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The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Células A549 , Animales , Niño , Preescolar , Femenino , Mutación con Ganancia de Función/genética , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Células Madre Embrionarias de Ratones , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Transducción de Señal , Dominios Homologos src/genéticaRESUMEN
PURPOSE: Rare genetic variants in the PURA gene cause the PURA-related neurodevelopmental disorder (PURA-NDD), characterized by neonatal abnormalities and developmental delay. Using genome-wide DNA methylation analysis on patients with PURA variants, we aim to establish a PURA-NDD-specific methylation profile and provide further insights on the molecular basis of the PURA-NDD. METHODS: Twenty three individuals (including 12 unpublished) carrying PURA variants were enrolled. We conducted the Illumina Infinium EPIC microarray analysis in 17 PURA-NDD individuals. In vitro experiments were performed to examine how PURA variants affect Pur-a expression. RESULTS: Additional phenotypes in 12 newly identified patients were described in this study. Genome-wide DNA methylation analysis unveiled distinctive methylation profiles to PURA-NDD, and the established classifier can reclassify PURA variants of uncertain significance. Patients bearing PURA hapoloinsufficient and missense variants have comparable DNA methylation profiles, and cells expressing these PURA variants showed consistent Pur-a downregulation, suggesting a haploinsufficiency mechanism. CONCLUSION: Patients with PURA-NDD exhibit a specific episignature, which has potential to aid identification and diagnosis of PURA-NDD patients and offer implications for further functional investigations.
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Metilación de ADN , Epigénesis Genética , Trastornos del Neurodesarrollo , Humanos , Trastornos del Neurodesarrollo/genética , Metilación de ADN/genética , Femenino , Epigénesis Genética/genética , Masculino , Niño , Preescolar , Estudio de Asociación del Genoma Completo , Fenotipo , Haploinsuficiencia/genética , LactanteRESUMEN
Mitochondrial diseases are a group of genetic diseases caused by mutations in mitochondrial DNA and nuclear DNA. However, the genetic spectrum of this disease is not yet complete. In this study, we identified a novel variant m.4344T>C in mitochondrial tRNAGln from a patient with developmental delay. The mutant loads of m.4344T>C were 95% and 89% in the patient's blood and oral epithelial cells, respectively. Multialignment analysis showed high evolutionary conservation of this nucleotide. TrRosettaRNA predicted that m.4344T>C variant would introduce an additional hydrogen bond and alter the conformation of the T-loop. The transmitochondrial cybrid-based study demonstrated that m.4344T>C variant impaired the steady-state level of mitochondrial tRNAGln and decreased the contents of mitochondrial OXPHOS complexes I, III, and IV, resulting in defective mitochondrial respiration, elevated mitochondrial ROS production, reduced mitochondrial membrane potential and decreased mitochondrial ATP levels. Altogether, this is the first report in patient carrying the m.4344T>C variant. Our data uncover the pathogenesis of the m.4344T>C variant and expand the genetic mutation spectrum of mitochondrial diseases, thus contributing to the clinical diagnosis of mitochondrial tRNAGln gene variants-associated mitochondrial diseases.
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ADN Mitocondrial , Discapacidades del Desarrollo , Enfermedades Mitocondriales , Humanos , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Mutación , Mitocondrias/genética , Mitocondrias/metabolismo , Masculino , Femenino , Potencial de la Membrana Mitocondrial/genética , Fosforilación Oxidativa , Preescolar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Rare variants of CCNK (cyclin K) give rise to a syndrome with intellectual disability. The purpose of this study was to describe the genotype-phenotype spectrum of CCNK-related syndrome and the underlying molecular mechanisms of pathogenesis. METHODS: We identified a number of de novo CCNK variants in unrelated patients. We generated patient-induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs) as disease models. In addition, we constructed NPC-specific Ccnk knockout (KO) mice and performed molecular and morphological analyses. RESULTS: We identified 2 new patients harboring CCNK missense variants and followed-up 3 previous reported patients, which constitute the largest patient population analysis of the disease. We demonstrate that both the patient-derived NPC models and the Ccnk KO mouse displayed deficient NPC proliferation and enhanced apoptotic cell death. RNA sequencing analyses of these NPC models uncovered transcriptomic signatures unique to CCNK-related syndrome, revealing significant changes in genes, including WNT5A, critical for progenitor proliferation and cell death. Further, to confirm WNT5A's role, we conducted rescue experiments using NPC and mouse models. We found that a Wnt5a inhibitor significantly increased proliferation and reduced apoptosis in NPCs derived from patients with CCNK-related syndrome and NPCs in the developing cortex of Ccnk KO mice. INTERPRETATION: We discussed the genotype-phenotype relationship of CCNK-related syndrome. Importantly, we demonstrated that CCNK plays critical roles in NPC proliferation and NPC apoptosis in vivo and in vitro. Together, our study highlights that Wnt5a may serve as a promising therapeutic target for the disease intervention. ANN NEUROL 2023;94:1136-1154.
