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Clinical short-read exome and genome sequencing approaches have positively impacted diagnostic testing for rare diseases. Yet, technical limitations associated with short reads challenge their use for the detection of disease-associated variation in complex regions of the genome. Long-read sequencing (LRS) technologies may overcome these challenges, potentially qualifying as a first-tier test for all rare diseases. To test this hypothesis, we performed LRS (30× high-fidelity [HiFi] genomes) for 100 samples with 145 known clinically relevant germline variants that are challenging to detect using short-read sequencing and necessitate a broad range of complementary test modalities in diagnostic laboratories. We show that relevant variant callers readily re-identified the majority of variants (120/145, 83%), including ∼90% of structural variants, SNVs/insertions or deletions (indels) in homologous sequences, and expansions of short tandem repeats. Another 10% (n = 14) was visually apparent in the data but not automatically detected. Our analyses also identified systematic challenges for the remaining 7% (n = 11) of variants, such as the detection of AG-rich repeat expansions. Titration analysis showed that 90% of all automatically called variants could also be identified using 15-fold coverage. Long-read genomes thus identified 93% of challenging pathogenic variants from our dataset. Even with reduced coverage, the vast majority of variants remained detectable, possibly enhancing cost-effective diagnostic implementation. Most importantly, we show the potential to use a single technology to accurately identify all types of clinically relevant variants., Competing Interests: Declaration of interests T.M., E.D., X.C., and M.A.E. are employees and shareholders of Pacific Biosciences, a company commercializing DNA sequencing technologies. Pacific Biosciences also kindly provided part of the reagents required for this study., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Clinical exome sequencing (ES) has facilitated genetic diagnosis in individuals with a rare genetic disorder by analysis of all protein-coding sequences in a single experiment. However, in 40-60% of patients, a conclusive diagnosis remains elusive. In 2-5% of these individuals, ES does identify a disease-associated monoallelic variant in a recessive disorder. We hypothesized that short-read genome sequencing (GS) might uncover a pathogenic variant on the second allele, thereby increasing diagnostic yield. We performed GS for 174 individuals in whom ES identified a monoallelic pathogenic variant in a gene associated with recessive disease related to their phenotype. GS interpretation was limited to the (non-)coding parts of the gene in which this first pathogenic variant was identified, focusing on splice-disrupting variants. Firstly, we uncovered a second pathogenic variant affecting coding sequence in five individuals, including two SNV/indel variants, two copy number variants, and one insertion. Secondly, for 24 individuals, we identified a total of 31 rare non-coding intronic SNV/indel variants, all predicted to disrupt splicing. Using functional follow-up assays, we confirmed an effect on splicing for three of these variants (in ABCA4, POLR3A and COL4A4) in three individuals. In summary, we identified a (likely) pathogenic second variant in 4.6% (8/174), and a possible diagnosis for 12.1% (21/174) of our cohort. Hence, when performing GS as first-tier diagnostic test, including the interpretation of SVs and rare intronic variants in known recessive disease genes, the overall diagnostic yield of rare disease will increase. The added diagnostic value of GS for recessive disease In our cohort of 174 individuals (84 males and 90 females) with a monoallelic pathogenic variant in genes associated with a wide and diverse range of recessive diseases (pie chart), using genome sequencing (GS) and a systematic approach (methods), we identified eight new diagnoses (4.6%). We identified a second likely pathogenic variant in eight individuals (results); In two a second coding variant was found, in three others, a rare non-coding SNV anticipated to disrupt splicing was uncovered, and in three individuals a structural rearrangement was identified (two copy number variants (CNV), and one structural variant (SV))., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: This study was approved by the Medical Review Ethics Committee Arnhem-Nijmegen under 2011/188 and 2020-7142., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

