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The PAX6 gene encodes a highly-conserved transcription factor involved in eye development. Heterozygous loss-of-function variants in PAX6 can cause a range of ophthalmic disorders including aniridia. A key molecular diagnostic challenge is that many PAX6 missense changes are presently classified as variants of uncertain significance. While computational tools can be used to assess the effect of genetic alterations, the accuracy of their predictions varies. Here, we evaluated and optimised the performance of computational prediction tools in relation to PAX6 missense variants. Through inspection of publicly available resources (including HGMD, ClinVar, LOVD and gnomAD), we identified 241 PAX6 missense variants that were used for model training and evaluation. The performance of ten commonly used computational tools was assessed and a threshold optimization approach was utilized to determine optimal cut-off values. Validation studies were subsequently undertaken using PAX6 variants from a local database. AlphaMissense, SIFT4G and REVEL emerged as the best-performing predictors; the optimized thresholds of these tools were 0.967, 0.025, and 0.772, respectively. Combining the prediction from these top-three tools resulted in lower performance compared to using AlphaMissense alone. Tailoring the use of computational tools by employing optimized thresholds specific to PAX6 can enhance algorithmic performance. Our findings have implications for PAX6 variant interpretation in clinical settings., (© 2024. The Author(s).)

Albinism is a clinically and genetically heterogeneous group of conditions characterised by visual abnormalities and variable degrees of hypopigmentation. Multiple studies have demonstrated the clinical utility of genetic investigations in individuals with suspected albinism. Despite this, the variation in the provision of genetic testing for albinism remains significant. One key issue is the lack of a standardised approach to the analysis of genomic data from affected individuals. For example, there is variation in how different clinical genetic laboratories approach genotypes that involve incompletely penetrant alleles, including the common, 'hypomorphic' TYR c.1205G>A (p.Arg402Gln) [rs1126809] variant. Here, we discuss the value of genetic testing as a frontline diagnostic tool in individuals with features of albinism and propose a practice pattern for the analysis of genomic data from affected families., Competing Interests: Competing interests: EB is a paid consultant and equity holder of Oxford Nanopore, a paid consultant to Dovetail, and a non-executive director of Genomics England, a limited company wholly owned by the UK Department of Health and Social Care. All other authors declare no competing interests. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Background: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient., Methods: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis., Results: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield., Conclusion: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Objective: Agitation in children in acute care settings poses significant patient and staff safety concerns. While behavioral approaches are central to reducing agitation and oral medications are preferred, parenteral medications are used when necessary to promote safety. The goal of this systematic review was to evaluate the effectiveness and safety of an ultra-short-acting parenteral medication, droperidol, for the management of acute, severe agitation in children in acute care settings., Methods: A systematic review of randomized controlled trials, observational studies, and case series/reports examined the effectiveness and safety of parenteral droperidol for management of acute agitation in patients ≤21 years old in acute care settings. Effectiveness outcomes included time to sedation and need for a subsequent dose of medication. Safety outcomes were adverse effects such as QTc prolongation, hypotension, respiratory depression, and dystonic reactions., Results: A total of 431 unique articles were identified. Six articles met inclusion criteria: two in the prehospital setting, one in the emergency department, and three in the inpatient hospital setting. The articles included a prospective observational study, three retrospective observational studies, and two case reports. The largest study reported a median time to sedation of 14 min (interquartile range 10-20 min); other studies reported a time to sedation of 15 min or less. Across studies, 8%-22% of patients required a second dose of medication for ongoing agitation. The most frequent adverse effects were dystonic reactions and transient hypotension. One patient had QTc prolongation and another developed respiratory depression, but both had significant comorbidities that may have contributed. The risk of bias in included studies ranged from moderate to critical., Conclusions: Existing data on droperidol for management of acute agitation in children suggest that droperidol is both effective and safe for acute, severe agitation in children. Data are limited by study designs that may introduce bias., (© 2022 The Authors. Academic Emergency Medicine published by Wiley Periodicals LLC on behalf of Society for Academic Emergency Medicine.)