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Discapacidad Intelectual , Células-Madre Neurales , Ratones , Animales , Humanos , Células-Madre Neurales/metabolismo , Transducción de Señal/genética , Ciclinas/metabolismo , ApoptosisRESUMEN
OBJECTIVES: Regions of homozygosity (ROH) could implicate uniparental disomy (UPD) on specific chromosomes associated with imprinting disorders. Though the algorithms for ROH detection in exome sequencing (ES) have been developed, optimal reporting thresholds and when to pursue confirmatory UPD testing for imprinting disorders remain in ambiguity. This study used a data-driven approach to assess optimal reporting thresholds of ROH in clinical practice. METHODS: ROH analysis was performed using Automap in a retrospective cohort of 8,219 patients and a prospective cohort of 1,964 patients with ES data. Cases with ROH on imprinting-disorders related chromosomes were selected for additional methylation-specific confirmatory testing. The diagnostic yield, the ROH pattern of eventually diagnosed cases and optimal thresholds for confirmatory testing were analyzed. RESULTS: In the retrospective analysis, 15 true UPD cases of imprinting disorders were confirmed among 51 suspected cases by ROH detection. Pattern of ROH differed between confirmed UPD and non-UPD cases. Maximized yield and minimized false discovery rate of confirmatory UPD testing was achieved at the thresholds of >20â¯Mb or >25â¯% chromosomal coverage for interstitial ROH, and >5â¯Mb for terminal ROH. Current recommendation by ACMG was nearly optimal, though refined thresholds as proposed in this study could reduce the workload by 31â¯% without losing any true UPD diagnosis. Our refined thresholds remained optimal after independent evaluation in a prospective cohort. CONCLUSIONS: ROH identified in ES could implicate the presence of clinically relevant UPD. This study recommended size and coverage thresholds for confirmatory UPD testing after ROH detection in ES, contributing to the development of evidence-based reporting guidelines.
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BACKGROUND: Primary adrenal insufficiency (PAI) is a rare but life-threatening condition. Differential diagnosis of numerous causes of PAI requires a thorough understanding of the condition. METHODS: To describe the genetic composition and presentations of PAI. The following data were collected retrospectively from 111 patients with non-21OHD with defined genetic diagnoses: demographic information, onset age, clinical manifestations, laboratory findings and genetic results. Patients were divided into four groups based on the underlying pathogenesis: (1) impaired steroidogenesis, (2) adrenal hypoplasia, (3) resistance to adrenocorticotropic hormone (ACTH) and (4) adrenal destruction. The age of onset was compared within the groups. RESULTS: Mutations in the following genes were identified: NR0B1 (n=39), STAR (n=33), CYP11B1 (n=12), ABCD1 (n=8), CYP17A1 (n=5), HSD3B2 (n=4), POR (n=4), MRAP (n=2), MC2R (n=1), CYP11A1 (n=1), LIPA (n=1) and SAMD9 (n=1). Frequent clinical manifestations included hyperpigmentation (73.0%), dehydration (49.5%), vomiting (37.8%) and abnormal external genitalia (23.4%). Patients with adrenal hypoplasia typically presented manifestations earlier than those with adrenal destruction but later than those with impaired steroidogenesis (both p<0.01). The elevated ACTH (92.6%) and decreased cortisol (73.5%) were the most common laboratory findings. We generated a differential diagnosis flowchart for PAI using the following clinical features: 17-hydroxyprogesterone, very-long-chain fatty acid, external genitalia, hypertension and skeletal malformation. This flowchart identified 84.8% of patients with PAI before next-generation DNA sequencing. CONCLUSIONS: STAR and NR0B1 were the most frequently mutated genes in patients with non-21OHD PAI. Age of onset and clinical characteristics were dependent on aetiology. Combining clinical features and molecular tests facilitates accurate diagnosis.
