Journal of Medical Genetics

ObjectiveReliable data on prevalence of differences of sex development (DSD) are lacking. We aimed to estimate population-based prevalence of DSD in Switzerland. DesignRetrospective population-based study including children and adolescents with DSD according to Chicago Consensus, born in Switzerland from 2000-2019. MethodsEndocrine care centers in ten Swiss Childrens Hospitals and eight private endocrine practices collected DSD data through the I-DSD registry or case report forms. We calculated prevalence for DSD diagnostic groups and analyzed trends in prevalence. ResultsOver the 20-year study period, we identified 561 individuals with DSD. Almost half (n=266, 47%) had sex chromosome DSD, 177 (32%) had 46,XY DSD and 118 (21%) had 46, XX DSD. Causes for 46,XY DSD were disturbed androgen synthesis or action (37/177, 21%), atypical gonadal development (28/177, 16%), or other causes (112/177, 63%). Causes for 46,XX DSD were androgen excess (99/118, 84%), atypical gonadal development (8/118, 7%), or other causes (11/118, 9%). On average, 28 new cases were born with DSD annually. Prevalence was 17 for sex chromosome DSD, 12 for 46,XY DSD and 8 for 46,XX DSD per 100000 live births and year. One per 7500 newborn girls had 46,XX congenital adrenal hyperplasia (CAH). ConclusionPrevalence of sex chromosome DSD was lower than expected because of underreporting due to late diagnosis. Prevalence of 46,XX CAH is similar to newborn screening data, suggesting good completeness of cases. For complex DSD cases, we expect complete coverage. This study provides a valuable resource for policymaking and (inter)national research on DSD.

Significance StatementThis study identifies microhomology-mediated end joining (MMEJ) as a novel mutational mechanism underlying a pathogenic deletion in the androgen receptor gene in a patient with complete androgen insensitivity syndrome. By mapping the genomic breakpoint at the nucleotide level, we demonstrate the presence of canonical features of MMEJ, thereby expanding the known mechanisms of genomic structural variation in AR. These findings underscore the importance of incorporating copy number variation (CNV) detection and breakpoint analysis into diagnostic workflows for 46,XY differences of sex development (DSD), enabling more accurate molecular classification and improved patient management. BackgroundCopy number variations in the androgen receptor gene are an underrecognized cause of androgen insensitivity syndrome. Understanding their mutational mechanisms can improve AIS diagnosis and genotype-phenotype correlation. ObjectiveTo investigate microhomology-mediated end joining (MMEJ) as a mutational mechanism underlying a structural variant in the AR gene and to assess the contribution of AR CNVs to AIS through comparative gene burden analysis. MethodsWhole-exome sequencing, Multiplex Ligation-dependent Probe Amplification, PCR, and Sanger sequencing were used to identify and refine a hemizygous deletion affecting exons 6-8 of the AR gene in a 46,XY individual with CAIS. Breakpoint mapping and local alignment were performed using R packages Biostrings and GenomicRanges. A literature and database review identified AR CNVs in AIS cases, which were compared to CNVs in the general population to assess AR-specific CNV burden. ResultsAn accurate genomic analysis revealed an 8-bp microhomology region flanking the genomic breakpoint of the CNV event found in this CAIS patient, consistent with microhomology-mediated end joining event. Among 991 AIS cases, 49 harbored AR CNVs, significantly enriched compared to controls (OR = 4.59, P = 9.2 x 10-{superscript 1}). Exon 2 was the most frequently affected region and most strongly associated with CAIS. ConclusionThis study provides the first molecular evidence that MMEJ can mediate pathogenic deletions in the AR gene. The significant enrichment of CNVs in AIS and their non-random distribution across functional domains support their role in disease pathogenesis and highlight the value of CNV-level analysis in the diagnostic evaluation of AIS.

