RESUMO
Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.
Assuntos
Regulação da Expressão Gênica , Ictiose/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/metabolismo , Modelos Biológicos , Criança , Pré-Escolar , Feminino , Proteínas Filagrinas , Humanos , Ictiose/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Lactente , Queratinócitos/patologia , MasculinoAssuntos
Ictiose , Neoplasias Cutâneas , Humanos , Receptor 2 Toll-Like , Transdução de Sinais , Queratinas/metabolismoRESUMO
We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/imunologia , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/genética , Linfo-Histiocitose Hemofagocítica/diagnóstico , Proteínas Musculares/genética , Fatores de Transcrição/genética , Doenças Assintomáticas , Degranulação Celular , Criança , Citometria de Fluxo , Testes Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/imunologia , Proteínas Musculares/metabolismo , Mutação/genética , Transplante de Órgãos , Guias de Prática Clínica como Assunto , Prognóstico , Estudos Prospectivos , Irmãos , Fatores de Transcrição/metabolismo , Sequenciamento do ExomaRESUMO
Tight junctions are important for skin barrier function. The tight junction protein claudin 1 (Cldn-1) has been reported to be down-regulated in nonlesional skin of atopic dermatitis (AD) patients. In contrast, we did not observe a significant down-regulation of Cldn-1 in nonlesional skin of the AD cohort used in this study. However, for the first time, a significant down-regulation of Cldn-1 in the upper and lower epidermal layers of lesional skin was detected. In addition, there was a significant up-regulation of Cldn-4 in nonlesional, but not lesional, AD skin. For occludin, no significant alterations were observed. In an AD-like allergic dermatitis mouse model, Cldn-1 down-regulation in eczema was significantly influenced by dermal inflammation, and significantly correlated with hallmarks of eczema (ie, increased keratinocyte proliferation, altered keratinocyte differentiation, increased epidermal thickness, and impaired barrier function). In human epidermal equivalents, the addition of IL-4, IL-13, and IL-31 resulted in a down-regulation of Cldn-1, and Cldn1 knockdown in keratinocytes resulted in abnormal differentiation. In summary, we provide the first evidence that Cldn-1 and Cldn-4 are differentially involved in AD pathogenesis. Our data suggest a role of Cldn-1 in AD eczema formation triggered by inflammation.
Assuntos
Claudina-1/metabolismo , Claudina-4/metabolismo , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Queratinócitos/patologia , Adulto , Regulação para Baixo , Feminino , Humanos , Interleucina-13/genética , Masculino , Pele/metabolismo , Pele/patologiaRESUMO
BACKGROUND: Dupuytren's disease is a benign fibromatosis of the hands and fingers that leads to flexion contractures. We hypothesized that multiple genetic and environmental factors influence susceptibility to this disease and sought to identify susceptibility genes to better understand its pathogenesis. METHODS: We conducted a genomewide association study of 960 Dutch persons with Dupuytren's disease and 3117 controls (the discovery set) to test for association between the disease and genetic markers. We tested the 35 single-nucleotide polymorphisms (SNPs) most strongly associated with Dupuytren's disease (P<1×10(-4)) in the discovery set in three additional, independent case series comprising a total of 1365 affected persons and 8445 controls from Germany, the United Kingdom, and The Netherlands. RESULTS: Initially, we observed a significant genomewide association between Dupuytren's disease and 8 SNPs at three loci. Tests of replication and joint analysis of all data from 2325 patients with Dupuytren's disease and 11,562 controls yielded an association with 11 SNPs from nine different loci (P<5.0×10(-8)). Six of these loci contain genes known to be involved in the Wnt-signaling pathway: WNT4 (rs7524102) (P=2.8×10(-9); odds ratio, 1.28), SFRP4 (rs16879765) (P=5.6×10(-39); odds ratio, 1.98), WNT2 (rs4730775) (P=3.0×10(-8); odds ratio, 0.83), RSPO2 (rs611744) (P=7.9×10(-15); odds ratio, 0.75), SULF1 (rs2912522) (P=2.0×10(-13); odds ratio, 0.72), and WNT7B (rs6519955) (P=3.2×10(-33); odds ratio, 1.54). CONCLUSIONS: This study implicates nine different loci involved in genetic susceptibility to Dupuytren's disease. The fact that six of these nine loci harbor genes encoding proteins in the Wnt-signaling pathway suggests that aberrations in this pathway are key to the process of fibromatosis in Dupuytren's disease.