While next generation sequencing has expanded the scientific understanding of Inborn Errors of Immunity (IEI), the clinical use and re-use of exome sequencing is still emerging. We revisited clinical exome data from 1300 IEI patients using an updated in silico IEI gene panel. Variants were classified and curated through expert review. The molecular diagnostic yield after standard exome analysis was 11.8 %. Through systematic reanalysis, we identified variants of interest in 5.2 % of undiagnosed patients, with 76.7 % being (candidate) disease-causing, providing a (candidate) diagnosis in 15.2 % of our cohort. We find a 1.7 percentage point increase in conclusive molecular diagnoses. We find a high degree of actionability in patients with a genetic diagnosis (76.4 %). Despite the modest absolute diagnostic gain, these data support the benefit of iterative exome reanalysis in IEI patients, conveying the notion that our current understanding of genes and variants involved in IEI is by far not saturated., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Usher syndrome (USH), characterized by bilateral sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP), prompts increased reliance on hearing due to progressive visual deterioration. It can be categorized into three subtypes: USH type 1 (USH1), characterized by severe to profound congenital SNHL, childhood-onset RP, and vestibular areflexia; USH type 2 (USH2), presenting with moderate to severe progressive SNHL and RP onset in the second decade, with or without vestibular dysfunction; and USH type 3 (USH3), featuring variable progressive SNHL beginning in childhood, variable RP onset, and diverse vestibular function. Previous studies evaluating cochlear implant (CI) outcomes in individuals with USH used varying or short follow-up durations, while others did not evaluate outcomes for each subtype separately. This study evaluates long-term CI performance in subjects with USH, at both short-term and long-term, considering each subtype separately., Design: This retrospective, observational cohort study identified 36 CI recipients (53 ears) who were categorized into four different groups: early-implanted USH1 (first CI at ≤7 years of age), late-implanted USH1 (first CI at ≥8 years of age), USH2 and USH3. Phoneme scores at 65 dB SPL with CI were evaluated at 1 year, ≥2 years (mid-term), and ≥5 years postimplantation (long-term). Each subtype was analyzed separately due to the significant variability in phenotype observed among the three subtypes., Results: Early-implanted USH1-subjects (N = 23 ears) achieved excellent long-term phoneme scores (100% [interquartile ranges {IQR} = 95 to 100]), with younger age at implantation significantly correlating with better CI outcomes. Simultaneously implanted subjects had significantly better outcomes than sequentially implanted subjects ( p = 0.028). Late-implanted USH1 subjects (N = 3 ears) used CI solely for sound detection and showed a mean phoneme discrimination score of 12% (IQR = 0 to 12), while still expressing satisfaction with ambient sound detection. In the USH2 group (N = 23 ears), a long-term mean phoneme score of 85% (IQR = 81 to 95) was found. Better outcomes were associated with younger age at implantation and higher preimplantation speech perception scores. USH3-subjects (N = 7 ears) achieved a mean postimplantation phoneme score of 71% (IQR = 45 to 91)., Conclusions: This study is currently one of the largest and most comprehensive studies evaluating CI outcomes in individuals with USH, demonstrating that overall, individuals with USH benefit from CI at both short- and long-term follow-up. Due to the considerable variability in phenotype observed among the three subtypes, each subtype was analyzed separately, resulting in smaller sample sizes. For USH1 subjects, optimal CI outcomes are expected with early simultaneous bilateral implantation. Late implantation in USH1 provides signaling function, but achieved speech recognition is insufficient for oral communication. In USH2 and USH3, favorable CI outcomes are expected, especially if individuals exhibit sufficient speech recognition with hearing aids and receive ample auditory stimulation preimplantation. Early implantation is recommended for USH2, given the progressive nature of hearing loss and concomitant severe visual impairment. In comparison with USH2, predicting outcomes in USH3 remains challenging due to the variability found. Counseling for USH2 and USH3 should highlight early implantation benefits and encourage hearing aid use., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Ear & Hearing is published on behalf of the American Auditory Society, by Wolters Kluwer Health, Inc.)