Background: Improving the clinical interpretation of missense variants can increase the diagnostic yield of genomic testing and lead to personalised management strategies. Currently, due to the imprecision of bioinformatic tools that aim to predict variant pathogenicity, their role in clinical guidelines remains limited. There is a clear need for more accurate prediction algorithms and this study aims to improve performance by harnessing structural biology insights. The focus of this work is missense variants in a subset of genes associated with X linked disorders., Methods: We have developed a pro tein- sp ecific variant interpret er (ProSper) that combines genetic and protein structural data. This algorithm predicts missense variant pathogenicity by applying machine learning approaches to the sequence and structural characteristics of variants., Results: ProSper outperformed seven previously described tools, including meta-predictors, in correctly evaluating whether or not variants are pathogenic; this was the case for 11 of the 21 genes associated with X linked disorders that met the inclusion criteria for this study. We also determined gene-specific pathogenicity thresholds that improved the performance of VEST4, REVEL and ClinPred, the three best-performing tools out of the seven that were evaluated; this was the case in 11, 11 and 12 different genes, respectively., Conclusion: ProSper can form the basis of a molecule-specific prediction tool that can be implemented into diagnostic strategies. It can allow the accurate prioritisation of missense variants associated with X linked disorders, aiding precise and timely diagnosis. In addition, we demonstrate that gene-specific pathogenicity thresholds for a range of missense prioritisation tools can lead to an increase in prediction accuracy., Competing Interests: Competing interests: NL is an employee of Congenica. All other authors declare that they have no conflict of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Paediatric neurology syndromes are a broad and complex group of conditions with a large spectrum of clinical phenotypes. Joubert syndrome is a genetically heterogeneous neurological ciliopathy syndrome with molar tooth sign as the neuroimaging hallmark. We reviewed the clinical, radiological and genetic data for several families with a clinical diagnosis of Joubert syndrome but negative genetic analysis. We detected biallelic pathogenic variants in LAMA1 , including novel alleles, in each of the four cases we report, thereby establishing a firm diagnosis of Poretti-Boltshauser syndrome. Analysis of brain MRI revealed cerebellar dysplasia and cerebellar cysts, associated with Poretti-Boltshauser syndrome and the absence of typical molar tooth signs. Using large UK patient cohorts, the relative prevalence of Joubert syndrome as a cause of intellectual disability was 0.2% and of Poretti-Boltshauser syndrome was 0.02%. We conclude that children with congenital brain disorders that mimic Joubert syndrome may have a delayed diagnosis due to poor recognition of key features on brain imaging and the lack of inclusion of LAMA1 on molecular genetic gene panels. We advocate the inclusion of LAMA1 genetic analysis on all intellectual disability and Joubert syndrome gene panels and promote a wider awareness of the clinical and radiological features of these syndromes., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Thirty percent of all inherited retinal disease (IRD) is accounted for by conditions with extra-ocular features. This study aimed to establish the genetic diagnostic pick-up rate for IRD patients with one or more extra-ocular features undergoing panel-based screening in a clinical setting. One hundred and six participants, tested on a gene panel which contained both isolated and syndromic IRD genes, were retrospectively ascertained from the Manchester Genomic Diagnostics Laboratory database spanning 6 years (2012-2017). Phenotypic features were extracted from the clinical notes and classified according to Human Phenotype Ontology; all identified genetic variants were interpreted in accordance to the American College of Medical Genetics and Genomics guidelines. Overall, 49% (n = 52) of patients received a probable genetic diagnosis. A further 6% (n = 6) had a single disease-associated variant in an autosomal recessive disease-relevant gene. Fifty-two percent (n = 55) of patients had a clinical diagnosis at the time of testing. Of these, 71% (n = 39) received a probable genetic diagnosis. By contrast, for those without a provisional clinical diagnosis (n = 51), only 25% (n = 13) received a probable genetic diagnosis. The clinical diagnosis of Usher (n = 33) and Bardet-Biedl syndrome (n = 10) was confirmed in 67% (n = 22) and 80% (n = 8), respectively. The testing diagnostic rate in patients with clinically diagnosed multisystemic IRD conditions was significantly higher than those without one (71% versus 25%; p value < 0.001). The lower pick-up rate in patients without a clinical diagnosis suggests that panel-based approaches are unlikely to be the most effective means of achieving a molecular diagnosis for this group. Here, we suggest that genome-wide approaches (whole exome or genome) are more appropriate.

Purpose: A key property to consider in all genetic tests is clinical utility, the ability of the test to influence patient management and health outcomes. Here we assess the current clinical utility of genetic testing in diverse pediatric inherited eye disorders (IEDs)., Methods: Two hundred one unrelated children (0-5 years old) with IEDs were ascertained through the database of the North West Genomic Laboratory Hub, Manchester, UK. The cohort was collected over a 7-year period (2011-2018) and included 74 children with bilateral cataracts, 8 with bilateral ectopia lentis, 28 with bilateral anterior segment dysgenesis, 32 with albinism, and 59 with inherited retinal disorders. All participants underwent panel-based genetic testing., Results: The diagnostic yield of genetic testing for the cohort was 64% (ranging from 39% to 91% depending on the condition). The test result led to altered management (including preventing additional investigations or resulting in the introduction of personalized surveillance measures) in 33% of probands (75% for ectopia lentis, 50% for cataracts, 33% for inherited retinal disorders, 7% for anterior segment dysgenesis, 3% for albinism)., Conclusion: Genetic testing helped identify an etiological diagnosis in the majority of preschool children with IEDs. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in significant subsets of patients.