Assuntos
Contratura de Dupuytren/genética , Loci Gênicos , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/genética , Proteínas Wnt/genética , Estudos de Casos e Controles , Europa (Continente) , Estudo de Associação Genômica Ampla , Humanos , Proteínas Wnt/metabolismoRESUMO
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.
Assuntos
Células Clonais , Metilação de DNA , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Ilhas de CpG , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.
Assuntos
Cistatina M/genética , Epiderme/crescimento & desenvolvimento , Lentivirus/genética , Morfogênese/genética , RNA Interferente Pequeno/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cistatina M/fisiologia , Células Epidérmicas , Epiderme/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Ictiose/patologia , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Fenótipo , RNA Interferente Pequeno/farmacologia , Pele Artificial , Alicerces TeciduaisRESUMO
The barrier function of the human epidermis is supposed to be governed by lipid composition and organization in the stratum corneum. Disorders of keratinization, namely ichthyoses, are typically associated with disturbed barrier activity. Using autozygosity mapping and exome sequencing, we have identified a homozygous missense mutation in CERS3 in patients with congenital ichthyosis characterized by collodion membranes at birth, generalized scaling of the skin, and mild erythroderma. We demonstrate that the mutation inactivates ceramide synthase 3 (CerS3), which is synthesized in skin and testis, in an assay of N-acylation with C26-CoA, both in patient keratinocytes and using recombinant mutant proteins. Moreover, we show a specific loss of ceramides with very long acyl chains from C26 up to C34 in terminally differentiating patient keratinocytes, which is in line with findings from a recent CerS3-deficient mouse model. Analysis of reconstructed patient skin reveals disturbance of epidermal differentiation with an earlier maturation and an impairment of epidermal barrier function. Our findings demonstrate that synthesis of very long chain ceramides by CerS3 is a crucial early step for the skin barrier formation and link disorders presenting with congenital ichthyosis to defects in sphingolipid metabolism and the epidermal lipid architecture.
Assuntos
Ceramidas/biossíntese , Ictiose Lamelar/genética , Ictiose Lamelar/patologia , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismo , Animais , Células Cultivadas , Ceramidas/química , Criança , Pré-Escolar , Modelos Animais de Doenças , Células Epidérmicas , Epiderme/patologia , Exoma/genética , Saúde da Família , Feminino , Fibroblastos/citologia , Genes Recessivos , Homozigoto , Humanos , Recém-Nascido , Queratinócitos/citologia , Masculino , Camundongos , Peso Molecular , Mutação de Sentido Incorreto , Linhagem , FenótipoRESUMO
In recent years several new genes for autosomal recessive congenital ichthyosis (ARCI) have been identified. However, little is known about the molecular epidemiology and pathophysiology of this genetically and clinically heterogeneous group of severe disorders of keratinization. ARCI is characterized by intense scaling of the whole integument often associated with erythema. We and others have shown that mutations in ALOX12B and ALOXE3, coding for the lipoxygenases 12R-LOX and eLOX-3 predominantly synthesized in the epidermis, can underlie this rare condition. Here we have surveyed a large group of 250 patients with ARCI for mutations in these two genes. We have identified 11 different previously unreported mutations in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that mutations in the two genes are the second most common cause for ARCI in this cohort of patients. Examination of the molecular data revealed allelic heterogeneity for ALOX12B and two mutational hotspots in ALOXE3. Functional analysis of all missense mutations and a splice site mutation demonstrated that complete loss of function of the enzymes underlies the phenotype. Our findings further establish the pivotal role of the 12-lipoxygenase pathway during epidermal differentiation.
