RESUMEN
BACKGROUND: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. METHODS: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. RESULTS: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. CONCLUSION: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.
Asunto(s)
Amelogénesis Imperfecta , Colágenos no Fibrilares , Humanos , Colágenos no Fibrilares/genética , Colágenos no Fibrilares/metabolismo , Autoantígenos/genética , Amelogénesis Imperfecta/genética , Heterocigoto , Fenotipo , Mutación/genéticaRESUMEN
BACKGROUND: Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease. METHODS: Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice. RESULTS: Rare biallelic pathogenic variants in plexin B2 (PLXNB2), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts. CONCLUSION: We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously.
Asunto(s)
Amelogénesis Imperfecta , Discapacidad Intelectual , Linaje , Humanos , Animales , Masculino , Femenino , Ratones , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Receptores de Superficie Celular/genética , Proteínas del Tejido Nervioso/genética , Alelos , Niño , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Adulto , Mutación/genética , Adolescente , Preescolar , FenotipoRESUMEN
The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.
Asunto(s)
Cromosomas Humanos Par 17/química , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Secuencia de Aminoácidos , Diferenciación Celular , Reprogramación Celular , Niño , Mapeo Cromosómico , Estudios de Cohortes , Elementos de Facilitación Genéticos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Genes Dominantes , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Proteínas Nucleares/metabolismo , Organoides/metabolismo , Organoides/patología , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo Genético , Cultivo Primario de Células , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Factores de Transcripción/metabolismo , Secuenciación Completa del GenomaRESUMEN
PURPOSE: To characterize the phenotype observed in a case series with macular disease and determine the cause. DESIGN: Multicenter case series. PARTICIPANTS: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. METHODS: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. MAIN OUTCOME MEASURES: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. RESULTS: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. CONCLUSIONS: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.
Asunto(s)
Degeneración Macular , Humanos , Hibridación Genómica Comparativa , Degeneración Macular/diagnóstico , Degeneración Macular/genética , Linaje , Fenotipo , Transactivadores/genética , Proteínas de Homeodominio/genéticaRESUMEN
Amelogenesis is the process of enamel formation. For amelogenesis to proceed, the cells of the inner enamel epithelium (IEE) must first proliferate and then differentiate into the enamel-producing ameloblasts. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective or absent tooth enamel. We identified a 2 bp variant c.817_818GC>AA in SP6, the gene encoding the SP6 transcription factor, in a Caucasian family with autosomal dominant hypoplastic AI. The resulting missense protein change, p.(Ala273Lys), is predicted to alter a DNA-binding residue in the first of three zinc fingers. SP6 has been shown to be crucial to both proliferation of the IEE and to its differentiation into ameloblasts. SP6 has also been implicated as an AI candidate gene through its study in rodent models. We investigated the effect of the missense variant in SP6 (p.(Ala273Lys)) using surface plasmon resonance protein-DNA binding studies. We identified a potential SP6 binding motif in the AMBN proximal promoter sequence and showed that wild-type (WT) SP6 binds more strongly to it than the mutant protein. We hypothesize that SP6 variants may be a very rare cause of AI due to the critical roles of SP6 in development and that the relatively mild effect of the missense variant identified in this study is sufficient to affect amelogenesis causing AI, but not so severe as to be incompatible with life. We suggest that current AI cohorts, both with autosomal recessive and dominant disease, be screened for SP6 variants.
Asunto(s)
Amelogénesis Imperfecta/genética , Proteínas de Unión al ADN/genética , Proteínas del Esmalte Dental/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Ameloblastos/metabolismo , Ameloblastos/patología , Amelogénesis Imperfecta/patología , Proteínas Relacionadas con la Autofagia/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/patología , Femenino , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Masculino , Mutación Missense/genética , Linaje , Regiones Promotoras Genéticas/genética , Diente/crecimiento & desarrollo , Diente/patología , Secuenciación del ExomaRESUMEN
Amelogenesis imperfecta (AI) describes a heterogeneous group of developmental enamel defects that typically have Mendelian inheritance. Exome sequencing of 10 families with recessive hypomaturation AI revealed four novel and one known variants in the matrix metallopeptidase 20 (MMP20) gene that were predicted to be pathogenic. MMP20 encodes a protease that cleaves the developing extracellular enamel matrix and is necessary for normal enamel crystal growth during amelogenesis. New homozygous missense changes were shared between four families of Pakistani heritage (c.625G>C; p.(Glu209Gln)) and two of Omani origin (c.710C>A; p.(Ser237Tyr)). In two families of UK origin and one from Costa Rica, affected individuals were homozygous for the previously reported c.954-2A>T; p.(Ile319Phefs*19) variant. For each of these variants, microsatellite haplotypes appeared to exclude a recent founder effect, but elements of haplotype were conserved, suggesting more distant founding ancestors. New compound heterozygous changes were identified in one family of the European heritage: c.809_811+12delinsCCAG; p.(?) and c.1122A>C; p.(Gln374His). This report further elucidates the mutation spectrum of MMP20 and the probable impact on protein function, confirms a consistent hypomaturation phenotype and shows that mutations in MMP20 are a common cause of autosomal recessive AI in some communities.