Objective: USH2A-related disorders are characterised by genetic and phenotypic heterogeneity, and are associated with a spectrum of sensory deficits, ranging from deaf blindness to blindness with normal hearing. It has been previously proposed that the presence of specific USH2A alleles can be predictive of unaffected hearing. This study reports the clinical and genetic findings in a group of patients with USH2A-related disease and evaluates the validity of the allelic hierarchy model., Patients and Intervention: USH2A variants from 27 adults with syndromic and nonsyndromic USH2A-related disease were analyzed according to a previously reported model of allelic hierarchy. The analysis was replicated on genotype-phenotype correlation information from 197 individuals previously reported in 2 external datasets., Main Outcome Measure: Genotype-phenotype correlations in USH2A-related disease., Results: A valid allelic hierarchy model was observed in 93% of individuals with nonsyndromic USH2A-retinopathy (n = 14/15) and in 100% of patients with classic Usher syndrome type IIa (n = 8/8). Furthermore, when two large external cohorts of cases were combined, the allelic hierarchy model was valid across 85.7% (n = 78/91) of individuals with nonsyndromic USH2A-retinopathy and 95% (n = 123/129) of individuals with classic Usher syndrome type II (p = 0.012, χ test). Notably, analysis of all three patient datasets revealed that USH2A protein truncating variants were reported most frequently in individuals with hearing loss., Conclusion: Genetic testing results in individuals suspected to have an USH2A-related disorder have the potential to facilitate personalized audiological surveillance and rehabilitation pathways.

Individuals who have ocular features of albinism and skin pigmentation in keeping with their familial background present a considerable diagnostic challenge. Timely diagnosis through genomic testing can help avert diagnostic odysseys and facilitates accurate genetic counselling and tailored specialist management. Here, we report the clinical and gene panel testing findings in 12 children with presumed ocular albinism. A definitive molecular diagnosis was made in 8/12 probands (67%) and a possible molecular diagnosis was identified in a further 3/12 probands (25%). TYR was the most commonly mutated gene in this cohort (75% of patients, 9/12). A disease-causing TYR haplotype comprised of two common, functional polymorphisms, TYR c.[575 C > A;1205 G > A] p.[(Ser192Tyr);(Arg402Gln)], was found to be particularly prevalent. One participant had GPR143-associated X-linked ocular albinism and another proband had biallelic variants in SLC38A8, a glutamine transporter gene associated with foveal hypoplasia and optic nerve misrouting without pigmentation defects. Intriguingly, 2/12 individuals had a single, rare, likely pathogenic variant in each of TYR and OCA2 - a significant enrichment compared to a control cohort of 4046 individuals from the 100,000 genomes project pilot dataset. Overall, our findings highlight that panel-based genetic testing is a clinically useful test with a high diagnostic yield in children with partial/ocular albinism.

This article is an update of the best practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes published in 2010 in BMC Medical Genetics [1]. The update takes into account developments in terms of techniques, differential diagnoses and (especially) reporting standards. It highlights the advantages and disadvantages of each method and moreover, is meant to facilitate the interpretation of the obtained results - leading to improved standardised reports.

