RESUMO
PURPOSE: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified. METHODS: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes. RESULTS: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect. CONCLUSION: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome.
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Síndrome de Angelman , Transtornos do Neurodesenvolvimento , Humanos , Síndrome de Angelman/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Transtornos do Neurodesenvolvimento/genética , FenótipoRESUMO
INTRODUCTION: Pompe disease is a rare, lysosomal disorder, characterized by intra-lysosomal glycogen accumulation due to an impaired function of α-glucosidase enzyme. The laboratory testing for Pompe is usually performed by enzyme activity, genetic test, or urine glucose tetrasaccharide (Glc4) screening by HPLC. Despite being a good preliminary marker, the Glc4 is not specific for Pompe. OBJECTIVE: The purpose of the present study was to develop a simple methodology using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for targeted quantitative analysis of Glc4 combined with untargeted metabolic profiling in a single analytical run to search for complementary biomarkers in Pompe disease. METHODS: We collected 21 urine specimens from 13 Pompe disease patients and compared their metabolic signatures with 21 control specimens. RESULTS: Multivariate statistical analyses on the untargeted profiling data revealed Glc4, creatine, sorbitol/mannitol, L-phenylalanine, N-acetyl-4-aminobutanal, N-acetyl-L-aspartic acid, and 2-aminobenzoic acid as significantly altered in Pompe disease. This panel of metabolites increased sample class prediction (Pompe disease versus control) compared with a single biomarker. CONCLUSION: This study has demonstrated the potential of combined acquisition methods in LC-HRMS for Pompe disease investigation, allowing for routine determination of an established biomarker and discovery of complementary candidate biomarkers that may increase diagnostic accuracy, or improve the risk stratification of patients with disparate clinical phenotypes.
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Doença de Depósito de Glicogênio Tipo II , Humanos , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/urina , Metabolômica/métodos , Biomarcadores/urina , Fenótipo , Espectrometria de Massas em TandemRESUMO
Heterozygous pathogenic variants in SLC12A2 are reported in patients with nonsyndromic hearing loss. Recently, homozygous loss-of-function variants have been reported in two patients with syndromic intellectual disability, with or without hearing loss. However, the clinical and molecular spectrum of SLC12A2 disease has yet to be characterized and confirmed. Using whole-exome sequencing, we detected a homozygous splicing variant in four patients from two independent families with severe developmental delay, microcephaly, respiratory abnormalities, and subtle dysmorphic features, with or without congenital hearing loss. We also reviewed the reported cases with pathogenic variants associated with autosomal dominant and recessive forms of the SLC12A2 disease. About 50% of the cases have syndromic and nonsyndromic congenital hearing loss. All patients harboring the recessive forms of the disease presented with severe global developmental delay. Interestingly, all reported variants are located in the c-terminal domain, suggesting a critical role of this domain for the proper function of the encoded co-transporter protein. In conclusion, our study provides an additional confirmation of the autosomal recessive SLC12A2 disease.
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Surdez/genética , Predisposição Genética para Doença , Deficiência Intelectual/genética , Membro 2 da Família 12 de Carreador de Soluto/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Pré-Escolar , Surdez/complicações , Surdez/diagnóstico por imagem , Surdez/patologia , Exoma/genética , Feminino , Genes Recessivos/genética , Homozigoto , Humanos , Lactente , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/patologia , Masculino , Mutação/genética , Linhagem , Fenótipo , Splicing de RNA/genética , Membro 2 da Família 12 de Carreador de Soluto/deficiência , Sequenciamento do ExomaRESUMO
Biallelic variants in the USP53 gene have recently been reported to segregate with normal gamma glutamyltransferase (GGT) cholestasis. Using whole-exome sequencing (WES), we detected two USP53 homozygous variants (c.951delT; p. Phe317fs and c.1744C>T; p. Arg582*) in five additional cases, including an unpublished cousin of a previously described family with intractable itching and normal GGT cholestasis. Three patients, a child and two adults, presented with recurrent episodes of normal GGT cholestasis, consistent with a diagnosis of benign recurrent intrahepatic cholestasis (BRIC). Cholangiopathic changes, possibly autoimmune in origin, were recognized in some patients. Additional phenotypic details in one patient included an enlarged left kidney, and speech/developmental delay. Notably, two patients exhibited a complete response to rifampicin, and one responded to ursodeoxycholic acid (UDCA). Two adult patients were suspected to have autoimmune liver disease and treated with steroids. This report describes new cases of USP53 disease presenting with normal GGT cholestasis or BRIC in three children and two adults. We also describe the novel finding of a dramatic response to rifampicin. The association of cholangiopathy with normal GGT cholestasis provides a diagnostic challenge and remains poorly understood.
