Clinical exome sequencing data from patients with inborn errors of immunity: Cohort level diagnostic yield and the benefit of systematic reanalysis.

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.

Uncovering recessive alleles in rare Mendelian disorders by genome sequencing of 174 individuals with monoallelic pathogenic variants.

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)).

Automatized detection of uniparental disomies in a large cohort.

Uniparental disomy (UPD) is the inheritance of both homologues of a chromosome from only one parent. The detection of UPDs in sequencing data is not well established and a common gap in genetic diagnostics. We applied our in-house UPD detection pipeline to evaluate a cohort of 9212 samples, including multigene panels as well as exome sequencing data in a single, duo or trio constellation. We used the results to inform the design of our publicly available web app altAFplotter. UPDs categorized as heterodisomy, whole chromosome or segmental isodisomy were identified and validated with microsatellites, multiplex ligation-dependent probe amplification as well as Sanger sequencing. We detected 14 previously undiagnosed UPDs including nine isodisomies, four segmental isodisomies as well as one heterodisomy on chromosome 22. We characterized eight findings as potentially causative through homozygous pathogenic variants or imprinting disorders. Overall, our study demonstrates the utility of our UPD detection pipeline with our web app, altAFplotter, to reliably identify UPDs. This not only increases the diagnostic yield of cases with growth and metabolic disturbances, as well as developmental delay, but also enhances the understanding of UPDs that may be relevant for recurrence risks and genetic counseling.

Detection of DNA Contamination in Prenatal Samples from Whole Exome Sequencing Data.

BACKGROUND: Maternal cell contamination (MCC) in prenatal samples poses a risk for misdiagnosis, and therefore, testing for contamination is necessary during genetic analysis of prenatal specimens. MCC testing is currently performed as a method separate from the diagnostic method. With the increasing application of whole exome sequencing (WES) in prenatal diagnosis, we sought to develop a method to estimate the level of contamination from WES data, aiming to eliminate the need for a separate MCC test. METHODS: To investigate the impact of MCC on the distribution of the variant allele fraction in WES data, contamination was both simulated in silico and artificially induced. Subsequently, a bioinformatic WES contamination method was developed and validated by comparing its performance to that of the gold standard (short tandem repeat [STR]) MCC test, validated for detecting ≥5% contamination. Finally, post-implementation performance was monitored for a 15-month period. RESULTS: During validation, 270 prenatal samples underwent analysis with both WES and the gold standard test. In 259 samples, the results were concordant (248 not contaminated, 11 contaminated with both tests). In 11 samples, contamination was only detected in WES data (2 of which contained ≥5% contamination with WES, which is above the detection limit of the gold standard test). The data of the post-implementation evaluation on 361 samples, of which 68 were contaminated, were in line with the validation data. CONCLUSIONS: Contamination can reliably be detected in WES data, rendering a separate contamination test unnecessary for the majority of samples.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

BACKGROUND: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects. METHODS: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. RESULTS: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. CONCLUSIONS: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity.

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary ß subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

Next-generation sequencing to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan.

Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.

Whole genome sequencing identifies elusive variants in genetically unsolved Italian inherited retinal disease patients.

Inherited retinal diseases (IRDs) are a group of rare monogenic diseases with high genetic heterogeneity (pathogenic variants identified in over 280 causative genes). The genetic diagnostic rate for IRDs is around 60%, mainly thanks to the routine application of next-generation sequencing (NGS) approaches such as extensive gene panels or whole exome analyses. Whole-genome sequencing (WGS) has been reported to improve this diagnostic rate by revealing elusive variants, such as structural variants (SVs) and deep intronic variants (DIVs). We performed WGS on 33 unsolved cases with suspected autosomal recessive IRD, aiming to identify causative genetic variants in non-coding regions or to detect SVs that were unexplored in the initial screening. Most of the selected cases (30 of 33, 90.9%) carried monoallelic pathogenic variants in genes associated with their clinical presentation, hence we first analyzed the non-coding regions of these candidate genes. Whenever additional pathogenic variants were not identified with this approach, we extended the search for SVs and DIVs to all IRD-associated genes. Overall, we identified the missing causative variants in 11 patients (11 of 33, 33.3%). These included three DIVs in ABCA4, CEP290 and RPGRIP1; one non-canonical splice site (NCSS) variant in PROM1 and three SVs (large deletions) in EYS, PCDH15 and USH2A. For the previously unreported DIV in CEP290 and for the NCCS variant in PROM1, we confirmed the effect on splicing by reverse transcription (RT)-PCR on patient-derived RNA. This study demonstrates the power and clinical utility of WGS as an all-in-one test to identify disease-causing variants missed by standard NGS diagnostic methodologies.

