RESUMEN
STUDY QUESTION: To what extent do self-reported sleep duration and non-daytime work schedules in either partner affect the rate of spontaneous abortion (SAB)? SUMMARY ANSWER: Incidence of SAB had little association with female sleep duration and a modest positive association with male short sleep duration, female work at night, and discrepant work schedules among partners. WHAT IS KNOWN ALREADY: Several studies have reported an association between short sleep duration in either partner and reproductive health outcomes, including fecundability. Moreover, certain types of female occupational exposures during pregnancy have been associated with an increased risk of SAB. No studies have evaluated SAB risk in relation to male sleep and work schedules, or joint exposures within a couple. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 9357 female participants and 2602 of their male partners residing in North America (June 2013 to April 2023). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants enrolled when they were attempting pregnancy and completed self-administered baseline questionnaires about their average sleep duration and work schedules. Among those who conceived, we ascertained SAB and gestational age at loss via follow-up questionnaires. We used multivariable Cox proportional hazards models with gestational weeks as the time scale to estimate hazard ratios (HRs) and 95% CIs relating SAB with sleep duration and non-daytime work schedules for female and male participants, and the couple. We used inverse probability weighting to account for potential selection bias due to the possibility of differential participation of male partners with respect to the exposures. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to female participants with recommended sleep (7-8.9 h), those reporting short sleep duration (<6 h) did not have a higher rate of SAB (HR 0.88, 95% CI 0.69, 1.13). Short self-reported sleep duration among male participants was modestly associated with a higher rate of SAB (adjusted and weighted HR 1.30, 95% CI 0.96, 1.75). Female night work at night (adjusted HR 1.19, 95% CI 1.02, 1.38) and male non-daytime work (adjusted and weighted HR 1.26, 95% CI 1.00, 1.59) were associated with modestly higher rates of SAB, whereas female rotating shift work was not (adjusted HR 0.91, 0.78, 1.05) compared with daytime workers. Couples in which work schedules were discrepant had an elevated rate of SAB if the male partner worked a non-daytime shift (adjusted and weighted HR 1.46, 95% CI 1.13, 1.88) compared with couples in which both members worked during the day. The corresponding HR if only the female partner worked a non-daytime shift was 1.21 (95% CI 0.92, 1.58). LIMITATIONS, REASONS FOR CAUTION: Data on sleep duration and work schedules were based on self-report, which is vulnerable to misclassification, particularly since participants were asked to report their average sleep duration during the past month. Work exposures were heterogeneous, as many different types of employment may require night and shift work and may have different associations with SAB. WIDER IMPLICATIONS OF THE FINDINGS: Our findings are consistent with previous research indicating that some types of female employment schedules may be associated with SAB incidence. This is the first study to indicate a relationship between SAB and male employment schedules, indicating that discrepant work schedules within a couple might be relevant. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development grants R01HD105863 (PIs: L.A.W. and M.L.E.), R01HD086742 (PIs: L.A.W. and E.E.H.), and R21HD072326 (PI: L.A.W.). PRESTO has received in-kind donations from Swiss Precision Diagnostics and Kindara.com for primary data collection. L.A.W. is a consultant for AbbVie, Inc. and the Gates Foundation. M.L.E. is an advisor for and holds stock in Ro, Hannah, Dadi, Underdog, Vseat, & Doveras. The other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.
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Aborto Espontáneo , Horario de Trabajo por Turnos , Embarazo , Niño , Humanos , Masculino , Femenino , Aborto Espontáneo/epidemiología , Aborto Espontáneo/etiología , Incidencia , Estudios Prospectivos , Duración del SueñoRESUMEN
The presence of SARS-CoV-2 in untreated sewage has been confirmed in many countries but its incidence and infection risk in contaminated waters is poorly understood. The River Thames in the UK receives untreated sewage from 57 Combined Sewer Overflows (CSOs), with many discharging dozens of times per year. This study investigated if such discharges provide a pathway for environmental transmission of SARS-CoV-2. Samples of wastewater, surface water, and sediment collected close to six CSOs on the River Thames were assayed over eight months for SARS-CoV-2 RNA and infectious virus. Bivalves were also sampled as an indicator species of viral bioaccumulation. Sediment and water samples from the Danube and Sava rivers in Serbia, where raw sewage is also discharged in high volumes, were assayed as a positive control. No evidence of SARS-CoV-2 RNA or infectious virus was found in UK samples, in contrast to RNA positive samples from Serbia. Furthermore, this study shows that infectious SARS-CoV-2 inoculum is stable in Thames water and sediment for <3 days, while SARS-CoV-2 RNA is detectable for at least seven days. This indicates that dilution of wastewater likely limits environmental transmission, and that detection of viral RNA alone is not an indication of pathogen spillover.