Asunto(s)
Amelogénesis Imperfecta , Metaloproteinasa 20 de la Matriz , Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Efecto Fundador , Homocigoto , Humanos , Metaloproteinasa 20 de la Matriz/genética , LinajeRESUMEN
Amelogenesis imperfecta (AI) is a heterogeneous group of genetic diseases characterised by dental enamel malformation. Pathogenic variants in at least 33 genes cause syndromic or non-syndromic AI. Recently variants in RELT, encoding an orphan receptor in the tumour necrosis factor (TNF) superfamily, were found to cause recessive AI, as part of a syndrome encompassing small stature and severe childhood infections. Here we describe four additional families with autosomal recessive hypomineralised AI due to previously unreported homozygous mutations in RELT. Three families carried a homozygous missense variant in the fourth exon (c.164C>T, p.(T55I)) and a fourth family carried a homozygous missense variant in the 11th exon (c.1264C>T, p.(R422W)). We found no evidence of additional syndromic symptoms in affected individuals. Analyses of tooth microstructure with computerised tomography and scanning electron microscopy suggest a role for RELT in ameloblasts' coordination and interaction with the enamel matrix. Microsatellite genotyping in families segregating the T55I variant reveals a shared founder haplotype. These findings extend the RELT pathogenic variant spectrum, reveal a founder mutation in the UK Pakistani population and provide detailed analysis of human teeth affected by this hypomineralised phenotype, but do not support a possible syndromic presentation in all those with RELT-variant associated AI.
Asunto(s)
Amelogénesis Imperfecta/genética , Predisposición Genética a la Enfermedad , Receptores del Factor de Necrosis Tumoral/genética , Desmineralización Dental/genética , Amelogénesis Imperfecta/diagnóstico por imagen , Amelogénesis Imperfecta/patología , Exones , Femenino , Homocigoto , Humanos , Masculino , Mutación Missense/genética , Linaje , Fenotipo , Desmineralización Dental/diagnóstico por imagen , Desmineralización Dental/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype.
Asunto(s)
Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/metabolismo , Ameloblastos/metabolismo , Animales , Esmalte Dental/metabolismo , Proteínas del Esmalte Dental/genética , Proteínas del Esmalte Dental/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación Puntual , Estrés Fisiológico , Respuesta de Proteína DesplegadaRESUMEN
Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.
Asunto(s)
Amelogénesis Imperfecta/genética , Mutación , Receptores Acoplados a Proteínas G/genética , Amelogénesis/genética , Animales , Secuencia de Bases , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/patología , Femenino , Homocigoto , Humanos , Concentración de Iones de Hidrógeno , Masculino , Linaje , Ratas , Receptores Acoplados a Proteínas G/análisisRESUMEN
OBJECTIVES: Variants in DLX3 cause tricho-dento-osseous syndrome (TDO, MIM #190320), a systemic condition with hair, nail and bony changes, taurodontism and amelogenesis imperfecta (AI), inherited in an autosomal dominant fashion. Different variants found within this gene are associated with different phenotypic presentations. To date, six different DLX3 variants have been reported in TDO. The aim of this paper was to explore and discuss three recently uncovered new variants in DLX3. SUBJECTS AND METHODS: Whole-exome sequencing identified a new DLX3 variant in one family, recruited as part of an ongoing study of genetic variants associated with AI. Targeted clinical exome sequencing of two further families revealed another new variant of DLX3 and complete heterozygous deletion of DLX3. For all three families, the phenotypes were shown to consist of AI and taurodontism, together with other attenuated features of TDO. RESULTS: c.574delG p.(E192Rfs*66), c.476G>T (p.R159L) and a heterozygous deletion of the entire DLX3 coding region were identified in our families. CONCLUSION: These previously unreported variants add to the growing literature surrounding AI, allowing for more accurate genetic testing and better understanding of the associated clinical consequences.
Asunto(s)
Amelogénesis Imperfecta/genética , Anomalías Craneofaciales/genética , Hipoplasia del Esmalte Dental/genética , Enfermedades del Cabello/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Femenino , Humanos , Masculino , LinajeRESUMEN
Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. Amelotin (AMTN) is a secreted protein thought to act as a promoter of matrix mineralization in the final stage of enamel development, and is strongly expressed, almost exclusively, in maturation stage ameloblasts. Amtn overexpression and Amtn knockout mouse models have defective enamel with no other associated phenotypes, highlighting AMTN as an excellent candidate gene for human AI. However, no AMTN mutations have yet been associated with human AI. Using whole exome sequencing, we identified an 8,678 bp heterozygous genomic deletion encompassing exons 3-6 of AMTN in a Costa Rican family segregating dominant hypomineralised AI. The deletion corresponds to an in-frame deletion of 92 amino acids, shortening the protein from 209 to 117 residues. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects at least some of which appeared to be associated with organic material as evidenced using elemental analysis. This study demonstrates for the first time that AMTN mutations cause non-syndromic human AI and explores the human phenotype, comparing it with that of mice with disrupted Amtn function.
