Ruth Charlton

Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant‐Filtering Interface

Targeted hybridization enrichment prior to next‐generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the methods diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer‐predisposing genes or (2) whole‐exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome‐based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Investigations on a clinically and functionally unusual and novel germline p53 mutation

This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an osteosarcoma (age 22). The results from this study, and others, suggest that it may be advisable to consider the possibility of a germline p53 mutation in adults presenting with choroid plexus tumours. In the current study automated DNA sequencing of genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient. The alteration in frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position 182 in the mutated protein. Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood lymphocytes from this individual. These results led us to carry out more detailed functional tests on the mutant protein. The mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes. Further, the mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21WAF1, bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells. However, surprisingly, data from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein retains significant ability to induce apoptosis.

GeneScreen: a program for high-throughput mutation detection in DNA sequence electropherograms

Background While massively parallel DNA sequencing methods continue to evolve rapidly, the benchmark technique for detection and verification of rare (particularly disease-causing) sequence variants remains four-colour dye-terminator sequencing by capillary electrophoresis. The high throughput and long read lengths currently available have shifted the bottleneck in mutation detection away from data generation to data analysis. While excellent computational methods have been developed for quantifying sequence accuracy and detecting variants, either during de novo sequence assembly or for single-nucleotide polymorphism detection, the identification, verification and annotation of very rare sequence variants remains a rather labour-intensive process for which few software aids exist. Aim To provide a freely available, intuitive software application for highly efficient mutation screening of large sequence batches. Methods and results The authors developed GeneScreen, a desktop program that analyses capillary electropherograms and compares their sequences with a known reference for identification of mutations. The detected sequence variants are then made available for rapid assessment and annotation via a graphical user interface, allowing chosen variants to be exported for reporting and archiving. The program was validated using more than 16 000 diagnostic laboratory sequence traces. Conclusion Using GeneScreen, a single user requires only a few minutes to identify rare mutations in hundreds of sequence traces, with comparable sensitivity to expensive commercial products.

Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients

BACKGROUND Congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene. OBJECTIVE To elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients. PATIENTS AND METHODS Mutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS The genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis. CONCLUSIONS The frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. Methods Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. Results Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). Conclusions Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.

Histopathology of melanocytic lesions in a family with an inherited BAP1 mutation

To the Editor, We read with interest the report by Marusic et al.1 who expanded on the histopathological features of melanocytic neoplasms in a family with an inherited BAP1 mutation. Marusic et al.1 described the presence of nuclear pseudoinclusions and multinucleated melanocytes within five of six nevi from this family. The lesions were mainly intradermal, composed of large epithelioid melanocytes, and showed loss of BAP1 expression by immunohistochemistry. We report similar histopathological findings in melanocytic lesions excised from members of an English family in which a germline pathogenic BAP1 mutation has been identified. The proband was diagnosed with melanoma at 26 years of age that was clinically bland and pink in appearance. This was initially considered to be a melanocytic lesion of uncertain malignant potential (MELTUMP) but on further assessment, showed significant cytological atypia and lacked maturation and was therefore treated as a melanoma. The Breslow thickness was 2.4 mm and there was no evidence of ulceration. This patient later developed a melanoma in situ. He had multiple pink, bland nevi one of which subsequently changed and this was excised in view of his significant history. Histopathological examination showed an intradermal melanocytic neoplasm composed of epithelioid melanocytes with abundant cytoplasm, some of which contained multinucleated cells (Fig. 1A) and nuclear pseudoinclusions (Fig. 1B). The proband’s maternal grandfather had occupational exposure to asbestos and had died of mesothelioma. His maternal uncle had a history of stage IIA melanoma, which later metastasized to a regional lymph node. Striking pleomorphism, multinucleated melanocytes and nuclear pseudoinclusions were readily identified in this nodal metastasis. The proband’s maternal aunt, presented at 44 years of age with a history of a recalcitrant scalp lesion, which had been curetted twice elsewhere in the belief that this was a cyst. The curettings had not been sent for histopathological assessment. Following a further recurrence, approximately 18 months after onset, the lesion was partially excised by general surgeons. Histopathology revealed a malignant blue nevus-like melanoma showing a highly cellular melanocytic tumor, at least 11 mm in thickness; composed of spindle cells and some dendritic-like melanocytes. Nuclear pseudoinclusions were noted, although distributed more sparsely within this lesion. To our knowledge, there is only one other reported case of a 64-year-old female with a BAP1 germline mutation who had a malignant blue nevus-like melanoma of the scalp.2 Our patient was also found to have a meningioma on computed tomography (CT) scanning. Following an invitation for screening, the proband’s maternal cousin had a pink lesion excised, due to a history of change. This was diagnosed as a spitzoid tumor of uncertain malignant potential (STUMP). The tumor consisted of a lobulated, intradermal nodule composed of pleomorphic epithelioid melanocytes, flanked by a more diffuse melanocytic lesion with hyperchromatic nuclei (Fig. 2A). Nuclear

Novel DLX3 variants in amelogenesis imperfecta with attenuated tricho-dento-osseous syndrome

Abstract Objectives Variants in DLX3 cause tricho‐dento‐osseous syndrome (TDO, MIM #190320), a systemic condition with hair, nail and bony changes, taurodontism and amelogenesis imperfecta (AI), inherited in an autosomal dominant fashion. Different variants found within this gene are associated with different phenotypic presentations. To date, six different DLX3 variants have been reported in TDO. The aim of this paper was to explore and discuss three recently uncovered new variants in DLX3. Subjects and Methods Whole‐exome sequencing identified a new DLX3 variant in one family, recruited as part of an ongoing study of genetic variants associated with AI. Targeted clinical exome sequencing of two further families revealed another new variant of DLX3 and complete heterozygous deletion of DLX3. For all three families, the phenotypes were shown to consist of AI and taurodontism, together with other attenuated features of TDO. Results c.574delG p.(E192Rfs*66), c.476G>T (p.R159L) and a heterozygous deletion of the entire DLX3 coding region were identified in our families. Conclusion These previously unreported variants add to the growing literature surrounding AI, allowing for more accurate genetic testing and better understanding of the associated clinical consequences.