Background: Fetal structural anomalies, which are detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs; which are detectable by chromosomal microarrays), and pathogenic sequence variants in developmental genes. Testing for aneuploidy and CNVs is routine during the investigation of fetal structural anomalies, but there is little information on the clinical usefulness of genome-wide next-generation sequencing in the prenatal setting. We therefore aimed to evaluate the proportion of fetuses with structural abnormalities that had identifiable variants in genes associated with developmental disorders when assessed with whole-exome sequencing (WES)., Methods: In this prospective cohort study, two groups in Birmingham and London recruited patients from 34 fetal medicine units in England and Scotland. We used whole-exome sequencing (WES) to evaluate the presence of genetic variants in developmental disorder genes (diagnostic genetic variants) in a cohort of fetuses with structural anomalies and samples from their parents, after exclusion of aneuploidy and large CNVs. Women were eligible for inclusion if they were undergoing invasive testing for identified nuchal translucency or structural anomalies in their fetus, as detected by ultrasound after 11 weeks of gestation. The partners of these women also had to consent to participate. Sequencing results were interpreted with a targeted virtual gene panel for developmental disorders that comprised 1628 genes. Genetic results related to fetal structural anomaly phenotypes were then validated and reported postnatally. The primary endpoint, which was assessed in all fetuses, was the detection of diagnostic genetic variants considered to have caused the fetal developmental anomaly., Findings: The cohort was recruited between Oct 22, 2014, and June 29, 2017, and clinical data were collected until March 31, 2018. After exclusion of fetuses with aneuploidy and CNVs, 610 fetuses with structural anomalies and 1202 matched parental samples (analysed as 596 fetus-parental trios, including two sets of twins, and 14 fetus-parent dyads) were analysed by WES. After bioinformatic filtering and prioritisation according to allele frequency and effect on protein and inheritance pattern, 321 genetic variants (representing 255 potential diagnoses) were selected as potentially pathogenic genetic variants (diagnostic genetic variants), and these variants were reviewed by a multidisciplinary clinical review panel. A diagnostic genetic variant was identified in 52 (8·5%; 95% CI 6·4-11·0) of 610 fetuses assessed and an additional 24 (3·9%) fetuses had a variant of uncertain significance that had potential clinical usefulness. Detection of diagnostic genetic variants enabled us to distinguish between syndromic and non-syndromic fetal anomalies (eg, congenital heart disease only vs a syndrome with congenital heart disease and learning disability). Diagnostic genetic variants were present in 22 (15·4%) of 143 fetuses with multisystem anomalies (ie, more than one fetal structural anomaly), nine (11·1%) of 81 fetuses with cardiac anomalies, and ten (15·4%) of 65 fetuses with skeletal anomalies; these phenotypes were most commonly associated with diagnostic variants. However, diagnostic genetic variants were least common in fetuses with isolated increased nuchal translucency (≥4·0 mm) in the first trimester (in three [3·2%] of 93 fetuses)., Interpretation: WES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness., Funding: UK Department of Health and Social Care and The Wellcome Trust., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Cardiofaciocutaneous (CFC) syndrome is a RASopathy characterized by intellectual disability, congenital heart defects, a characteristic facial appearance, gastro-intestinal complications, ectodermal abnormalities and growth failure. The RASopathies result from germline mutations in the Ras/Mitogen-activated-protein-kinase (MAPK) pathway. CFC is associated with mutations in BRAF, KRAS, MEK1 and MEK2. CFC has been considered a "sporadic" disorder, with minimal recurrence risk to siblings. In recent years, vertical transmission of CFC has been seen in mutations involving the MEK2 and KRAS genes, but has not previously been reported with BRAF mutations. Two brothers with clinical features of CFC and mutations in BRAF (c.770A > G, p.Gln257Arg) are described. Neither parent (both phenotypically normal) had the BRAF mutation in their leukocyte DNA. Although this mutation is one of the most common mutations in CFC, to our knowledge, this is the first molecularly confirmed BRAF mutation causing CFC in siblings. This observation also likely represents the first description of germ cell mosaicism in CFC and so it is important to provide optimal genetic counselling to families regarding the risk of reoccurrence., (© 2018 Wiley Periodicals, Inc.)