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Enfermedad de Addison , Insuficiencia Suprarrenal , Humanos , Enfermedad de Addison/genética , Estudios Retrospectivos , Hormona Adrenocorticotrópica , China , Insuficiencia Suprarrenal/diagnóstico , Insuficiencia Suprarrenal/genética , Péptidos y Proteínas de Señalización IntracelularRESUMEN
BACKGROUND: Idiopathic short stature (ISS) is characterized by short stature with unknown causes. Recent studies showed different gut microbiota flora and reduced fecal short-chain fatty acids in ISS children. However, the roles of the microbiome and metabolites in the pathogenesis of ISS remains largely unknown. METHODS: We recruited 51 Chinese subjects, comprising 26 ISS children and 25 normal-height control individuals. Untargeted metabolomics was performed to explore the fecal metabolic profiles between groups. A shotgun metagenomic sequencing approach was used to investigate the microbiome at the strains level. Mediation analyses were done to reveal correlations between the height standard deviation (SD) value, the gut microbiome and metabolites. RESULTS: We detected marked differences in the composition of fecal metabolites in the ISS group, particularly a significant increase in erucic acid and a decrease in spermidine, adenosine and L-5-Hydroxytryptophan, when compared to those of controls. We further identified specific groups of bacterial strains to be associated with the different metabolic profile. Through mediation analysis, 50 linkages were established. KEGG pathway analysis of microbiota and metabolites indicated nutritional disturbances. 13 selected features were able to accurately distinguish the ISS children from the controls (AUC = 0.933 [95%CI, 79.9-100%]) by receiver operating characteristic (ROC) analysis. CONCLUSION: Our study suggests that the microbiome and the microbial-derived metabolites play certain roles in children's growth. These findings provide a new research direction for better understanding the mechanism(s) underlying ISS.
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Heces , Microbioma Gastrointestinal , Humanos , Niño , Masculino , Femenino , Heces/microbiología , Estudios de Casos y Controles , Adolescente , Estatura , Trastornos del Crecimiento/microbiología , Trastornos del Crecimiento/metabolismo , Metabolómica/métodos , MetabolomaRESUMEN
Variant prioritization is a crucial step in the analysis of exome and genome sequencing. Multiple phenotype-driven tools have been developed to automate the variant prioritization process, but the efficacy of these tools in clinical setting with fuzzy phenotypic information and whether ensemble of these tools could outperform single algorithm remains to be assessed. A large rare disease cohort with heterogeneous phenotypic information, including a primary cohort of 1614 patients and a replication cohort of 1904 patients referred to exome sequencing, were recruited to assess the efficacy of variant prioritization and their ensemble. Three freely available tools-Exomiser, Xrare, and DeepPVP-and their ensemble were evaluated. The performance of all three tools was influenced by the attributes of phenotypic input. When combining these three tools by weighted-sum entropy method (EWE3), the ensemble outperformed any single algorithm, achieving a rate of 78% diagnostic variants in top 3 (13% improvement over current best performer, compared to Exomiser: 63%, Xrare: 65%, and DeepPVP: 51%), 88% in top 10 and 96% in top 30. The results were replicated in another independent cohort. Our study supports using entropy-weighted ensemble of multiple tools to improve variant prioritization and accelerate molecular diagnosis in exome/genome sequencing.
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Algoritmos , Exoma , Humanos , Exoma/genética , Entropía , Fenotipo , Enfermedades Raras/genética , Programas InformáticosRESUMEN
21 hydroxylase deficiency (21-OHD), the most common form of congenital adrenal hyperplasia, is caused by defects in CYP21A2 gene, which encodes the cytochrome P450 oxidase (P450C21) involved in glucocorticoid and mineralocorticoid synthesis. The diagnosis of 21-OHD is based on the comprehensive evaluation of clinical manifestation, biochemical alteration and molecular genetics results. Due to the complex structure of CYP21A2, special techniques are required to perform delicate analysis to avoid the interference of its pseudogene. Recently, the state-of-the-art diagnostic methods were applied to the clinic gradually, including the steroid hormone profiling and third generation sequencing. To standardize the laboratory diagnosis of 21-OHD, this consensus was drafted on the basis of the extensive knowledge, the updated progress and the published consensuses and guidelines worldwide by expert discussion organized by Rare Diseases Group of Pediatric Branch of Chinese Medical Association, Medical Genetics Branch of Chinese Medical Doctor Association, Birth Defect Prevention and Molecular Genetics Branch of China Maternal and Child Health Association. and Molecular Diagnosis Branch of Shanghai Medical Association.