Although more than 140 genes have been associated with non-syndromic hereditary hearing loss (HL), at least half of the cases remain unexplained in medical genetic testing. One reason is that pathogenic variants are located in 'novel' deafness genes. A variant prioritization approach was used to identify novel (candidate) genes for HL. Exome-wide sequencing data were assessed for subjects with presumed hereditary HL that remained unexplained in medical genetic testing by gene-panel analysis. Cases in group AD had presumed autosomal dominantly inherited HL (n = 124), and in group AR, presumed autosomal recessive HL (n = 337). Variants in known and candidate deafness genes were prioritized based on allele frequencies and predicted effects. Selected variants were tested for their co-segregation with HL. Two cases were solved by variants in recently identified deafness genes (ABHD12, TRRAP). Variant prioritization also revealed potentially causative variants in candidate genes associated with recessive and X-linked HL. Importantly, missense variants in IKZF2 were found to co-segregate with dominantly inherited non-syndromic HL in three families. These variants specifically affected Zn 2+ -coordinating cysteine or histidine residues of the zinc finger motifs 2 and 3 of the encoded protein Helios. This finding indicates a complex genotype-phenotype correlation for IKZF2 defects, as this gene was previously associated with non-syndromic dysfunction of the immune system and ICHAD syndrome, including HL. The designed strategy for variant prioritization revealed that IKZF2 variants can underlie non-syndromic HL. The large number of candidate genes for HL and variants therein stress the importance of inclusion of family members for variant prioritization., (© 2024. The Author(s).)

Objective: Maternal cell contamination (MCC) poses a risk for misdiagnosis in prenatal genetic testing, and is examined in accredited diagnostic laboratories However, the awareness of possible MCC in perinatal/postnatal genetic testing, mainly of umbilical cord blood (CB), is lower., Method: We investigated the rate of MCC in DNA from both umbilical CB samples and umbilical cord samples that were sent to our diagnostic laboratory for diagnostic testing between 1995 and 2021 (n = 236)., Results: MCC was detected in 4% of umbilical CB samples, and in one umbilical cord sample. Particularly tests enriching for a specific variant are very sensitive for low amounts of MCC, as we emphasize here with a false positive diagnosis of myotonic dystrophy type 1 in a newborn., Conclusions: Overall, with appropriate collection and use, umbilical CB and umbilical cord samples are suitable for genetic testing based on the low rates of MCC and misdiagnosis. These findings do however underline the importance of routine MCC testing in umbilical CB samples and umbilical cord samples for both requesting clinicians and diagnostic genetic laboratories., (© 2024 The Author(s). Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Background: To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease., Methods: We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels < 50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥ 98% was considered the threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected., Results: Overall, 95% (1206/1271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79-100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected., Conclusions: GS can capture clinically relevant germline variants in a 'GS-first strategy' for the majority of clinical indications in a genetics diagnostic lab., (© 2024. The Author(s).)

Objectives: Non-normative uncertainty (uncertainty about empirical facts) and normative uncertainty (uncertainty about moral values or beliefs) regarding unsolicited findings (UFs) might play an important role in clinical genetics. Identifying normative uncertainty is of special interest since it might guide towards novel directions for counseling practice. This study aims to gain insight into the role of non-normative and normative uncertainty regarding UFs, as expressed by counselees and counselors., Methods: We performed a secondary qualitative analysis of interviews with counselees (n = 20) and counselors (n = 20) who had been confronted with UFs. Following a deductive approach, we used Han et al.'s existing theoretical framework of uncertainty, in which we additionally incorporated normative uncertainty., Results: Major issues of non-normative uncertainty were practical and personal for counselees, whilst counselors' uncertainty pertained mainly to scientific issues. Normative uncertainty was a major theme throughout the interviews. We encountered the moral conflicts of autonomy vs. beneficence and non-maleficence and of autonomy vs. truthfulness., Conclusion: Non-normative uncertainty regarding UFs highlights the need to gain more insight in their penetrance and clinical utility. This study suggests moral conflicts are a major source of feelings of uncertainty in clinical genetics., Practice Implications: Exploring counselees' non-normative uncertainties and normative conflicts seems a prerequisite to optimize genetic counseling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Rapid exome sequencing (rES) has become the first-choice genetic test for critically ill patients, mostly neonates, young infants, or fetuses in prenatal care, in time-sensitive situations and when it is expected that the genetic test result may guide clinical decision making. The implementation of rES has revolutionized medicine by enabling timely identification of genetic causes for various rare diseases. The utilization of rES has increasingly been recognized as an essential diagnostic tool for the identification of complex and undiagnosed genetic disorders. Methods: We conducted a retrospective evaluation of our experiences with rES performed on 575 critically ill patients from various age groups (prenatal to adulthood), over a four-year period (2016-2019). These patients presented with a wide spectrum of rare diseases, including but not limited to neurological disorders, severe combined immune deficiency, and cancer. Results: During the study period, there was a significant increase in rES referrals, with a rise from a total of two referrals in Q1-2016 to 10 referrals per week in Q4-2019. The median turnaround time (TAT) decreased from 17 to 11 days in the period 2016-2019, with an overall median TAT of 11 days (IQR 8-15 days). The overall diagnostic yield for this cohort was 30.4%, and did not significantly differ between the different age groups (e.g. adults 22.2% vs children 31.0%; p -value 0.35). However, variability in yield was observed between clinical entities: craniofacial anomalies yielded 58.3%, while for three clinical entities (severe combined immune deficiency, aneurysm, and hypogonadotropic hypogonadism) no diagnoses were obtained. Discussion: Importantly, whereas clinical significance is often only attributed to a conclusive diagnosis, we also observed impact on clinical decision-making for individuals in whom no genetic diagnosis was established. Hence, our experience shows that rES has an important role for patients of all ages and across the broad spectrum of rare diseases to impact clinical outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Marouane, Neveling, Deden, van den Heuvel, Zafeiropoulou, Castelein, van de Veerdonk, Koolen, Simons, Rodenburg, Westra, Mensenkamp, de Leeuw, Ligtenberg, Matthijsse, Pfundt, Kamsteeg, Brunner, Gilissen, Feenstra, de Boode, Yntema, van Zelst-Stams, Nelen and Vissers.)

