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1.
Lab Invest ; 100(1): 135-146, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31273287

RESUMO

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

2.
Hum Mutat ; 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31663672

RESUMO

The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule long-read sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

4.
J Obstet Gynaecol ; 39(3): 328-334, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30714504

RESUMO

Molecular diagnostic investigations, following the identification of foetal abnormalities, are routinely performed using array comparative genomic hybridisation (aCGH). Despite the utility of this technique, contemporary approaches for the detection of copy number variation are typically based on next-generation sequencing (NGS). We sought to compare an in-house NGS-based workflow (CNVseq) with aCGH, for invasively obtained foetal samples from pregnancies complicated by foetal structural abnormality. DNA from 40 foetuses was screened using both 8 × 60 K aCGH oligoarrays and low-coverage whole genome sequencing. Sequencer-compatible libraries were combined in a ten-sample multiplex and sequenced using an Illumina HiSeq2500. The mean resolution of CNVseq was 29 kb, compared to 60 kb for aCGH analyses. Four clinically significant, concordant, copy number imbalances were detected using both techniques, however, genomic breakpoints were more precisely defined by CNVseq. This data indicates CNVseq is a robust and sensitive alternative to aCGH, for the prenatal investigation of foetuses with structural abnormalities. Impact statement What is already known about this subject? Copy number variant analysis using next-generation sequencing has been successfully applied to investigations of tumour specimens and patients with developmental delays. The application of our approach, to a prospective prenatal diagnosis cohort, has not hitherto been assessed. What do the results of this study add? Next-generation sequencing has a comparable turnaround time and assay sensitivity to copy number variant analysis performed using array CGH. We demonstrate that having established a next-generation sequencing facility, high-throughput CNVseq sample processing and analysis can be undertaken within the framework of a regional diagnostic service. What are the implications of these findings for clinical practice and/or further research? Array CGH is a legacy technology which is likely to be superseded by low-coverage whole genome sequencing, for the detection of copy number variants, in the prenatal diagnosis of structural abnormalities.


Assuntos
Hibridização Genômica Comparativa/normas , Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala/normas , Diagnóstico Pré-Natal/métodos , Feminino , Humanos , Gravidez , Estudos Prospectivos
5.
Am J Hum Genet ; 103(5): 727-739, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388400

RESUMO

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.


Assuntos
Transporte Biológico/genética , Flagelos/genética , Depuração Mucociliar/genética , Mutação/genética , Proteínas/genética , Adulto , Alelos , Axonema/genética , Linhagem Celular , Chlamydomonas/genética , Cílios/genética , Dineínas/genética , Células Epiteliais/patologia , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Fenótipo , Trypanosoma brucei brucei/genética
6.
Endocrine ; 59(3): 677-684, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29327300

RESUMO

CONTEXT: Pseudoacromegaly describes conditions with an acromegaly related physical appearance without abnormalities in the growth hormone (GH) axis. Acromegaloid facies, together with hypertrichosis, are typical manifestations of Cantú syndrome. CASE DESCRIPTION: We present a three-generation family with 5 affected members, with marked acromegaloid facies and prominent hypertrichosis, due to a novel missense variant in the ABCC9 gene. The proband, a 2-year-old girl, was referred due to marked hypertrichosis, noticed soon after birth, associated with coarsening of her facial appearance. Her endocrine assessment, including of the GH axis, was normal. The proband's father, paternal aunt, and half-sibling were referred to the Endocrine department for exclusion of acromegaly. Although the GH axis was normal in all, two subjects had clinically non-functioning pituitary macroadenomas, a feature which has not previously been associated with Cantú syndrome. CONCLUSIONS: Activating mutations in the ABCC9 and, less commonly, KCNJ8 genes-representing the two subunits of the ATP-sensitive potassium channel-have been linked with Cantú syndrome. Interestingly, minoxidil, a well-known ATP-sensitive potassium channel agonist, can cause a similar phenotype. There is no clear explanation why activating this channel would lead to acromegaloid features or hypertrichosis. This report raises awareness for this complex condition, especially for adult or pediatric endocrinologists who might see these patients referred for evaluation of acromegaloid features or hirsutism. The link between Cantú syndrome and pituitary adenomas is currently unclear.