Assuntos
Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Eritrodermia Ictiosiforme Congênita/genética , Lipoxigenase/genética , Lipoxigenase/metabolismo , Mutação , Alelos , Estudos de Coortes , Análise Mutacional de DNA , Éxons , Genes Recessivos , Haplótipos , Humanos , Modelos Genéticos , Fenótipo , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Myosin XVA is an unconventional myosin which has been implicated in autosomal recessive nonsyndromic hearing impairment (ARNSHI) in humans. In Myo15A mouse models, vestibular dysfunction accompanies the autosomal recessive hearing loss. Genomewide homozygosity mapping and subsequent fine mapping in two Turkish families with ARNSHI revealed significant linkage to a critical interval harboring a known deafness gene MYO15A on chromosome 17p13.1-17q11.2. Subsequent sequencing of the MYO15A gene led to the identification of a novel missense mutation, c.5492G-->T (p.Gly1831Val) and a novel splice site mutation, c.8968-1G-->C. These mutations were not detected in additional 64 unrelated ARNSHI index patients and in 230 Turkish control chromosomes. Gly1831 is a conserved residue located in the motor domains of the different classes of myosins of different species. Molecular modeling of the motor head domain of the human myosin XVa protein suggests that the Gly1831Val mutation inhibits the powerstroke by reducing backbone flexibility and weakening the hydrophobic interactions necessary for signal transmission to the converter domain.
Assuntos
Perda Auditiva/genética , Mutação , Miosinas/química , Miosinas/genética , Sequência de Bases , Análise Mutacional de DNA , Haplótipos , Homozigoto , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Estrutura Terciária de Proteína , Turquia , Valina/genética , Valina/metabolismoAssuntos
Antígenos Ly/genética , Homozigoto , Mutação de Sentido Incorreto , Ativador de Plasminogênio Tipo Uroquinase/genética , Acitretina/uso terapêutico , Adulto , Anti-Infecciosos/uso terapêutico , Biópsia , Humanos , Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/tratamento farmacológico , Ceratodermia Palmar e Plantar/genética , Ceratolíticos/uso terapêutico , Masculino , FenótipoRESUMO
Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.
Assuntos
Mapeamento Cromossômico , Haplótipos , Rim em Esponja Medular/genética , Análise Mutacional de DNA , Humanos , Repetições de Microssatélites/genéticaRESUMO
BACKGROUND: Nephrolithiasis is a complex, multifactorial disease resulting from genetic and environmental interaction. The pathogenesis of nephrolithiasis is far from being understood. So far, no gene locus for autosomal dominant nephrolithiasis only has been described. We here identified a new suggestive gene locus for autosomal dominant nephrolithiasis by a genome-wide search for linkage in a Spanish kindred with nephrolithiasis. METHODS: Clinical data, blood and urine samples of 18 individuals from a Spanish kindred with nephrolithiasis were collected. We performed a genome-wide search for linkage using 380 polymorphic microsatellite markers. RESULTS: Nephrolithiasis segregated in this Spanish kindred in a pattern compatible with autosomal dominant inheritance. The total genome search yielded the highest two-point LOD score of Z(max) = 1.99 (theta = 0) for marker D9S159 on chromosome 9q33.2-q34.2. Multipoint analysis of 24 polymorphic markers used for further fine mapping resulted in a LOD score of Z(max) = 2.7 (theta = 0) for markers D9S1881-D9S164, thereby identifying a new gene locus for autosomal dominant nephrolithiasis (NPL1). Two recombination events define D9S1850 as the centromeric flanking marker and D9S1818 as the telomeric flanking marker, restricting the NPL1 locus to a 14 Mb interval. CONCLUSION: We here identified a new suggestive gene locus (NPL1) for autosomal dominant nephrolithiasis. It is localized on chromosome 9q33.2-q34.2. The identification of the responsible gene will provide new insights into the molecular basis of nephrolithiasis.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Ligação Genética , Cálculos Renais/genética , Idoso , Feminino , Genoma , Humanos , Escore Lod , Masculino , Repetições de Microssatélites , Pessoa de Meia-IdadeRESUMO
