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1.
Am J Med Genet A ; 179(12): 2494-2499, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31595668

RESUMO

Myhre syndrome is a rare multisystem connective tissue disorder, characterized by short stature, facial dysmorphology, variable intellectual disability, skeletal abnormalities, arthropathy, cardiopathy, laryngotracheal anomalies, and stiff skin. So far, all molecularly confirmed cases harbored a de novo heterozygous gain-of-function mutation in SMAD4, encoding the SMAD4 transducer protein required for both transforming growth factor-beta and bone morphogenic proteins signaling. We report on four novel patients (one female proband and her two affected children, and one male proband) with Myhre syndrome harboring the recurrent c.1486C>T (p.Arg496Cys) mutation in SMAD4. The female proband presented with a congenital heart defect, vertebral anomalies, and facial dysmorphic features. She developed severe tracheal stenosis requiring a total laryngectomy. With assisted reproductive treatment, she gave birth to two affected children. The second proband presented with visual impairment following lensectomy in childhood, short stature, brachydactyly, stiff skin, and decreased peripheral sensitivity. Transmission electron microscopy (TEM) of the dermis shows irregular elastin cores with globular deposits and almost absent surrounding microfibrils and suggests age-related increased collagen deposition. We report on the first familial case of Myhre syndrome and illustrate the variable clinical spectrum of the disorder. Despite the primarily fibrotic nature of the disease, TEM analysis mainly indicates elastic fiber anomalies.

2.
Genet Med ; 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31316167

RESUMO

PURPOSE: Congenital contractural arachnodactyly (CCA) is an autosomal dominant connective tissue disorder manifesting joint contractures, arachnodactyly, crumpled ears, and kyphoscoliosis as main features. Due to its rarity, rather aspecific clinical presentation, and overlap with other conditions including Marfan syndrome, the diagnosis is challenging, but important for prognosis and clinical management. CCA is caused by pathogenic variants in FBN2, encoding fibrillin-2, but locus heterogeneity has been suggested. We designed a clinical scoring system and diagnostic criteria to support the diagnostic process and guide molecular genetic testing. METHODS: In this retrospective study, we assessed 167 probands referred for FBN2 analysis and classified them into a FBN2-positive (n = 44) and FBN2-negative group (n = 123) following molecular analysis. We developed a 20-point weighted clinical scoring system based on the prevalence of ten main clinical characteristics of CCA in both groups. RESULTS: The total score was significantly different between the groups (P < 0.001) and was indicative for classifying patients into unlikely CCA (total score <7) and likely CCA (total score ≥7) groups. CONCLUSIONS: Our clinical score is helpful for clinical guidance for patients suspected to have CCA, and provides a quantitative tool for phenotyping in research settings.

3.
Sci Rep ; 8(1): 15845, 2018 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-30374100

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

4.
Dis Model Mech ; 11(10)2018 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-30355591

RESUMO

Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.

6.
Methods Mol Biol ; 1865: 83-90, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151760

RESUMO

Due to its simple nature, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technique is massively used nowadays to modify genomic loci in a wide range of model systems. The possibility to interrogate gene function on a genome-wide scale is revolutionizing fundamental life sciences and will lead to new clinical breakthroughs. Its strength is even more pronounced when it is used in tandem with next-generation sequencing (NGS). The high throughput and low cost cause NGS to be the method of choice for exploring CRISPR-Cas9 experimental results. To analyze the NGS reads from genome editing experiments only few bioinformatics tools are available. BATCH-GE is a flexible and easy-to-use tool, which is especially useful for dealing with large amounts of data. It detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel.

