Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 84
Filter
Add more filters

Country/Region as subject
Publication year range
1.
J Med Genet ; 61(4): 363-368, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38290823

ABSTRACT

BACKGROUND: SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, rare heterozygous loss-of-function variants in SMAD6 were demonstrated to increase the risk of disparate clinical disorders including cardiovascular disease, craniosynostosis and radioulnar synostosis. Only two unrelated patients harbouring biallelic SMAD6 variants presenting a complex cardiovascular phenotype and facial dysmorphism have been described. CASES: Here, we present the first two patients with craniosynostosis harbouring homozygous SMAD6 variants. The male probands, both born to healthy consanguineous parents, were diagnosed with metopic synostosis and bilateral or unilateral radioulnar synostosis. Additionally, one proband had global developmental delay. Echocardiographic evaluation did not reveal cardiac or outflow tract abnormalities. MOLECULAR ANALYSES: The novel missense (c.[584T>G];[584T>G], p.[(Val195Gly)];[(Val195Gly)]) and missense/splice-site variant (c.[817G>A];[817G>A], r.[(817g>a,817delins[a;817+2_817+228])];[(817g>a,817delins[a;817+2_817+228])], p.[(Glu273Lys,Glu273Serfs*72)];[(Glu273Lys,Glu273Serfs*72)]) both locate in the functional MH1 domain of the protein and have not been reported in gnomAD database. Functional analyses of the variants showed reduced inhibition of BMP signalling or abnormal splicing, respectively, consistent with a hypomorphic mechanism of action. CONCLUSION: Our data expand the spectrum of variants and phenotypic spectrum associated with homozygous variants of SMAD6 to include craniosynostosis.


Subject(s)
Craniosynostoses , Radius/abnormalities , Synostosis , Ulna/abnormalities , Humans , Male , Craniosynostoses/diagnosis , Craniosynostoses/genetics , Radius/metabolism , Ulna/metabolism , Mutation, Missense/genetics , Smad6 Protein/genetics , Smad6 Protein/metabolism
2.
J Clin Immunol ; 43(7): 1543-1556, 2023 10.
Article in English | MEDLINE | ID: mdl-37246174

ABSTRACT

BACKGROUND: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. OBJECTIVE: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. METHODS: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. RESULTS: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. CONCLUSION: CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.


Subject(s)
Antigens, CD19 , Plasma Cells , Humans , Plasma Cells/metabolism , Antigens, CD19/genetics , Antigens, CD19/metabolism , Proto-Oncogene Proteins c-akt/metabolism , B-Lymphocytes , Receptors, Antigen, B-Cell , Adaptor Proteins, Signal Transducing/metabolism , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism
3.
J Med Genet ; 59(11): 1082-1086, 2022 11.
Article in English | MEDLINE | ID: mdl-35414530

ABSTRACT

Background The ever-increasing capacity of short-read sequencing instruments is driving the adoption of whole genome sequencing (WGS) as a universal approach to the diagnosis of rare genetic disorders. However, many challenging genomic regions remain, for which alternative technologies must be deployed in order to address the clinical question satisfactorily. Methods Here we report the use of long-read sequencing to resolve ambiguity over a suspected diagnosis of Angelman syndrome. Results Despite a normal chromosomal microarray result and methylation studies at the imprinted 15q11q13 locus, the continued clinical suspicion of Angelman Syndrome prompted trio WGS of the proband and his parents. A de novo heterozygous frameshift variant, c.2370_2373del (NM_130838.2) p.(Asp790Glufs*7), in UBE3A was identified. To determine the parental allele on which this variant arose, long-read sequencing of the flanking genomic region was performed. Comparison of the resulting haplotypes allowed us to determine that the pathogenic frameshift variant arose on the maternal allele, confirming a diagnosis of Angelman syndrome in this case. Conclusion Long-read nanopore sequencing provides significant clinical utility when assessing the parental origin of de novo variants.