Assuntos
Adenoma/complicações , Cardiomegalia/complicações , Hipertricose/complicações , Osteocondrodisplasias/complicações , Neoplasias Hipofisárias/complicações , Adenoma/diagnóstico por imagem , Adenoma/genética , Adulto , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/genética , Pré-Escolar , Feminino , Humanos , Hipertricose/diagnóstico por imagem , Hipertricose/genética , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/genética , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/genética , Receptores Sulfonilureia/genética , Adulto Jovem
7.
Mol Diagn Ther ; 21(6): 685-692, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986857

RESUMO

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.


Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Neoplasias/genética , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , PTEN Fosfo-Hidrolase/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
8.
J Mol Diagn ; 19(6): 933-940, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28867604

RESUMO

Like many clinical diagnostic laboratories, the Yorkshire Regional Genetics Service undertakes routine investigation of cancer-predisposed individuals by high-throughput sequencing of patient DNA that has been target-enriched for genes associated with hereditary cancer. Accurate diagnosis using such reagents requires alertness regarding rare nonpathogenic variants that may interfere with variant calling. In a cohort of 2042 such cases, we identified 5 that initially appeared to be carriers of a 95-bp deletion of SMAD4 intron 6. More detailed analysis indicated that these individuals all carried one copy of a SMAD4 processed gene. Because of its interference with diagnostic analysis, we characterized this processed gene in detail. Whole-genome sequencing and confirmatory Sanger sequencing of junction PCR products were used to show that in each of the 5 cases, the SMAD4 processed gene was integrated at the same position on chromosome 9, located within the last intron of the SCAI gene. This rare polymorphic processed gene therefore reflects the occurrence of a single ancestral retrotransposition event. Compared to the reference SMAD4 mRNA sequence NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide), the 5' and 3' untranslated regions of the processed gene are both truncated, but its open reading frame is unaltered. Our experience leads us to advocate the use of an RNA-seq aligner as part of diagnostic assay quality assurance, since this allows recognition of processed pseudogenes in a comparatively facile automated fashion.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Proteína Smad4/genética , Fatores de Transcrição/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 9/genética , Genômica , Heterozigoto , Humanos , Íntrons/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Patologia Molecular/métodos , Pseudogenes/genética , Sequenciamento Completo do Genoma
9.
PLoS One ; 12(9): e0185678, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28957425

RESUMO

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.


Assuntos
Alelos , Proteínas de Ciclo Celular/genética , Regiões Promotoras Genéticas , Retroelementos , Elementos Nucleotídeos Curtos e Dispersos/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Linfócitos B/metabolismo , Ilhas de CpG , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Transcrição Genética
10.
RNA ; 23(10): 1493-1501, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28724534

RESUMO

Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.


Assuntos
Adenosina/análogos & derivados , Biologia Computacional/métodos , Análise de Sequência de RNA/métodos , Software , Adenosina/análise , Interface Usuário-Computador
11.
PLoS One ; 12(4): e0174264, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28388629

RESUMO

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.


Assuntos
Encéfalo/embriologia , Espermatogênese/genética , Tubulina (Proteína)/genética , Animais , Exoma , Homozigoto , Camundongos , Camundongos Knockout
12.
J Neurosurg Pediatr ; 19(6): 675-683, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28362186