Background: Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD., Methods: Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory., Results: We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques., Conclusion: Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations., Competing Interests: Competing interests: CT is an employee of Congenica Ltd. All other authors declare no competing interests., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Inborn errors of metabolism (IEMs) underlie a substantial proportion of paediatric disease burden but their genetic diagnosis can be challenging using the traditional approaches., Methods: We designed and validated a next-generation sequencing (NGS) panel of 226 IEM genes, created six overlapping phenotype-based subpanels and tested 102 individuals, who presented clinically with suspected childhood-onset IEMs., Results: In 51/102 individuals, NGS fully or partially established the molecular cause or identified other actionable diagnoses. Causal mutations were identified significantly more frequently when the biochemical phenotype suggested a specific IEM or a group of IEMs (p<0.0001), demonstrating the pivotal role of prior biochemical testing in guiding NGS analysis. The NGS panel helped to avoid further invasive, hazardous, lengthy or expensive investigations in 69% individuals (p<0.0001). Additional functional testing due to novel or unexpected findings had to be undertaken in only 3% of subjects, demonstrating that the use of NGS does not significantly increase the burden of subsequent follow-up testing. Even where a molecular diagnosis could not be achieved, NGS-based approach assisted in the management and counselling by reducing the likelihood of a high-penetrant genetic cause., Conclusion: NGS has significant clinical utility for the diagnosis of IEMs. Biochemical testing and NGS analysis play complementary roles in the diagnosis of IEMs. Incorporating NGS into the diagnostic algorithm of IEMs can improve the accuracy of diagnosis., Competing Interests: Competing interests: Arunabha Ghosh reports travel grants from Shire Plc, Biomarin Pharmaceutical, outside the submitted work. Alexander Broomfield reports consulting fees from Genzyme, Synageva, outside the submitted work. Christian Hendriksz is director of FYMCA Ltd and reports consulting fees and travel grants from Actelion, Alexion, Amicus, Biomarin, Inventiva, Sanofi Genzyme and Shire and research grants from Actelion, Amicus, Biomarin, Sanofi Genzyme and Shire, outside the submitted work. Gisela Wilcox reports travel grants from Biomarin, Genzyme, Shire, Vitaflo and Actelion, outside the submitted work. Simon Jones reports travel grants, research grants and consultancy fees from Genzyme, Shire, Biomarin, Ultragenyx, Alexion, and PTC, outside the submitted work. Helene Schlecht, Lesley Heptinstall, John Bassett, Eleanor Cartwright, Sanjeev Bhaskar, Jill Urquhart, Andrew Morris, Elisabeth Jameson, Bernd Schwahn, John Walter, Sofia Douzgou, Helen Murphy, Reena Sharma, Ellen Crushell, Ardeshir Monavari, Richard Martin, Anne Doolan, Senthil Senniappan, Simon Ramsden and Siddharth Banka have nothing to disclose., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGR ORF15 ). Despite successful RPGR ORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGR ORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr -/y and C57BL/6J Rd9/Boc ) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess the clinical usefulness of genetic testing in a pediatric population with inherited retinal disease (IRD)., Design: Single-center retrospective case series., Participants: Eighty-five unrelated children with a diagnosis of isolated or syndromic IRD who were referred for clinical genetic testing between January 2014 and July 2016., Methods: Participants underwent a detailed ophthalmic examination, accompanied by electrodiagnostic testing (EDT) and dysmorphologic assessment where appropriate. Ocular and extraocular features were recorded using Human Phenotype Ontology terms. Subsequently, multigene panel testing (105 or 177 IRD-associated genes) was performed in an accredited diagnostic laboratory, followed by clinical variant interpretation., Main Outcome Measures: Diagnostic yield and clinical usefulness of genetic testing., Results: Overall, 78.8% of patients (n = 67) received a probable molecular diagnosis; 7.5% (n = 5) of these had autosomal dominant disease, 25.4% (n = 17) had X-linked disease, and 67.2% (n = 45) had autosomal recessive disease. In a further 5.9% of patients (n = 5), a single heterozygous ABCA4 variant was identified; all these participants had a spectrum of clinical features consistent with ABCA4 retinopathy. Most participants (84.7%; n = 72) had undergone EDT and 81.9% (n = 59) of these patients received a probable molecular diagnosis. The genes most frequently mutated in the present cohort were CACNA1F and ABCA4, accounting for 14.9% (n = 10) and 11.9% (n = 8) of diagnoses respectively. Notably, in many cases, genetic testing helped to distinguish stationary from progressive IRD subtypes and to establish a precise diagnosis in a timely fashion., Conclusions: Multigene panel testing pointed to a molecular diagnosis in 84.7% of children with IRD. The diagnostic yield in the study population was significantly higher compared with that in previously reported unselected IRD cohorts. Approaches similar to the one described herein are expected to become a standard component of care in pediatric ophthalmology. We propose the introduction of genetic testing early in the diagnostic pathway in children with clinical and/or electrophysiologic findings, suggestive of IRD., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Although a common cause of disease, copy number variants (CNVs) have not routinely been identified from next-generation sequencing (NGS) data in a clinical context. This study aimed to examine the sensitivity and specificity of a widely used software package, ExomeDepth, to identify CNVs from targeted NGS data sets. We benchmarked the accuracy of CNV detection using ExomeDepth v1.1.6 applied to targeted NGS data sets by comparison to CNV events detected through whole-genome sequencing for 25 individuals and determined the sensitivity and specificity of ExomeDepth applied to these targeted NGS data sets to be 100% and 99.8%, respectively. To define quality assurance metrics for CNV surveillance through ExomeDepth, we undertook simulation of single-exon (n=1000) and multiple-exon heterozygous deletion events (n=1749), determining a sensitivity of 97% (n=2749). We identified that the extent of sequencing coverage, the inter- and intra-sample variability in the depth of sequencing coverage and the composition of analysis regions are all important determinants of successful CNV surveillance through ExomeDepth. We then applied these quality assurance metrics during CNV surveillance for 140 individuals across 12 distinct clinical areas, encompassing over 500 potential rare disease diagnoses. All 140 individuals lacked molecular diagnoses after routine clinical NGS testing, and by application of ExomeDepth, we identified 17 CNVs contributing to the cause of a Mendelian disorder. Our findings support the integration of CNV detection using ExomeDepth v1.1.6 with routine targeted NGS diagnostic services for Mendelian disorders. Implementation of this strategy increases diagnostic yields and enhances clinical care.

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., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Although the majority of small in-frame insertions/deletions (indels) has no/little affect on protein function, a small subset of these changes has been causally associated with genetic disorders. Notably, the molecular mechanisms and frequency by which they give rise to disease phenotypes remain largely unknown. The aim of this study is to provide insights into the role of in-frame indels (≤21 nucleotides) in two genetically heterogeneous eye disorders., Results: One hundred eighty-one probands with childhood cataracts and 486 probands with retinal dystrophy underwent multigene panel testing in a clinical diagnostic laboratory. In-frame indels were collected and evaluated both clinically and in silico. Variants that could be modeled in the context of protein structure were identified and analysed using integrative structural modeling. Overall, 55 small in-frame indels were detected in 112 of 667 probands (16.8 %); 17 of these changes were novel to this study and 18 variants were reported clinically. A reliable model of the corresponding protein sequence could be generated for 8 variants. Structural modeling indicated a diverse range of molecular mechanisms of disease including disruption of secondary and tertiary protein structure and alteration of protein-DNA binding sites., Conclusions: In childhood cataract and retinal dystrophy subjects, one small in-frame indel is clinically reported in every ~37 individuals tested. The clinical utility of computational tools evaluating these changes increases when the full complexity of the involved molecular mechanisms is embraced.