Background: The spiral ganglion hypothesis suggests that pathogenic variants in genes preferentially expressed in the spiral ganglion nerves (SGN), may lead to poor cochlear implant (CI) performance. It was long thought that TMPRSS3 was particularly expressed in the SGNs. However, this is not in line with recent reviews evaluating CI performance in subjects with TMPRSS3-associated sensorineural hearing loss (SNHL) reporting overall beneficial outcomes. These outcomes are, however, based on variable follow-up times of, in general, 1 year or less. Therefore, we aimed to 1. evaluate long-term outcomes after CI implantation of speech recognition in quiet in subjects with TMPRSS3-associated SNHL, and 2. test the spiral ganglion hypothesis using the TMPRSS3-group., Methods: This retrospective, multicentre study evaluated long-term CI performance in a Dutch population with TMPRSS3-associated SNHL. The phoneme scores at 70 dB with CI in the TMPRSS3-group were compared to a control group of fully genotyped cochlear implant users with post-lingual SNHL without genes affecting the SGN, or severe anatomical inner ear malformations. CI-recipients with a phoneme score ≤ 70% at least 1-year post-implantation were considered poor performers and were evaluated in more detail., Results: The TMPRSS3 group consisted of 29 subjects (N = 33 ears), and the control group of 62 subjects (N = 67 ears). For the TMPRSS3-group, we found an average phoneme score of 89% after 5 years, which remained stable up to 10 years post-implantation. At both 5 and 10-year follow-up, no difference was found in speech recognition in quiet between both groups (p = 0.830 and p = 0.987, respectively). Despite these overall adequate CI outcomes, six CI recipients had a phoneme score of ≤ 70% and were considered poor performers. The latter was observed in subjects with residual hearing post-implantation or older age at implantation., Conclusion: Subjects with TMPRSS3-associated SNHL have adequate and stable long-term outcomes after cochlear implantation, equal to the performance of genotyped patient with affected genes not expressed in the SGN. These findings are not in line with the spiral ganglion hypothesis. However, more recent studies showed that TMPRSS3 is mainly expressed in the hair cells with only limited SGN expression. Therefore, we cannot confirm nor refute the spiral ganglion hypothesis., (© 2023. The Author(s).)

Competing Interests: Competing interests: None declared.

Background: Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques., Results: We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection., Conclusion: We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques., (© 2023. The Author(s).)