IntroductionLynch syndrome (LS) is a hereditary cancer syndrome caused by (likely) pathogenic variants (LP/P) in DNA mismatch repair genes, including MSH6. It is associated with elevated lifetime risks for colorectal cancer (CRC), endometrial cancer (EC), and other malignancies. However, cancer risks specific to MSH6-associated LS, particularly for non-colorectal cancers, remain poorly defined. This study aims to provide refined cancer risk estimates for individuals with MSH6 LP/P. MethodsWe conducted a retrospective cohort study of 360 families with 1117 known MSH6 LP/P carriers identified in the Netherlands between 1995 and 2020. Pedigree data were collected from multiple clinical centers, and cancer diagnoses were confirmed through medical records. Age- and sex-specific hazard ratios (HRs) and cumulative risks (CRs) were estimated using segregation analysis, appropriately adjusted for ascertainment. ResultsCR by age 80 for MSH6 LP/P carriers were 36% in males (95% CI:25-48%) and 21% in females (95% CI 13-32%) for CRC, and 23% in females (95% CI:15-43%) for EC. Elevated risks were observed for ovarian cancer (OC) (6.4%, 95% CI:3-14.8%; HR 5.58, p=0.00037), urinary tract cancers (10.1% in males, 4.1% in females; HR 2.52, p=0.012), and biliary tract cancers (4.9% in males, 4.2% in females; HR 2.76, p=0.031). No increased risks were identified for prostate or breast cancer. ConclusionThis study refines cancer risk estimates for MSH6 LP/P carriers, suggesting the need for delayed CRC screening in males and females and proactive discussions regarding prophylactic surgery for females to address elevated risks for EC and OC.

ObjectiveTo identify rare and common CNVs associated with strabismus and amblyopia and to determine whether these variants reveal overlapping genetic mechanisms between the two disorders. DesignCase-control association study using structural variant calls from short-read whole- genome sequencing. Subject, participants and controls1,141 adults with strabismus, 566 with amblyopia (157 with both), and controls (95,806 for strabismus; 96,381 for amblyopia) enrolled in the All of Us Research Program and with available structural variant calls. MethodsCNVs were called using the GATK-SV pipeline from short-read whole genome sequencing (srWGS). After instituting a variety of quality control measures, including requiring two types of evidence and being identified by two different calling algorithms, CNVs present in 20 or more affected individuals were divided into rare (<1% population frequency) and common (>1% population frequency). The rates of each CNV were compared between affected individuals and controls. Significant CNVs were manually verified in IGV. Functional effects were annotated using Varient Effect Predictor from Ensembl. Main Outcome MeasuresOdds ratios for CNV carrier status in cases versus controls, adjusted for multiple testing (Benjamini-Hochberg FDR < 0.05); functional annotation, dosage sensitivity, regulatory element overlap, and pathway enrichment. ResultsFourteen rare and 29 common CNVs were significantly associated with strabismus; 1 rare and 2 common CNVs were associated with amblyopia (45 unique CNVs total). The rare CNV associated with amblyopia is an intronic deletion in MDGA2. Two common intronic deletions (GRIN2B; CACNA1B) were associated with both conditions and highly predictive of comorbid strabismus and amblyopia (47% comorbidity when both present, p < .001). Implicated genes predominantly affect synapse formation and function (e.g., CSMD1, GRIN2B, CACNA1B, RIMS1), neuronal migration (e.g., TUBB, EML1), and neurodevelopment; 64% have known neurodevelopmental phenotypes and 27% have been linked to mental health disorders. ConclusionsCNVs highlight synaptic and neurodevelopmental pathways as central to strabismus and amblyopia etiology and provide the first evidence of shared genetic risk. The combination of GRIN2B and CACNA1B deletions identify strabismus patients at high risk for amblyopia.

This study aimed to evaluate the outcome of germline clinical genetic testing in uveal melanoma (UM) patients who met the National Comprehensive Cancer Network (NCCN) guidelines for genetic testing. A retrospective chart review was conducted on UM patients seen in The Ohio State University Cancer Genetics Clinic between 5/1/2021-9/19/2025. Seventy individuals underwent clinical genetic testing, primarily via large multi-gene panels. Ten UM patients, including two related individuals, had pathogenic or likely pathogenic (P/LP) variants in known cancer genes (BAP1, BRCA1, BRCA2, MBD4, MUTYH, POT1, XRCC2). Among unrelated individuals, the positive rate was12.9% (8/70). Excluding carrier genes, the rate was10% (7/70) Eight patients would have been missed if only tested for BAP1 per ASCO 2024 recommendations. There was no association between tumor size, stage and germline P/LP variants. In summary, NCCN guidelines are useful in the prioritization of UM patients for genetic testing. Additionally, large panel testing, rather than BAP1 single gene testing, is recommended.

