Simon C. Ramsden

Angelman syndrome phenotype associated with mutations in MECP2, a gene encoding a methyl CpG binding protein.

Angelman syndrome (AS) is a neurodevelopmental disorder characterised by severe mental retardation, absent speech, ataxia, sociable affect, and dysmorphic facial features. Eighty five percent of patients with AS have an identifiable genetic abnormality of chromosome 15q11-13. Mutations within the X linkedMECP2 gene have been identified in patients with Rett syndrome (RTT), a neurodevelopmental disorder which affects females almost exclusively and which shares phenotypic overlap with AS. RTT is usually associated with normal development in infancy followed by loss of acquired skills and evolution of characteristic hand wringing movements and episodes of hyperventilation. A panel of 25 female and 22 male patients with a clinical diagnosis of AS and no molecular abnormality of 15q11-13 were screened for MECP2 mutations and these were identified in four females and one male. Following the diagnosis, it was possible to elicit a history of regression in three of these patients, who by then were showing features suggestive of Rett syndrome. In the remaining two subjects the clinical phenotype was still considered to be Angelman-like. These findings illustrate the phenotypic overlap between the two conditions and suggest that screening forMECP2 mutations should be considered in AS patients without a demonstrable molecular or cytogenetic abnormality of 15q11-13. Since MECP2 mutations almost always occur de novo, their identification will substantially affect genetic counselling for the families concerned.

Somatic mutation in MECP2 as a non-fatal neurodevelopmental disorder in males

Rett syndrome is a cause of severe learning disability in girls and is associated with a characteristic history and movement disorder. It is an X-linked dominant condition associated with mutations of the MECP2 gene on the distal part of the X-chromosome. If present in a male conceptus, the mutation is usually lethal. We present evidence to show that males can be affected by Rett syndrome. In the boy presented, this situation came about because cells containing the MECP2 mutation existed alongside a normal cell line. Somatic mosaicism could explain the occurrence of other X-linked dominant disorders in males, when they would normally be lethal.

Quality control in molecular genetic testing

DNA-based testing for genetic diseases has developed from nothing into a principal part of laboratory medicine over the past 15 years. In the rush to bring these powerful new technologies into medical use, issues of quality have not always been given sufficient attention. Efforts are now being made to assess the quality of the output of genetic testing laboratories, and the results show that there is room for improvement.

A paradigm shift in the delivery of services for diagnosis of inherited retinal disease

Objectives Current technologies for delivering gene testing are labour-intensive and expensive. Over the last 3 years, new high-throughput DNA sequencing techniques (next generation sequencing; NGS), with the capability to analyse multiple genes or entire genomes, have been rapidly adopted into research. This study examines the possibility of incorporating NGS into a clinical UK service context. Methods The study applied NGS of 105 genes to 50 patients known to be affected by inherited forms of blindness in the setting of a UK National Health Service-accredited diagnostic molecular genetics laboratory. The study assessed the ability of an NGS protocol to identify likely disease-causing genetic variants when compared with current methodologies available through UK diagnostic laboratories. Results Conventional testing is only applicable to the minority of patients with inherited retinal disease and identifies mutations in fewer than one in four of those patients tested. By contrast, the NGS assay is directed at all patients with such disorders and identifies disease-causing mutations in 50–55%, which is a dramatic increase. This includes patients with apparently ‘sporadic’ disease, and those for whom clinical management and prognosis are altered as a consequence of defining their disease at a molecular level. Conclusions The new NGS approach delivers a step change in the diagnosis of inherited eye disease, provides precise diagnostic information and extends the possibility of targeted treatments including gene therapy. The approach represents an exemplar that illustrates the opportunity that NGS provides for broadening the availability of genetic testing. The technology will be applied to many conditions that are associated with high levels of genetic heterogeneity.

Practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes

BackgroundPrader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurodevelopmental genetic disorders that map to 15q11-q13. The primary phenotypes are attributable to loss of expression of imprinted genes within this region which can arise by means of a number of mechanisms. The most sensitive single approach to diagnosing both PWS and AS is to study methylation patterns within 15q11-q13; however many techniques exist for this purpose. Given the diversity of techniques available, there is a need for consensus testing and reporting guidelines.MethodsTesting and reporting guidelines have been drawn up and agreed in accordance with the procedures of the UK Clinical Molecular Genetics Society and the European Molecular Genetics Quality Network.ResultsA practical set of molecular genetic testing and reporting guidelines has been developed for these two disorders. In addition, advice is given on appropriate reporting policies, including advice on test sensitivity and recurrence risks. In considering test sensitivity, the possibility of differential diagnoses is discussed.ConclusionAn agreed set of practice guidelines has been developed for the diagnostic molecular genetic testing of PWS and AS.