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COVID-19 , Aguas del Alcantarillado , Humanos , Aguas Residuales , SARS-CoV-2 , ARN Viral , Monitoreo del Ambiente , COVID-19/epidemiología , AguaAsunto(s)
Antivirales/uso terapéutico , Betacoronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Hidroxicloroquina/uso terapéutico , Modelos Biológicos , Neumonía Viral/tratamiento farmacológico , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/patogenicidad , COVID-19 , Cricetinae , Humanos , Ratones , Pandemias , Primates , SARS-CoV-2 , Insuficiencia del Tratamiento , Tratamiento Farmacológico de COVID-19RESUMEN
The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296 ± 52 µm and a pore size of 512 ± 22 µm × 476 ± 25 µm × 180 ± 30 µm. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50 µg/ml fibronectin and a cell seeding density of 25 × 10(6) cells/ml and glycosaminoglycan (GAG) accumulation after 28 days culture in the presence of TGFß3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12 weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair.
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Cartílago/lesiones , Cartílago/metabolismo , Condrogénesis , Células Madre Mesenquimatosas/metabolismo , Poliésteres , Polietilenglicoles , Andamios del Tejido , Animales , Cartílago/inervación , Células Madre Mesenquimatosas/patología , ConejosRESUMEN
Three hundred forty-seven crossbred heifers (330 +/- 11 kg initial BW) were used in a randomized complete block study to identify the optimal level of dried corn distillers grains with solubles (DGS) in flaked corn finishing diets. Fifty-four pens were used, with 9 pens per treatment and 6 to 7 heifers per pen. Finishing diets were steam-flaked corn-based and were fed once daily for 148 d. Dietary treatments consisted of 6 levels of DGS (0, 15, 30, 45, 60, and 75%, DM basis). Dry matter intake, ADG, and final BW responded quadratically (P < or = 0.03) to increasing levels of DGS and were maximized at 15% DGS. However, G:F decreased linearly (P = 0.01) as level of DGS increased. Longissimus muscle areas were not different (P > or = 0.27), whereas 12th-rib fat thicknesses decreased linearly (P = 0.05) for heifers fed increasing levels of DGS. Marbling score and USDA yield grades were not different (P > or = 0.06) for heifers fed different levels of DGS. Number of carcasses grading USDA Prime or Choice were not different (P > or = 0.07), whereas number of carcasses grading USDA Select increased (P = 0.02; linear) as dietary level of DGS increased from 0 to 75%. Myofibrillar and overall tenderness increased linearly (P = 0.01) as dietary level of DGS increased from 0 to 75%. Juiciness, off-flavor intensity, and thiobarbituric acid reactive substances were not different (P > or = 0.16) among treatments. Redness of steaks (i.e., a*) was not different (P > or = 0.13) for steaks collected from heifers fed different levels of DGS as evidenced by similar instrumental color measurements after d 0, 3, and 5 of display. However, on d 7, steak color was less red (P = 0.04) and had more metmyoglobin. Concentration of linoleic acid (18:2n-6cis), total n-6 fatty acids, and total PUFA linearly increased (P = 0.01) with increasing levels of DGS.