Genome sequencing as a generic diagnostic strategy for rare disease.

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.

Early detection of active Human CytomegaloVirus (hCMV) infection in pregnant women using data generated for noninvasive fetal aneuploidy testing.

BACKGROUND: Prenatal hCMV infections can lead to severe embryopathy and neurological sequelae in neonates. Screening during pregnancy is not recommended by global societies, as there is no effective therapy. Recently, several groups showed that maternal-fetal hCMV transmission can be strongly reduced by administering anti-viral agents early in pregnancy. This calls for a screening method to identify at risk pregnancies at an appropriate gestational age, with the possibility for large-scale enrolment. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidy screening early in pregnancy is already implemented in many countries and performed on a large-scale basis. We investigated the use of whole genome cell-free DNA (cfDNA) sequencing data, generated for the purpose of NIPT, as (pre-)screening tool to identify women with active hCMV-infections, eligible for therapy. METHODS: Coded raw sequencing NIPT data from 204,818 pregnant women from three testing laboratories were analyzed for the presence of hCMV-cfDNA. Samples were stratified by cfDNA-hCMV load. For validation and interpretation, diagnostic hCMV-qPCR and serology testing were performed on a subset of cfDNA-hCMV-positive (n = 112) and -negative (n = 127) samples. FINDINGS: In 1930 samples (0.94%) hCMV fragments were detected. Validation by hCMV-qPCR showed that samples with high cfDNA-hCMV load tested positive and cfDNA-hCMV-negative samples tested negative. In 32/112 cfDNA-hCMV-positive samples (28.6%) the serological profile suggested a recent primary infection: this was more likely in samples with high cfDNA-hCMV load (78.6%) than in samples with low cfDNA-hCMV load (11.0%). In none of the cfDNA-hCMV-negative samples serology was indicative of a recent primary infection. INTERPRETATION: Our study shows that large-scale (pre-)screening for both genetic fetal aberrations and active maternal hCMV infections during pregnancy can be combined in one cfDNA sequencing test, performed on a single blood sample, drawn in the first trimester of pregnancy. FUNDING: This work was partly funded by the Prenatal Screening Foundation Nijmegen, the Netherlands.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects. Methods: Evolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating that AFF3 LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Mobile element insertions in rare diseases: a comparative benchmark and reanalysis of 60,000 exome samples.

Mobile element insertions (MEIs) are a known cause of genetic disease but have been underexplored due to technical limitations of genetic testing methods. Various bioinformatic tools have been developed to identify MEIs in Next Generation Sequencing data. However, most tools have been developed specifically for genome sequencing (GS) data rather than exome sequencing (ES) data, which remains more widely used for routine diagnostic testing. In this study, we benchmarked six MEI detection tools (ERVcaller, MELT, Mobster, SCRAMble, TEMP2 and xTea) on ES data and on GS data from publicly available genomic samples (HG002, NA12878). For all the tools we evaluated sensitivity and precision of different filtering strategies. Results show that there were substantial differences in tool performance between ES and GS data. MELT performed best with ES data and its combination with SCRAMble increased substantially the detection rate of MEIs. By applying both tools to 10,890 ES samples from Solve-RD and 52,624 samples from Radboudumc we were able to diagnose 10 patients who had remained undiagnosed by conventional ES analysis until now. Our study shows that MELT and SCRAMble can be used reliably to identify clinically relevant MEIs in ES data. This may lead to an additional diagnosis for 1 in 3000 to 4000 patients in routine clinical ES.