Purpose: To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD)., Design: Case series., Participants: A total of 562 patients diagnosed with IRD., Methods: We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data., Main Outcome Measures: Diagnostic yield of genomic testing., Results: Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield., Conclusions: We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS., (Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Mutations in the ciliary transporter gene IFT140, usually associated with a severe syndromic ciliopathy, may also cause isolated retinal dystrophy. A series of patients with nonsyndromic retinitis pigmentosa (RP) due to IFT140 was investigated in this study., Methods: Five probands and available affected family members underwent detailed phenotyping including retinal imaging and electrophysiology. Whole exome sequencing was performed on two probands, a targeted sequencing panel of 176 retinal genes on a further two, and whole genome sequencing on the fifth. Missense mutations of IFT140 were further investigated in vitro using transient plasmid transfection of hTERT-RPE1 cells., Results: Eight affected patients from five families had preserved visual acuity until at least the second decade; all had normal development without skeletal manifestations or renal failure at age 13 to 67 years (mean, 42 years; median, 44.5 years). Bi-allelic mutations in IFT140 were identified in all families including two novel mutations: c.2815T > C (p.Ser939Pro) and c.1422&#95;23insAA (p.Arg475Asnfs*14). Expression studies demonstrated a significantly reduced number of cells showing localization of mutant IFT140 with the basal body for two nonsyndromic mutations and two syndromic mutations compared with the wild type and a polymorphism., Conclusions: This study highlights the phenotype of nonsyndromic RP due to mutations in IFT140 with milder retinal dystrophy than that associated with the syndromic disease.

Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) are chondrodysplasias resulting in short-limbed dwarfism, joint pain and stiffness and early onset osteoarthritis. All PSACH, and the largest proportion of MED, result from mutations in cartilage oligomeric matrix protein (COMP). The first mutations in COMP were identified in 1995 in patients with both PSACH and MED and subsequently there has been over 30 publications describing COMP mutations in at least 250 PSACH-MED patients. However, despite these discoveries, a methodical analysis of the relationship between COMP mutations and phenotypes has not been undertaken. In particular, there has, to date, been little correlation between the type and location of a COMP mutation and the resulting phenotype of PSACH or MED. To determine if genotype to phenotype correlations could be derived for COMP, we collated 300 COMP mutations, including 25 recently identified novel mutations. The results of this analysis demonstrate that mutations in specific residues and/or regions of the type III repeats of COMP are significantly associated with either PSACH or MED. This newly derived genotype to phenotype correlation may aid in determining the prognosis of PSACH and MED, including the prediction of disease severity, and in the long term guide genetic counselling and contribute to the clinical management of patients with these diseases.

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., (Copyright © 2014 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To present a detailed phenotypic and molecular study of a series of 18 patients from 11 families with retinal dystrophies consequent on mutations in the cone-rod homeobox (CRX) gene and to report a novel phenotype., Methods: Families were ascertained from a tertiary clinic in the United Kingdom and enrolled into retinal dystrophy studies investigating the phenotype and molecular basis of inherited retinal disease. Eleven patients were ascertained from the study cohorts and a further seven from investigation of affected relatives. Detailed phenotyping included electrodiagnostic testing and retinal imaging. Bidirectional Sanger sequencing of all exons and intron-exon boundaries of CRX was performed on all 18 reported patients and segregation confirmed in available relatives., Results: Based on clinical characteristics and electrophysiology, four patients had Leber congenital amaurosis (LCA), two had rod-cone dystrophy (RCD), five had cone-rod dystrophy (CORD), one had cone dystrophy (COD), and six had macular dystrophy with different phenotypes observed within 5 of 11 families. The macular dystrophy patients presented between 35 to 50 years of age and had visual acuities at last review ranging from 0.2 to 1.5 logMAR (20/32 to 20/630 Snellen). All 18 patients were heterozygous for a mutation in CRX with seven novel mutations identified. There was no evident association between age of onset and position or type of CRX mutation. De novo mutations were confirmed in three patients., Conclusions: Mutations in CRX demonstrate significant phenotypic heterogeneity both between and within pedigrees. A novel, adult-onset, macular dystrophy phenotype is characterized, further extending our knowledge of the etiology of dominant macular dystrophies., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