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Alimentación Animal/análisis , Composición Corporal/efectos de los fármacos , Dieta/veterinaria , Carne/normas , Zea mays , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Bovinos/crecimiento & desarrollo , Culinaria , MasculinoRESUMEN
In Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPHu) hydroxylates both tryptophan to yield 5-hydroxytryptophan, the first step in serotonin synthesis, and phenylalanine, to generate tyrosine. Analysis of the sequenced Drosophila genome identified an additional enzyme with extensive homology to mammalian tryptophan hydroxylase (TPH), which we have termed DTRHn. We have shown that DTRHn can hydroxylate tryptophan in vitro but displays differential activity relative to DTPHu when using tryptophan as a substrate. Recent studies in mice identified the presence of two TPH genes, Tph1 and Tph2, from distinct genetic loci. Tph1 represents the non-neuronal TPH gene, and Tph2 is expressed exclusively in the brain. In this article, we show that DTRHn is neuronal in expression and function and thus represents the Drosophila homologue of Tph2. Using a DTRHn-null mutation, we show that diminished neuronal serotonin affects locomotor, olfactory and feeding behaviors, as well as heart rate. We also show that DTPHu functions in vivo as a phenylalanine hydroxylase in addition to its role as the peripheral TPH in Drosophila, and is critical for non-neuronal developmental events.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Fenilalanina Hidroxilasa/metabolismo , Triptófano Hidroxilasa/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Cuerpo Adiposo/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Estadios del Ciclo de Vida , Mutación/genética , Fenilalanina Hidroxilasa/genética , Serotonina/metabolismo , Distribución Tisular , Triptófano Hidroxilasa/genéticaRESUMEN
Congenital obstructive nephropathy is a major cause of renal insufficiency in children. Osteopontin (OPN) is a phosphoprotein produced by the kidney that mediates cell adhesion and migration. We investigated the role of OPN in the renal response to unilateral ureteral obstruction (UUO) in neonatal mice. OPN null mutant (-/-) and wild-type (+/+) mice were subjected to sham operation or UUO within the first 2 days of life. At 7 and 21 days of age, fibroblasts (fibroblast-specific protein (FSP)-1), myofibroblasts (alpha-smooth muscle actin (SMA)), and macrophages (F4/80) were identified by immunohistochemical staining. Apoptotic cells were detected by terminal deoxy transferase uridine triphosphate nick end-labeling technique and interstitial collagen by Masson trichrome or picrosirius red stain. Compared to sham-operated or contralateral kidneys, obstructed kidneys showed increases in all parameters by 7 days, with further increases by 21 days. After 21 days UUO, there was an increase in tubular and interstitial apoptosis in OPN -/- mice as compared to +/+ animals (P<0.05). However, FSP-1- and alpha-SMA-positive cells and collagen in the obstructed kidney were decreased in OPN -/- compared to +/+ mice (P<0.05), whereas the interstitial macrophage population did not differ between groups. We conclude that OPN plays a significant role in the recruitment and activation of interstitial fibroblasts to myofibroblasts in the progression of interstitial fibrosis in the developing hydronephrotic kidney. However, OPN also suppresses apoptosis. Future approaches to limit the progression of obstructive nephropathy in the developing kidney will require targeting of specific renal compartments.
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Apoptosis/fisiología , Riñón/patología , Osteopontina/metabolismo , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Animales , Animales Recién Nacidos , Colágeno/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Riñón/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Osteopontina/genética , Obstrucción Ureteral/fisiopatologíaRESUMEN
We examined BMP regulation of the gap junction gene Gjal (Cx43alpha1) using a series of lacZ reporter constructs containing up to 6.7 kbs of mouse Cx43alpha1 promoter sequence. Using transient transfection assays, we showed that BMP2, BMP4, and GDF5, but not BMP6 or BMP7, can modulate Cx43alpha1 promoter activity in the osteosarcoma cell line ROS17/2.8. Positive regulatory elements were found at the proximal and distal ends of the 6.7 kb promoter fragment, while negative regulatory elements were found in the intervening region. Comparison of Cx43alpha1 promoter sequences from the human vs. mouse showed five regions with significant sequence conservation, two of which contained Smad binding elements in conjunction with a BMP response element. Analysis of a transgenic mouse line containing a Cx43alpha1 promoter driven lacZ reporter construct revealed lacZ expression in the developing joints, an expression pattern similar to that previously reported for Gdf5. LacZ expression was also observed in axial regions of the skeletal anlage, which in situ hybridization analysis confirmed as sites of Gdf5 transcript expression. When the Cx43alpha1 promoter driven lacZ transgene was bred into the brachypodism mouse Gdf5(bpJ)(bp) harboring a Gdf5 loss of function mutation, lacZ expression was extinguished. This was observed in homozygous and heterozygous bp animals, suggesting that Cx43alpha1 promoter regulation by GDF5 is subject to haploinsufficiency. Overall, these observations are consistent with recent studies by others indicating a role for Cx43alpha1 in osteogenesis and osteoblastic function during mouse development.
