergic phenotypes. We tested significant associations in other cohorts using a burden test. The effects of p.L406P on STAT6 protein function were examined in cell lines and by comparing CD4+ T-cell responses from carriers and non-carriers of the variant. RESULTS: p.L406P associated with reduced plasma levels of STAT6 and IgE as well as with lower eosinophil and basophil counts in blood. It also protected against asthma, mostly driven by severe T2 high asthma. We showed that p.L406P led to lower IL-4-induced activation in luciferase reporter assays and lower levels of STAT6 in CD4+ T cells. We identified multiple genes with expression that was affected by the p.L406P genotype upon IL-4 treatment of CD4+ T cells; the effect was consistent with a weaker IL-4 response in carriers than non-carriers of p.L406P. CONCLUSIONS: We report a partial loss-of-function variant in STAT6, resulting in dampened IL-4 responses and protection from T2 high asthma, implicating STAT6 as an attractive therapeutic target.

Conditional heterozygous loss of Kit receptor tyrosine kinase in neural crest cell lineage is associated with midline cleft lip and bifid nose deformity.

OBJECTIVES: The receptor tyrosine kinase Kit is expressed in cells derived from the trunk neural crest (NC), such as melanocytes; however, its role in cranial NC cell development is not fully understood. METHODS: We investigated the effects of the heterozygous loss of Kit in NC cells during embryonic development by mating Kit2lox/+ mice with Wnt1-Cre mice to produce Wnt1-Cre; Kit2lox/+ embryos. In addition, Wnt1-Cre mice were mated with Rosa26R-yellow fluorescent protein (YFP) mice to visualize the tissue regions expressing Cre recombinase. Histological studies of the craniofacial regions of these mice were performed using samples from embryonic day (E) 12.5 and postnatal day (P) 1. Cellular apoptosis and proliferation were both analyzed through the immunostaining of tissue sections collected on E13.5 and E14.5 using anti-cleaved caspase 3 (CC3) to detect apoptosis and anti-Ki67 to detect proliferation. Cells from YFP-positive tissue regions of the facial areas of Wnt1-Cre; Kit+/+; Rosa26R-YFP embryos and Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos collected on E12.5 and E15.5 were cultured and evaluated for cell proliferation. RESULTS: Compared with control littermates, Wnt1-Cre; Kit2lox/+ embryos exhibited midline cleft lip and bifid nose deformities. Substantial early (P1) postnatal lethality was observed in Wnt1-Cre; Kit2lox/+ mice, with none surviving to 3 weeks of age. YFP-positive cells from the maxillary regions of Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos exhibited defective cell growth and self-renewal in vitro. CONCLUSION: Conditional heterozygous loss of Kit in Wnt1-Cre; Kit2lox/+ embryos is associated with craniofacial dysplasia and exhibit defective NC development in vitro and in vivo.

Catalytic activities of wild-type C. elegans DAF-2 kinase and dauer-associated mutants.

DAF-2, the Caenorhabditis elegans insulin-like receptor homolog, regulates larval development, metabolism, stress response, and lifespan. The availability of numerous daf-2 mutant alleles has made it possible to elucidate the genetic mechanisms underlying these physiological processes. The DAF-2 pathway is significantly conserved with the human insulin/IGF-1 signaling pathway; it includes proteins homologous to human IRS, GRB-2, and PI3K, making it an important model to investigate human pathological conditions. We expressed and purified the kinase domain of wild-type DAF-2 to examine the catalytic activity and substrate specificity of the enzyme. Like the human insulin receptor kinase, DAF-2 kinase phosphorylates tyrosines within specific YxN or YxxM motifs, which are important for recruiting downstream effectors. DAF-2 kinase phosphorylated peptides derived from the YxxM and YxN motifs located in the C-terminal extension of the receptor tyrosine kinase, consistent with the idea that the DAF-2 receptor may possess independent signaling capacity. Unlike the human insulin or IGF-1 receptor kinases, DAF-2 kinase was poorly inhibited by the small-molecule inhibitor linsitinib. We also expressed and purified mutant kinases corresponding to daf-2 alleles that result in partial loss-of-function phenotypes in C. elegans. These mutations caused a complete loss of kinase function in vitro. Our biochemical investigations provide new insights into DAF-2 kinase function, and the approach should be useful for studying other mutations to shed light on DAF-2 signaling in C. elegans physiology.