A diagnosis of choroideraemia (CHM) can be made clinically, based on the fundus examination and a family history consistent with X-linked inheritance. Molecular genetic testing offers a means of confirming the clinical diagnosis, establishing carrier status and allows presymptomatic diagnosis for families who wish to pursue these options. The aim of this study was to examine the uptake and assess the results from a diagnostic molecular genetics service for CHM. We have carried out a comprehensive audit of all molecular genetic results of UK NHS patients and families referred to the North West Regional Molecular Genetics Laboratory in Manchester, UK over a 55 month period. 110 people were referred to this service for testing including diagnostic, carrier and predictive requests. Putative pathogenic mutations were identified in 65/83 (78%) of male index cases. The identification of a familial pathogenic change enabled carrier testing in 16 asymptomatic females and predictive testing in 3 males. Case examples illustrate the range of cases referred for testing and also reflect the need for genetic counselling that results from offering a molecular diagnostic service such as this. Clinical molecular testing for CHM is available clinically and can be used to support the clinical diagnosis and management of patients with choroideraemia as well as their families. Case studies demonstrate the need to provide genetic testing to families and the potential clinical utility of testing., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Purpose: The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa., Methods: Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance., Results: Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic., Conclusion: Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.

Aims: To describe the UK NEQAS for Molecular Genetics scheme for gastrointestinal stromal tumour (GIST) and to report and interpret the findings of four rounds of circulation of this quality assurance programme for KIT/PDGFRA mutation analyses., Methods: Samples of GISTs from formalin-fixed paraffin-embedded tissue blocks were circulated to registered participants of the UK NEQAS for Molecular Genetics scheme for GIST. Three samples were provided per annual circulation from 2008 to 2011 inclusive. The participants were required to analyse the samples for KIT and/or PDGFRA mutations using their routine protocols, and the anonymised participants' reports were assessed and an annual scheme report issued., Results: The genotyping error rates for the 2008, 2009, 2010 and 2011 circulations were 13%, 33%, 19% and 4%, respectively. These errors were either missed or incorrectly described mutations. There was an overall false negative rate of 2% and false positive rate of 0%. The main recommendations that arose from these circulations were: (1) inclusion of reference accession numbers in reports; (2) avoidance of the term 'heterozygous' when analysing DNA from tumour tissue unless there was certainty that only neoplastic DNA was studied; and (3) the need to screen KIT exons 9, 11, 13 and 17 and PDGFRA exons 12, 14 and 18 before classifying a GIST as 'wild type'., Conclusions: The UK NEQAS for Molecular Genetics scheme emphasises the potential complexities of KIT/PDGFRA mutation analyses for GISTs and provides recommendations to help optimise such genotyping and reporting. The scheme has also demonstrated its educational value among participating laboratories.

Advancements in genetic testing to identify predisposition for hereditary breast cancer (HBC) mean that it is important to understand the incremental costs and benefits of the new technologies compared with current testing strategies. This study aimed to (1) identify and critically appraise existing economic evidence for BRCA1/2 mutation testing for HBC and (2) establish whether economic evidence was used to inform national guidance in England and Wales. A telephone interview with diagnostic laboratories (n=14) offering BRCA1/2 mutation testing identified that 9 (64%) used Sanger DNA sequencing with multiplex ligation-dependent probe amplification and two offered next generation sequencing. A systematic review identified 15 economic studies that evaluated: genetic testing for HBC (5 studies); preventive management options for women at risk of HBC (8 studies); and different laboratory approaches for BRCA1 testing (2 studies). These evaluations were not relevant to U.K. practice, and therefore the development of national guidance using a risk threshold to trigger BRCA1/2 testing has not been informed by existing economic evidence. The lack of economic evidence supporting the current risk threshold for national guidance has implications for the efficient use of healthcare resources and the design of economic evaluations of new technologies for BRCA1/2 testing.

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.

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., (© 2011 Wiley Periodicals, Inc.)

Prader Willi and Angelman syndromes are clinically distinct genetic disorders both mapping to chromosome region 15q11-q13, which are caused by a loss of function of paternally or maternally inherited genes in the region, respectively. With clinical diagnosis often being difficult, particularly in infancy, confirmatory genetic diagnosis is essential to enable clinical intervention. However, the latter is challenged by the complex genetics behind both disorders and the unmet need for characterised reference materials to aid accurate molecular diagnosis. With this in mind, a panel of six genotyping reference materials for Prader Willi and Angelman syndromes was developed, which should be stable for many years and available to all diagnostic laboratories. The panel comprises three Prader Willi syndrome materials (two with different paternal deletions, and one with maternal uniparental disomy (UPD)) and three Angelman syndrome materials (one with a maternal deletion, one with paternal UPD or an epigenetic imprinting centre defect, and one with a UBE3A point mutation). Genomic DNA was bulk-extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines established from consenting patients, and freeze-dried as aliquots in glass ampoules. In total, 37 laboratories from 26 countries participated in a collaborative study to assess the suitability of the panel. Participants evaluated the blinded, triplicate materials using their routine diagnostic methods against in-house controls or externally sourced uncertified reference materials. The panel was established by the Expert Committee on Biological Standardization of the World Health Organization as the first International Genetic Reference Panel for Prader Willi and Angelman syndromes.