Systematic analysis of paralogous regions in 41,755 exomes uncovers clinically relevant variation.

The short lengths of short-read sequencing reads challenge the analysis of paralogous genomic regions in exome and genome sequencing data. Most genetic variants within these homologous regions therefore remain unidentified in standard analyses. Here, we present a method (Chameleolyser) that accurately identifies single nucleotide variants and small insertions/deletions (SNVs/Indels), copy number variants and ectopic gene conversion events in duplicated genomic regions using whole-exome sequencing data. Application to a cohort of 41,755 exome samples yields 20,432 rare homozygous deletions and 2,529,791 rare SNVs/Indels, of which we show that 338,084 are due to gene conversion events. None of the SNVs/Indels are detectable using regular analysis techniques. Validation by high-fidelity long-read sequencing in 20 samples confirms >88% of called variants. Focusing on variation in known disease genes leads to a direct molecular diagnosis in 25 previously undiagnosed patients. Our method can readily be applied to existing exome data.

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors.

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility.

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for genetic factors underlying hereditary disease predisposition.

Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients.

Anophthalmia and microphthalmia (A/M) are among the most severe congenital developmental eye disorders. Despite the advancements in genome screening technologies, more than half of A/M patients do not receive a molecular diagnosis. We included seven consanguineous families affected with A/M from Pakistani cohort and an unknown molecular basis. Single gene testing of FOXE3 was performed, followed by genome sequencing for unsolved probands in order to establish a genetic diagnosis for these families. All seven families were provided with a genetic diagnosis. The identified variants were all homozygous, classified as (likely) pathogenic and present in an A/M-associated gene. Targeted FOXE3 sequencing revealed two previously reported pathogenic FOXE3 variants in four families. In the remaining families, genome sequencing revealed a known pathogenic PXDN variant, a novel 13bp deletion in VSX2, and one novel deep intronic splice variant in PXDN. An in vitro splice assay was performed for the PXDN splice variant which revealed a severe splicing defect. Our study confirmed the utility of genome sequencing as a diagnostic tool for A/M-affected individuals. Furthermore, the identification of a novel deep intronic pathogenic variant in PXDN highlights the role of non-coding variants in A/M-disorders and the value of genome sequencing for the identification of this type of variants.

Evolution of age-related mutation-driven clonal haematopoiesis over 20 years is associated with metabolic dysfunction in obesity.

BACKGROUND: Haematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF) increase with age and are linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones (VAF<2%) are also associated with adverse outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes in individuals with obesity treated by usual care or bariatric surgery (a treatment that improves metabolic status), and to examine the expansion of clones in relation to age and metabolic dysregulation over up to 20 years. METHODS: Clonal haematopoiesis-driver mutations (CHDMs) were identified in blood samples from participants of the Swedish Obese Subjects intervention study. Using an ultrasensitive assay, we analysed single-timepoint samples from 1050 individuals treated by usual care and 841 individuals who had undergone bariatric surgery, and multiple-timepoint samples taken over 20 years from a subset (n = 40) of the individuals treated by usual care. FINDINGS: In this explorative study, prevalence of CHDMs was similar in the single-timepoint usual care and bariatric surgery groups (20.6% and 22.5%, respectively, P = 0.330), with VAF ranging from 0.01% to 31.15%. Clone sizes increased with age in individuals with obesity, but not in those who underwent bariatric surgery. In the multiple-timepoint analysis, VAF increased by on average 7% (range -4% to 24%) per year and rate of clone growth was negatively associated with HDL-cholesterol (R = -0.68, 1.74 E-04). INTERPRETATION: Low HDL-C was associated with growth of haematopoietic clones in individuals with obesity treated by usual care. FUNDING: The Swedish Research Council, The Swedish state under an agreement between the Swedish government and the county councils, the ALF (Avtal om Läkarutbildning och Forskning) agreement, The Swedish Heart-Lung Foundation, The Novo Nordisk Foundation, The European Research Council, The Netherlands Organisation for Scientific Research.