Subject(s)
Angelman Syndrome , Humans , Angelman Syndrome/diagnosis , Angelman Syndrome/genetics , Frameshift Mutation/genetics , Haplotypes , Whole Genome Sequencing , Ubiquitin-Protein Ligases/genetics
4.
Prenat Diagn ; 42(2): 226-232, 2022 Feb.
Article in English | MEDLINE | ID: mdl-35014072

ABSTRACT

BACKGROUND: The adoption of massively parallel short-read DNA sequencing methods has greatly expanded the scope and availability of genetic testing for inherited diseases. Indeed, the power of these methods has encouraged the integration of whole genome sequencing, the most comprehensive single approach to genomic analysis, into clinical practice. Despite these advances, diagnostic techniques that incompletely resolve the precise molecular boundaries of pathogenic sequence variants continue to be routinely deployed. This can present a barrier for certain prenatal diagnostic approaches. For example, the pre-referral workup for couples seeking preimplantation genetic diagnosis requires intragenic dosage variants to be characterised at nucleotide resolution. OBJECTIVE: We sought to assess the use of long-read nanopore sequencing to rapidly characterise an apparent heterozygous RB1 exon 23 deletion that was initially identified by multiplex ligation-dependent probe amplification (MLPA), in a patient with bilateral retinoblastoma. METHODS: Target enrichment was performed by long-range polymerase chain reaction (PCR) amplification prior to Flongle sequencing on a MinION long-read sequencer. RESULTS: Characterisation of the deletion breakpoint included an unexpected 85-bp insertion which duplicated RB1 exon 24 (and was undetected by MLPA). The long-read sequence permitted design of a multiplex PCR assay, which confirmed that the mutation arose de novo. CONCLUSION: Our experience demonstrates the diagnostic utility of long-read technology for the precise characterisation of structural variants, and highlights how this technology can be efficiently deployed to enable onward referral to reproductive medicine services.


Subject(s)
Gene Deletion , Gene Duplication , Genetic Testing/methods , Nanopore Sequencing/methods , Preimplantation Diagnosis/methods , Retinoblastoma/diagnosis , Sequence Analysis, DNA/methods , Female , Genetic Markers , Humans , Infant , Male , Pregnancy , Retinoblastoma/genetics , Retinoblastoma Binding Proteins/genetics , Ubiquitin-Protein Ligases/genetics
5.
Lab Invest ; 101(4): 442-449, 2021 04.
Article in English | MEDLINE | ID: mdl-32989232

ABSTRACT

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.


Subject(s)
DNA Mutational Analysis/methods , Interspersed Repetitive Sequences/genetics , Mutagenesis, Insertional/genetics , Nanopore Sequencing/methods , DNA/analysis , DNA/genetics , Databases, Genetic , High-Throughput Nucleotide Sequencing/methods , Humans , Neoplasms/genetics
6.
Am J Hum Genet ; 103(5): 727-739, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30388400

ABSTRACT

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.


Subject(s)
Biological Transport/genetics , Flagella/genetics , Mucociliary Clearance/genetics , Mutation/genetics , Proteins/genetics , Adult , Alleles , Axoneme/genetics , Cell Line , Chlamydomonas/genetics , Cilia/genetics , Dyneins/genetics , Epithelial Cells/pathology , Female , HEK293 Cells , Humans , Infant , Male , Phenotype , Trypanosoma brucei brucei/genetics
7.
Hum Mutat ; 41(2): 525-531, 2020 02.
Article in English | MEDLINE | ID: mdl-31663672

ABSTRACT

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.


Subject(s)
Ciliary Motility Disorders/diagnosis , Ciliary Motility Disorders/genetics , Encephalocele/diagnosis , Encephalocele/genetics , High-Throughput Nucleotide Sequencing , Membrane Proteins/genetics , Polycystic Kidney Diseases/diagnosis , Polycystic Kidney Diseases/genetics , Retinitis Pigmentosa/diagnosis , Retinitis Pigmentosa/genetics , Base Sequence , Exons , Genetic Association Studies , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Sequence Analysis, DNA
8.
Lab Invest ; 100(1): 135-146, 2020 01.
Article in English | MEDLINE | ID: mdl-31273287

ABSTRACT

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.


Subject(s)
Autism Spectrum Disorder/genetics , Cytoskeletal Proteins/genetics , Dystrophin/genetics , Gene Duplication , Molecular Chaperones/genetics , Nanopore Sequencing/methods , Adolescent , CRISPR-Associated Protein 9 , Child, Preschool , Comparative Genomic Hybridization , Female , Humans , Male
9.
Am J Hum Genet ; 98(4): 735-43, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-27058446

ABSTRACT

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.