Pseudoachondroplasia and multiple epiphyseal dysplasia: A 7‐year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short‐limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD‐MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD‐MED is genetically heterogenous and can also result from mutations in matrilin‐3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on‐line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED. Hum Mutat 33:144–157, 2012.

Personalized diagnosis and management of congenital cataract by next-generation sequencing.

PURPOSE To assess the utility of integrating genomic data from next-generation sequencing and phenotypic data to enhance the diagnosis of bilateral congenital cataract (CC). DESIGN Evaluation of diagnostic technology. PARTICIPANTS Thirty-six individuals diagnosed with nonsyndromic or syndromic bilateral congenital cataract were selected for investigation through a single ophthalmic genetics clinic. METHODS Participants underwent a detailed ophthalmic examination, accompanied by dysmorphology assessment where appropriate. Lenticular, ocular, and systemic phenotypes were recorded. Mutations were detected using a custom-designed target enrichment that permitted parallel analysis of 115 genes associated with CC by high-throughput, next-generation DNA sequencing (NGS). Thirty-six patients and a known positive control were tested. Suspected pathogenic variants were confirmed by bidirectional Sanger sequencing in relevant probands and other affected family members. MAIN OUTCOME MEASURES Molecular genetic results and details of clinical phenotypes were identified. RESULTS Next-generation DNA sequencing technologies are able to determine the precise genetic cause of CC in 75% of individuals, and 85% patients with nonsyndromic CC were found to have likely pathogenic mutations, all of which occurred in highly conserved domains known to be vital for normal protein function. The pick-up rate in patients with syndromic CC also was high, with 63% having potential disease-causing mutations. CONCLUSIONS This analysis demonstrates the clinical utility of this test, providing examples where it altered clinical management, directed care pathways, and enabled more accurate genetic counseling. This comprehensive screen will extend access to genetic testing and lead to improved diagnostic and management outcomes through a stratified medicine approach. Establishing more robust genotype-phenotype correlations will advance knowledge of cataract-forming mechanisms.

A pragmatic randomized controlled trial of thiopurine methyltransferase genotyping prior to azathioprine treatment: the TARGET study.

AIM To conduct a pragmatic, randomized controlled trial to assess whether thiopurine methyltransferase (TPMT) genotyping prior to azathioprine reduces adverse drug reactions (ADRs). METHODS A total of 333 participants were randomized 1:1 to undergo TPMT genotyping prior to azathioprine or to commence treatment without genotyping. RESULTS There was no difference in the primary outcome of stopping azathioprine due to an adverse reaction (ADR, p = 0.59) between the two study arms. ADRs were more common in older patients (p = 0.01). There was no increase in stopping azathioprine due to ADRs in TPMT heterozygotes compared with wild-type individuals. The single individual with TPMT variant homozygosity experienced severe neutropenia. CONCLUSION Our work supports the strong evidence that individuals with TPMT variant homozygosity are at high risk of severe neutropenia, whereas TPMT heterozygotes are not at increased risk of ADRs at standard doses of azathioprine.

COMP mutation screening as an aid for the clinical diagnosis and counselling of patients with a suspected diagnosis of pseudoachondroplasia or multiple epiphyseal dysplasia.

The skeletal dysplasias are a clinically and genetically heterogeneous group of conditions affecting the development of the osseous skeleton and fall into the category of rare genetic diseases in which the diagnosis can be difficult for the nonexpert. Two such diseases are pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which result in varying degrees of short stature, joint pain and stiffness and often resulting in early onset osteoarthritis. PSACH and some forms of MED result from mutations in the cartilage oligomeric matrix protein (COMP) gene and to aid the clinical diagnosis and counselling of patients with a suspected diagnosis of PSACH or MED, we developed an efficient and accurate molecular diagnostic service for the COMP gene. In a 36-month period, 100 families were screened for a mutation in COMP and we identified disease-causing mutations in 78% of PSACH families and 36% of MED families. Furthermore, in several of these families, the identification of a disease-causing mutation provided information that was immediately used to direct reproductive decision-making.

Molecular findings from 537 individuals with inherited retinal disease

Background Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. Methods We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). Results Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. Conclusions Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.