Unsolicited findings (UFs) from diagnostic genetic testing are a subject of debate. The emerging consensus is that some UFs from genetic testing should be disclosed, but recommendations on UF disclosure generally leave room for variation in practice. This study aimed to explore clinical geneticists' views on and experiences with UFs during pretest counseling and UF disclosure. We interviewed 20 certified clinical genetics medical specialists and clinical genetics residents, working in 7 Dutch genetic centers. Participants indicated that discussing the probability of detecting UFs is an integral part of pretest counseling and informed consent. However, they expressed doubts about the degree to which this discussion should occur and about what information they should share with patients. They argued that the contents of their counseling should depend on the individual patient's capacity to understand information. These results endorse the importance of tailored pretest counseling alongside informed consent for optimal genetic consultations. While "medical actionability" is broadly accepted as an important criterion for the disclosure of UFs, participants experienced substantial uncertainty regarding this concept. This study underscores the need for further demarcation of what exactly constitutes medical actionability. Installation of an expert panel to help healthcare professionals decide what variants to disclose will support them when facing the dilemmas presented by UFs., (© 2022 The Authors. Journal of Genetic Counseling published by Wiley Periodicals LLC on behalf of National Society of Genetic Counselors.)

The aim of this study is to contribute to a better description of the genotypic and phenotypic spectrum of DFNA6/14/38 and aid in counseling future patients identified with this variant. Therefore, we describe the genotype and phenotype in a large Dutch-German family (W21-1472) with autosomal dominant non-syndromic, low-frequency sensorineural hearing loss (LFSNHL). Exome sequencing and targeted analysis of a hearing impairment gene panel were used to genetically screen the proband. Co-segregation of the identified variant with hearing loss was assessed by Sanger sequencing. The phenotypic evaluation consisted of anamnesis, clinical questionnaires, physical examination and examination of audiovestibular function. A novel likely pathogenic WFS1 variant (NM&#95;006005.3:c.2512C>T p.(Pro838Ser)) was identified in the proband and found to co-segregate with LFSNHL, characteristic of DFNA6/14/38, in this family. The self-reported age of onset of hearing loss (HL) ranged from congenital to 50 years of age. In the young subjects, HL was demonstrated in early childhood. At all ages, an LFSNHL (0.25-2 kHz) of about 50-60 decibel hearing level (dB HL) was observed. HL in the higher frequencies showed inter-individual variability. The dizziness handicap inventory (DHI) was completed by eight affected subjects and indicated a moderate handicap in two of them (aged 77 and 70). Vestibular examinations ( n = 4) showed abnormalities, particularly in otolith function. In conclusion, we identified a novel WFS1 variant that co-segregates with DFNA6/14/38 in this family. We found indications of mild vestibular dysfunction, although it is uncertain whether this is related to the identified WFS1 variant or is an incidental finding. We would like to emphasize that conventional neonatal hearing screening programs are not sensitive to HL in DFNA6/14/38 patients, because high-frequency hearing thresholds are initially preserved. Therefore, we suggest screening newborns in DFNA6/14/38 families with more frequency-specific methods.

Genome sequencing (GS) can identify novel diagnoses for patients who remain undiagnosed after routine diagnostic procedures. We tested whether GS is a better first-tier genetic diagnostic test than current standard of care (SOC) by assessing the technical and clinical validity of GS for patients with neurodevelopmental disorders (NDD). We performed both GS and exome sequencing in 150 consecutive NDD patient-parent trios. The primary outcome was diagnostic yield, calculated from disease-causing variants affecting exonic sequence of known NDD genes. GS (30%, n = 45) and SOC (28.7%, n = 43) had similar diagnostic yield. All 43 conclusive diagnoses obtained with SOC testing were also identified by GS. SOC, however, required integration of multiple test results to obtain these diagnoses. GS yielded two more conclusive diagnoses, and four more possible diagnoses than ES-based SOC (35 vs. 31). Interestingly, these six variants detected only by GS were copy number variants (CNVs). Our data demonstrate the technical and clinical validity of GS to serve as routine first-tier genetic test for patients with NDD. Although the additional diagnostic yield from GS is limited, GS comprehensively identified all variants in a single experiment, suggesting that GS constitutes a more efficient genetic diagnostic workflow., (© 2022. The Author(s).)

