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ABSTRACT: UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5' and 3' of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype.
European journal of human genetics: EJHG 12/2011; 20(5):552-8. · 3.56 Impact Factor
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ABSTRACT: Originally prenatal diagnosis was confined to the diagnosis of metabolic disorders and depended on assaying enzyme levels in amniotic fluid. With the development of recombinant DNA technology, molecular diagnosis became possible for some genetic conditions late in the 1970s. Here we briefly review the history of molecular prenatal diagnostic testing, using Duchenne muscular dystrophy as an example, and describe how over the last 30 years we have moved from offering testing to a few affected individuals using techniques, such as Southern blotting to identify deletions, to more rapid and accurate PCR-based testing which identifies the precise change in dystrophin for a greater number of families. We discuss the potential for safer, earlier prenatal genetic diagnosis using cell free fetal DNA in maternal blood before concluding by speculating on how more recent techniques, such as next generation sequencing, might further impact on the potential for molecular prenatal testing. Progress is not without its challenges, and as cytogenetics and molecular genetics begin to unite into one, we foresee the main challenge will not be in identifying the genetic change, but rather in interpreting its significance, particularly in the prenatal setting where we frequently have no phenotype on which to base interpretation.
Prenatal Diagnosis 07/2010; 30(7):674-81. · 2.11 Impact Factor
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Allan J Richards,
Annie McNinch,
Howard Martin,
Kim Oakhill,
Harjeet Rai,
Sarah Waller,
Becky Treacy, Joanne Whittaker,
Sarah Meredith,
Arabella Poulson,
Martin P Snead
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ABSTRACT: Stickler syndrome is a dominantly inherited disorder affecting the fibrillar type II/XI collagen molecules expressed in vitreous and cartilage. Mutations have been found in COL2A1, COL11A1 and COL11A2. It has a highly variable phenotype that can include midline clefting, hearing loss, premature osteoarthritis, congenital high myopia and blindness through retinal detachment. Although the systemic phenotype is highly variable, the vitreous phenotype has been used successfully to differentiate between patients with mutations in these different genes. Mutations in COL2A1 usually result in a congenital membranous vitreous anomaly. In contrast mutations in COL11A1 result in a different vitreous phenotype where the lamellae have an irregular and beaded appearance. However, it is now apparent that a new sub-group of COL2A1 mutations is emerging that result in a different phenotype with a hypoplastic vitreous that fills the posterior chamber of the eye, and is either optically empty or has sparse irregular lamellae. Here we characterise a further 89 families with Stickler syndrome or a type II collagenopathy, and correlate the mutations with the vitreous phenotype. We have identified 57 novel mutations including missense changes in both COL2A1 and COL11A1 and have also detected two cases of complete COL2A1 gene deletions using MLPA.
Human Mutation 06/2010; 31(6):E1461-71. · 5.69 Impact Factor
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ABSTRACT: The UK Genetic Testing Network (UKGTN) established a process for the evaluation of genetic tests for entry onto the National Health Service (NHS) Directory of Molecular Genetic Testing. The Network requested the development and piloting of a prioritization framework that could be used for the commissioning of genetic tests by the NHS.
A selected working group developed and piloted a multi-criteria prioritization process using 10 genetic tests evaluated by the UKGTN.
The framework was able to rank the 10 genetic tests used in the pilot. The rankings were also consistent with the commissioning recommendations for these genetic tests by the UKGTN.
A set of criteria for the prioritization of genetic tests has been developed. The results from the pilot suggest that the methodology is valid and robust but requires considerable resources to implement. Further development of the process is needed before the framework could be used to influence commissioning decisions for clinical genetic services in the NHS.
Public Health Genomics 03/2010; 13(7-8):538-43. · 2.33 Impact Factor
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ABSTRACT: The UK Department of Health supported the establishment of the UK Genetic Testing Network (UKGTN) in 2002. The UKGTN is a collaborative network of NHS molecular genetic laboratories that offer tests for human single gene germ-line disorders. Its objective is to provide high quality and equitable services for patients and their families who require genetic advice, diagnosis and management. The UKGTN has developed a 'Gene Dossier' process to evaluate genetic tests and recommend which tests will be provided by the National Health Service. This paper describes the UKGTN organisation and the 'Gene Dossier' process. A brief review of the UKGTN genetic test evaluation experience is presented.