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Proteínas Morfogenéticas Óseas/metabolismo , Conexina 43/biosíntesis , Conexina 43/genética , Regulación del Desarrollo de la Expresión Génica , Osteoblastos/metabolismo , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 6 , Proteína Morfogenética Ósea 7 , Secuencia Conservada , Genes Reporteros , Factor 5 de Diferenciación de Crecimiento , Heterocigoto , Homocigoto , Humanos , Hibridación in Situ , Operón Lac , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Transfección , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The molecular basis of Meesmann's epithelial corneal dystrophy (MECD) has recently been attributed to mutations in the cornea specific keratin genes KRT3 and KRT12. The mechanisms by which these mutations cause the Meesmann's phenotype are not clear. This study presents new data, examines clinical, histological, ultrastructural, and molecular aspects of MECD, and compares the features seen in this condition with those observed in other well studied keratin diseases such as epidermolysis bullosa simplex. METHODS: A two generation family with typical features of Meesmann's epithelial corneal dystrophy (MECD) was studied. All family members were examined under a slit lamp. Biopsy material from elective keratoplasty was studied by histopathological and ultrastructural analysis using standard techniques. Direct automated sequencing of genomic DNA was used for mutation detection, mutations were confirmed by restriction digest analysis. RESULTS: The abnormal corneal epithelium was acanthotic and contained numerous dyskeratotic cells and intraepithelial vesicles. By electron microscopy abnormally aggregated and clumped keratin filament bundles were detected in basal and suprabasal keratinocytes from the centre of the cornea. Direct sequencing of the patients' genomic DNA revealed a novel missense mutation (423T>G) in exon 1 of the cornea specific keratin 12 (KRT12) gene. This mutation predicts the amino acid change N133K within the helix initiation motif of the K12 polypeptide. Comparative studies with well established keratin disorders of other human epithelia underscore the pathogenic relevance of K3 and K12 gene mutations in Meesmann's epithelial corneal dystrophy. The morphological data presented here illustrate the disruptive effects of keratin gene mutations on the integrity of corneal keratinocytes. CONCLUSIONS: A clinical, histopathological, and ultrastructural study of a previously unreported family with MECD is presented. In this family the disease is ascribed to a novel mutation in KRT12. A molecular mechanism is proposed for MECD based on the comparison with other well characterised keratin diseases.
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Distrofias Hereditarias de la Córnea/genética , Mutación Missense , Adulto , Membrana Basal/ultraestructura , Distrofias Hereditarias de la Córnea/patología , Distrofias Hereditarias de la Córnea/cirugía , Trasplante de Córnea , Epitelio Corneal/ultraestructura , Humanos , Queratinocitos/ultraestructura , Masculino , Trasplante HomólogoRESUMEN
Pachyonychia congenita type 2 is an inherited ectodermal dysplasia characterized by hypertrophic nail dystrophy and multiple pilosebaceous cysts. Focal nonepidermolytic palmoplantar keratoderma, natal teeth, and pili torti may also be present. Epithelial tissues affected in pachyonychia congenita type 2 express the keratin pair K6b/K17. Here, we report three novel heterozygous mutations in the K17 gene (KRT17A) in patients presenting with pachyonychia congenita type 2. These mutations, R94-98del (deletion of the peptide sequence RLASY) and missense mutations R94P and L95Q, are all within the 1A domain hotspot for pathogenic keratin mutations.
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Displasia Ectodérmica/genética , Queratinas/genética , Mutación , Secuencia de Bases/genética , Preescolar , Displasia Ectodérmica/patología , Femenino , Eliminación de Gen , Humanos , Hiperplasia , Datos de Secuencia Molecular , Mutación/genética , Mutación Missense , Uñas/patologíaRESUMEN
Naegeli-Franceschetti-Jadassohn syndrome is a rare autosomal dominant form of ectodermal dysplasia affecting sweat glands, nails, teeth, and skin. We have studied a multigeneration family of Anglo-Saxon British descent using microsatellite markers to screen candidate loci, including the epidermal differentiation complex on 1q, the keratin gene clusters on chromosomes 12q and 17q and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 17q was observed using marker D17S 1787, with a maximum two-point LOD score of 4.166 at a recombination fraction of theta = 0. Recombination events in the family place the gene in a 26.97 cM interval between markers D17S798 and D17S957, a region known to contain the type I keratin gene cluster and other genes expressed in epithelia. Keratins K15, K19, and K20, plakoglobin, and MEOX1 were excluded as candidates by direct sequencing of genomic polymerase chain reaction products.