Background and aimsA substantial proportion of patients with adenomatous polyposis have no germline pathogenic variant in APC. The aim of this study was to determine the prevalence of APC mosaicism in these patients with unexplained polyposis and to draft guidelines for APC mosaicism testing and surveillance. MethodsAPC mosaicism was analyzed by targeted Next-Generation sequencing in 542 patients with a broad spectrum of polyposis phenotypes. ResultsThe rate of APC mosaicism was 9.4%. This rate was 14.3% (46/322) in patients who meet the scope of national hereditary polyposis testing guidelines ([&ge;]10 adenomas before the age of 60 or with [&ge;]20 adenomas before the age of 70). In patients who do not meet the scope of national guidelines, the detection rate was 2.3% (5/219). In patients with [&ge;]20 adenomas before the age of 60, or [&ge;]30 adenomas before the age of 70 the detection rate was [&ge;]10%. Of 34 mosaic patients who underwent an esophagogastroduodenoscopy, 26% were diagnosed with gastroduodenal polyps. In one patient, the mosaic variant was detected in semen, but none of the children tested in this cohort inherited the mosaic variant. ConclusionWe recommend APC mosaicism testing at least in patients negative for germline pathogenic variants with (1) [&ge;]20 adenomas before the age of 60 or (2) [&ge;]30 adenomas before the age of 70. Regular colonoscopy and at least one gastroduodenoscopy should be offered to APC mosaics, with frequency of follow-up based on findings. Offering germline testing for offspring should be considered.

Four members of a three-generation family with early-onset chorioretinal dystrophy were shown to be heterozygous carriers of the n.37C>T in MIR204. The identification of this previously reported pathogenic variant confirms the existence of a distinct clinical entity caused by a sequence change in MIR204. The chorioretinal dystrophy was variably associated with iris coloboma, congenital glaucoma, and premature cataracts extending the phenotypic range of the condition. In silico analysis of the n.37C>T variant revealed 713 novel targets. Additionally, family members were shown to be affected by albinism resulting from biallelic pathogenic OCA2 variants.

APC c.3920T>A; p.Ile1307Lys (I1307K), prevalent in individuals of Ashkenazi Jewish (AJ) origin, has been associated with a modestly increased colorectal cancer (CRC) risk. Clinical recommendations for I1307K heterozygotes vary across countries and expert groups, reflecting differences in population frequencies, modest risk estimates, and limited data in non-AJ individuals. We analyzed UK Biobank data comprising 466,315 individuals (8,727 with CRC), using genomic analysis to classify AJ and non-AJ ancestries. I1307K was identified in 7.1% of AJ and 0.08% of non-AJ white participants. No significant association with CRC was observed in AJ (OR: 0.71; 95%CI: 0.17-2.95) or non-AJ white individuals (OR: 1.05; 95%CI: 0.50- 2.22). The previously established OR of 1.7-1.8 for AJ individuals lies within our 95% CI, indicating underpowered results due to limited CRC cases. No significant associations were detected for other cancers. Unbiased, adequately powered CRC case-control studies in non-AJ populations would require cohorts far larger than current resources for feasible analysis. Clinical actionability of I1307K should prioritize risk stratification based on overall CRC risk and ancestry-dependent variant detection rates. However, management strategies need not differ by ancestry once a carrier is identified, as the biological impact of I1307K should be consistent across populations.