Subject(s)
Corpus Striatum/pathology , Hyperkinesis/genetics , Mutation , Phosphoric Diester Hydrolases/genetics , Alleles , Amino Acid Sequence , Animals , Disease Models, Animal , Gene Expression Regulation , Genetic Variation , HEK293 Cells , Humans , Hyperkinesis/diagnosis , Hyperkinesis/pathology , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pedigree , Phosphodiesterase Inhibitors/metabolism , Sequence Alignment
10.
RNA ; 23(10): 1493-1501, 2017 10.
Article in English | MEDLINE | ID: mdl-28724534

ABSTRACT

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.


Subject(s)
Adenosine/analogs & derivatives , Computational Biology/methods , Sequence Analysis, RNA/methods , Software , Adenosine/analysis , User-Computer Interface
11.
J Obstet Gynaecol ; 39(3): 328-334, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30714504

ABSTRACT

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.


Subject(s)
Comparative Genomic Hybridization/standards , DNA Copy Number Variations , High-Throughput Nucleotide Sequencing/standards , Prenatal Diagnosis/methods , Female , Humans , Pregnancy , Prospective Studies
12.
J Pathol ; 241(2): 119-122, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27859271

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/adverse effects , DNA Damage/drug effects , Genomics , Inflammatory Bowel Diseases/therapy , Mutation/genetics , Animals , Genetic Testing , Humans , Inflammatory Bowel Diseases/genetics
13.
EMBO J ; 32(18): 2454-62, 2013 Sep 11.
Article in English | MEDLINE | ID: mdl-23872947

ABSTRACT

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1(-/-) cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism.


Subject(s)
Autoimmune Diseases of the Nervous System/metabolism , HIV Infections/physiopathology , HIV-1/physiology , Monomeric GTP-Binding Proteins/metabolism , Nervous System Malformations/metabolism , Reverse Transcription/physiology , Animals , Autoimmune Diseases of the Nervous System/genetics , Cell Line , Genetic Vectors/genetics , HIV Infections/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Interferon Type I/metabolism , Mice , Mice, Knockout , Monomeric GTP-Binding Proteins/genetics , Nervous System Malformations/genetics , Nucleotides/metabolism , Reverse Transcription/genetics , SAM Domain and HD Domain-Containing Protein 1
14.
J Med Genet ; 53(4): 264-9, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26733463

ABSTRACT

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.


Subject(s)
Arthrogryposis/genetics , Fetal Growth Retardation/genetics , High-Throughput Nucleotide Sequencing , MyoD Protein/genetics , Aborted Fetus , Animals , Arthrogryposis/pathology , Exome/genetics , Female , Fetal Growth Retardation/pathology , Humans , Lung/pathology , Mice , Mutation , Pedigree , Phenotype , Pregnancy
15.
PLoS Genet ; 10(9): e1004577, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25232951

ABSTRACT

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.


Subject(s)
Cilia/metabolism , Cilia/physiology , Proteins/metabolism , Animals , Axonemal Dyneins , Axoneme/genetics , Axoneme/metabolism , Binding Sites/genetics , Cell Line , Child, Preschool , Cilia/genetics , Ciliary Motility Disorders/genetics , Ciliary Motility Disorders/metabolism , Drosophila/genetics , Drosophila/metabolism , Dyneins/genetics , Dyneins/metabolism , Female , Humans , Kartagener Syndrome/genetics , Kartagener Syndrome/metabolism , Male , Mutation/genetics , Pedigree , Phenotype , Proteins/genetics , Transcription, Genetic/genetics
16.
Am J Hum Genet ; 93(6): 1135-42, 2013 Dec 05.
Article in English | MEDLINE | ID: mdl-24290375

ABSTRACT

Short stature, auditory canal atresia, mandibular hypoplasia, and skeletal abnormalities (SAMS) has been reported previously to be a rare, autosomal-recessive developmental disorder with other, unique rhizomelic skeletal anomalies. These include bilateral humeral hypoplasia, humeroscapular synostosis, pelvic abnormalities, and proximal defects of the femora. To identify the genetic basis of SAMS, we used molecular karyotyping and whole-exome sequencing (WES) to study small, unrelated families. Filtering of variants from the WES data included segregation analysis followed by comparison of in-house exomes. We identified a homozygous 306 kb microdeletion and homozygous predicted null mutations of GSC, encoding Goosecoid homeobox protein, a paired-like homeodomain transcription factor. This confirms that SAMS is a human malformation syndrome resulting from GSC mutations. Previously, Goosecoid has been shown to be a determinant at the Xenopus gastrula organizer region and a segment-polarity determinant in Drosophila. In the present report, we present data on Goosecoid protein localization in staged mouse embryos. These data and the SAMS clinical phenotype both suggest that Goosecoid is a downstream effector of the regulatory networks that define neural-crest cell-fate specification and subsequent mesoderm cell lineages in mammals, particularly during shoulder and hip formation. Our findings confirm that Goosecoid has an essential role in human craniofacial and joint development and suggest that Goosecoid is an essential regulator of mesodermal patterning in mammals and that it has specific functions in neural crest cell derivatives.