RESUMO

OBJECTIVE Complications have been used extensively to facilitate evaluation of craniosynostosis practice. However, description of complications tends to be nonstandardized, making comparison difficult. The authors propose a new pragmatic classification of complications that relies on prospective data collection, is geared to capture significant morbidity as well as any "near misses" in a systematic fashion, and can be used as a quality improvement tool. METHODS Data on complications for all patients undergoing surgery for nonsyndromic craniosynostosis between 2010 and 2015 were collected from a prospective craniofacial audit database maintained at the authors' institution. Information on comorbidities, details of surgery, and follow-up was extracted from medical records, anesthetic and operation charts, and electronic databases. Complications were defined as any unexpected event that resulted or could have resulted in a temporary or permanent damage to the child. RESULTS A total of 108 operations for the treatment of nonsyndromic craniosynostosis were performed in 103 patients during the 5-year study period. Complications were divided into 6 types: 0) perioperative occurrences; 1) inpatient complications; 2) outpatient complications not requiring readmission; 3) complications requiring readmission; 4) unexpected long-term deficit; and 5) mortality. These types were further subdivided according to the length of stay and time after discharge. The overall complication rate was found to be 35.9%. CONCLUSIONS The proportion of children with some sort of complication using the proposed definition was much higher than commonly reported, predominantly due to the inclusion of problems often dismissed as minor. The authors believe that these complications should be included in determining complication rates, as they will cause distress to families and may point to potential areas for improving a surgical service.


Assuntos
Craniossinostoses/cirurgia , Procedimentos Neurocirúrgicos/efeitos adversos , Complicações Pós-Operatórias/classificação , Pré-Escolar , Comorbidade , Craniossinostoses/epidemiologia , Bases de Dados Factuais , Feminino , Seguimentos , Humanos , Lactente , Tempo de Internação , Masculino , Procedimentos Neurocirúrgicos/estatística & dados numéricos , Readmissão do Paciente , Complicações Pós-Operatórias/epidemiologia , Estudos Prospectivos , Estudos Retrospectivos
13.
J Pathol ; 241(2): 119-122, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27859271

RESUMO

The analytical power of modern methods for DNA analysis has outstripped our capability to interpret and understand the data generated. To make good use of this genomic data in a biomedical setting (whether for research or diagnosis), it is vital that we understand the mechanisms through which mutations affect biochemical pathways and physiological systems. This lies at the centre of what genetics is all about, and it is the reason why genetics and genomics should go hand in hand whenever possible. In this Annual Review Issue of The Journal of Pathology, we have assembled a collection of 16 expert reviews covering a wide range of topics. Through these, we illustrate the power of genetic analysis to improve our understanding of normal physiology and disease pathology, and thereby to think in rational ways about clinical management. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Antineoplásicos/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Genômica , Doenças Inflamatórias Intestinais/terapia , Mutação/genética , Animais , Testes Genéticos , Humanos , Doenças Inflamatórias Intestinais/genética
14.
PLoS One ; 11(6): e0157075, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27272187

RESUMO

Next generation sequencing methodologies are facilitating the rapid characterisation of novel structural variants at nucleotide resolution. These approaches are particularly applicable to variants initially identified using alternative molecular methods. We report a child born with bilateral postaxial syndactyly of the feet and bilateral fifth finger clinodactyly. This was presumed to be an autosomal recessive syndrome, due to the family history of consanguinity. Karyotype analysis revealed a homozygous pericentric inversion of chromosome 7 (46,XX,inv(7)(p15q21)x2) which was confirmed to be heterozygous in both unaffected parents. Since the resolution of the karyotype was insufficient to identify any putatively causative gene, we undertook medium-coverage whole genome sequencing using paired-end reads, in order to elucidate the molecular breakpoints. In a two-step analysis, we first narrowed down the region by identifying discordant read-pairs, and then determined the precise molecular breakpoint by analysing the mapping locations of "soft-clipped" breakpoint-spanning reads. PCR and Sanger sequencing confirmed the identified breakpoints, both of which were located in intergenic regions. Significantly, the 7p15 breakpoint was located 523 kb upstream of HOXA13, the locus for hand-foot-genital syndrome. By inference from studies of HOXA locus control in the mouse, we suggest that the inversion has delocalised a HOXA13 enhancer to produce the phenotype observed in our patient. This study demonstrates how modern genetic diagnostic approach can characterise structural variants at nucleotide resolution and provide potential insights into functional regulation.