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Cromosomas Humanos Par 17 , Displasia Ectodérmica/genética , Mapeo Cromosómico , Proteínas del Citoesqueleto/genética , Desmoplaquinas , Femenino , Genes Dominantes , Humanos , Queratinas/genética , Masculino , Repeticiones de Microsatélite/genética , Linaje , gamma CateninaRESUMEN
PURPOSE: Meesmann corneal dystrophy is an autosomal dominant disorder characterized by fragility of the anterior corneal epithelium. We have previously demonstrated that this disease can be caused by mutations in the genes encoding keratins K3 or K12, the major intermediate filament proteins expressed in corneal epithelial cells. Here, we have carried out mutation analysis in a United States kindred presenting with typical features of Meesmann corneal dystrophy. METHODS: Exons 1 and 6 of the K12 gene (KRT12) were polymerase chain reaction amplified from the proband's and control DNA and subjected to direct automated sequencing. RESULTS: A heterozygous missense mutation 1300A-->G was detected in exon 6 of KRT12, predicting amino acid substitution 1426V in the helix termination motif of the K12 polypeptide. The mutation was confirmed in the proband and excluded from 50 normal individuals by restriction enzyme analysis of polymerase chain reaction products. CONCLUSION: We report a novel mutation in a critical molecular overlap region of K12 in a United States family with Meesmann corneal dystrophy. The results confirm that mutations in the corneal keratins (K3 or K12) can underlie Meesmann corneal dystrophy.
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Distrofias Hereditarias de la Córnea/genética , Queratinas/genética , Mutación Missense , Mutación Puntual , Regiones Terminadoras Genéticas , Adulto , Distrofias Hereditarias de la Córnea/patología , Análisis Mutacional de ADN , Cartilla de ADN/química , Familia , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
Pachyonychia congenita (PC) is a group of inherited ectodermal dysplasias, the characteristic phenotype being hypertrophic nail dystrophy. Two main clinical subtypes, PC-1 and PC-2, are inherited as autosomal dominant disorders, but other less well characterized clinical forms also exist. The PC-1 phenotype may be distinguished by the absence of the epidermal cysts found in PC-2, and it has been shown to be caused by mutations in either keratin K16 or its expression partner, the K6a isoform of K6. Mutations in K16 have also been shown to cause a milder related phenotype, focal non-epidermolytic palmoplantar keratoderma. Recently, we have developed a long-range polymerase chain reaction (PCR) strategy which allows specific amplification of the entire functional K16 gene (KRT16A), without amplification of the two K16 pseudogenes (psiKRT16B and psiKRT16C), enabling mutation analysis based on genomic DNA. Here, using this methodology, we describe novel mutations R127P and Q122P in the helix 1A domain of K16 in two families presenting with PC-1. Both mutations were excluded from 50 normal unrelated individuals by restriction enzyme analysis of K16 PCR fragments. In one family, ultrastructural analysis was performed, revealing distinctive tonofilament abnormalities. Specifically, keratin filament bundles were greatly condensed, but did not form the dense amorphous aggregates seen in a number of other keratin disorders. In the second kindred, autosomal dominant cataract was present in some but not all members affected by PC. As the cataract phenotype did not fully cosegregate with the K16 mutation, and given that K16 is not expressed in the lens, these two phenotypes may be coincidental.
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Displasia Ectodérmica/genética , Queratinas/genética , Mutación Missense , Uñas Malformadas , Prolina/genética , Catarata/genética , Displasia Ectodérmica/patología , Femenino , Humanos , Lactante , Masculino , Linaje , Piel/ultraestructuraRESUMEN
Hypotrichosis of Marie Unna (MU) is an autosomal dominant hair-loss disorder with onset in childhood. A genomewide search for the gene was performed in a large Dutch family using 400 fluorescent microsatellite markers. Linkage was detected with marker D8S258, and analysis of this family and a further British kindred with additional markers in the region gave a combined maximum two-point LOD score of 13.42, with D8S560. Informative recombinants placed the MU gene in a 2.4-cM interval between markers D8S258 and D8S298. Recently, recessive mutations in the hr gene were reported in families with congenital atrichia, and this gene was previously mapped close to the MU interval. By radiation-hybrid mapping, we placed the hr gene close to D8S298 but were unable to exclude it from the MU interval. This, with the existence of the semidominant murine hr allele, prompted us to perform mutation analysis for this gene. Full-length sequencing of hr cDNA obtained from an affected individual showed no mutations. Similarly, screening of all exons of the hr gene amplified from the genomic DNA of an affected individual revealed no mutations. Analysis of expressed sequences and positional cloning of the MU locus is underway.