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Hiperplasia Suprarrenal Congénita , Niño , Humanos , Hiperplasia Suprarrenal Congénita/diagnóstico , Hiperplasia Suprarrenal Congénita/genética , Esteroide 21-Hidroxilasa/genética , Consenso , China , Técnicas de Laboratorio Clínico , MutaciónRESUMEN
Dystrophinopathies, including Duchenne muscular dystrophy, Becker muscular dystrophy and dilated cardiomyopathy, are X-linked recessive genetic disorders due to variants of the dystrophin gene, which can seriously affect quality of life and health. Genetic diagnosis plays a crucial role in their diagnosis, treatment, and prevention. How to rationally select and standardize the use of various genetic techniques is a skill that clinicians must acquire. By compiling expertise of experts from the relevant areas and guidelines published home and abroad, this consensus has provided a guidance from the perspective of genetic diagnosis for the selection of genetic techniques, testing strategies, and detection process for dystrophinopathies.
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Cardiomiopatía Dilatada , Distrofia Muscular de Duchenne , Humanos , Calidad de Vida , Consenso , Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Cardiomiopatía Dilatada/genética , ElectrocardiografíaRESUMEN
Glycogen storage disease (GSD) Type VI is a glycogenolysis disorder caused by variants of PYGL. Knowledge about this disease is limited because only approximately 50 cases have been reported. We investigated the clinical profiles, molecular diagnosis, and treatment outcomes in patients with GSD VI from 2000 to 2021. The main initial clinical features of this cohort include hepatomegaly, short stature, elevated liver transaminases, hypertriglyceridemia, fasting hypoglycemia, and hyperuricemia. After uncooked cornstarch treatment, the stature and biochemical parameters improved significantly (p < 0.05). However, hyperuricemia recurred in most patients during adolescence. Among the 56 GSD VI patients, 54 biallelic variants and two single allelic variants of PYGL were identified, of which 43 were novel. There were two hotspot variants, c.1621-258_2178-23del and c.2467C>T p.(Gln823*), mainly in patients from Southwest and South China. c.1621-258_2178-23del is a 3.6 kb deletion that results in an out-of-frame deletion r.1621_2177del and an in-frame deletion r.1621_2265del. Our data show for the first time that long-term monitoring of uric acid is recommended for older GSD VI patients. This study also broadens the variant spectrum of PYGL and indicates that there are two hot-spot variants in China.
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Enfermedad del Almacenamiento de Glucógeno Tipo VI , Enfermedad del Almacenamiento de Glucógeno , Hiperuricemia , Adolescente , Estudios de Seguimiento , Glucógeno Fosforilasa de Forma Hepática , Enfermedad del Almacenamiento de Glucógeno/diagnóstico , Enfermedad del Almacenamiento de Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno Tipo VI/diagnóstico , HumanosRESUMEN
Genome sequencing (GS) has been used in the diagnosis of global developmental delay (GDD)/intellectual disability (ID). However, the performance of GS in patients with inconclusive results from chromosomal microarray analysis (CMA) and exome sequencing (ES) is unknown. We recruited 100 pediatric GDD/ID patients from multiple sites in China from February 2018 to August 2020 for GS. Patients have received at least one genomic diagnostic test before enrollment. Reanalysis of their CMA/ES data was performed. The yield of GS was calculated and explanations for missed diagnoses by CMA/ES were investigated. Clinical utility was assessed by interviewing the parents by phone. The overall diagnostic yield of GS was 21%. Seven cases could have been solved with reanalysis of ES data. Thirteen families were missed by previous CMA/ES due to improper methodology. Two remained unsolved after ES reanalysis due to complex variants missed by ES, and a CNV in untranslated regions. Follow-up of the diagnosed families revealed that nine families experienced changes in clinical management, including identification of targeted treatments, cessation of unnecessary treatment, and considerations for family planning. GS demonstrated high diagnostic yield and clinical utility in this undiagnosed GDD/ID cohort, detecting a wide range of variant types of different sizes in a single workflow.