7.
Am J Hum Genet ; 103(2): 245-260, 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30057031

RESUMO

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

8.
Circ Genom Precis Med ; 11(6): e002039, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29875124

RESUMO

BACKGROUND: The introduction of next-generation sequencing techniques has substantially increased the identification of new genetic variants and hence the necessity of accurate variant interpretation. In 2015, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology proposed new variant interpretation guidelines. Gene-specific characteristics were, however, not considered, sometimes leading to inconsistent variant interpretation. METHODS: To allow a more uniform interpretation of variants in the FBN1 (fibrillin-1) gene, causing Marfan syndrome, we tailored these guidelines to this gene and disease. We adapted 15 of the 28 general criteria and classified 713 FBN1 variants previously identified in our laboratory as causal mutation or variant of uncertain significance according to these adapted guidelines. We then compared the agreement between previous methods and the adapted American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. RESULTS: Agreement between the methods was 86.4% (K-alpha, 0.6). Application of the tailored guidelines resulted in an increased number of variants of uncertain significance (14.5% to 24.2%). Of the 85 variants that were downscaled to likely benign or variant of uncertain significance, 59.7% were missense variants outside a well-established functional site. Available clinical- or segregation data, necessary to further classify these types of variants, were in many cases insufficient to aid the classification. CONCLUSIONS: Our study shows that classification of variants remains challenging and may change over time. Currently, a higher level of evidence is necessary to classify a variant as pathogenic. Gene-specific guidelines may be useful to allow a more precise and uniform interpretation of the variants to accurately support clinical decision-making.

9.
Acta Clin Belg ; 73(1): 7-10, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29384039

RESUMO

INTRODUCTION: In recent decades, we witnessed a revolution in genetic technology. Some 20 years ago, analysing a single gene was quite laborious and time-consuming. In addition, diagnostic testing was only available for selected genes. Nowadays, whole exome analysis - a technique enabling sequencing of all protein coding sequences in the entire genome - is gradually introduced in a clinical setting. Whole genome sequencing forms the ultimate exponent of this evolution and offers an even broader application. METHODS: A review of the application of these technologies in a diagnostic setting is presented. RESULTS: Whole exome sequencing has a prominent place in modern clinical diagnostics. It offers a cost- and time-efficient way to interrogate all protein coding portions of the genome leading to a quick and adequate diagnosis, also in cases of phenotypic heterogeneity. As sequencing costs continue to drop, whole genome sequencing will take over in the near future guaranteeing a further improvement of the quality of genetic testing. CONCLUSION: Due to technological advances in the past decades, the field of clinical diagnostics has changed dramatically. With techniques such as whole exome and whole genome sequencing, the diagnostic yield increases serving both the patient and the health care system.


Assuntos
Testes Genéticos/tendências , Sequenciamento Completo do Genoma , Humanos
10.
Am J Med Genet A ; 173(4): 1047-1050, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28261977

RESUMO

Type I collagen is the predominant protein of connective tissues such as skin and bone. Mutations in the type I collagen genes (COL1A1 and COL1A2) mainly cause osteogenesis imperfecta (OI). We describe a patient with clinical signs of Ehlers-Danlos syndrome (EDS), including fragile skin, easy bruising, recurrent luxations, and fractures resembling mild OI. Biochemical collagen analysis of the patients' dermal fibroblasts showed faint overmodification of the type I collagen bands, a finding specific for structural defects in type I collagen. Bidirectional Sanger sequencing detected an in-frame deletion in exon 44 of COL1A1 (c.3150_3158del), resulting in the deletion of three amino acids (p.Ala1053_Gly1055del) in the collagen triple helix. This COL1A1 mutation was hitherto identified in four probands with lethal OI, and never in EDS patients. As the peaks on the electropherogram corresponding to the mutant allele were decreased in intensity, we performed next generation sequencing of COL1A1 to study mosaicism in skin and blood. While approximately 9% of the reads originating from fibroblast gDNA harbored the COL1A1 deletion, the deletion was not detected in gDNA from blood. Most likely, the mild clinical symptoms observed in our patient can be explained by the mosaic state of the mutation.


Assuntos
Colágeno Tipo I/genética , Síndrome de Ehlers-Danlos/genética , Mosaicismo , Mutação , Osteogênese Imperfeita/genética , Sequência de Bases , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/patologia , Éxons , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Especificidade de Órgãos , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/patologia , Pele/metabolismo , Pele/patologia , Adulto Jovem
11.
Am J Hum Genet ; 100(2): 216-227, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28065471

RESUMO

Defects of the V-type proton (H+) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V1 domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.