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Cromosomas Humanos Par 8/genética , Análisis Mutacional de ADN , Hipotricosis/genética , Repeticiones de Microsatélite , Mapeo Físico de Cromosoma , Proteínas/genética , Factores de Transcripción , Alopecia/genética , Inglaterra , Salud de la Familia , Femenino , Genes Dominantes , Genotipo , Haplotipos , Humanos , Células Híbridas , Escala de Lod , Masculino , Mutación/genética , Países Bajos , LinajeRESUMEN
Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by diffuse keratoderma, typically with an erythematous border. Histologically, palmoplantar epidermis shows suprabasal cytolysis and ultrastructurally, tonofilament aggregation with overlying epidermolytic hyperkeratosis. Mutations in the KRT9 gene, encoding keratin 9 (K9), a cytoskeletal protein expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis, have been reported to cause EPPK. To date, all KRT9 defects reported in EPPK have been missense mutations in exon 1, which encodes the start of the alpha-helical rod domain. However, based on studies of other keratin disorders, it was postulated that mutations at the other end of the rod domain might also produce the EPPK phenotype. Here, we report the first mutation in the 2B domain of KRT9, 1362ins3, leading to an insertion of histidine in the helix termination motif of the K9 polypeptide. Insertional mutations have not been previously described in keratins. The phenotype of this case is similar to EPPK caused by 1A domain mutations, demonstrating that mutations in either of the helix boundary motif sequences of K9 are detrimental to keratin function and keratinocyte structure.
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Queratinas/genética , Queratodermia Palmoplantar/genética , Mutación/genética , Adulto , Análisis Mutacional de ADN , Elementos Transponibles de ADN , Humanos , Queratodermia Palmoplantar/sangre , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dysplasia characterized by hypertrophic nail dystrophy, focal non-epidermolytic palmoplantar keratoderma and variable features of oral leukokeratosis and follicular keratosis. Previously, we have shown that this disease can be caused by mutations in type I keratin K16 and one mutation has been reported in its type II keratin expression partner, K6a. Mutation analysis for K6a has been hampered by the presence of multiple copies of the K6 gene in the human genome, of which some are expressed and others are pseudogenes. Here, we describe a mutation detection strategy where the entire KRT6A gene, approximately 7 kb, is specifically amplified by long-range PCR. Using this technique, we have detected two novel mutations in the 1A domain of the K6a polypeptide, N171K and F174S. Mutations were confirmed in the affected individuals and were excluded from 50 unaffected unrelated individuals by restriction enzyme analysis of KRT6A PCR products. Additionally, mutation N171K was confirmed by RT-PCR in mRNA derived from lesional palmoplantar epidermis of an affected individual, confirming the specificity of the genomic PCR for the functional K6a gene. This, together with a similar strategy which we have developed for the K16 gene, provide a robust system for mutation detection and prenatal diagnosis for patients with PC-1.
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Displasia Ectodérmica/genética , Queratinas/genética , Mutación Missense , Uñas Malformadas/genética , Enfermedades de la Piel/genética , Sustitución de Aminoácidos , Secuencia de Bases , Exones , Femenino , Genoma Humano , Humanos , Masculino , Familia de Multigenes , LinajeRESUMEN
Type I and type II keratins form the heteropolymeric intermediate filament cytoskeleton, which is the main stress-bearing structure within epithelial cells. Pachyonychia congenita (PC) is a group of autosomal dominant disorders whose most prominent phenotype is hypertrophic nail dystrophy accompanied by other features of ectodermal dysplasia. It has been shown previously that mutations in either K16 or K6a, which form a keratin expression pair, produce the PC-1 variant (MIM 184510). Mutations in K17 alone, an unpaired accessory keratin, result in the PC-2 phenotype (MIM 184500). Here, we describe a family with PC-2 in which the K17 locus on 17q was excluded and linkage to the type II keratin locus on 12q was obtained (Z max 3.31 at straight theta = 0). Mutation analysis of candidate keratins revealed the first reported missense mutation in K6b, implying that this keratin is the previously unknown expression partner of K17, analogous to the K6a/K16 pair. Co-expression of these genes was confirmed by in situ hybridization and immunohistochemical staining. These results reveal the hitherto unknown role of the K6b isoform in epithelial biology, as well as genetic heterogeneity in PC-2.