Early-onset colorectal cancer (EOCRC) incidence is increasing rapidly worldwide. However, the majority of EOCRCs are not substantiated by germline variants in the main colorectal cancer (CRC) predisposition genes (the "DIGE" panel). To investigate a potential genetic transmission of EOCRC (dominant, recessive and oligogenic hypotheses) and thus identify potentially novel EOCRC-specific predisposition genes, we conducted an analysis of 585 cancer pathway genes on an EOCRC patient cohort (n=87 patients diagnosed at [&le;] 40 years of age, DIGE-) with or without a CRC family history. By comparing this germline variant spectrum to the GnomAD cancer-free database, we identified high impact variants (HVs) in 15 genes significantly over-represented in the EOCRC cohort. Among the 32 unrelated patients with a CRC family history (i.e. with a potentially dominant transmission pattern), nine presented HVs in ten of the genes tested, four of these genes had a DNA repair function. A potentially recessive transmission of EOCRC in patients without a CRC family history cannot be supported by our results nor can an oligogenic transmission. We subsequently sequenced these 15 genes in a cohort of 82 late-onset CRCs (cancer diagnosis [&ge;]50 years, DIGE-) and found variants in 11 of these genes to be specific to EOCRC. To evaluate whether variants in these 11 genes would allow to specifically detect EOCRC patients, we screened our patient database (n=6482), which only contained 2% of EOCRCs (DIGE-), and identified two other EOCRC cases diagnosed after the constitution of our cohort, with individual HVs in RECQL4 and NUTM1. Altogether, we showed that 37.5% and 18.75% of heterozygous NUTM1 and RECQL4 HVs of our database were diagnosed with EOCRC. Our work has identified a pattern of germline gene variants not previously associated with EOCRC. This paves the way to addressing the contribution of these variants to EOCRC risk and oncogenesis. Author SummaryEarly-onset colorectal cancer (diagnosed at [&le;] 40 years of age) is a rare disease that can in part be explained by a hereditary genetic predisposition. To identify novel gene variants potentially associated with EOCRC risk, we analysed a panel of 585 genes in 87 patients with early-onset colorectal cancer unexplained by conventional genetic tests. This first analysis highlighted 15 genes of interest. To evaluate if this genetic profile is specific to early onset, we sequenced these 15 genes in a population of late-onset colorectal cancers (diagnosed after 50 years of age). Variants in 11 of these genes were specific to the early-onset population. To assess if this genetic pattern allows to identify other early-onset cases, we screened these genes in our whole database of 6482 patients and identified two new early-onset cases. Our results need to be confirmed, and validated in larger cohorts but pave the way for future research into early-onset colorectal cancer and the possibility of improving screening or treatment options for these patients and their family members.

Retinoblastoma (Rb) is a rare childhood eye cancer. Almost half of cases are heritable, associated with germline RB1 pathogenic variants that pre-dispose to Rb and extraocular cancers. This study aimed to investigate the prevalence and penetrance of RB1-heritable Rb in two adult population cohorts. We screened participants with whole genome sequencing in the UK Biobank (UKB) (n=490,413) and All of Us (AoU) (n=317,964) cohorts for predicted loss-of-function (pLoF) and/or ClinVar pathogenic/likely pathogenic RB1 variants. Electronic health records and questionnaires were used to screen participants for Rb-associated features. In the UKB we generated a stringent and permissive phenotype category, ranging from Rb to Rb-associated extraocular cancers; in AoU we included participants with Rb or ocular cancer. A total of 22 pathogenic pLoF RB1 variants were detected in the UKB (n=12) and AoU (n=13) participants. In the UKB, only 25.0% (3/12) of variant carriers reported developing Rb by the age of 60, increasing to 50.0% when including ocular/extraocular cancers. Similarly, 30.8% (4/13) AoU participants developed Rb and/or ocular cancer by the age of 60. Overall, this results in a combined penetrance estimate of 28.0% (7/25). We found 21 and 28 individuals with Rb in the UKB and AoU, respectively, which is within published prevalence estimates, suggesting these cohorts are not depleted of Rb cases. Notably, the penetrance of pathogenic RB1 variants in >800,000 clinically unselected adults was substantially lower than the near complete penetrance reported in clinical cohorts. This has important implications for counselling families following a positive newborn screening result.

We sequenced nine trios in which the probands in an underserved population were affected by a rare and undiagnosed disorder with neurological features. Sequencing was performed with one trio per flowcell on a benchtop sequencing instrument, leveraging the design of sequencing the proband at twice the coverage of the parents. The reduced coverage in the parents led to sequencing efficiencies while retaining the benefits of trio sequencing: the ability to discover de novo variants and the ability to trace inheritance patterns of rare variants. Once the sequencing data was generated, our two teams used independent informatics pipelines for variant calling and interpretation. In five of the nine cases, both teams found a single SNV or small indel that was deemed causal pending clinical validation. In three of the nine cases, neither team had a significant finding. In the final case, an additional scan for large CNVs performed by one of the teams identified a de novo deletion and duplication in the proband which is the likely cause of the underlying disease. The results across cases with significant findings showed a variety of affected genes (CFAP52, DYNC1H1, FANCE, TCF4, and TOP3A), variant types, and inheritance patterns. All findings were clinically validated, after which the families were counseled about disease management, current research studies (i.e. gene therapy), and family planning. With six of nine families receiving findings, the study demonstrated an efficient and effective trio sequencing design strategy.