Gene co-expression networks reveal sex-biased differences in musculoskeletal ageing.

Aging is a universal and progressive process involving the deterioration of physiological functions and the accumulation of cellular damage. Gene regulation programs influence how phenotypes respond to environmental and intrinsic changes during aging. Although several factors, including sex, are known to impact this process, the underlying mechanisms remain incompletely understood. Here, we investigate the functional organization patterns of skeletal muscle genes across different sexes and ages using gene co-expression networks (GCNs) to explore their influence on aging. We constructed GCNs for three different age groups for male and female samples, analyzed topological similarities and differences, inferred significant associated processes for each network, and constructed null models to provide statistically robust results. We found that each network is topologically and functionally distinct, with young women having the most associated processes, likely due to reproductive tasks. The functional organization and modularity of genes decline with age, starting from middle age, potentially leading to age-related deterioration. Women maintain better gene functional organization throughout life compared to men, especially in processes like macroautophagy and sarcomere organization. The study suggests that the loss of gene co-expression could be a universal aging marker. This research offers insights into how gene organization changes with age and sex, providing a complementary method to analyze aging.

Clinical report and genetic analysis of a Chinese family with retinitis pigmentosa 79 caused by a novel loss-of-function HK1 variant.

BACKGROUND: Retinitis pigmentosa (RP) is a genetically heterogeneous disease. RP 79 has been associated with heterozygous variants of hexokinase 1 (HK1). Only two missense HK1 variants have been reported in 11 families. OBJECTIVE: To discover the molecular pathogenic mechanism of RP and validate the biological harm of HK1 through in vitro experiments. METHODS: We conducted a genetic analysis of a 3-year-old female patient with RP and her family. We also evaluated the ocular phenotypes caused by HK1 (the identified variant). Peripheral blood samples were collected from the patient, her parents, and her brother, and trio whole-exome sequencing was performed. A protein structure analysis was performed to assess the functional impact of the variant, and a mutant plasmid was constructed for the quantitative polymerase chain reaction (qPCR) and western blot (WB) analysis of the effects of the variant on transcription and protein translation. RESULTS: The patient harbored the NM_000188.3: c.613del (p.Ala205Leufs*3) variant, which is a heterozygous variant of HK1. Sanger sequencing confirmed the presence of this variant in the patient; however, the patient's parents and brother had the wild-type variant. The protein structure analysis indicated that the variant resulted in a truncated protein caused by premature termination of amino acid coding. The qPCR results indicated that the variant may not have affected the transcription process. However, the WB analysis demonstrated that the mutant HK-1 protein was not expressed and that the wild-type group exhibited normal expression. CONCLUSIONS: Our patient had a loss-of-function (LoF) variant of HK1, which may be the genetic cause of typical features of RP that are observed at an early age. These findings expand the spectrum of HK1 variants and phenotypes and suggest that LoF variants of HK1 may represent a specific pathogenic mechanism of RP.

Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib.

BACKGROUND: Loss of heterozygosity (LOH) diminishes genetic diversity within cancer genomes. A tumour arising in an individual heterozygous for a functional and a loss-of-function (LoF) allele of a gene occasionally retain only the LoF allele. This can result in deficiency of specific protein activities in cancer cells, creating unique differences between tumour cells and normal cells of the individual. Such differences may constitute vulnerabilities that can be exploited through allele-specific therapies. METHODS: To discover frequently lost genes with prevalent LoF alleles, we mined the 1000 Genomes dataset for SNVs causing protein truncation through base substitution, indels or splice site disruptions, resulting in 60 LoF variants in 60 genes. From these, the variant rs3892097 in the liver enzyme CYP2D6 was selected because it is located within a genomic region that frequently undergoes LOH in several tumor types including hepatocellular cancers. To evaluate the relationship between CYP2D6 activity and the toxicities of anticancer agents, we screened 525 compounds currently in clinical use or undergoing clinical trials using cell model systems with or without CYP2D6 activity. FINDINGS: We identified 12 compounds, AZD-3463, CYC-116, etoposide, everolimus, GDC-0349, lenvatinib, MK-8776, PHA-680632, talazoparib, tyrphostin 9, VX-702, and WZ-3146, using an engineered HEK293T cell model. Of these, talazoparib and MK-8776 demonstrated consistently heightened cytotoxic effects against cells with compromised CYP2D6 activity in engineered hepatocellular cancer cell models. Moreover, talazoparib displayed CYP2D6 genotype dependent effects on primary hepatocellular carcinoma organoids. INTERPRETATION: Exploiting the loss of drug-metabolizing enzyme gene activity in tumor cells following loss of heterozygosity could present a promising therapeutic strategy for targeted cancer treatment. FUNDING: This work was funded by Barncancerfonden (T.S, PR2022-0099 and PR2020-0171, X.Z, TJ2021-0111), Cancerfonden (T.S, 211719Pj and D.G, 222449Pj), Vetenskapsrådet (T.S, 2020-02371 and D.G, 2020-04707), and the Erling Persson Foundation (T.S, 2020-0037 and T.S, 2023-0113).

