RESUMEN
Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.
Asunto(s)
Anomalías Múltiples/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Mutación , Transducción de Señal , Femenino , Humanos , Masculino , Linaje , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , SíndromeRESUMEN
Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.
Asunto(s)
Anomalías Múltiples , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Aparato Lagrimal/anomalías , Deformidades Congénitas de las Extremidades , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Glándulas Salivales/anomalías , Transducción de Señal/fisiología , Anomalías Dentarias , Línea Celular , Factor 10 de Crecimiento de Fibroblastos/genética , Humanos , Modelos Moleculares , Conformación Proteica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , SíndromeRESUMEN
Mutations in fibroblast growth factor receptor 2 (FGFR2) and its ligand, FGF10, are known to cause lacrimo-auriculo-dento-digital (LADD) syndrome. Multiple gain-of-function mutations in FGF receptors have been implicated in a variety of severe skeletal disorders and in many cancers. We aimed to elucidate the mechanism by which a missense mutation in the tyrosine kinase domain of FGFR2, described in the sporadic case of LADD syndrome, leads to reduced tyrosine kinase activity. In this report, we describe the crystal structure of a FGFR2 A628T LADD mutant in complex with a nucleotide analog. We demonstrate that the A628T LADD mutation alters the configuration of key residues in the catalytic pocket that are essential for substrate coordination, resulting in reduced tyrosine kinase activity. Further comparison of the structures of WT FGFR2 and WT FGFR1 kinases revealed that FGFR2 uses a less stringent mode of autoinhibition than FGFR1, which was also manifested in faster in vitro autophosphorylation kinetics. Moreover, the nearly identical conformation of WT FGFR2 kinase and the A628T LADD mutant to either the phosphorylated FGFR2 or FGFR2 harboring pathological activating mutations in the kinase hinge region suggests that FGFR autoinhibition and activation are better explained by changes in the conformational dynamics of the kinase rather than by static crystallographic snapshots of minor structural variations.
Asunto(s)
Anomalías Múltiples , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Adenosina Trifosfato/metabolismo , Alanina/genética , Alanina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación/genética , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Homología Estructural de Proteína , Especificidad por Sustrato , SíndromeRESUMEN
In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.
Asunto(s)
Mutación del Sistema de Lectura , Pérdida Auditiva Bilateral/genética , Pérdida Auditiva Sensorineural/genética , Proteínas de la Membrana/genética , Mutación Missense , Secuencia de Aminoácidos , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Consanguinidad , Análisis Mutacional de ADN , Femenino , Ligamiento Genético , Haplotipos , Pérdida Auditiva Bilateral/diagnóstico , Pérdida Auditiva Sensorineural/diagnóstico , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Alineación de SecuenciaRESUMEN
A reliable method to provide molecular biology products free of contaminating DNA is of forensic interest. Ethylene oxide (EO) treatment has been demonstrated as an effective method in published studies. This study aimed to address some additional experiments that are closer to forensic practice. In the first part of this study, different consumables such as cotton swabs, latex gloves and micro test tubes were spiked with saliva, blood and skin cells to mimic a real-life contamination scenario. EO treatment was performed for a period of 3, 5, 7, and 10h, respectively. For comparison, gamma and electron beam treatment was applied. In the second part of this study, a cell culture line (K562) was used to apply defined cell counts on cotton swabs followed by EO treatment for 3 and 5h. After extraction of samples, the DNA content was quantified using a real-time PCR based system. STR analysis was performed using a latest generation STR kit to meet current sensitivity limits. A good correlation of real-time PCR results and STR results was observed. This work confirmed the findings of earlier studies showing that chemical EO treatment is much more successful in reducing the amount of PCR-amplifiable DNA than ionising radiation. Furthermore, the efficacy of EO treatment is affected by the nature of the samples. DNA in saliva was more susceptible to damage by EO gas than DNA in blood. Our results show, that accessibility of the sample to EO gas has a strong influence on the method's efficiency. While treatment of samples on cotton swabs packed into gas-permeable bags was very successful, samples inside a closed micro test tube were resistant to the same treatment conditions. Our work with defined K562 cell numbers and multi-copy quantitative PCR could show that a 5h EO treatment results in a 10(5) fold reduction of PCR-amplifiable DNA. Corresponding STR-PCR results also show only sporadic allele calls in the Mini-loci range, providing a reliable interpretation of forensic analysis. Finally, we do recommend an EO treatment of forensic consumables and a multi-copy quantitative PCR approach to establish reliable treatment conditions.
Asunto(s)
ADN/genética , Óxido de Etileno/química , Genética Forense , Reacción en Cadena de la Polimerasa/métodos , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5-10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.