BackgroundPrevious studies identified a strong linkage signal for non-syndromic persistent developmental stuttering on chromosome 3q13.2-3q13.33 in a large consanguineous family. To identify the causative genetic variant at this locus, including we performed further analysis, including whole exome, whole genome, and targeting Sanger sequencing. ResultsWe identified a homozygous rare c.2155G>A variant in ZBTB20 in individuals who stutter. This mutation encodes Isoleucine in place of a highly conserved Valine at amino acid position 719 in ZBTB20 that co-segregates (LOD = 4.23) with stuttering under a model of recessive inheritance with reduced penetrance in this family. Coding variants in this gene were significantly more frequent in a multiethnic cohort of unrelated individuals who stutter than in individuals in large population databases comprised of our normal control subjects and gnomAD database subjects matched for ethnicity. ZBTB20 encodes a zinc-finger transcription factor, and luciferase reporter constructs using genes known to be regulated by ZBTB20 showed that the Ile719 mutant form of the protein displays altered transcriptional regulation in vitro. Although homozygosity for mutations in ZBTB20 has not previously been observed, dominant mutations in ZBTB20 have been reported in Primrose syndrome, a rare Mendelian dominant disorder characterized by tall stature, developmental delay, dysgenesis of the corpus callosum, but generally not speech disorders. Clinical re-examination of the affected family members ten years after their original ascertainment revealed only some suggestive signs associated with Primrose syndrome. ConclusionsOur findings support variants in ZBTB20 as a cause of stuttering in a large family, and rarely in the wider population. Previous studies in mice have demonstrated that Zbtb20 is required for the development of astrocytes, a brain cell type previously implicated in an animal model of stuttering. Our findings support our hypothesis that astrocyte pathology is involved in this disorder. Our findings also broaden the medical genetic view of disorders of ZBTB20, and demonstrate that individuals carrying a homozygous mutation in this gene can be viable, and that they can primarily display persistent developmental stuttering.

To date, a disease-causing mutation can be found in 15-30% of families with hereditary breast and ovarian cancer (HBOC) and it is believed that more than half of the cases still remain unsolved. Usually it is intended to perform genetic analyses in the family member with the most severe phenotype, which, however, may not always be possible. Moreover, no standard criteria have been established to define the person who is most suitable for genetic testing within a family: the best index case. We therefore established clinical criteria to identify the best index case in HBOC and analyzed the impact on genetic testing. 130 patients who presented at our department from 2016 to 2018 were divided into two groups. In group A (N = 98) genetic analyses were performed in the best index case based on our criteria. In group B (N = 32) at least one other family member was considered a better index case. The detection rate of expected mutations was significantly higher for group A (64.3% vs. 32.0%, p = 0.034) while there was no significant difference of calculated mutation carrier risks between these groups. We conclude that the mutation detection rate in families with HBOC is notably higher after identifying the best index case for genetic testing according to the clinical selection criteria reported here.

PMS2, a Lynch Syndrome gene, presents challenges in genetic testing due to the existence of multiple pseudogenes. This study aims to describe a series of cases harboring a rare LoF variant in the PMS2CL pseudogene that has been incorrectly assigned to PMS2 with different nomenclatures. We reviewed data from 647 Brazilian patients who underwent multigene genetic testing at a single center to identify those harboring the PMS2 V1:c.2186_2187delTC or V2:c.2182_2184delACTinsG variants, allegedly located at PMS2 exon 13. Gene-specific PCR and transcript sequencing was performed. Among the 647 individuals, 1.8% (12) carried the investigated variants, with variant allele frequencies ranging from 15 to 34%. By visually inspecting the alignments, we confirmed that both V1 and V2 represented the same variant and through gene-specific PCR and PMS2 transcript analysis, we demonstrated that V1/V2 is actually located in the PMS2CL pseudogene. Genomic databases (ExAC and gnomAD) report an incidence of 2.5% - 5.3% of this variant in the African population. Currently, V1 is classified as "uncertain significance" and V2 as "conflicting" in ClinVar, with several laboratories classifying them as "pathogenic". We identified a frequent African PMS2CL LoF variant in the Brazilian population that is misclassified as a PMS2 variant. It is likely that V1/V2 have been erroneously assigned to PMS2 in several manuscripts and by clinical laboratories, underscoring a disparity-induced matter. Considering the limitations of short-read NGS differentiating between certain regions of PMS2 and PMS2CL, using complementary methodologies is imperative to provide an accurate diagnosis.