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.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

Background: Prader-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., Methods: Testing 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., Results: A 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., Conclusion: An agreed set of practice guidelines has been developed for the diagnostic molecular genetic testing of PWS and AS.

Aim: For certain drugs, pharmacogenetic tests can reduce adverse drug reactions and improve treatment efficacy. However, the adoption of pharmacogenetics into clinical practice has been relatively slow. One potential barrier is the capacity of laboratories to meet the demands of a clinical pharmacogenetic service. We aimed to establish the range, capacity to deliver, and demand for germline pharmacogenetic testing in the United Kingdom and Ireland, through an e-survey of 34 molecular genetics and 28 histocompatibility and immunogenetics (H&I) laboratories., Results: Thirty-five percent of molecular genetics laboratories and 54% of H&I laboratories responded to the survey. The majority of H&I laboratories (93%) offered pharmacogenetic testing, whereas only one molecular genetics laboratory provided a pharmacogenetic service. HLA-B*5701 was most commonly tested to identify those at risk of abacavir hypersensitivity among patients with HIV. A number of barriers to testing were identified, including lack of clinician knowledge and a lack of scientific evidence. All molecular genetics laboratories believed that national coordination of clinical pharmacogenetic services was required, whereas only 50% of H&I laboratories supported this view., Conclusions: In the United Kingdom, pharmacogenetic testing is currently being predominantly provided through H&I laboratories for a limited number of indications. The number of laboratories offering pharmacogenetic tests is increasing and is likely to continue to increase over the coming years.

Background: FCI is an R code for analyzing data from real-time PCR experiments. This algorithm estimates standard curve features as well as nucleic acid concentrations and confidence intervals according to Fieller's theorem., Results: In order to describe the features of FCI four situations were selected from real data collected during an international external quality assessment program for quantitative assays based on real-time PCR. The code generates a diagnostic figure suitable for assessing the quality of the quantification process., Conclusion: We have provided a freeware programme using this algorithm specifically designed to increase the information content of the real-time PCR assay.

Objectives: Prenatal diagnosis using rapid molecular genetic techniques is now a widely used method for detecting the most prevalent chromosomal aneuploidies. The object of this work was to develop a methodology for delivering external quality assessment (EQA) appropriate to the needs of routine diagnostic testing laboratories., Methods: We have provided three rounds of EQA using 15 different samples over 3 years. The scheme has developed to assess both the genotyping accuracy of the results and the appropriateness of the clinical reports issued to the referring clinician., Results: Participation in the EQA scheme has increased from 9 to 27 laboratories from across Europe over the three sample distributions. All laboratories have used quantitative fluorescence-PCR (QF-PCR) to analyse these samples except for a sole participant in 2006 who used multiplex ligation-dependent probe amplification (MLPA). In total 265 samples have been distributed, of which four (1.5%) were not reported due to technical failures and one (0.4%) was reported incorrectly and must be regarded as a genotyping error., Conclusions: We have demonstrated a significant and increasing demand for EQA in the rapid detection of aneuploidies in UK and other European laboratories. Using the methodologies described, we have had a very low rate of technical failures and demonstrated a high level of genotyping accuracy. However, the quality of the clinical reports was variable and suggestions are made for improvement.

Molecular genetic techniques have entered many areas of clinical practice. Public expectations from this technology are understandably high. To maintain confidence in this technology, laboratories must implement the highest standards of quality assurance (QA). External quality assessment (EQA) is recognized as an essential component of QA. The United Kingdom National External Quality Assessment Service (UKNEQAS) for Molecular Genetics, first set up in 1991, is currently the longest provider of EQA to molecular genetic testing laboratories in the UK, The Netherlands, and Ireland. Errors in the scheme are sporadic events. However, evidence from this and other EQA schemes suggests that a residual error rate persists, which should be taken into account in clinical practice. This EQA scheme has evolved from the respective scientific bodies of the constituent countries and retains a strong emphasis on collective peer review. It is essential that the steps taken to ensure quality in this rapidly expanding field are clear and transparent to participants and public alike. We describe the procedures developed and the governance imposed to monitor and improve analytical and reporting standards in participant laboratories and we compare our experiences with those of equivalent EQA services in the United States.

Background: Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR., Methods: The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number., Results: The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants., Conclusions: This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.

Congenital adrenal hyperplasia (CAH) is an inherited disorder of steroidogenesis with a wide spectrum of expression. In about 95% of cases, the disease is the result of 21-hydroxylase deficiency, an autosomal recessive condition that maps to the major histocompatibility complex (MHC) on 6p21.3 (1). Classical CAH results in excessive androgen production. Females with this disorder are frequently diagnosed at birth because of ambiguous development of external genitalia, whereas males may not present until age 4-7 when they begin to manifest inappropriate virilization. Approximately 30% of individuals with classical CAH have this simple virilizing form of the disease. The remaining 70% in addition manifest the potentially life-threatening salt-wasting form of classical CAH characterized by an inability to retain dietary sodium.