Pathogenic variants in the OPN1LW/OPN1MW gene cluster are causal for a range of mild to severe visual impairments with color deficiencies. The widely utilized short-read next-generation sequencing (NGS) is inappropriate for the analysis of the OPN1LW/OPN1MW gene cluster and many patients with pathogenic variants stay underdiagnosed. A diagnostic genetic assay was developed for the OPN1LW/OPN1MW gene cluster, consisting of copy number analysis via multiplex ligation-dependent probe amplification and sequence analysis via long-read circular consensus sequencing. Performance was determined on 50 clinical samples referred for genetic confirmation of the clinical diagnosis (n = 43) or carrier status analysis (n = 7). A broad range of pathogenic haplotypes were detected, including deletions, hybrid genes, single variants and combinations of variants. The developed genetic assay for the OPN1LW/OPN1MW gene cluster is a diagnostic test that can detect both structural and nucleotide variants with a straightforward analysis, improving diagnostic care of patients with visual impairment., (© 2022. The Author(s).)

Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is highly heterogeneous both clinically and genetically. Recently, variants in the arylsulfatase G (ARSG) gene have been reported to underlie USH type IV. This distinct type of USH is characterized by late-onset RP with predominantly pericentral and macular changes, and late onset SNHL without vestibular dysfunction. In this study, we describe the USH type IV phenotype in three unrelated subjects. We identified three novel pathogenic variants, two novel likely pathogenic variants, and one previously described pathogenic variant in ARSG. Functional experiments indicated a loss of sulfatase activity of the mutant proteins. Our findings confirm that ARSG variants cause the newly defined USH type IV and support the proposed extension of the phenotypic USH classification., (© 2022. The Author(s).)

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results., (© 2022. The Author(s).)

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Background: Approximately two third of patients with a rare genetic disease remain undiagnosed after exome sequencing (ES). As part of our post-test counseling procedures, patients without a conclusive diagnosis are advised to recontact their referring clinician to discuss new diagnostic opportunities in due time. We performed a systematic study of genetically undiagnosed patients 5 years after their initial negative ES report to determine the efficiency of diverse reanalysis strategies., Methods: We revisited a cohort of 150 pediatric neurology patients originally enrolled at Radboud University Medical Center, of whom 103 initially remained genetically undiagnosed. We monitored uptake of physician-initiated routine clinical and/or genetic re-evaluation (ad hoc re-evaluation) and performed systematic reanalysis, including ES-based resequencing, of all genetically undiagnosed patients (systematic re-evaluation)., Results: Ad hoc re-evaluation was initiated for 45 of 103 patients and yielded 18 diagnoses (including 1 non-genetic). Subsequent systematic re-evaluation identified another 14 diagnoses, increasing the diagnostic yield in our cohort from 31% (47/150) to 53% (79/150). New genetic diagnoses were established by reclassification of previously identified variants (10%, 3/31), reanalysis with enhanced bioinformatic pipelines (19%, 6/31), improved coverage after resequencing (29%, 9/31), and new disease-gene associations (42%, 13/31). Crucially, our systematic study also showed that 11 of the 14 further conclusive genetic diagnoses were made in patients without a genetic diagnosis that did not recontact their referring clinician., Conclusions: We find that upon re-evaluation of undiagnosed patients, both reanalysis of existing ES data as well as resequencing strategies are needed to identify additional genetic diagnoses. Importantly, not all patients are routinely re-evaluated in clinical care, prolonging their diagnostic trajectory, unless systematic reanalysis is facilitated. We have translated our observations into considerations for systematic and ad hoc reanalysis in routine genetic care., (© 2022. The Author(s).)