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Colangite/tratamento farmacológico , Colestase/tratamento farmacológico , Homozigoto , Mutação , Rifampina/farmacologia , Proteases Específicas de Ubiquitina/genética , gama-Glutamiltransferase/metabolismo , Adolescente , Adulto , Criança , Colangite/genética , Colangite/patologia , Colestase/genética , Colestase/patologia , Feminino , Humanos , Lactente , Masculino , Inibidores da Síntese de Ácido Nucleico/farmacologia , Linhagem , Prognóstico , Sequenciamento do ExomaRESUMO
Protein disulfide isomerase A6 (PDIA6) is an unfolded protein response (UPR)-regulating protein. PDIA6 regulates the UPR sensing proteins, Inositol requiring enzyme 1, and EIF2AK3. Biallelic inactivation of the two genes in mice and humans resulted in embryonic lethality, diabetes, skeletal defects, and renal insufficiency. We recently showed that PDIA6 inactivation in mice caused embryonic and early lethality, diabetes and immunodeficiency. Here, we present a case with asphyxiating thoracic dystrophy (ATD) syndrome and infantile-onset diabetes. Whole exome sequencing revealed a homozygous frameshift variant in the PDIA6 gene. RNA expression was reduced in a gene dosage-dependent manner, supporting a loss-of-function effect of this variant. Phenotypic correlation with the mouse model recapitulated the growth defect and delay, early lethality, coagulation, diabetes, immunological, and polycystic kidney disease phenotypes. In general, the phenotype of the current patient is consistent with phenotypes associated with the disruption of PDIA6 and the sensors of UPR in mice and humans. This is the first study to associate ATD to the UPR gene, PDIA6. We recommend screening ATD cases with or without insulin-dependent diabetes for variants in PDIA6.
Assuntos
Síndrome de Ellis-Van Creveld/genética , Doenças do Prematuro/genética , Mutação com Perda de Função , Isomerases de Dissulfetos de Proteínas/genética , Resposta a Proteínas não Dobradas/genética , Anormalidades Múltiplas/genética , Alelos , Animais , Consanguinidade , Síndrome de Ellis-Van Creveld/diagnóstico por imagem , Técnicas de Inativação de Genes , Idade Gestacional , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , LinhagemRESUMO
PURPOSE: Asparagine synthetase deficiency (ASNSD) is a rare neurometabolic disease. Patients may not demonstrate low asparagine levels, which highlights the advantage of molecular over biochemical testing in the initial work-up of ASNSD. We aimed to further delineate the ASNSD variant and phenotypic spectrum and determine the value of biochemical testing as a frontline investigation in ASNSD. METHODS: We retrospectively collected the clinical and molecular information on 13 families with ASNSD from the major metabolic clinics in Saudi Arabia. RESULTS: The major phenotypes included congenital microcephaly (100%), facial dysmorphism (100%), global developmental delay (100%), brain abnormalities (100%), spasticity (86%), and infantile-onset seizures (93%). Additional unreported phenotypes included umbilical hernia, osteopenia, eczema, lung hypoplasia, and hearing loss. Overall, seven homozygous variants accounted for ASNSD. The p.Tyr398Cys and p.Asn75Ile variants accounted for 54% of the cases. The clinical sensitivity and specificity of the proposed biochemical analysis of cerebrospinal fluid (CSF) for the detection of patients with ASNSD were 83% and 98%, respectively. CONCLUSION: Our study describes the largest reported ASNSD cohort with clinical, molecular, and biochemical characterization. Taking into consideration the suboptimal sensitivity of biochemical screening, the delineation of the phenotype variant spectrum is of diagnostic utility for accurate diagnosis, prognosis, counseling, and carrier screening.
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Aspartato-Amônia Ligase , Deficiência Intelectual , Microcefalia , Aspartato-Amônia Ligase/genética , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Estudos Retrospectivos , Arábia Saudita/epidemiologiaRESUMO
Adult-onset noncirrhotic hyperammonemia (NCH) is poorly understood and has a high morbidity and mortality. To elucidate the etiology and management of NCH, we performed a retrospective analysis of 23 adults (median age 51) with NCH treated between 2014 and 2020 at two academic medical centers. Hyperammonemia was diagnosed in all cases during the evaluation of altered mental status, with 22% presenting with seizures. Peak ammonia levels were >200 µmol/L in 70% of cases. Defects in ammonia metabolism were assessed using urea cycle biochemical testing, germline genetic testing, and testing for urease-producing infectious agents. Ammonia metabolism defects in these cases appear attributable to four major sources: (a) infection with urease-producing organism (n = 5); (b) previously undiagnosed inborn errors of metabolism (IEMs) (n = 4); (c) clinical exposures causing acquired urea cycle dysfunction (n = 6); and (d) unexplained acquired urea cycle dysfunction (uaUCD) (n = 8), as evidenced by biochemical signatures of urea cycle dysfunction without a genetic or clinical exposure. Severe protein malnutrition appeared to be a reversible risk factor for uaUCD. Overall, 13% of our cohort died prior to resolution of hyperammonemia, 26% died after hyperammonemia resolution, 57% survived after having reversible neurological changes, and 4% survived with irreversible neurological changes. Renal replacement therapy for ammonia clearance was often utilized for patients with an ammonia level above 250 µmol/L and patients were frequently empirically treated with antibiotics targeting urea-splitting organisms. Our study demonstrates that acquired urea cycle dysfunction, IEMs and urease-producing infections are major sources of adult-onset NCH and highlights successful management strategies for adult-onset NCH.