European Journal of HumanGenetics 10/2007; 15(9):917-21. · 4.40 Impact Factor
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Aaron N Cranston,
Cristiana Carniti,
Kim Oakhill,
Elzbieta Radzio-Andzelm,
Eric A Stone,
Andrew S McCallion,
Shirley Hodgson,
Sue Clarke,
Piera Mondellini,
Jean Leyland,
Marco A Pierotti, Joanne Whittaker,
Susan S Taylor,
Italia Bongarzone,
Bruce A J Ponder
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ABSTRACT: Constitutive activation of the RET receptor tyrosine kinase underlies the genesis and progression of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer predisposition. Importantly, although kinase activation represents a common theme in neoplasias, not all activating mutations are functionally equivalent. Consistent with this, we ascertained a patient with classical features of MEN 2B, but lacking either of the classical mutations in RET (M918T or A883F). Instead, the patient harbors a novel pair of germ line missense mutations in cis at codons 804 and 805. We evaluated the potential physiochemical effects of these substitutions in silico, predicting both to be moderately deleterious in isolation, but severely deleterious in combination. Consistent with this postulate, we show that the identified tandem mutations (V804M/E805K) are biologically active, transforming cells in culture and that their transforming capacity in combination is distinctly synergistic. Furthermore, the V804M/E805K tandem lesion confers resistance to the small molecule receptor tyrosine kinase inhibitor, PP1, suggesting a mode of action distinct from that known for classical MEN 2B mutations. To address this question, we used homology molecular modeling in silico to model the active site of RET. We predict that RET804 constitutes a critical gatekeeper residue that, when mutated in combination with RET805, induces a conformational change in the hinge region that locks the active site in a position permissive for ATP hydrolysis. Our findings have implications both in the clinic and in the successful development of novel kinase-targeted anticancer drugs.
Cancer Research 11/2006; 66(20):10179-87. · 7.86 Impact Factor
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Allan J Richards,
Maureen Laidlaw, Joanne Whittaker,
Becky Treacy,
Harjeet Rai,
Philip Bearcroft,
David M Baguley,
Arabella Poulson,
Alan Ang,
John D Scott,
Martin P Snead
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ABSTRACT: Stickler syndrome is a genetically heterogeneous disorder that affects the ocular, skeletal, and auditory systems. To date three genes, COL2A1, COL11A1, and COL11A2, encoding the heterotypic type II/XI collagen fibrils present in vitreous and cartilage have been shown to have mutations that result in Stickler syndrome. As systemic features in this disorder are variable we have used an ophthalmic examination to differentiate those patients with a membranous vitreous phenotype associated with mutations in COL2A1, from other patients who may have mutations in other genes. Gene amplification and exon sequencing was used to screen 50 families or sporadic cases with this membranous phenotype, for mutations in COL2A1. Mutations were detected in 47 (94%) cases consisting of 166 affected and 78 unaffected individuals. We also demonstrate that the predominantly ocular form of type 1 Stickler syndrome is not confined to mutations in the alternatively spliced exon 2. Using splicing reporter constructs we demonstrate that a mutant GC donor splice site in intron 51 can be spliced normally; this contributed to the predominantly ocular phenotype in the family in which it occurred.