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Discapacidad Intelectual , Niño , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/genética , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Análisis por Micromatrices/métodos , Estudios Prospectivos , Secuenciación del ExomaRESUMEN
PURPOSE: To compare the clinical characteristics of pediatric urolithiasis patients with positive and negative molecular diagnoses. METHODS: The clinical characteristics corresponding to pediatric urolithiasis patients that had undergone exome sequencing at our hospital between January 2016 and May 2021 were collected. Genetic analysis results were used to separate patients into positive and negative molecular diagnosis groups. Multivariate logistic regression analyses adjusted for visiting age, sex, ethnicity, province, and body mass index were used to compare differences in medical history, diagnostic imaging findings, and renal function between individuals with and without molecular diagnoses. RESULTS: In total, 194 patients with pediatric urolithiasis of unknown etiology underwent exome sequencing and were included in the present study, of whom 63 obtained urolithiasis-related molecular diagnoses. Relative to cases without a molecular diagnosis, those with a positive molecular diagnosis were more likely to be associated with a positive family history (OR 2.84, 95% CI 1.29-6.29, p = 0.008), consanguineous parents (OR 24.7, 95% CI 1.34-454, p = 0.002), early onset (OR 1.26, 95% CI 1.09-1.45, p < 0.001), nephrocalcinosis (OR 10.6, 95% CI 3.06-36.6, p < 0.001), cast stone (OR 18.9, 95% CI 4.40-81.1, p < 0.001), multiple stones (OR 13.9, 95% CI 6.39-30.2, p < 0.001), bilateral stones (OR 7.04, 95% CI 3.47-14.2, p < 0.001), a lower estimated glomerular filtration rate (OR 1.17, 95% CI 1.07-1.28, p < 0.001), and chronic kidney disease (OR 26.9, 95% CI 1.42-526, p < 0.001). CONCLUSION: A positive family history, consanguineous parents, early onset, nephrocalcinosis, severe stone burden, and impaired renal function are signals of concern that are suggestive of inherited urolithiasis.
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Nefrocalcinosis , Insuficiencia Renal Crónica , Urolitiasis , Niño , Femenino , Humanos , Masculino , Estudios Retrospectivos , Urolitiasis/diagnóstico , Urolitiasis/genéticaRESUMEN
Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.
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Anomalías Múltiples/genética , Anomalías Craneofaciales/genética , Ciclinas/genética , Discapacidades del Desarrollo/genética , Atrofia Muscular/genética , Mutación/genética , Trastornos del Neurodesarrollo/genética , Adolescente , Animales , Niño , Preescolar , Femenino , Haploinsuficiencia/genética , Heterocigoto , Humanos , Hipertelorismo/genética , Discapacidad Intelectual/genética , Masculino , Anomalías Musculoesqueléticas/genética , Malformaciones del Sistema Nervioso/genética , Fenotipo , Síndrome , Pez CebraRESUMEN
Long continuous stretches of homozygosity (LCSH) are associated with risk of recessive disorders. Though LCSH can be detected by SNP microarrays, additional testing is necessary to clarify the clinical significance. This study is to assess the yield of additional exome sequencing (ES) after LCSH detection and inform the likelihood of eventual diagnosis. In 2226 patients referred to SNP microarrays, 35 patients met the criteria of indicative LCSH. These patients were recruited and went through additional ES. The diagnostic yield was analyzed, and the LCSH pattern was compared between eventually diagnosed cases and those undiagnosed. The results showed additional ES attained a diagnostic yield of 31.4% (11/35), but only one-third of the yield (11.4%, 4/35) was relevant to LCSH. In contrast, two-thirds of the diagnostic variants (20%, 7/35) were de novo or dominantly inherited, irrelevant to the original LCSH finding. No particular LCSH pattern, including the chromosomal coverage or LCSH size, was found to associate with the diagnostic outcome. We concluded that additional ES after LCSH detection could reveal diagnostic variants, but it is strongly recommended to consider all possible inheritance mode, as the diagnostic variants may be irrelevant to the original LCSH finding.