Subject(s)
Abnormalities, Multiple/genetics , Bone and Bones/abnormalities , Dwarfism/genetics , Ear Canal/abnormalities , Goosecoid Protein/genetics , Mandible/abnormalities , Mutation , Abnormalities, Multiple/diagnosis , Adult , Animals , Child , DNA Mutational Analysis , Female , Genetic Association Studies , Homozygote , Humans , Male , Mice , Pedigree , Phenotype , Syndrome , Young Adult
17.
Bioinformatics ; 31(16): 2728-35, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-25861967

ABSTRACT

MOTIVATION: In attempts to determine the genetic causes of human disease, researchers are often faced with a large number of candidate genes. Linkage studies can point to a genomic region containing hundreds of genes, while the high-throughput sequencing approach will often identify a great number of non-synonymous genetic variants. Since systematic experimental verification of each such candidate gene is not feasible, a method is needed to decide which genes are worth investigating further. Computational gene prioritization presents itself as a solution to this problem, systematically analyzing and sorting each gene from the most to least likely to be the disease-causing gene, in a fraction of the time it would take a researcher to perform such queries manually. RESULTS: Here, we present Gene TIssue Expression Ranker (GeneTIER), a new web-based application for candidate gene prioritization. GeneTIER replaces knowledge-based inference traditionally used in candidate disease gene prioritization applications with experimental data from tissue-specific gene expression datasets and thus largely overcomes the bias toward the better characterized genes/diseases that commonly afflict other methods. We show that our approach is capable of accurate candidate gene prioritization and illustrate its strengths and weaknesses using case study examples. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://dna.leeds.ac.uk/GeneTIER/. CONTACT: umaan@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Algorithms , Disease/genetics , Genetic Association Studies/methods , Organ Specificity/genetics , Transcriptome/genetics , Area Under Curve , Humans , ROC Curve
18.
Bioinformatics ; 31(23): 3822-9, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26272982

ABSTRACT

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.


Subject(s)
Algorithms , Biological Ontologies , Computational Biology/methods , Disease/genetics , Phenotype , Polymorphism, Single Nucleotide/genetics , Exome/genetics , Genotype , Humans , Transcriptome
19.
BMC Med Genet ; 17: 1, 2016 Jan 04.
Article in English | MEDLINE | ID: mdl-26729329

ABSTRACT

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.


Subject(s)
Cerebellum/abnormalities , Chromosome Mapping , Ciliary Motility Disorders/diagnosis , Ciliary Motility Disorders/genetics , Encephalocele/diagnosis , Encephalocele/genetics , Exome , Polycystic Kidney Diseases/diagnosis , Polycystic Kidney Diseases/genetics , Retina/abnormalities , Abnormalities, Multiple/diagnosis , Abnormalities, Multiple/genetics , Alleles , Cohort Studies , DNA Copy Number Variations , Exons , Eye Abnormalities/diagnosis , Eye Abnormalities/genetics , Genetic Testing , High-Throughput Nucleotide Sequencing , Humans , Kidney Diseases, Cystic/diagnosis , Kidney Diseases, Cystic/genetics , Prenatal Diagnosis , Retinitis Pigmentosa , Sequence Analysis, DNA , Sequence Deletion
20.
J Med Genet ; 52(12): 797-803, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26424145

ABSTRACT

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.


Subject(s)
Neurodevelopmental Disorders/genetics , Ubiquitin-Protein Ligases/deficiency , Cells, Cultured , Child , Child, Preschool , DNA Mutational Analysis , Female , Genes, Recessive , Humans , Infant , Male , Pedigree , Polymorphism, Single Nucleotide , Syndrome , Ubiquitin-Protein Ligases/genetics
SELECTION OF CITATIONS
SEARCH DETAIL