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1.
NPJ Genom Med ; 8(1): 16, 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37419908

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of kidney failure and is primarily associated with PKD1 or PKD2. Approximately 10% of patients remain undiagnosed after standard genetic testing. We aimed to utilise short and long-read genome sequencing and RNA studies to investigate undiagnosed families. Patients with typical ADPKD phenotype and undiagnosed after genetic diagnostics were recruited. Probands underwent short-read genome sequencing, PKD1 and PKD2 coding and non-coding analyses and then genome-wide analysis. Targeted RNA studies investigated variants suspected to impact splicing. Those undiagnosed then underwent Oxford Nanopore Technologies long-read genome sequencing. From over 172 probands, 9 met inclusion criteria and consented. A genetic diagnosis was made in 8 of 9 (89%) families undiagnosed on prior genetic testing. Six had variants impacting splicing, five in non-coding regions of PKD1. Short-read genome sequencing identified novel branchpoint, AG-exclusion zone and missense variants generating cryptic splice sites and a deletion causing critical intron shortening. Long-read sequencing confirmed the diagnosis in one family. Most undiagnosed families with typical ADPKD have splice-impacting variants in PKD1. We describe a pragmatic method for diagnostic laboratories to assess PKD1 and PKD2 non-coding regions and validate suspected splicing variants through targeted RNA studies.

2.
Genome Biol ; 24(1): 118, 2023 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-37198692

RESUMO

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .


Assuntos
Sítios de Splice de RNA , Splicing de RNA , Humanos , Íntrons , Aprendizado de Máquina , Mutação
3.
J Neurosci Res ; 90(6): 1126-35, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22354615

RESUMO

Neuropeptide Y (NPY) and peptide YY (PYY) are differentially expressed throughout the olfactory neuroepithelium (ON), with NPY expression present in sustentacular cells, olfactory ensheathing cells, and olfactory receptor neurons and PYY expressed only in sustentacular cells. Examination of the anatomical morphology of the ON in NPY knockout (NPY⁻/⁻) and PYY knockout (PYY⁻/⁻) mice shows that there are significantly more neurons in PYY⁻/⁻ mice and significantly fewer neurons in NPY⁻/⁻ mice. Interestingly, the mature neurons of NPY⁻/⁻ mice were undergoing apoptosis. The transcription factor Mash1, which is critical in the production of olfactory precursors, is also differentially expressed in NPY⁻/⁻ and PYY⁻/⁻ ON. It is upregulated in the neurons of NPY⁻/⁻ mice and unchanged in PYY⁻/⁻ mice. Furthermore, significantly fewer olfactory neurospheres are present in cultures prepared from PYY⁻/⁻ mice in the first 2 weeks compared with NPY⁻/⁻ and wild-type mice. Together these results suggest that, during olfactory neurogenesis, NPY acts as a trophic factor for the maturation and survival of olfactory receptor neurons, whereas PYY has an important role in the regulation of olfactory neuron differentiation.


Assuntos
Neurogênese/genética , Neuropeptídeo Y/fisiologia , Condutos Olfatórios/citologia , Neurônios Receptores Olfatórios/metabolismo , Peptídeo YY/fisiologia , Células-Tronco Adultas/fisiologia , Animais , Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caspase 3/metabolismo , Contagem de Células/métodos , Proliferação de Células , Regulação da Expressão Gênica/genética , Marcação In Situ das Extremidades Cortadas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeo Y/deficiência , Peptídeo YY/deficiência , RNA Mensageiro/metabolismo
4.
BMC Neurosci ; 13: 134, 2012 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-23113950

RESUMO

BACKGROUND: The olfactory neuroepithelium lines the upper nasal cavity and is in direct contact with the external environment and the olfactory bulbs. The ability to self-renew throughout life and the reproducible recovery after injury, make it a model tissue to study mechanisms underlying neurogenesis. In this study, X-rays were used to disrupt proliferating olfactory stem cell populations and to assess their role in the cellular and morphological changes involved in olfactory neurogenic processes. RESULTS: We have analysed the histological and functional effects of a sub-lethal dose of X-rays on the adult mouse olfactory neuroepithelium at 2 hours, 24 hours, 1 week, 2 weeks and 5 weeks. We have shown an immediate cessation of proliferating olfactory stem cells as shown by BrdU, Ki67 and pH3 expression. At 24 hours there was an increase in the neural transcription factors Mash1 and Pax6 expression, and a disruption of the basal lamina and increase in glandular cell marker expression at 1 week post-irradiation. Coincident with these changes was an impairment of the olfactory function in vivo. CONCLUSIONS: We have shown significant changes in basal cell proliferation as well as morphological changes in the olfactory neuroepithelium following X-ray irradiation. There is involvement of the basal lamina as well as a clear role for glandular and sustentacular cells.