ME2 Deficiency Is Associated With Recessive Neurodevelopmental Disorder.

Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2-associated recessive NDD.

MARK2 variants cause autism spectrum disorder via the downregulation of WNT/ß-catenin signaling pathway.

Microtubule affinity-regulating kinase 2 (MARK2) contributes to establishing neuronal polarity and developing dendritic spines. Although large-scale sequencing studies have associated MARK2 variants with autism spectrum disorder (ASD), the clinical features and variant spectrum in affected individuals with MARK2 variants, early developmental phenotypes in mutant human neurons, and the pathogenic mechanism underlying effects on neuronal development have remained unclear. Here, we report 31 individuals with MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and distinctive facial features. Loss-of-function (LoF) variants predominate (81%) in affected individuals, while computational analysis and in vitro expression assay of missense variants supported the effect of MARK2 loss. Using proband-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), we show that MARK2 loss leads to early neuronal developmental and functional deficits, including anomalous polarity and dis-organization in neural rosettes, as well as imbalanced proliferation and differentiation in neural progenitor cells (NPCs). Mark2+/- mice showed abnormal cortical formation and partition and ASD-like behavior. Through the use of RNA sequencing (RNA-seq) and lithium treatment, we link MARK2 loss to downregulation of the WNT/ß-catenin signaling pathway and identify lithium as a potential drug for treating MARK2-associated ASD.

Contrasting Genomic Diversity and Inbreeding Levels Among Two Closely Related Falcon Species With Overlapping Geographic Distributions.

Genomic resources are valuable to examine historical demographic patterns and their effects to better inform management and conservation of threatened species. We evaluated population trends and genome-wide variation in the near-threatened Orange-breasted Falcon (Falco deiroleucus) and its more common sister species, the Bat Falcon (F. rufigularis), to explore how the two species differ in genomic diversity as influenced by their contrasting long-term demographic histories. We generated and aligned whole genome resequencing data for 12 Orange-breasted Falcons and 9 Bat Falcons to an annotated Gyrfalcon (F. rusticolus) reference genome that retained approximately 22.4 million biallelic autosomal SNPs (chromosomes 1-22). Our analyses indicated much lower genomic diversity in Orange-breasted Falcons compared to Bat Falcons. All sampled Orange-breasted Falcons were significantly more inbred than the sampled Bat Falcons, with values similar to those observed in island-mainland species comparisons. The distribution of runs of homozygosity showed variation suggesting long-term low population size and the possibility of bottlenecks in Orange-breasted Falcons contrasting with consistently larger populations in Bat Falcons. Analysis of genetic load suggests that Orange-breasted Falcons are less likely to experience inbreeding depression than Bat Falcons due to reduced inbreeding load but are at elevated risk from fixation of deleterious gene variants and perhaps a reduced adaptive potential. These genomic analyses highlight differences in the historical demography of two closely related species that have influenced their current genomic diversity and should result in differing strategies for their continued conservation.