Pathogenic variants in SLC26A4 have been associated with autosomal recessive hearing loss (arHL) and a unilateral or bilateral enlarged vestibular aqueduct (EVA). SLC26A4 is the second most frequently mutated gene in arHL. Despite the strong genotype-phenotype correlation, a significant part of cases remains genetically unresolved. In this study, we investigated a cohort of 28 Dutch index cases diagnosed with HL in combination with an EVA but without (M0) or with a single (M1) pathogenic variant in SLC26A4. To explore the missing heritability, we first determined the presence of the previously described EVA-associated haplotype (Caucasian EVA (CEVA)), characterized by 12 single nucleotide variants located upstream of SLC26A4. We found this haplotype and a delimited V1-CEVA haplotype to be significantly enriched in our M1 patient cohort (10/16 cases). The CEVA haplotype was also present in two M0 cases (2/12). Short- and long-read whole genome sequencing and optical genome mapping could not prioritize any of the variants present within the CEVA haplotype as the likely pathogenic defect. Short-read whole-genome sequencing of the six M1 cases without this haplotype and the two M0/CEVA cases only revealed previously overlooked or misinterpreted splice-altering SLC26A4 variants in two cases, who are now genetically explained. No deep-intronic or structural variants were identified in any of the M1 subjects. With this study, we have provided important insights that will pave the way for elucidating the missing heritability in M0 and M1 SLC26A4 cases. For pinpointing the pathogenic effect of the CEVA haplotype, additional analyses are required addressing defect(s) at the RNA, protein, or epigenetic level., (© 2021. The Author(s).)

Unsolicited findings (UFs) are uncovered unintentionally and predispose to a disease unrelated to the clinical question. The frequency and nature of UFs uncovered in clinical practice remain largely unexplored. We here evaluated UFs identified during a 5-year period in which 16,482 index patients received clinical whole-exome sequencing (WES). UFs were identified in 0.58% (95/16,482) of index patients, indicating that the overall frequency of UFs in clinical WES is low. Fewer UFs were identified using restricted disease-gene panels (0.03%) than when using whole-exome/Mendeliome analysis (1.03%). The UF was disclosed to 86 of 95 individuals, for reasons of medical actionability. Only 61% of these UFs reside in a gene that is listed on the "ACMG59"-list, representing a list of 59 genes for which the American College of Medical Genetics recommends UF disclosure. The remaining 39% were grouped into four categories: disorders similar to "ACMG59"-listed disorders (25%); disorders for which disease manifestation could be influenced (7%); UFs providing reproductive options (2%); and UFs with pharmacogenetic implications (5%). Hence, our experience shows that UFs predisposing to medically actionable disorders affect a broader range of genes than listed on the "ACMG59", advocating that a pre-defined gene list is too restrictive, and that UFs may require ad hoc evaluation of medical actionability. While both the identification and disclosure of UFs depend on local policy, our lessons learned provide general essential insight into the nature and odds of UFs in clinical exome sequencing., (© 2021. The Author(s).)

We recently described a novel missense variant [c.2090T>G:p.(Leu697Trp)] in the MYO3A gene, found in two Brazilian families with late-onset autosomal dominant nonsyndromic hearing loss (ADNSHL). Since then, with the objective of evaluating its contribution to ADNSHL in Brazil, the variant was screened in additional 101 pedigrees with probable ADNSHL without conclusive molecular diagnosis. The variant was found in three additional families, explaining 3/101 (~3%) of cases with ADNSHL in our Brazilian pedigree collection. In order to identify the origin of the variant, 21 individuals from the five families were genotyped with a high-density SNP array (~600 K SNPs- Axiom Human Origins; ThermoFisher). The identity by descent (IBD) approach revealed that many pairs of individuals from the different families have a kinship coefficient equivalent to that of second cousins, and all share a minimum haplotype of ~607 kb which includes the c.2090T>G variant suggesting it probably arose in a common ancestor. We inferred that the mutation occurred in a chromosomal segment of European ancestry and the time since the most common ancestor was estimated in 1100 years (CI = 775-1425). This variant was also reported in a Dutch family, which shares a 87,121 bp haplotype with the Brazilian samples, suggesting that Dutch colonists may have brought it to Northeastern Brazil in the 17th century. Therefore, the present study opens new avenues to investigate this variant not only in Brazilians but also in European families with ADNSHL., (© 2021. The Author(s), under exclusive licence to European Society of Human Genetics.)