Steroid-resistant nephrotic syndrome (SRNS) leads to end-stage renal disease (ESRD) in childhood or young adulthood. Positional cloning for genes causing SRNS has opened the first insights into the understanding of its pathogenesis. This study reports a genome-wide search for linkage in a consanguineous Palestinian kindred with SRNS and deafness and detection of a region of homozygosity on chromosome 14q24.2. Multipoint analysis of 12 markers used for further fine mapping resulted in a LOD score Z(max) of 4.12 (theta = 0) for marker D14S1025 and a two-point LOD score of Z(max) = 3.46 (theta = 0) for marker D14S77. Lack of homozygosity defined D14S1065 and D14S273 as flanking markers to a 10.7 cM interval. The identification of the responsible gene will provide new insights into the molecular basis of nephrotic syndrome and sensorineural deafness.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Perda Auditiva Neurossensorial/genética , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/genética , Esteroides/uso terapêutico , Alelos , Criança , Pré-Escolar , Resistência a Medicamentos/genética , Feminino , Marcadores Genéticos , Haplótipos , Homozigoto , Humanos , Lactente , Escore Lod , Masculino , Síndrome Nefrótica/fisiopatologia , LinhagemRESUMO
Nephronophthisis (NPHP) comprises a group of autosomal recessive cystic kidney diseases, which constitute the most frequent genetic cause for end-stage renal failure in children and young adults. The most prominent histologic feature of NPHP consists of development of renal fibrosis, which, in chronic renal failure of any origin, represents the pathogenic event correlated most strongly to loss of renal function. Four gene loci for NPHP have been mapped to chromosomes 2q13 (NPHP1), 9q22 (NPHP2), 3q22 (NPHP3), and 1p36 (NPHP4). At all four loci, linkage has also been demonstrated in families with the association of NPHP and retinitis pigmentosa, known as "Senior-Løken syndrome" (SLS). Identification of the gene for NPHP type 1 had revealed nephrocystin as a novel docking protein, providing new insights into mechanisms of cell-cell and cell-matrix signaling. We here report identification of the gene (NPHP4) causing NPHP type 4, by use of high-resolution haplotype analysis and by demonstration of nine likely loss-of-function mutations in six affected families. NPHP4 encodes a novel protein, nephroretinin, that is conserved in evolution--for example, in the nematode Caenorhabditis elegans. In addition, we demonstrate two loss-of-function mutations of NPHP4 in patients from two families with SLS. Thus, we have identified a novel gene with critical roles in renal tissue architecture and ophthalmic function.
Assuntos
Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Proteínas , Retinose Pigmentar/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Citoesqueleto , Haplótipos , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , Especificidade de Órgãos , Análise de Sequência de DNARESUMO
BACKGROUND: Autosomal-dominant medullary cystic kidney disease type 1 (MCKD1) is a tubulointerstitial nephropathy that causes renal salt wasting and end-stage renal failure in the sixth decade of life. The chromosomal locus for MCKD1 was localized to chromosome 1q21 in a Cyprotic kindred. In this report we describe further refinement of the critical genetic region by a recombination in a Belgian kindred. METHODS: Clinical data and blood samples of 33 individuals from a large Belgian kindred were collected and high-resolution haplotype analysis was performed. RESULTS: In the Belgian kindred linkage to the MCKD1 locus on chromosome 1q21 was found with a logarithm of odds (LOD) score significant for linkage. A recombination in individual III:7 for marker D1S2624 refines the critical genetic region to 2.1 Mb. In this kindred a wide variety of clinical symptoms and age of onset of renal failure was detected. CONCLUSION: We confirm the MCKD1 locus on chromosome 1q21 and show further refinement of the MCKD1 locus to 2.1 Mb. This allowed us to exclude another 17 genes as positional candidate genes.