Heterozygous COL17A1 variants are a frequent cause of amelogenesis imperfecta.

BACKGROUND: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. METHODS: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. RESULTS: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. CONCLUSION: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.

Screening of potential novel candidate genes in schwannomatosis patients.

Schwannomatosis comprises a group of hereditary tumor predisposition syndromes characterized by, usually benign, multiple nerve sheath tumors, which frequently cause severe pain that does not typically respond to drug treatments. The most common schwannomatosis-associated gene is NF2, but SMARCB1 and LZTR1 are also associated. There are still many cases in which no pathogenic variants (PVs) have been identified, suggesting the existence of as yet unidentified genetic risk factors. In this study, we performed extended genetic screening of 75 unrelated schwannomatosis patients without identified germline PVs in NF2, LZTR1, or SMARCB1. Screening of the coding region of DGCR8, COQ6, CDKN2A, and CDKN2B was carried out, based on previous reports that point to these genes as potential candidate genes for schwannomatosis. Deletions or duplications in CDKN2A, CDKN2B, and adjacent chromosome 9 region were assessed by multiplex ligation-dependent probe amplification analysis. Sequencing analysis of a patient with multiple schwannomas and melanomas identified a novel duplication in the coding region of CDKN2A, disrupting both p14ARF and p16INK4a. Our results suggest that none of these genes are major contributors to schwannomatosis risk but the possibility remains that they may have a role in more complex mechanisms for tumor predisposition.

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

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.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

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 method's 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.

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.

Half of germline pathogenic and likely pathogenic variants found on panel tests do not fulfil NHS testing criteria.

Genetic testing for cancer predisposition has been curtailed by the cost of sequencing, and testing has been restricted by eligibility criteria. As the cost of sequencing decreases, the question of expanding multi-gene cancer panels to a broader population arises. We evaluated how many additional actionable genetic variants are returned by unrestricted panel testing in the private sector compared to those which would be returned by adhering to current NHS eligibility criteria. We reviewed 152 patients referred for multi-gene cancer panels in the private sector between 2014 and 2016. Genetic counselling and disclosure of all results was standard of care provided by the Consultant. Every panel conducted was compared to current eligibility criteria. A germline pathogenic / likely pathogenic variant (P/LP), in a gene relevant to the personal or family history of cancer, was detected in 15 patients (detection rate of 10%). 46.7% of those found to have the P/LP variants (7 of 15), or 4.6% of the entire set (7 of 152), did not fulfil NHS eligibility criteria. 46.7% of P/LP variants in this study would have been missed by national testing guidelines, all of which were actionable. However, patients who do not fulfil eligibility criteria have a higher Variant of Uncertain Significance (VUS) burden. We demonstrated that the current England NHS threshold for genetic testing is missing pathogenic variants which would alter management in 4.6%, nearly 1 in 20 individuals. However, the clinical service burden that would ensue is a detection of VUS of 34%.

Innovative recruitment and clinical orientation programme to manage NHS junior doctor shortfall: A district hospital experience.

BACKGROUND: International medical graduates (IMGs) contribute significantly towards the NHS care provision. No standardised clinical orientation programme (COP) for IMGs new to the NHS exists. OBJECTIVE: Our objective was to describe recruitment and retention strategies for junior doctors (JDs) in general medicine and develop a framework to anticipate outcomes of these interventions using the realist evaluation methodology. METHODS: We performed quality improvement interventions of recruitment and COP for new entrant IMGs in our organisation employed between December 2017 and April 2019. RESULTS: Twenty-three IMGs were recruited, 96% successfully completed the COP with a mean contract duration of 13±5 months. From the academic year 2017/18 to 2018/19, mean JD post occupancy increased from 54±3 to 73±4 JDs (p<0.001) and JD locum spend fell by £1.9 million. CONCLUSION: Our structured COP provides a stable, trained and financially sustainable JD workforce. Application in broader NHS settings is recommended.

Extensive gene conversion at the PMS2 DNA mismatch repair locus.

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens.

Characterization and Genomic Localization of a SMAD4 Processed Pseudogene.

Like many clinical diagnostic laboratories, the Yorkshire Regional Genetics Service undertakes routine investigation of cancer-predisposed individuals by high-throughput sequencing of patient DNA that has been target-enriched for genes associated with hereditary cancer. Accurate diagnosis using such reagents requires alertness regarding rare nonpathogenic variants that may interfere with variant calling. In a cohort of 2042 such cases, we identified 5 that initially appeared to be carriers of a 95-bp deletion of SMAD4 intron 6. More detailed analysis indicated that these individuals all carried one copy of a SMAD4 processed gene. Because of its interference with diagnostic analysis, we characterized this processed gene in detail. Whole-genome sequencing and confirmatory Sanger sequencing of junction PCR products were used to show that in each of the 5 cases, the SMAD4 processed gene was integrated at the same position on chromosome 9, located within the last intron of the SCAI gene. This rare polymorphic processed gene therefore reflects the occurrence of a single ancestral retrotransposition event. Compared to the reference SMAD4 mRNA sequence NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide), the 5' and 3' untranslated regions of the processed gene are both truncated, but its open reading frame is unaltered. Our experience leads us to advocate the use of an RNA-seq aligner as part of diagnostic assay quality assurance, since this allows recognition of processed pseudogenes in a comparatively facile automated fashion.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.

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.

The common TMC1 mutation c.100C>T (p.Arg34X) is not a significant cause of deafness in British Asians.