ImportanceRET pathogenic variants cause Multiple Endocrine Neoplasia type 2 (MEN2), characterised by medullary thyroid cancer (MTC). With increasing incidental identification of these variants in asymptomatic individuals outside family screening, their risk of MTC and all-cause mortality without intervention remain unknown in this context. ObjectiveTo determine the risk of MTC and all-cause mortality in clinically unselected individuals and assess how the risk of MTC differ from clinically ascertained cases. Design, Setting, and ParticipantsProspective cohort study of 383,914 unrelated individuals from the clinically unselected UK population (UK Biobank) and 122,640 from the US health system (Geisinger cohort). We compared MTC risk in these cohorts to 1,078 individuals who were clinically ascertained with suspicion of MEN2 from UK routine practice. ExposuresRET pathogenic variants causing MEN2 Main Outcomes and MeasuresFrequency and the spectrum of pathogenic RET variants, Risk of clinically presented MTC, all-cause mortality without thyroidectomy. ResultsPathogenic RET variants were found in 0.04% of individuals from UK population cohort and 0.08% of individuals from US health system cohort. They were predominantly from moderate-risk category as per American Thyroid Association guideline (99.4% and 94.8% respectively). MTC risk by age 75 in variant carriers in the UK population was 2.2% (95% CI 0.7-6.8) and 19% (95% CI 5.7-30) in US health system cohort. This was significantly lower than the clinically ascertained cohort with the matched variants (95.7%, 95% CI 82.1-99.7 p<0.0001). In the UK Biobank, most variant carriers (98.2%) did not undergo thyroidectomy and their all-cause mortality by age 75 was similar to non-carriers (6.1%, 95% CI 2.7-13.8 vs 5.7%, 5.6-5.8, p=0.79), with consistent findings in the US health system cohort. Conclusions and RelevanceModerate-risk RET variants are most common in incidental cases. These variants carry substantially lower MTC risk than clinically ascertained cases. This evidence addresses a current knowledge gap, enabling more informed clinical decision-making.

Although rare genetic conditions are mostly caused by DNA sequence alterations that functionally disrupt individual genes, large-scale studies using genome sequencing have started to unmask additional complexity. Understanding how combinations of variants in different genes shape human phenotypes is expected to provide important insights into the clinical and genetic heterogeneity of rare disorders. We used albinism, an archetypal rare condition associated with hypopigmentation, as an exemplar for the study of genetic interactions. We analysed data from the Genomics England 100,000 Genomes Project alongside a cohort of 1,120 individuals with albinism, and investigated the effect of dual heterozygosity for the combination of two established albinism-related variants: TYR:c.1205G>A (p.Arg402Gln) [rs1126809] and OCA2:c.1327G>A (p.Val443Ile) [rs74653330]. As each of these changes alone is insufficient to cause disease when present in the heterozygous state, we sought evidence of synergistic effects. We found that, when both variants are present, the probability of receiving a diagnosis of albinism is significantly increased (odds ratio 12.8; 95% confidence interval 6.0 - 24.7; p-value 2.1 x 10-8). Further analyses in an independent cohort, the UK Biobank, supported this finding and highlighted that heterozygosity for the TYR:c.1205G>A and OCA2:c.1327G>A variant combination is associated with statistically significant alterations in visual acuity and central retinal thickness (traits that are considered albinism endophenotypes). The approach discussed in this report opens up new avenues for the investigation of oligogenic patterns in apparently Mendelian disorders.