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Genes Recesivos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Homocigoto , Polimorfismo de Nucleótido Simple , Variaciones en el Número de Copia de ADN , Humanos , Secuenciación del ExomaRESUMEN
PURPOSE: To investigate the prevalence of inherited causes in an early onset urolithiasis cohort and each metabolic subgroup. METHODS: A retrospective analysis of both metabolic and genomic data was performed for the first 105 pediatric urolithiasis patients who underwent exome sequencing at our hospital from February 2016 to October 2018. Measurements included the diagnostic yield of exome sequencing in the entire cohort and each metabolic subgroup (hyperoxaluria, hypocitraturia, hypercalciuria, hyperuricosuria and cystine stone subgroups). The conformity between molecular diagnoses and metabolic evaluation was also evaluated. RESULTS: The present study involved a cohort of 105 pediatric patients with urolithiasis, from which diagnostic variants were identified in 38 patients (36%), including 27 primary hyperoxaluria and 11 cystinuria. In the metabolic subgroup analyses, 41% hyperoxaluria cases were primary hyperoxaluria caused by monogenic defects, and 100% of the causes of cystine stones could be explained by monogenic defects. However, no appropriate inherited causes were identified for hypocitraturia, hypercalciuria, or hyperuricosuria in the cohort. A high conformity (100%) was obtained between the molecular diagnoses and metabolic evaluation. CONCLUSION: Exome sequencing in a cohort of 105 pediatric patients with urolithiasis yielded a genetic diagnosis in 36% of cases and the molecular diagnostic yield varies substantially across different metabolic abnormalities.
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Urolitiasis/diagnóstico , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios Retrospectivos , Urolitiasis/genética , Urolitiasis/metabolismo , Secuenciación del ExomaRESUMEN
Haploinsufficiency of ARID1B (AT-rich interaction domain 1B) has been involved in autism spectrum disorder, nonsyndromic and syndromic intellectual disability, and corpus callosum agenesis. Growth impairment is a major clinical feature caused by ARID1B mutations; however, the mechanistic link has not been elucidated. Here, we confirm that growth delay is a common characteristic of patients with ARID1B mutations, which may be associated with dysregulation of the Wnt/ß-catenin signaling pathway. An analysis of patients harboring pathogenic variants of ARID1B revealed that nearly half had short stature and nearly all had below-average height. Moreover, the percentage of patients with short stature increased with age. Knockdown of arid1b in zebrafish embryos markedly reduced body length and perturbed the expression of both chondrogenic and osteogenic genes including sox9a, col2a1a, runx2b, and col10a1. Knockout of Arid1b in chondrogenic ATDC5 cells inhibited chondrocyte proliferation and differentiation. Finally, Wnt/ß-catenin signaling was perturbed in Arid1b-depleted zebrafish embryos and Arid1b knockout ATDC5 cells. These data indicate that ARID1B modulates bone growth possibly via regulation of the Wnt/ß-catenin pathway, and may be an appropriate target for gene therapy in disorders of growth and development.
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Proteínas de Unión al ADN/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Trastornos del Crecimiento/diagnóstico , Trastornos del Crecimiento/genética , Mutación , Factores de Transcripción/genética , Vía de Señalización Wnt , Alelos , Animales , Animales Modificados Genéticamente , Pesos y Medidas Corporales , Diferenciación Celular/genética , Preescolar , Proteínas de Unión al ADN/metabolismo , Facies , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Estudios de Asociación Genética/métodos , Genotipo , Gráficos de Crecimiento , Trastornos del Crecimiento/metabolismo , Humanos , Mutación con Pérdida de Función , Masculino , Fenotipo , Factores de Transcripción/metabolismo , Pez CebraRESUMEN
BACKGROUND: Variants perturbing the normal splicing of pre-mRNA can lead to human diseases. The splice-altering effect and eventual consequence on gene function was sometimes uncertain and hinders a definitive molecular diagnosis. METHODS: The impact of four rare intronic variants on splicing was analyzed through reverse transcription - polymerase chain reaction (RT-PCR) analysis of mRNA derived from the peripheral blood of patients. The results were compared with in-silico prediction. Potential implication on molecular diagnosis was discussed. RESULTS: Four rare intronic variants of SLC9A6, DLG3, GAA, and OCRL were identified in patients with suspected disorders, respectively. Although these four variants were all predicted to alter splicing by in-silico tools, RT-PCR analysis of mRNA derived from peripheral blood showed these variants affected splicing in different ways: c.899+3_899+6del of SLC9A6 resulted in one-exon skipping and an out-of-frame transcript; c.905-2A > G of DLG3 resulted in a mix of in-frame transcripts; c.1195-11T > A of GAA resulted in the in-frame insertion of nine nucleotides; c.723-2A > C of OCRL resulted in one-exon skipping and in-frame deletion of 102 nucleotides. The consequence revealed by mRNA analysis is essential for accurate interpretation of pathogenicity. CONCLUSION: Four intronic variants all caused aberrant mRNA splicing. For intronic variants with uncertain impact on splicing, mRNA analysis is helpful for ascertainment of alternative splicing and accurate interpretation of pathogenicity.
