Di Milnes

Objective: Prenatal exome sequencing (pES) is increasingly to standard care for diagnosing the etiology of fetal structural anomalies (FSA). We evaluated trio pES outcomes in the national multicentre, prospectively recruited Australian Government‐funded cohort, including diagnostic yield, frequency of variants of uncertain significance (VOUS), incidental findings and impacts on pregnancy management. Method: Prospective referral and analysis were undertaken for 275 consented families nation‐wide for trio pES. Eligibility was based on predefined FSA criteria and an uninformative chromosome microarray. Sequencing was performed in one of three clinically accredited national referral laboratories. Results: The pES diagnostic yield for pathogenic/likely pathogenic variants was 31.6% (87/275, 95% CI; 26.4–37.4). VOUS and incidental findings occurred at a rate of 4.4% (12/275) and 1.5% (4/275) respectively. Termination of pregnancy (TOP) occurred more frequently in diagnosed families, whereas live births were more common in the uninformative cohort. Conclusion: pES demonstrated a high diagnostic yield with low VOUS and incidental finding rates. Outcomes differed between diagnosed and uninformative families, with TOP more frequent after genomic diagnoses. These findings support pES as a core diagnostic test for antenatal FSA evaluation and emphasise the importance of consistent phenotype ontology, body system classification, and eligibility criteria for fetal genomic sequencing.

Background: Structural variants (SVs) that disrupt topologically associating domains (TADs) can cause disease by rewiring enhancer-promoter interactions. Duplications involving GPR101 are known to cause X-linked acrogigantism (X-LAG) by enabling aberrant expression of GPR101 through hijacking of enhancers at VGLL1 . However, not all GPR101 -containing duplications are pathogenic, presenting a diagnostic challenge, especially in the prenatal setting. Methods: We evaluated POSTRE, a tool designed to predict the regulatory impact of SVs, to distinguish pathogenic from benign GPR101 duplications. We analyzed six non-pathogenic duplications, and 27 known X-LAG associated pathogenic duplications. Tissue-specific enhancer maps built using H3K27ac ChIP-seq and ATAC-seq data as well as gene expression data derived from human anterior pituitary samples were integrated into POSTRE to enable predictions in a X-LAG relevant tissue context. Results: POSTRE correctly classified all 33 duplications as benign or pathogenic. In addition, one X-LAG case with mild clinical features (e.g., severe GH hypersecretion in the absence of pituitary tumorigenesis) was found to include only 2/5 VGLL1 enhancers (also predicted to be the weakest enhancers), whereas all 26 typical X-LAG cases had 24 enhancers duplicated. This suggests that milder enhancer hijacking at VGLL1 could explain the different clinical features of X-LAG in this individual. Conclusions: These findings support the utility of POSTRE to support diagnostic pipelines when interpreting SVs affecting chromatin architecture in pituitary disease. By accurately modelling enhancer adoption in a cell type-specific context, POSTRE could help to reduce uncertainty in genetic counselling and offers a rapid alternative to performing chromatin conformation capture experiments.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n=135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n=137; 80xCRCs, 33xECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Aim: Periodontal Ehlers‐Danlos syndrome (pEDS) is a monogenic type of Ehlers‐Danlos syndrome characterized by periodontal destruction at a young age. The present study aimed to document the oral phenotype of pEDS based on prospective clinical investigations.Materials and Methods: Thirty‐five adult individuals from 13 families with a clinically and genetically confirmed diagnosis of pEDS underwent a systematic oral assessment.Results: Periodontitis stage 3 or 4 or edentulism due to periodontal destruction were diagnosed in 94% of the individuals. First permanent tooth loss was reported at the age of 21.5 years (median; range 13–43 years). Deep periodontal pockets were infrequent, with 94% measuring <4 mm. However, there was increased clinical attachment loss (CAL) averaging 8 mm (range 4–13 mm), and the probability of being edentate between the age of 35 and 44 years was 28–47% compared with less than 0.25% of the general population. Radiographic anomalous findings were only found in a portion of subjects and consisted of fused roots of maxillary second molars (81%), root hypoplasia (57%), taurodontism (26%) and tooth rotation of premolars (67%). As such, radiographic findings are not considered common characteristics of pEDS.Conclusions: Characteristic oral traits of pEDS in adults are severe CAL with shallow probing depths and marked gingival recession. This is complemented by a lack of attached gingiva. These indications need to be paralleled by genetic analyses to diagnose pEDS unambiguously.

Missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques and joint hypermobility. We report extensive functional studies of sixteen C1R mutations associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). None of the pEDS mutants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Overexpression experiments showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense mutation disabling a C1q binding site, pEDS mutations had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression results are in line with western blot analyses of patient fibroblasts showing decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS appear to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.