PurposeConventional polygenic scores predict an individuals phenotype based on their genetics. By contrast, variance polygenic scores (vPGS) quantify genetic predisposition to phenotypic variance. We tested the hypothesis that a vPGS for refractive error can identify individuals with increased susceptibility to environmental risk factors for myopia. MethodsSix vPGS construction strategies were evaluated in UK Biobank participants: three variance heterogeneity genome-wide association studies (vGWAS) methods and two reweighting schemes. vPGS performance was assessed using two metrics: (i) Diff - difference in phenotypic variance in vPGS decile one vs. ten; (ii) Spearman correlation of phenotypic variance vs. vPGS decile. The optimal vPGS was used to test for vPGS x time spent reading or vPGS x time spent outdoors interactions in children aged 15 years (ALSPAC cohort; n=3471). ResultsOf the vGWAS methods, conditional quantile regression outperformed SCAMPI and Levenes Test. Of the re-weighting schemes, LDpred2 outperformed pruning and thresholding (P+T). In an independent sample of UK Biobank participants (n=19470), the top-performing vPGS successfully stratified individuals into groups with increasing variance in refractive error, even after adjusting for a conventional PGS (Diff: 2.55, 95% CI: 1.64-3.47; Spearman correlation: 0.87, 95% CI: 0.43-0.93). However, in ALSPAC participants, there was minimal support for vPGS interactions with time reading (P=0.80) or time outdoors (P=0.89). ConclusionA novel vPGS successfully stratified individuals into groups with relatively high or low genetic susceptibility to refractive error variance. However, the vPGS could not identify individuals at enhanced risk from lifestyle risk factors for myopia.

Common genetic variation throughout the genome together with rare coding variants identified to date explain about a half of the inherited genetic component of epithelial ovarian cancer risk. It is likely that rare variation in the non-coding genome will explain some of the unexplained heritability, but identifying such variants is challenging. The primary problem is lack of statistical power to identifying individual risk variants by association as power is a function of sample size, effect size and allele frequency. Power can be increased by using burden tests which test for association of carriers of any variant in a specified genomic region. This has the effect of increasing the putative effect allele frequency. PAX8 is a transcription factor that plays a critical role in tumour progression, migration and invasion. Furthermore, regulatory elements proximal to target genes of PAX8 are enriched for common ovarian cancer risk variants. We hypothesised that rare variation in PAX8 binding sites are also associated with ovarian cancer risk, but unlikely to be associated with risk of breast, colorectal or endometrial cancer. We have used publicly-available, whole-genome sequencing data from the UK 100,000 Genomes Project to evaluate the burden of rare variation in PAX8 binding sites across the genome. Data were available for 522 ovarian cancers, 2560 breast cancers, 2465 colorectal cancers and 729 endometrial cancers and 2253 non-cancer controls. Active binding sites were defined using data from multiple PAX8 and H3K27 ChIPseq experiments. We found no association between the burden of rare variation in PAX8 binding sites (defined in several ways) and risk of ovarian, breast or endometrial cancer. An apparent association with colorectal cancer was likely to be a technical artefact as a similar association was also detected for rare variation in random regions of the genome. Despite the null result this study provides a proof-of -principle for using burden testing to identify rare, non-coding germline genetic variation associated with disease. Larger sample sizes available from large-scale sequencing projects together with improved understanding of the function of the non-coding genome will increase the potential of similar studies in the future.