Human Mutation 08/2006; 27(7):696-704. · 5.69 Impact Factor
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ABSTRACT: Germ line missense mutations in the RET proto-oncogene are responsible for the inherited cancer syndrome multiple endocrine neoplasia type 2A (MEN2A). The clinical presentation of the disease and the age at onset varies even within families, where patients carry the same mutation. These variations in phenotypes suggest a role for genetic modifiers, and recently, it has been reported that polymorphisms within RET (G691S/S904S) may have such a modifier effect on the age at onset. Here, we investigate whether this observed association could be confirmed in a larger set of 384 individuals from MEN2 families from four different European populations. In addition, we tested as modifiers four other single nucleotide polymorphisms (SNPs), which we have found in a previous association study of RET, its coreceptors, and ligands to be associated with the risk of developing sporadic medullary thyroid carcinoma. We could not replicate the association between G691S/S904S and modifier effects in MEN2A families in any of the four European families analyzed. Of the other SNPs tested, only RET A432A showed a positive weak effect on tumor spectrum within MEN2A, which requires replication in a larger series.
Cancer Research 02/2006; 66(2):1177-80. · 7.86 Impact Factor
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Arancha Cebrian,
Fabienne Lesueur,
Sam Martin,
Jean Leyland,
Shahana Ahmed,
Craig Luccarini,
Paula L Smith,
Robert Luben, Joanne Whittaker,
Paul D Pharoah,
Alison M Dunning,
Bruce A J Ponder
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ABSTRACT: Medullary thyroid carcinoma (MTC) is a characteristic tumor occurring in individuals with multiple endocrine neoplasia type 2 who carry germ-line mutations in RET (rearranged during transfection). However, most MTC occur in individuals without a family history.
The objective of this study was to explore the possibility that susceptibility in these cases results from low penetrance alleles of RET, its coreceptors, and ligands.
We carried out an association study in 135 sporadic MTC (sMTC) patients and 533 controls from the United Kingdom population.
We analyzed 33 polymorphisms in all nine genes involved in the glial cell line-derived neurotropic factor receptor-alpha (GFRalpha)-RET complex. This is the first association study in which all genes involved in this complex have been investigated for susceptibility to sMTC. We did not find any association between single nucleotide polymorphisms in the exonic regions of the GFRalpha2, GFRalpha3, GFRalpha4, glial cell line-derived neurotropic factor, neurturin, or persephin genes and risk of developing sMTC. We found a strong association between the disease and specific haplotypes of RET. We not only confirmed the previously described association with G691S and S904S (for heterozygotes: odds ratio, 1.85; range, 1.22-2.82; P = 0.004), but we found a novel protective effect associated with a specific haplotype (odds ratio, 0.39; range, 0.21-0.72; P = 0.005) revealing the existence of different genetic variants in the RET oncogene that either increase or decrease risk of sMTC.
Journal of Clinical Endocrinology & Metabolism 12/2005; 90(11):6268-74. · 6.50 Impact Factor
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ABSTRACT: The association of the Noonan phenotype with neurofibromatosis type 1 (NF1) was first noted by Allanson et al. [Am J Med Genet 1985;21:457-462.] and 30 further cases have subsequently been reported. It has been suggested that this phenotype is more common than previously appreciated, as Colley et al. [Clin Genet 1996;49:59-64.] examined 94 sequentially identified patients with NF1 from their genetic register and found Noonan features in 12. A 3-bp deletion of exon 17 of the NF1 neurofibromin gene was described in one family by Carey et al. [Proc Greenwood Genet Center 1997;17:52-53]. However, it remains unclear whether Neurofibromatosis-Noonan syndrome (NFNS) represents a form of NF1 (with mutations in the NF1 neurofibromin gene) or a separate syndrome. We have used a new, rapid sequence analysis technique-comparative sequence analysis (CSA)-to examine the NF1 gene in six patients with NFNS. None of the six patients had the previously identified mutation, nor did we observe other mutations within this exon. However, two other mutations were found: in exon 25, a 3-bp deletion 4312 del GAA, and in exon 23-2, a 2-bp insertion 4095 ins TG. The PTPN11 gene, now known to cause over 50% of Noonan syndrome was also examined in four cases of NFNS, and no mutations were found. These results show that NFNS can in some cases result from different mutations in the NF1 gene and therefore represents a variant form of NF1.
American Journal of Medical Genetics Part A 06/2003; 119A(1):1-8. · 2.39 Impact Factor