Assuntos
Células Neuroepiteliais/citologia , Células Neuroepiteliais/efeitos da radiação , Neurogênese/efeitos da radiação , Bulbo Olfatório/efeitos da radiação , Neurônios Receptores Olfatórios/efeitos da radiação , Olfato/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Proteínas do Olho/biossíntese , Proteínas de Homeodomínio/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/citologia , Proteína de Marcador Olfatório/biossíntese , Neurônios Receptores Olfatórios/citologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Proteínas Repressoras/biossíntese , Células-Tronco/efeitos da radiação , Fatores de Tempo
5.
Eur J Hum Genet ; 29(5): 760-770, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33437033

RESUMO

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is common, with a prevalence of 1/1000 and predominantly caused by disease-causing variants in PKD1 or PKD2. Clinical diagnosis is usually by age-dependent imaging criteria, which is challenging in patients with atypical clinical features, without family history, or younger age. However, there is increasing need for definitive diagnosis of ADPKD with new treatments available. Sequencing is complicated by six pseudogenes that share 97% homology to PKD1 and by recently identified phenocopy genes. Whole-genome sequencing can definitively diagnose ADPKD, but requires validation for clinical use. We initially performed a validation study, in which 42 ADPKD patients underwent sequencing of PKD1 and PKD2 by both whole-genome and Sanger sequencing, using a blinded, cross-over method. Whole-genome sequencing identified all PKD1 and PKD2 germline pathogenic variants in the validation study (sensitivity and specificity 100%). Two mosaic variants outside pipeline thresholds were not detected. We then examined the first 144 samples referred to a clinically-accredited diagnostic laboratory for clinical whole-genome sequencing, with targeted-analysis to a polycystic kidney disease gene-panel. In this unselected, diagnostic cohort (71 males :73 females), the diagnostic rate was 70%, including a diagnostic rate of 81% in patients with typical ADPKD (98% with PKD1/PKD2 variants) and 60% in those with atypical features (56% PKD1/PKD2; 44% PKHD1/HNF1B/GANAB/ DNAJB11/PRKCSH/TSC2). Most patients with atypical disease did not have clinical features that predicted likelihood of a genetic diagnosis. These results suggest clinicians should consider diagnostic genomics as part of their assessment in polycystic kidney disease, particularly in atypical disease.


Assuntos
Frequência do Gene , Testes Genéticos/métodos , Doenças Renais Policísticas/genética , Sequenciamento Completo do Genoma/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Testes Genéticos/normas , Glucosidases/genética , Proteínas de Choque Térmico HSP40/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Doenças Renais Policísticas/diagnóstico , Receptores de Superfície Celular/genética , Sensibilidade e Especificidade , Canais de Cátion TRPP/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Sequenciamento Completo do Genoma/normas
6.
J Neurochem ; 105(3): 641-52, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18088353

RESUMO

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.


Assuntos
Proliferação de Células , Neuropeptídeo Y/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores de Neuropeptídeo Y/fisiologia , Células-Tronco/metabolismo , Fatores Etários , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Forma Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração Nervosa/genética , Plasticidade Neuronal/genética , Neuropeptídeo Y/genética , Mucosa Olfatória/citologia , Receptores de Neuropeptídeo Y/genética , Transdução de Sinais/genética , Esferoides Celulares
7.
Neuropeptides ; 68: 90-98, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29530408

RESUMO

The mouse olfactory neuroepithelium (ON) is comprised of anatomically distinct populations of cells in separate regions; apical (sustentacular and microvillar), neuronal (olfactory sensory neurons) and basal (horizontal and globose basal cells). The existence of microvillar cells (MVCs) is well documented but their nature and function remains unclear. An important transcription factor for the differentiation of MVCs is Skn-1a, with loss of function of Skn-1a in mice resulting in a complete loss of Trpm-5 expressing MVCs, while olfactory sensory neuron differentiation is normal. Our previous research has shown that neuropeptide Y (NPY) is expressed in MVCs and is important in the neuroproliferation of olfactory precursors. This study showed that following X-ray irradiation of the snout of wildtype mice, which decreases the proliferation of basal precursor cells, the numbers of Trpm-5-positive MVCs is increased at 2 and 5 weeks post-irradiation compared to controls. Skn-1a expression in the ON following X-ray irradiation also increases at 2 weeks post-irradiation in a regionally specific manner matching the expression pattern of Trpm-5-positive MVCs. In parallel, NPYCre knock-in mice were used to examine the expression of Skn-1a following activation of NPY unilaterally in the ON (unilateral nasal irrigation of AAV-NPY-FLEX). These experiments demonstrated that Skn-1a is only expressed when NPY is activated in MVCs. Therefore the expression of NPY is necessary for the transcription factor-mediated differentiation of olfactory MVCs.