Assuntos
Anormalidades Múltiplas/genética , Inversão Cromossômica , Cromossomos Humanos Par 7/genética , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Proteínas de Homeodomínio/genética , Anormalidades Urogenitais/genética , Pontos de Quebra do Cromossomo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Análise de Sequência de DNA
15.
Am J Hum Genet ; 98(4): 735-43, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058446

RESUMO

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.


Assuntos
Corpo Estriado/patologia , Hipercinese/genética , Mutação , Diester Fosfórico Hidrolases/genética , Alelos , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Variação Genética , Células HEK293 , Humanos , Hipercinese/diagnóstico , Hipercinese/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linhagem , Inibidores de Fosfodiesterase/metabolismo , Alinhamento de Sequência
16.
BMC Med Genet ; 17: 1, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26729329

RESUMO

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.


Assuntos
Cerebelo/anormalidades , Mapeamento Cromossômico , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/genética , Encefalocele/diagnóstico , Encefalocele/genética , Exoma , Doenças Renais Policísticas/diagnóstico , Doenças Renais Policísticas/genética , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Estudos de Coortes , Variações do Número de Cópias de DNA , Éxons , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Diagnóstico Pré-Natal , Retinite Pigmentosa , Análise de Sequência de DNA , Deleção de Sequência
17.
J Med Genet ; 53(4): 264-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26733463

RESUMO

BACKGROUND: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. METHODS: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. RESULTS: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. CONCLUSIONS: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme.


Assuntos
Artrogripose/genética , Retardo do Crescimento Fetal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteína MyoD/genética , Feto Abortado , Animais , Artrogripose/patologia , Exoma/genética , Feminino , Retardo do Crescimento Fetal/patologia , Humanos , Pulmão/patologia , Camundongos , Mutação , Linhagem , Fenótipo , Gravidez
19.
J Med Genet ; 52(12): 797-803, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26424145

RESUMO

BACKGROUND: The genetic aetiology of neurodevelopmental defects is extremely diverse, and the lack of distinctive phenotypic features means that genetic criteria are often required for accurate diagnostic classification. We aimed to identify the causative genetic lesions in two families in which eight affected individuals displayed variable learning disability, spasticity and abnormal gait. METHODS: Autosomal recessive inheritance was suggested by consanguinity in one family and by sibling recurrences with normal parents in the second. Autozygosity mapping and exome sequencing, respectively, were used to identify the causative gene. RESULTS: In both families, biallelic loss-of-function mutations in HACE1 were identified. HACE1 is an E3 ubiquitin ligase that regulates the activity of cellular GTPases, including Rac1 and members of the Rab family. In the consanguineous family, a homozygous mutation p.R219* predicted a truncated protein entirely lacking its catalytic domain. In the other family, compound heterozygosity for nonsense mutation p.R748* and a 20-nt insertion interrupting the catalytic homologous to the E6-AP carboxyl terminus (HECT) domain was present; western blot analysis of patient cells revealed an absence of detectable HACE1 protein. CONCLUSION: HACE1 mutations underlie a new autosomal recessive neurodevelopmental disorder. Previous studies have implicated HACE1 as a tumour suppressor gene; however, since cancer predisposition was not observed either in homozygous or heterozygous mutation carriers, this concept may require re-evaluation.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/deficiência , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Lactente , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Síndrome , Ubiquitina-Proteína Ligases/genética
20.
Bioinformatics ; 31(23): 3822-9, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26272982

RESUMO

MOTIVATION: Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. RESULTS: Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype.We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool. AVAILABILITY AND IMPLEMENTATION: OVA is freely accessible at http://dna2.leeds.ac.uk:8080/OVA/index.jsp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: umaan@leeds.ac.uk.


Assuntos
Algoritmos , Ontologias Biológicas , Biologia Computacional/métodos , Doença/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Exoma/genética , Genótipo , Humanos , Transcriptoma
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