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Hiperamonemia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Adulto , Idade de Início , Idoso , Amônia/sangue , Feminino , Humanos , Hiperamonemia/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Convulsões/complicações , Análise de Sobrevida , Ureia/metabolismo , Adulto JovemAssuntos
Acidose Láctica/etiologia , Arritmias Cardíacas/etiologia , Hiperamonemia/etiologia , Erros Inatos do Metabolismo Lipídico/diagnóstico , Acidose Láctica/sangue , Acidose Láctica/diagnóstico , Arritmias Cardíacas/sangue , Arritmias Cardíacas/diagnóstico , Carnitina/análogos & derivados , Carnitina/sangue , Feminino , Humanos , Hiperamonemia/sangue , Hiperamonemia/diagnóstico , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/complicações , Erros Inatos do Metabolismo Lipídico/genética , Proteínas de Membrana Transportadoras/genética , MutaçãoAssuntos
Anormalidades Múltiplas/diagnóstico , Acidose/etiologia , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Hipoglicemia/etiologia , Tioléster Hidrolases/deficiência , Anormalidades Múltiplas/genética , Acidose/genética , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/genética , Feminino , Humanos , Hipoglicemia/genética , Lactente , Mutação , Fenótipo , Tioléster Hidrolases/genéticaRESUMO
BACKGROUND: Neosaxitoxin (NeoSTX) is a site-1 sodium channel blocker that produces prolonged local anesthesia in animals and humans. Under a Food and Drug Administration-approved phase 1 Investigational New Drug trial, the authors evaluated safety and efficacy of NeoSTX alone and combined with 0.2% bupivacaine (Bup) with and without epinephrine. METHODS: The authors conducted a double-blind, randomized, controlled trial involving healthy male volunteers aged 18 to 35 yr receiving two 10-ml subcutaneous injections. Control sites received Bup. In part 1, active sites received (1) 5 to 40 µg NeoSTX+Saline (NeoSTX-Saline), (2) 5 to 40 µg NeoSTX+Bup (NeoSTX-Bup), or (3) placebo (Saline). In part 2, active sites received 10 or 30 µg NeoSTX+Bup+Epinephrine (NeoSTX-Bup-Epi) or placebo. Primary outcome measures were safety and adverse events associated with NeoSTX. Secondary outcomes included clinical biochemistry, NeoSTX pharmacokinetics, and cutaneous hypoesthesia. RESULTS: A total of 84 subjects were randomized and completed the two-part trial with no serious adverse events or clinically significant physiologic impairments. Perioral numbness and tingling increased with NeoSTX dose for NeoSTX-Saline and NeoSTX-Bup. All symptoms resolved without intervention. NeoSTX-Bup-Epi dramatically reduced symptoms compared with other NeoSTX combinations (tingling: 0 vs. 70%, P = 0.004; numbness: 0 vs. 60%, P = 0.013) at the same dose. Mean peak plasma NeoSTX concentration for NeoSTX-Bup-Epi was reduced at least two-fold compared with NeoSTX-Saline and NeoSTX-Bup (67 ± 14, 134 ± 63, and 164 ± 81 pg/ml, respectively; P = 0.016). NeoSTX-Bup showed prolonged cutaneous block duration compared with Bup, NeoSTX-Saline, or placebo, at all doses. Median time to near-complete recovery for 10 µg NeoSTX-Bup-Epi was almost five-fold longer compared with Bup (50 vs. 10 h, P = 0.007). CONCLUSION: NeoSTX combinations have a tolerable side effect profile and appear promising for prolonged local anesthesia.