Assuntos
Cútis Laxa/genética , Mutação de Sentido Incorreto , ATPases Vacuolares Próton-Translocadoras/genética , Adolescente , Alelos , Sequência de Aminoácidos , Estudos de Casos e Controles , Criança , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Conformação Proteica , Transporte Proteico , Espectrometria de Massas em Tandem
12.
Genet Med ; 19(4): 457-466, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27608171

RESUMO

PURPOSE: Our goal was to design a customized microarray, arrEYE, for high-resolution copy number variant (CNV) analysis of known and candidate genes for inherited retinal dystrophy (iRD) and retina-expressed noncoding RNAs (ncRNAs). METHODS: arrEYE contains probes for the full genomic region of 106 known iRD genes, including those implicated in retinitis pigmentosa (RP) (the most frequent iRD), cone-rod dystrophies, macular dystrophies, and an additional 60 candidate iRD genes and 196 ncRNAs. Eight CNVs in iRD genes identified by other techniques were used as positive controls. The test cohort consisted of 57 patients with autosomal dominant, X-linked, or simplex RP. RESULTS: In an RP patient, a novel heterozygous deletion of exons 7 and 8 of the HGSNAT gene was identified: c.634-408_820+338delinsAGAATATG, p.(Glu212Glyfs*2). A known variant was found on the second allele: c.1843G>A, p.(Ala615Thr). Furthermore, we expanded the allelic spectrum of USH2A and RCBTB1 with novel CNVs. CONCLUSION: The arrEYE platform revealed subtle single-exon to larger CNVs in iRD genes that could be characterized at the nucleotide level, facilitated by the high resolution of the platform. We report the first CNV in HGSNAT that, combined with another mutation, leads to RP, further supporting its recently identified role in nonsyndromic iRD.Genet Med 19 4, 457-466.


Assuntos
Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Distrofias Retinianas/genética , Acetiltransferases/genética , Proteínas da Matriz Extracelular/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , RNA não Traduzido/genética , Deleção de Sequência
13.
Sci Rep ; 6: 35264, 2016 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-27739525

RESUMO

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Retinoblastoma/genética , Xenopus/genética , Animais , Modelos Animais de Doenças , Neoplasias Oculares/genética , Neoplasias Oculares/patologia , Técnicas de Inativação de Genes/métodos , Retinoblastoma/patologia , Proteína do Retinoblastoma/genética
14.
BMC Med Genet ; 17: 13, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26880286

RESUMO

BACKGROUND: Nonphotosensitive trichothiodystrophy (TTDN) is a rare autosomal recessive disorder of neuroectodermal origin. The condition is marked by hair abnormalities, intellectual impairment, nail dystrophies and susceptibility to infections but with no UV sensitivity. METHODS: We identified three consanguineous Pakistani families with varied TTDN features and used homozygosity mapping, linkage analysis, and Sanger and exome sequencing in order to identify pathogenic variants. Haplotype analysis was performed and haplotype age estimated. A splicing assay was used to validate the effect of the MPLKIP splice variant on expression. RESULTS: Affected individuals from all families exhibit several TTDN features along with a heart-specific feature, i.e. mitral regurgitation. Exome sequencing in the probands from families ED168 and ED241 identified a homozygous splice mutation c.339 + 1G > A within MPLKIP. The same splice variant co-segregates with TTDN in a third family ED210. The MPLKIP splice variant was not found in public databases, e.g. the Exome Aggregation Consortium, and in unrelated Pakistani controls. Functional analysis of the splice variant confirmed intron retention, which leads to protein truncation and loss of a phosphorylation site. Haplotype analysis identified a 585.1-kb haplotype which includes the MPLKIP variant, supporting the existence of a founder haplotype that is estimated to be 25,900 years old. CONCLUSION: This study extends the allelic and phenotypic spectra of MPLKIP-related TTDN, to include a splice variant that causes cardiomyopathy as part of the TTDN phenotype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Insuficiência da Valva Mitral/genética , Processamento de RNA , Síndromes de Tricotiodistrofia/genética , Adolescente , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Criança , Clonagem Molecular , Exoma , Feminino , Ligação Genética , Células HEK293 , Haplótipos , Homozigoto , Humanos , Íntrons , Masculino , Paquistão , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Adulto Jovem
15.
Dis Markers ; 2015: 828970, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26504261