Re-analysis of Next-generation Sequencing Data in Patients with Hypertrophic Cardiomyopathy: Contribution of Spliceogenic MYBPC3 Variants in an Italian Cohort.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of disease-causing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40-60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the in-silico detection of intronic variants in MYBPC3 that may escape detection by algorithms normally used with tagged diagnostic panels. We included 142 HCM probands with negative results in Illumina TruSight Cardio panel analysis, including exonic regions of 174 cardiomyopathy genes. Raw data were re-analyzed using existing bioinformatics tools. The spliceogenic variant c.1224-80G>A was detected in three patients (2.1%), leading us to reconsider their molecular diagnosis. These patients showed late onset and mild symptoms, although no peculiar phenotypic characteristics were shared. Collectively, rare spliceogenic MYBPC3 variants may play a role in causing HCM, and their systematic detection should be performed to provide more comprehensive solutions in genetic testing using multigenic panels.

Expression analysis of genes including Zfhx4 in mice and zebrafish reveals a temporospatial conserved molecular basis underlying craniofacial development.

BACKGROUND: Embryonic craniofacial development involves several cellular and molecular events that are evolutionarily conserved among vertebrates. Vertebrate models such as mice and zebrafish have been used to investigate the molecular and cellular etiologies underlying human craniofacial disorders, including orofacial clefts. However, the molecular mechanisms underlying embryonic development in these two species are unknown. Therefore, elucidating the shared mechanisms of craniofacial development between disease models is crucial to understanding the underlying mechanisms of phenotypes in individual species. RESULTS: We selected mice and zebrafish as model organisms to compare various events during embryonic craniofacial development. We identified genes (Sox9, Zfhx3 and 4, Cjun, and Six1) exhibiting similar temporal expression patterns between these species through comprehensive and stage-matched gene expression analyses. Expression analysis revealed similar gene expression in hypothetically corresponding tissues, such as the mice palate and zebrafish ethmoid plate. Furthermore, loss-of-function analysis of Zfhx4/zfhx4, a causative gene of human craniofacial anomalies including orofacial cleft, in both species resulted in deformed skeletal elements such as the palatine and ethmoid plate in mice and zebrafish, respectively. CONCLUSIONS: These results demonstrate that these disease models share common molecular mechanisms, highlighting their usefulness in modeling craniofacial defects in humans.

Homozygous mutation of KISS1 receptor (KISS1R) gene identified in a Chinese patient with congenital hypogonadotropic hypogonadism (CHH): case report and literature review.

OBJECTIVES: Congenital hypogonadotropic hypogonadism (CHH) is a rare condition caused by a defect in the production, secretion or action of gonadotropin-releasing hormone. The absence of puberty and varying degrees of gonadotropic deficiency are common symptoms of this disorder. Heterogeneity exists in the clinical presentation of the different clinical subtypes and multiple genes have been implicated in CHH. A number of genetic defects have been identified as causes normosmic CHH, including mutations of GnRHR, GNRH1, KISS1R, KISS1, TACR3 and TAC3. Loss-of-function mutations in KISS1R gene are a rare cause of normosmic CHH. CASE PRESENTATION: We described an 11.5 years old Chinese patient who presented at birth with micropenis, microorchidia and bilateral cryptorchidism. Whole-exome sequencing was also performed and identified a homozygous mutation of KISS1R gene, c.1010_1028del (p.V337Afs*82). The variant was predicted as "deleterious" and classified as "likely pathogenic". This variant has never been reported in patients with CHH. Furthermore, we summarized the clinical presentations and analyzed the phenotype-genotype correlation between CHH and KISS1R mutations in previous reports. CONCLUSIONS: This study details the clinical phenotypes and hormone levels of the patient and expands the spectrum of mutations in the KISS1R gene associated with CHH.

WDR44 Loss-of-Function Promoter Deletion in a Male Newborn With a Ciliopathy Phenotype.

Gain-of-function variants in the WDR44 gene have recently been associated with an X-linked ciliopathy-related neurodevelopmental phenotype. Here, we report on a WDR44 loss-of-function (LOF) variant identified in the genome sequence from a male fetus enrolled in the Prenatal Genetic Diagnosis by Genomic Sequencing (PrenatalSEQ) multicenter study. The phenotype is consistent with the described X-linked ciliopathy that includes developmental delay, microcephaly, congenital heart defects, kidney abnormalities, cryptorchidism, musculoskeletal abnormalities, craniofacial dysmorphism, and effusions. This is the first report of a WDR44 LOF variant in an affected individual with a prenatal presentation and supports LOF as a mechanism for the X-linked WDR44 ciliopathy-related phenotype.

Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects.

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.