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Anestesia Local/métodos , Bupivacaína/administração & dosagem , Epinefrina/administração & dosagem , Saxitoxina/análogos & derivados , Adulto , Anestesia Local/efeitos adversos , Anestésicos Locais , Bupivacaína/efeitos adversos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Quimioterapia Combinada , Epinefrina/efeitos adversos , Humanos , Hipestesia/induzido quimicamente , Masculino , Medição da Dor/efeitos adversos , Medição da Dor/métodos , Saxitoxina/administração & dosagem , Saxitoxina/efeitos adversos , Adulto JovemRESUMO
Immunosuppressant drugs (ISDs) are commonly prescribed to solid organ transplant patients. Their narrow therapeutic index and potential for toxicity necessitates careful monitoring of blood concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are increasingly used for ISD measurement. However, there remain many challenges with this methodology, particularly regarding interassay variability. The Thermo Scientific Prelude is an online extraction/liquid chromatography platform that uses turbulent flow technology coupled with MS/MS. A multicenter evaluation of the Prelude for the measurement of cyclosporine A, tacrolimus, and sirolimus is described. ISDs were measured at each site using standardized protocols. Sample preparation liquid chromatography-MS/MS was performed using the Prelude coupled to a TSQ Vantage. Chromatography was achieved with a Cyclone-P TurboFlow/Accucore C8 column combination using a multisolvent loading and eluting pump system. Mass spectrometry acquisitions were performed in selective reaction monitoring mode and data processed using TraceFinder (version 3.1). Multisite mean imprecision for cyclosporine A ranged from 8.8% (54 mcg/L) to 9.8% (450 mcg/L); for tacrolimus, 4.7% (15.5 mcg/L) to 12.6% (2.5 mcg/L); for sirolimus, 7.4% (19.9 mcg/L) to 16.5% (2.6 mcg/L). Approximately 110 specimens were used for method comparison. For cyclosporine A, mean bias against the multisite mean ranged from -18% to 1%; for tacrolimus, values ranged from -7% to 4%; for sirolimus, values ranged from -4% to 2%. Comparisons of multisite mean Prelude results with routine ISD method results was also performed for cyclosporine A (slope = 0.7878, intercept = 24.16, r = 0.98), tacrolimus (slope = 0.9391, intercept = 0.1017, r = 98), and sirolimus (slope = 0.9618, intercept = 0.1483, r = 0.97). The Prelude ISD method offers acceptable and comparable multisite performance. This study has also highlighted the importance of adopting standardized protocols and LC-MS/MS methods for better comparability between ISD assays.
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Cromatografia Líquida/métodos , Monitoramento de Medicamentos/métodos , Imunossupressores/sangue , Espectrometria de Massas em Tandem/métodos , Ciclosporina/sangue , Humanos , Sirolimo/sangue , Tacrolimo/sangueAssuntos
Insuficiência Adrenal/etiologia , Deficiências do Desenvolvimento/etiologia , Glicerol Quinase/deficiência , Glicerol/metabolismo , Deficiência Intelectual/etiologia , Insuficiência Adrenal/metabolismo , Pré-Escolar , Receptor Nuclear Órfão DAX-1/genética , Deficiências do Desenvolvimento/metabolismo , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Humanos , Recém-Nascido , Deficiência Intelectual/metabolismo , MasculinoAssuntos
Anemia Sideroblástica/genética , DNA Mitocondrial/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação de Sentido Incorreto , Adolescente , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Criança , Análise Mutacional de DNA/métodos , Feminino , Estudos de Associação Genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Lactente , Masculino , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação Puntual , Recidiva , SíndromeRESUMO
BACKGROUND: Mucopolysaccharidoses (MPSs) are inherited genetic diseases caused by an absence or deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Undiagnosed patients, or those without adequate treatment in early life, can be severely and irreversibly affected by the disease. In this study, we applied liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to identify potential biomarkers for MPS disorders to better understand how MPS may affect the metabolome of patients. METHODS: Urine samples from 37 MPS patients (types I, II, III, IV, and VI; untreated and treated with enzyme replacement therapy (ERT)) and 38 controls were analyzed by LC-HRMS. Data were processed by an untargeted metabolomics workflow and submitted to multivariate statistical analyses to reveal significant differences between the MPS and control groups. RESULTS: A total of 12 increased metabolites common to all MPS types were identified. Dipeptides, amino acids and derivatives were increased in the MPS group compared to controls. N-acetylgalactosamines 4- or 6-sulfate, important constituents of GAGs, were also elevated in MPS patients, most prominently in those with MPS VI. Notably, treated patients exhibited lower levels of the aforementioned acylaminosugars than untreated patients in all MPS types. CONCLUSIONS: Untargeted metabolomics has enabled the detection of metabolites that could improve our understanding of MPS physiopathology. These potential biomarkers can be utilized in screening methods to support diagnosis and ERT monitoring.