RESUMO

Joint hypermobility is a common, mostly benign, finding in the general population. In a subset of individuals, however, it causes a range of clinical problems, mainly affecting the musculoskeletal system. Joint hypermobility often appears as a familial trait and is shared by several heritable connective tissue disorders, including the hypermobility subtype of the Ehlers-Danlos syndrome (EDS-HT) or benign joint hypermobility syndrome (BJHS). These hereditary conditions provide unique models for the study of the genetic basis of joint hypermobility. Nevertheless, these studies are largely hampered by the great variability in clinical presentation and the often vague mode of inheritance in many families. Here, we performed a genome-wide linkage scan in a unique three-generation family with an autosomal dominant EDS-HT phenotype and identified a linkage interval on chromosome 8p22-8p21.1, with a maximum two-point LOD score of 4.73. Subsequent whole exome sequencing revealed the presence of a unique missense variant in the LZTS1 gene, located within the candidate region. Subsequent analysis of 230 EDS-HT/BJHS patients resulted in the identification of three additional rare variants. This is the first reported genome-wide linkage analysis in an EDS-HT family, thereby providing an opportunity to identify a new disease gene for this condition.


Assuntos
Cromossomos Humanos Par 8/genética , Síndrome de Ehlers-Danlos/genética , Linhagem , Adulto , Células Cultivadas , Proteínas de Ligação a DNA/genética , Síndrome de Ehlers-Danlos/diagnóstico , Exoma , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Fenótipo , Proteínas Supressoras de Tumor/genética
16.
Am J Hum Genet ; 97(4): 521-34, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26365339

RESUMO

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.


Assuntos
Cílios/genética , Transtornos da Motilidade Ciliar/genética , Anormalidades Craniofaciais/genética , Proteínas de Membrana/genética , Mutação/genética , Ossificação Heterotópica/genética , Osteocondrodisplasias/genética , Sequência de Aminoácidos , Animais , Padronização Corporal , Diferenciação Celular , Movimento Celular , Cílios/metabolismo , Cílios/patologia , Embrião não Mamífero/anormalidades , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Masculino , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Crista Neural/citologia , Crista Neural/metabolismo , Linhagem , Transporte Proteico , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética
17.
Neurology ; 84(17): 1760-6, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25841028

RESUMO

OBJECTIVE: To identify the genetic cause in 2 Belgian families with autosomal recessive Huntington-like disorder (HDL). METHODS: Homozygosity mapping and whole-exome sequencing in a consanguineous family as well as Sanger sequencing of the candidate gene in an independent family with HDL followed by genotype-phenotype correlation studies. RESULTS: We identified a homozygous mutation in the gene RNF216 p.(Gly456Glu) within a shared 4.8-Mb homozygous region at 7p22.3 in 2 affected siblings of a consanguineous HDL family. In an independent family, 2 siblings with HDL were compound heterozygous for mutations in RNF216 p.(Gln302*) and p.(Tyr539Cys). Chorea, behavioral problems, and severe dementia were the core clinical signs in all patients. Brain imaging consistently showed white matter lesions. Low gonadotropin serum levels and cerebellar atrophy could be demonstrated in the index family. CONCLUSIONS: Mutations in RNF216 have recently been found in families with Gordon Holmes syndrome, a condition defined by hypogonadotropic hypogonadism and cerebellar ataxia. The mode of inheritance was proposed to be oligogenic for most families. We describe novel RNF216 mutations causing an HDL phenotype with pure monogenic recessive inheritance. Subclinical serum evidence of hypogonadotropic hypogonadism links this disorder to Gordon Holmes syndrome. Our study thus challenges the oligogenic inheritance model and emphasizes chorea as an essential clinical feature in RNF216-mediated neurodegeneration.