TMC1, a second-tier deafness gene below GJB2, is an appreciable cause of recessive nonsyndromic hearing loss (DFNB7/11) in North Africa, the Middle East, and parts of South Asia. Additionally, a single founder mutation, c.100C>T (p.Arg34X), dominates the TMC1 mutation spectrum. We investigated the frequency of TMC1 c.100C>T in a large set of British Asians with hearing loss, collectively a group with high prevalence of genetic deafness and limited routine clinical testing options beyond GJB2, on a candidate basis. An estimate of 0.21% (95% confidence interval, 0.04%-1.18%) was gained, indicating no significant enrichment in our set. Identification of the common non-GJB2 deafness genes and mutations in British Asian communities would require data from autozygosity mapping and/or massively parallel sequencing of gene panels.

Three-year follow-up of a randomised clinical trial of intravenous versus oral iron for anaemia in pregnancy.

BACKGROUND: To date, there are no data available concerning the impact of iron therapy on the long-term well-being and health-related quality of life (HRQoL) in pregnancy. OBJECTIVE: To assess the long-term effect of iron therapy on HRQoL in pregnancy. DESIGN: This is a follow-up study conducted between January 2010 and January 2011 of an earlier randomised open-label clinical trial of intravenous and oral iron versus oral iron for pregnancy-related iron deficiency anaemia. We used a modified version of the SF-36 questionnaire together with the original prospective HRQoL data collected during and after pregnancy. PARTICIPANTS AND INTERVENTIONS: Of the original evaluable 183 pregnant Caucasian women randomised to receive oral iron or a single intravenous iron polymaltose infusion followed by oral iron maintenance, 126 women completed the follow-up HRQoL study. METHODS: The participants were followed up 4 weeks after treatment, predelivery and postdelivery for a median period of 32 months (range, 26-42) with a well-being and HRQoL questionnaire using a modified SF-36 QoL-survey and child growth charts as set by the Australasian Paediatric Endocrine Group (APEG). RESULTS: Patients who received intravenous iron demonstrated significantly higher haemoglobin and serum ferritin levels (p<0.001). There were strong associations between iron status and a number of the HRQoL parameters, with improved general health (p<0.001), improved vitality (physical energy) (p<0.001), less psychological downheartedness (p=0.005), less clinical depression (p=0.003) and overall improved mental health (p<0.001). The duration of breastfeeding was longer (p=0.046) in the intravenous iron group. The babies born in both groups recorded similarly on APEG growth chart assessments. CONCLUSIONS: Our data suggest that HRQoL is improved until after pregnancy in anaemic pregnant women by repletion of their iron stores during pregnancy. About 80% of the intravenous iron group showed a maintained normal ferritin until delivery with long-term benefits. Further studies to confirm these findings are warranted.

Low prevalence of DFNB1 (connexin 26) mutations in British Pakistani children with non-syndromic sensorineural hearing loss.

OBJECTIVE: To determine the clinical sensitivity of DFNB1 genetic testing (analysis of the connexin 26 gene GJB2) for non-syndromic sensorineural hearing loss (SNHL) in British Pakistani children and extend to a comparison with British White children and literature data. DESIGN: Retrospective cohort study. SETTING: City of Bradford, UK. PATIENTS: Overall, 177 children (152 families) were eligible; 147 children (123 families) were British Pakistani, and 30 children (29 families) were British White. INTERVENTIONS: DFNB1 testing was offered. MAIN OUTCOME MEASURES: Detection rate for pathogenic bi-allelic GJB2 mutations. RESULTS: DFNB1 testing yielded positive results in 6.9% British Pakistani families compared with 15.4% British White families. Of 65 British Pakistani children tested (from 58 families), five children (from four families) were found to be homozygous for the common South Asian GJB2 mutation p.Trp24X. Of 14 British White children tested (from 13 families), bi-allelic pathogenic GJB2 mutations were seen in two children (from two families). CONCLUSIONS: The contribution of DFNB1 to non-syndromic SNHL in the Bradford British Pakistani children appears to be low when compared with a White peer group and White populations in general. The high prevalence of genetic deafness in this community, attributed to family structure and immigration history, points to a dilution effect in favour of other recessive deafness genes/loci.

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.

PMS2 mutations in childhood cancer.

Until recently, the PMS2 DNA mismatch repair gene has only rarely been implicated as a cancer susceptibility locus. New studies have shown, however, that earlier analyses of this gene have had technical limitations and also that the genetic behavior of mutant PMS2 alleles is unusual, in that, unlike MLH1 or MSH2 mutations, PMS2 mutations show low heterozygote penetrance. As a result, a dominantly inherited cancer predisposition has not been a feature reported in families with PMS2 mutations. Such families have instead been ascertained through childhood-onset cancers in homozygotes or through apparently sporadic colorectal cancer in heterozygotes. We present further information on the phenotype associated with homozygous PMS2 deficiency in 13 patients from six families of Pakistani origin living in the United Kingdom. This syndrome is characterized by café-au-lait skin pigmentation and a characteristic tumor spectrum, including leukemias, lymphomas, cerebral malignancies (such as supratentorial primitive neuroectodermal tumors, astrocytomas, and glioblastomas), and colorectal neoplasia with an onset in early adult life. We present evidence for a founder effect in five families, all of which carried the same R802-->X mutation (i.e., arginine-802 to stop) in PMS2. This cancer syndrome can be mistaken for neurofibromatosis type 1, with important management implications including the risk of the disorder occurring in siblings and the likelihood of tumor development in affected individuals.