Purpose: Expansions of a subset of short tandem repeats (STRs) have been implicated in approximately 30 different human genetic disorders. Despite extensive application of exome sequencing (ES) in routine diagnostic genetic testing, STRs are not routinely identified from these data., Methods: We assessed diagnostic utility of STR analysis in exome sequencing by applying ExpansionHunter to 2,867 exomes from movement disorder patients and 35,228 other clinical exomes., Results: We identified 38 movement disorder patients with a possible aberrant STR length. Validation by polymerase chain reaction (PCR) and/or repeat-primed PCR technologies confirmed the presence of aberrant expansion alleles for 13 (34%). For seven of these patients the genotype was compatible with the phenotypic description, resulting in a molecular diagnosis. We subsequently tested the remainder of our diagnostic ES cohort, including over 30 clinically and genetically heterogeneous disorders. Optimized manual curation yielded 167 samples with a likely aberrant STR length. Validations confirmed 93/167 (56%) aberrant expansion alleles, of which 48 were in the pathogenic range and 45 in the premutation range., Conclusion: Our work provides guidance for the implementation of STR analysis in clinical ES. Our results show that systematic STR evaluation may increase diagnostic ES yield by 0.2%, and recommend making STR evaluation a routine part of ES interpretation in genetic testing laboratories., (© 2021. The Author(s), under exclusive licence to the American College of Medical Genetics and Genomics.)

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A . Seven unexplained cases were selected for future analysis with whole genome sequencing.

Unsolicited findings (UFs) in clinical exome sequencing are variants that are unrelated to the initial clinical question the DNA test was performed for, but that may nonetheless be of medical relevance to patients and/or their families. There is limited knowledge about the impact of UFs on patients' lives. In order to characterise patient perceptions of the impact of an UF, we conducted 20 semi-structured face-to-face interviews with patients and/or their relatives to whom an UF predisposing to oncological disease (n = 10) or predisposing to a cardiac condition (n = 10) had been disclosed. We have identified a psychological, physical and financial aspect of the perceived impact of UF disclosure in exome sequencing. Actionability, understanding, patients' pre-test health and social context were influencing factors, according to our participants. Although most expressed considerable psychological impact initially, all but one participant would choose to undergo genetic testing again, knowing what they know now. These novel findings provide insight in patients' perspectives on the impact of UF disclosure. Our study highlights the value of incorporating patients' perceptions in UF disclosure policy.

The number and distribution of recessive alleles in the population for various diseases are not known at genome-wide-scale. Based on 6,447 exome sequences of healthy, genetically unrelated Europeans of two distinct ancestries, we estimate that every individual is a carrier of at least 2 pathogenic variants in currently known autosomal-recessive (AR) genes and that 0.8%-1% of European couples are at risk of having a child affected with a severe AR genetic disorder. This risk is 16.5-fold higher for first cousins but is significantly more increased for skeletal disorders and intellectual disabilities due to their distinct genetic architecture., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Loss of functional UBE3A, an E3 protein ubiquitin ligase, causes Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, speech impairment, epilepsy, movement or balance disorder, and a characteristic behavioral pattern. We identified a novel UBE3A sequence variant in a large family with eight affected individuals, who did not meet the clinical AS criteria., Methods: Detailed clinical examination and genetic analysis was performed to establish the phenotypic diversity and the genetic cause. The function of the mutant UBE3A protein was assessed with respect to its subcellular localization, stability, and E3 ubiquitin ligase activity., Results: All eight affected individuals showed the presence of a novel maternally inherited UBE3A sequence variant (NM&#95;130838.4(UBE3A):c.1018-1020del, p.(Asn340del), which is in line with a genetic AS diagnosis. Although they presented with moderate to severe intellectual disability, the phenotype did not match the clinical criteria for AS. In line with this, functional analysis of the UBE3A p.Asn340del mutant protein revealed no major deficits in UBE3A protein localization, stability, or E3 ubiquitin ligase activity., Conclusion: The p.(Asn340del) mutant protein behaves distinctly different from previously described AS-linked missense mutations in UBE3A, and causes a phenotype that is markedly different from AS. This study further extends the range of phenotypes that are associated with UBE3A loss, duplication, or mutation., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

De novo mutations in protein-coding genes are a well-established cause of developmental disorders 1 . However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations 1,2 . Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.