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1.
Am J Hum Genet ; 105(4): 836-843, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31564437

RESUMO

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.

2.
BMC Med Genet ; 18(1): 53, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28499369

RESUMO

BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.


Assuntos
Carcinoma de Células Renais/genética , Genes Supressores de Tumor , Neoplasias Renais/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Processamento de RNA , Proteínas Supressoras de Tumor/genética , Carcinoma de Células Renais/diagnóstico por imagem , Humanos , Neoplasias Renais/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
3.
Clin Nephrol ; 88(1): 45-51, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28502323

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of dialysis-requiring end-stage renal disease in adults and is characterized by the slowly progressing replacement of renal tissue by focal macrocysts. Alport syndrome (AS; hereditary nephritis) is a rare, inherited disorder of the basement membrane associated with hematuria, proteinuria, and loss of kidney function as well as sensorineural hearing loss and ocular abnormalities. Here, we report on a family in which both ADPKD and AS are present. In a male patient, both -ADPKD and AS coincided. This patient shows the very rare coexistence of two severe, inherited renal disorders and illustrates the importance of considering additional diagnoses in the setting of positive family history for a common hereditary disorder.
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Assuntos
Nefrite Hereditária/complicações , Rim Policístico Autossômico Dominante/complicações , Adulto , Humanos , Masculino
4.
PLoS Genet ; 10(4): e1004267, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24699222

RESUMO

Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.


Assuntos
Encéfalo/metabolismo , Ciclinas/genética , Deficiência Intelectual/genética , Magnésio/metabolismo , Mutação de Sentido Incorreto/genética , Convulsões/genética , Adolescente , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/metabolismo , Rim/metabolismo , Masculino , Fenótipo , Convulsões/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
5.
Eur J Med Genet ; 56(8): 458-62, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23811035

RESUMO

Distal deletion 9p is associated with gonadal dysfunction in XY individuals. Little is known about the gonadal function and fertility of XX females with this condition. We report on an affected 31-year-old infertile woman presenting with premature ovarian failure, mild dysmorphic features, a history of mild developmental delay and an otherwise normal female phenotype. Cytogenetic analysis showed a deletion 9p with the karyotype 46,XX,del(9)(p23-24) in lymphocytes. The subsequent oligonucleotide array-based CGH analysis with genomic DNA from peripheral blood revealed a terminal deletion of approximately 7.6 Mb. SNP microarray analyses of the patient and her unaffected parents confirmed the deletion breakpoint and revealed a de novo mutation of paternal origin. This is apparently the first description of an adult woman with a cytogenetically visible terminal deletion of chromosome 9p. The fertility problems observed in this patient complement earlier findings in prepubertal and pubertal 46,XX-girls with 9p deletions, who displayed a phenotype ranging from primary ovarian dysfunction and mild gonadotropin hyperresponses to positive menses. DMRT1 is hemizygous in our patient. We discuss the role of DMRT1 in female gonadal development.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 9 , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/genética , Adulto , Bandeamento Cromossômico , Hibridização Genômica Comparativa , Feminino , Humanos
6.
PLoS One ; 8(3): e60264, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23527311

RESUMO

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.


Assuntos
Neoplasias da Mama/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Sequência de Bases , Neoplasias da Mama/fisiopatologia , Análise Mutacional de DNA , Primers do DNA/genética , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Dosagem de Genes/genética , Estudos de Associação Genética , Alemanha , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Multiplex , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia
7.
Eur J Hum Genet ; 21(2): 162-72, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22781098

RESUMO

Identification of mutations in the HOGA1 gene as the cause of autosomal recessive primary hyperoxaluria (PH) type III has revitalized research in the field of PH and related stone disease. In contrast to the well-characterized entities of PH type I and type II, the pathophysiology and prevalence of type III is largely unknown. In this study, we analyzed a large cohort of subjects previously tested negative for type I/II by complete HOGA1 sequencing. Seven distinct mutations, among them four novel, were found in 15 patients. In patients of non-consanguineous European descent the previously reported c.700+5G>T splice-site mutation was predominant and represents a potential founder mutation, while in consanguineous families private homozygous mutations were identified throughout the gene. Furthermore, we identified a family where a homozygous mutation in HOGA1 (p.P190L) segregated in two siblings with an additional AGXT mutation (p.D201E). The two girls exhibiting triallelic inheritance presented a more severe phenotype than their only mildly affected p.P190L homozygous father. In silico analysis of five mutations reveals that HOGA1 deficiency is causing type III, yet reduced HOGA1 expression or aberrant subcellular protein targeting is unlikely to be the responsible pathomechanism. Our results strongly suggest HOGA1 as a major cause of PH, indicate a greater genetic heterogeneity of hyperoxaluria, and point to a favorable outcome of type III in the context of PH despite incomplete or absent biochemical remission. Multiallelic inheritance could have implications for genetic testing strategies and might represent an unrecognized mechanism for phenotype variability in PH.


Assuntos
Testes Genéticos , Hiperoxalúria Primária , Oxo-Ácido-Liases/genética , Adolescente , Adulto , Técnicas de Cultura de Células , Feminino , Expressão Gênica , Humanos , Hiperoxalúria Primária/diagnóstico , Hiperoxalúria Primária/genética , Cálculos Renais/genética , Cálculos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , Oxo-Ácido-Liases/metabolismo , Linhagem
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