ObjectivesMitochondrial DNA (mtDNA) mutations account for up to 5% of hereditary hearing loss cases. Most commonly, the m.3243A>G mtDNA variant contributes to rare monogenic MIDD (Maternally Inherited Diabetes and Deafness) or MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes) syndromes. Different proportions of the mutated mtDNA (heteroplasmy) among the affected tissues result in variability in the clinical manifestation and severity of the phenotype. The aim of the presented study was to establish the prevalence of the m.3243A>G variant in large cohorts of hearing-impaired and diabetic patients in Slovakia and to evaluate the genotype-phenotype correlations and long-term cochlear implantation outcomes. DesignProbands (n=5957) were recruited via three independent nationwide studies on hereditary hearing loss (n=1145) and diabetes (unselected diabetes group, n=4158 and Monogenic diabetes group, n=654; total n=4812). DNA from peripheral blood and/or buccal mucosa was tested for the presence of the m.3243A>G variant using two PCR methods - qPCR and dPCR. Audiological and other clinical data of the identified variant carriers were also collected for phenotype evaluation. ResultsWe identified 25 probands/families harboring the m.3243A>G variant (0.42%). The prevalence was higher in the groups where monogenic disorder was suspected - 0.79% in the Hearing loss group and 1.68% in the Monogenic diabetes group versus 0.14% in the general diabetes group (p < 0.001). Heteroplasmy levels assessed by dPCR ranged between 0.04% and 76% in peripheral blood and 0.01% and 92% in buccal samples. In most individuals, the symptoms manifested in the fourth decade of life in affected subjects with the MIDD phenotype or isolated hearing loss/diabetes, but as early as in the second decade in the probands with MELAS. We observed high phenotype variability, ranging from severe multisystemic involvement through isolated symptoms to asymptomatic young "dormant" or very low heteroplasmy carriers. Only 54% of individuals with the m.3243A>G variant had both diabetes and hearing loss. The heteroplasmy levels from buccal swabs showed a better correlation with the age of onset of both hearing loss and diabetes than the age-adjusted blood heteroplasmy. On the other hand, the age-adjusted blood heteroplasmy was associated with overall severity of the disease (i.e., with a higher number of clinical symptoms). We show that the most typical audiogram configurations are flat and sloping. Three individuals identified as cochlear implant recipients showed excellent and long-term stable functional outcomes. In addition, the authors report the first case of successful stapes surgery in a patient with confirmed mitochondrial disorder. ConclusionsThe diagnostic yield was higher in the deafness and monogenic diabetes groups than in the unselected diabetes group. Implementation of rigorous inclusion criteria requiring the presence of both diabetes and hearing loss may lead to a lower detection rate due to different or incomplete phenotype manifestation. Age-adjusted blood heteroplasmy levels seem to be a good predictor of overall severity of m.3243A>G-associated diseases, but buccal mucosa heteroplasmy better predicted the age of hearing loss and diabetes onset. We further confirm that cochlear implantation and stapedectomy are safe and efficient options for hearing restoration and rehabilitation in m.3243A>G carriers.

ImportanceAnophthalmia and microphthalmia (A/M) are rare congenital eye disorders with a low molecular diagnosis rate, which limits clinical management and genetic counselling. Improved detection and interpretation of pathogenic variants is essential for advancing diagnosis and care in affected individuals. ObjectiveTo improve the molecular diagnostic yield in A/M patients by refining the methodology of variant investigation and association using an updated rigorously curated gene panel, and a refined bioinformatic pipeline incorporating structural variant detection, in silico Artificial Intelligence assisted predictive tools, and molecular dynamics simulations. MethodologyWe curated an updated A/M gene panel through a systematic literature review and screened for rare variants in these genes using data from the UKs 100,000 Genomes Project, a national whole-genome sequencing initiative conducted by Genomics England. The cohort comprised 306 individuals recruited to the Rare Disease programme with a clinical diagnosis of anophthalmia or microphthalmia, recorded either as the primary phenotype or within HPO, SNOMED, or ICD-10 terms. Variants, including loss-of-function, missense, RNA splicing, and structural variants, were annotated with deep learning tools (AlphaMissense, SpliceAI), and missense variants were further assessed using REVEL, Missense3D, and molecular dynamics simulations. ResultsWe identified pathogenic or likely pathogenic variants in 37 (12.1%) individuals, with an additional 23 (7.5%) harbouring strong candidate variants of uncertain significance. Our literature review identified the biggest contributors to A/M phenotypes to be MFRP, OTX2, PRSS56 and SOX2, each with over 100 patients reported in the literature, with a total number of 124 genes found be associated to A/M. Variants from our screen were most often found in genes with high A/M association, but also included novel findings within genes with a weaker association to A/M such as ACTG1, HDAC6, RERE and SIX3; adding support to their disease relevance. Conclusions and RelevanceThis study increased the diagnostic yield in A/M patients recruited to the 100KGP, and provides further evidence of genotype-phenotype associations within the aetiology of A/M. We also provide an updated framework for enhancing clinical genetic diagnosis in A/M that may inform broader strategies for other complex congenital disorders. However, as molecular diagnosis of A/M remains low, further research in understanding the genetic aetiology of A/M is necessary.