Assuntos
Diferenciação Celular , Neuropeptídeo Y/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Mucosa Olfatória/efeitos da radiação
8.
Eur J Hum Genet ; 24(11): 1584-1590, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27165007

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder and is due to disease-causing variants in PKD1 or PKD2. Strong genotype-phenotype correlation exists although diagnostic sequencing is not part of routine clinical practice. This is because PKD1 bears 97.7% sequence similarity with six pseudogenes, requiring laborious and error-prone long-range PCR and Sanger sequencing to overcome. We hypothesised that whole-genome sequencing (WGS) would be able to overcome the problem of this sequence homology, because of 150 bp, paired-end reads and avoidance of capture bias that arises from targeted sequencing. We prospectively recruited a cohort of 28 unique pedigrees with ADPKD phenotype. Standard DNA extraction, library preparation and WGS were performed using Illumina HiSeq X and variants were classified following standard guidelines. Molecular diagnosis was made in 24 patients (86%), with 100% variant confirmation by current gold standard of long-range PCR and Sanger sequencing. We demonstrated unique alignment of sequencing reads over the pseudogene-homologous region. In addition to identifying function-affecting single-nucleotide variants and indels, we identified single- and multi-exon deletions affecting PKD1 and PKD2, which would have been challenging to identify using exome sequencing. We report the first use of WGS to diagnose ADPKD. This method overcomes pseudogene homology, provides uniform coverage, detects all variant types in a single test and is less labour-intensive than current techniques. This technique is translatable to a diagnostic setting, allows clinicians to make better-informed management decisions and has implications for other disease groups that are challenged by regions of confounding sequence homology.


Assuntos
Testes Genéticos/métodos , Genoma Humano , Rim Policístico Autossômico Dominante/genética , Pseudogenes , Homologia de Sequência , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Deleção de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Rim Policístico Autossômico Dominante/diagnóstico , Análise de Sequência de DNA/métodos , Canais de Cátion TRPP/genética
9.
Brain Res Mol Brain Res ; 107(2): 195-200, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12487125

RESUMO

The GALR1 galanin receptor is expressed at high levels within the central nervous system. To determine which specific actions of galanin are mediated by GALR1, we have developed mice with an insertional inactivating mutation within the gene encoding GALR1 (Galr1). Homozygous Galr1-/- mice are viable and capable of breeding. They exhibit no significant difference in growth rate relative to Galr1+/+ controls but have reduced circulating levels of insulin-like growth factor-I (IGF-I) and exhibit spontaneous tonic-clonic seizures. The phenotype of these mice identifies a critical role for GALR1 in neuroendocrine regulation and in mediating the anti-seizure activity of galanin.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Galanina/metabolismo , Sistemas Neurossecretores/crescimento & desenvolvimento , Receptores de Neuropeptídeos/deficiência , Convulsões/genética , Animais , Comportamento Animal/fisiologia , Peso Corporal/genética , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Regulação para Baixo/genética , Feminino , Fator de Crescimento Insulin-Like I/deficiência , Lactação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/fisiopatologia , Fenótipo , Receptores de Galanina , Receptores de Neuropeptídeos/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Caracteres Sexuais , Distribuição por Sexo
10.
J Bone Miner Res ; 25(8): 1736-47, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20200977

RESUMO

The neuropeptide Y (NPY) system has been implicated in the regulation of bone homeostasis and osteoblast activity, but the mechanism behind this is unclear. Here we show that Y1 receptor signaling is directly involved in the differentiation of mesenchymal progenitor cells isolated from bone tissue, as well as the activity of mature osteoblasts. Importantly, the mRNA levels of two key osteogenic transcription factors, runx2 and osterix, as well as the adipogenic transcription factor PPAR-gamma, were increased in long bones of Y1(-/-) mice compared with wild-type mice. In vitro, bone marrow stromal cells (BMSCs) isolated from Y1(-/-) mice formed a greater number of mineralized nodules under osteogenic conditions and a greater number of adipocytes under adipogenic conditions than controls. In addition, both the number and size of fibroblast colony-forming units formed in vitro by purified osteoprogenitor cells were increased in the absence of the Y1 receptors, suggestive of enhanced proliferation and osteogenesis. Furthermore, the ability of two specific populations of mesenchymal progenitor cells isolated from bone tissue, an immature mesenchymal stem cell population and a more committed osteoprogenitor cell population, to differentiate into osteoblasts and adipocytes in vitro was enhanced in the absence of Y1 receptor signaling. Finally, Y1 receptor deletion also enhanced the mineral-producing ability of mature osteoblasts, as shown by increased in vitro mineralization by BMSCs isolated from osteoblast-specific Y1(-/-) mice. Together these data demonstrate that the NPY system, via the Y1 receptor, directly inhibits the differentiation of mesenchymal progenitor cells as well as the activity of mature osteoblasts, constituting a likely mechanism for the high-bone-mass phenotype evident in Y1(-/-) mice.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Adipócitos/citologia , Adipogenia , Animais , Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Calcificação Fisiológica , Contagem de Células , Ensaio de Unidades Formadoras de Colônias , Feminino , Deleção de Genes , Masculino , Camundongos , Neuropeptídeo Y/deficiência , Neuropeptídeo Y/metabolismo , Osteogênese/genética , Receptores de Neuropeptídeo Y/deficiência , Células Estromais/citologia , Células Estromais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
11.
Stem Cells ; 25(3): 621-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17110620