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Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Mutación , Empalme del ARN , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Preescolar , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Humanos , Lactante , Masculino , Microcefalia/genética , Microcefalia/patología , Proteínas Nucleares/genética , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/patología , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Pronóstico , ARN Mensajero/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Transcripción/genética , alfa-Glucosidasas/genéticaRESUMEN
BACKGROUND: Capture sequencing (CS) is widely applied to detect small genetic variations such as single nucleotide variants or indels. Algorithms based on depth comparison are becoming available for detecting copy number variation (CNV) from CS data. However, a systematic evaluation with a large sample size has not been conducted to evaluate the efficacy of CS-based CNV detection in clinical diagnosis. METHODS: We retrospectively studied 3010 samples referred to our diagnostic laboratory for CS testing. We used 68 chromosomal microarray analysis-positive samples (true set [TS]) and 1520 reference samples to build a robust CS-CNV pipeline. The pipeline was used to detect candidate clinically relevant CNVs in 1422 undiagnosed samples (undiagnosed set [UDS]). The candidate CNVs were confirmed by an alternative method. RESULTS: The CS-CNV pipeline detected 78 of 79 clinically relevant CNVs in TS samples, with analytical sensitivity of 98.7% and positive predictive value of 49.4%. Candidate clinically relevant CNVs were identified in 106 UDS samples. CNVs were confirmed in 96 patients (90.6%). The diagnostic yield was 6.8%. The molecular etiology includes aneuploid (n = 7), microdeletion/microduplication syndrome (n = 40), and Mendelian disorders (n = 49). CONCLUSIONS: These findings demonstrate the high yield of CS-based CNV. With further improvement of our CS-CNV pipeline, the method may have clinical utility for simultaneous evaluation of CNVs and small variations in samples referred for pre- or postnatal analysis.
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Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Algoritmos , Aneuploidia , Anomalías Congénitas/diagnóstico , Exoma , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios RetrospectivosRESUMEN
BACKGROUND: Hyperphenylalaninemia is the most common genetic metabolic disease. Early treatment prevents brain injury effectively. The present study aimed to detect the exact amino acid status of patients with hyperphenylalaninemia before treatment. METHODS: Data of 116 newborn patients from our Newborn Screening Center and 161 older patients from our clinic before treatment were collected. The content of 17 amino acids in their blood was determined by tandem mass spectrometry and compared with normal controls. Relationship between phenylalanine and other amino acids in patients was analyzed using the smoothing curve fitting and threshold effect analysis. RESULTS: Most amino acids in the blood of patients were within the normal range; however, they were different significantly from those of the normal children. Newborn patients showed higher phenylalanine (346.30 vs 45.90 µmol/L), valine (121.50 vs 110.30 µmol/L), citrulline, ornithine and lower tyrosine (52.97 vs 66.12 µmol/L), threonine (68.68 vs 78.21 µmol/L), glutamine levels than observed in normal newborns. Older patients showed significantly higher phenylalanine (844.00 vs 51.82 µmol/L), valine (117.60 vs 110.90 µmol/L), histidine, serine and lower tyrosine (55.97 vs 67.31 µmol/L), threonine (35.94 vs 51.89 µmol/L), alanine, asparagine, glutamic acid, methionine, arginine, glycine, ornithine, glutamine content than found in matched normal children. Tyrosine, valine, ornithine, and threonine in newborn patients and tyrosine, glycine, glutamine, and threonine in older patients had a nonlinear correlation with phenylalanine levels with obvious threshold effect and clear inflection points. CONCLUSION: Significant difference was observed in the amino acid status between pretherapeutic hyperphenylalaninemia patients and normal children. Some amino acids showed notable threshold effect with phenylalanine level in a nonlinear pattern.