Assuntos
Doença de Huntington/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idade de Início , Idoso , Bélgica , Cerebelo/patologia , Consanguinidade , Feminino , Genes Recessivos , Gonadotropinas/sangue , Humanos , Doença de Huntington/sangue , Doença de Huntington/patologia , Masculino , Pessoa de Meia-Idade , Herança Multifatorial , Mutação , Linhagem
18.
Hum Mutat ; 36(3): 379-87, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25504618

RESUMO

The release of benchtop next-generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost-efficient workflows is high. We used singleplex-PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing-based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost-efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189-accreditation was obtained for the workflow. A major asset of the singleplex PCR-based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.


Assuntos
Doenças Genéticas Inatas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Prognóstico , Sensibilidade e Especificidade
19.
J Invest Dermatol ; 135(4): 992-998, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25264593

RESUMO

The molecular etiology of pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in ATP-binding cassette family C member 6 (ABCC6) but also ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and gamma-glutamyl carboxylase (GGCX) can cause resembling phenotypes. Identification of modifier genes, such as vascular endothelial growth factor A, has further contributed to the molecular heterogeneity of PXE. In such heterogeneous diseases, next-generation sequencing (NGS) allows to perform mutation screening of several genes in a single reaction. We explored whole-exome sequencing (WES) as an efficient diagnostic tool to identify the causal mutations in ABCC6, GGCX, ENPP1, and vitamin K epoxide reductase complex, subunit 1 (VKORC1) in 16 PXE patients. WES identified a causal ABCC6 mutation in 30 out of 32 alleles and one GGCX mutation, whereas no causal mutations in ENPP1 or VKORC1 were detected. Exomes with insufficient reads (⩽20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found. The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative. Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.


Assuntos
Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Pseudoxantoma Elástico/diagnóstico , Pseudoxantoma Elástico/genética , Análise de Sequência de DNA , Adulto , Alelos , Carbono-Carbono Ligases/genética , Feminino , Variação Genética , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Patologia Molecular , Fenótipo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Vitamina K Epóxido Redutases/genética
20.
Mol Genet Metab ; 113(3): 230-5, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25240749

RESUMO

INTRODUCTION: Stickler syndrome is caused by mutations in genes encoding type II and type XI collagens. About 85% of the pathogenic variants is found in COL2A1 (Stickler type 1), whereas a minority of mutations has been reported in COL11A1 (Stickler type 2) and COL11A2 (Stickler type 3). Beside the typical skeletal and orofacial manifestations, ocular anomalies are predominantly present in type 1 and type 2, while hearing loss is more pronounced in type 2 and type 3. METHODS: We performed COL11A1 mutation analysis for 40 type 2 Stickler patients and COL11A2 mutation analysis for five type 3 Stickler patients, previously all COL2A1 mutation-negative, using targeted next-generation sequencing (NGS) whereas whole-exome sequencing (WES) was performed in parallel for two patients. Three patients were analyzed for both genes due to unclear ocular findings. RESULTS: In total 14 COL11A1 and two COL11A2 mutations could be identified, seven of which are novel. Splice site alterations are the most frequent mutation type, followed by glycine substitutions. In addition, six variants of unknown significance (VUS) have been found. Identical mutations and variants were identified with both NGS techniques. CONCLUSION: We expand the mutation spectrum of COL11A1 and COL11A2 in Stickler syndrome patients and show that targeted NGS is an efficient and cost-effective molecular tool in the genetic diagnosis of Stickler syndrome, whereas the more standardized WES might be an alternative approach.


Assuntos
Colágeno Tipo XI/genética , Artrite , Doenças do Colágeno/genética , Doenças do Tecido Conjuntivo , Análise Mutacional de DNA , Exoma , Estudos de Associação Genética , Perda Auditiva Neurossensorial , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linhagem , Descolamento Retiniano
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