Asunto(s)
Amelogénesis Imperfecta/genética , Proteínas del Esmalte Dental/genética , Esmalte Dental/patología , Predisposición Genética a la Enfermedad , Amelogénesis Imperfecta/fisiopatología , Secuencia de Aminoácidos/genética , Animales , Esmalte Dental/crecimiento & desarrollo , Modelos Animales de Enfermedad , Exones/genética , Humanos , Ratones , Ratones Noqueados , Fenotipo , Eliminación de Secuencia/genéticaRESUMEN
Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.
Asunto(s)
Adenosina Trifosfatasas/genética , Amelogénesis Imperfecta/genética , Fibroblastos/patología , Pérdida Auditiva Sensorineural/genética , Proteínas de la Membrana/genética , Mutación/genética , Uñas Malformadas/genética , Peroxisomas/patología , ATPasas Asociadas con Actividades Celulares Diversas , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Masculino , Linaje , Peroxisomas/metabolismo , Fenotipo , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.
Asunto(s)
Amelogénesis Imperfecta/patología , Proteínas del Esmalte Dental/genética , Eliminación de Secuencia , Diente/ultraestructura , Amelogénesis Imperfecta/genética , Secuencia de Aminoácidos , Animales , Exones , Femenino , Humanos , Masculino , Ratones , Linaje , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Diente/patologíaRESUMEN
We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-ß6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell-matrix interaction or compromised ITGB6 activation of transforming growth factor-ß (TGF-ß) impacting indirectly on ameloblast-ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell-matrix interactions during enamel formation.
Asunto(s)
Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Genes Recesivos , Cadenas beta de Integrinas/fisiología , Mutación Missense/genética , Amelogénesis Imperfecta/metabolismo , Secuencia de Aminoácidos , Animales , ADN/genética , Esmalte Dental/metabolismo , Esmalte Dental/patología , Exoma/genética , Humanos , Técnicas para Inmunoenzimas , Ratones , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , Microtomografía por Rayos XRESUMEN
BACKGROUND: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. METHODS: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. RESULTS: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. CONCLUSIONS: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations.
Asunto(s)
Anomalías Múltiples/genética , Quinasa de la Caseína I/genética , Fisura del Paladar/genética , Exoftalmia/genética , Proteínas de la Matriz Extracelular/genética , Microcefalia/genética , Anomalías de la Boca/complicaciones , Mutación , Osteosclerosis/genética , Linaje , Fenotipo , Anomalías Dentarias/complicaciones , Adolescente , Secuencia de Bases , Niño , Preescolar , Fisura del Paladar/complicaciones , Exoftalmia/complicaciones , Femenino , Humanos , Masculino , Microcefalia/complicaciones , Osteosclerosis/complicaciones , Adulto JovenRESUMEN
BACKGROUND: The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole - exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow - up whole - genome sequencing or the time - consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost-effective, long-range sequencing method to characterise deletions. METHODS: Genomic DNA was amplified with primers spanning the deletion using long-range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing. RESULTS: Four cases with deletions detected following comparative read-depth analysis of targeted short-read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS, CNGA1 and CNGB1 and a heterozygous deletion in PRPF31. All breakpoints were verified by Sanger sequencing. CONCLUSIONS: In this study, a quick, accurate and cost - effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing.
Asunto(s)
Secuenciación de Nanoporos , Humanos , Secuenciación de Nanoporos/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Exones , Exoma , Secuenciación Completa del Genoma , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Proteínas del OjoRESUMEN
Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular "antennae" in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.
RESUMEN
The imprinted gene PLAGL1 is an important regulator of apoptosis and cell cycle arrest. Loss of its expression has been implicated in tumorigenesis in a range of different cancers, and overexpression during fetal development causes transient neonatal diabetes mellitus (TNDM). PLAGL1 lies within an imprinted region of chromosome 6q24, and monoallelic expression from the major, differentially methylated promoter (P1) occurs in most human tissues. However, in peripheral blood leukocytes, the active promoter (P2) is non-imprinted and drives biallelic transcription. We report here a novel PLAGL1 promoter (P5) derived from the insertion of a primate-specific, MIR3 SINE retrotransposon. P5 is highly utilized in lymphocytes, particularly in T cells, and like P2, directs biallelic transcription. Our results show that it is important to consider P5 in relation to PLAGL1 function in T cells when investigating the dysregulation of this gene.
Asunto(s)
Alelos , Proteínas de Ciclo Celular/genética , Regiones Promotoras Genéticas , Retroelementos , Elementos de Nucleótido Esparcido Corto/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Linfocitos B/metabolismo , Islas de CpG , Humanos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Transcripción GenéticaRESUMEN
Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the possibility of novel treatments and prevention strategies for AI.