RESUMO

Many forms of deafness result from degeneration of the sensory cells for hearing, the hair cells in the cochlea. Stem cells offer a potential cell-based therapy for the treatment of deafness. Here, we investigate whether adult olfactory precursor cells can differentiate into hair cells in culture. Precursor cells were isolated from mouse olfactory neuroepithelium, were sphere-forming, showed proliferative capacity, and contained cells expressing neuronal and non-neuronal proteins. To induce differentiation, precursor cells were cocultured with cochlear cells and/or cochlear supernatant. Differentiated precursor cells were immunopositive for specific hair cell markers, including myosin VIIa, FM1-43, calretinin, phalloidin, and espin, and resembled hair cells anatomically and immunocytochemically in culture. The results demonstrate for the first time that adult olfactory precursor cells can differentiate into hair cell-like cells, thus providing a potential autotransplantation therapy for hearing loss.


Assuntos
Diferenciação Celular/fisiologia , Cóclea/citologia , Células Ciliadas Auditivas/citologia , Mucosa Olfatória/citologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos CBA
12.
Clin Endocrinol (Oxf) ; 56(3): 397-403, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11940053

RESUMO

OBJECTIVES: Galanin (GAL) is a neuropeptide widely expressed in the central and peripheral nervous system and in neuroendocrine tissue, including the adenohypophysis where, in humans, it is expressed in corticotrophs and in ACTH-producing adenomas. Previous analyses of human tissue have used antiserum against porcine GAL for detection of GAL immunoreactivity (GAL-IR) and no pathophysiological correlates have been reported. Given significant differences between the sequence of porcine and human GAL peptides, the aim of this study was to use antiserum raised against synthetic human GAL to investigate GAL-IR in non tumorous pituitaries and in pituitary adenomas, and to correlate GAL-IR with the clinical and hormonal characteristics of patients with Cushing's disease. PATIENTS: Six nontumorous pituitaries were obtained from autopsy and 151 pituitary adenomas, comprising 62 functioning (16 corticotroph, 26 somatotroph, 19 lactotroph and one thyrotroph) and 89 nonfunctioning adenomas, were obtained by surgery. RESULTS: All non tumorous pituitary glands showed GAL-IR in corticotrophs, in basophil cells within the neurohypophysis and in nerve fibres of the neurohypophysis. GAL-IR was found in a subset (10 of 16) of patients with ACTH-secreting tumours causing Cushing's syndrome. GAL-IR was rarely expressed in somatotroph adenomas and prolactinomas, but was expressed in approximately one-third of nonfunctioning tumours. GAL-IR was found in almost 90% of nonfunctioning tumours that were positive for ACTH. There were no significant differences in sex ratio, age at presentation or 24-h urinary free cortisol secretion in the subset of patients with Cushing's disease positive (n = 10) or negative (n = 6) for GAL-IR. However, Cushing's patients positive for GAL-IR tended to have smaller tumours and achieved a higher cure rate than those without (100 vs. 50%, P = 0.017). CONCLUSIONS: Galanin is present in normal and tumorous human pituitaries. In addition, GAL colocalizes exclusively in corticotrophs of normal pituitaries and is coexpressed almost exclusively in corticotrophs from functioning and nonfunctioning tumours. The finding that corticotroph adenomas can function irrespective of the presence of GAL suggests that GAL may not play a pathophysiological role in Cushing's disease. However, the better surgical outcome observed in patients with Cushing's disease who had tumours positive for GAL-IR suggests that the expression of GAL confers a less aggressive tumour phenotype.


Assuntos
Adenoma/química , Biomarcadores Tumorais/análise , Galanina/análise , Hipófise/química , Neoplasias Hipofisárias/química , Adenoma/complicações , Adenoma/cirurgia , Hormônio Adrenocorticotrópico/análise , Adulto , Síndrome de Cushing/etiologia , Síndrome de Cushing/metabolismo , Síndrome de Cushing/cirurgia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/cirurgia , Prognóstico
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