Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency.

Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.

A Human Homozygous HELQ Missense Variant Does Not Cause Premature Ovarian Insufficiency in a Mouse Model.

Disruption of meiosis and DNA repair genes is associated with female fertility disorders like premature ovarian insufficiency (POI). In this study, we identified a homozygous missense variant in the HELQ gene (c.596 A>C; p.Gln199Pro) through whole exome sequencing in a POI patient, a condition associated with disrupted ovarian function and female infertility. HELQ, an enzyme involved in DNA repair, plays a crucial role in repairing DNA cross-links and has been linked to germ cell maintenance, fertility, and tumour suppression in mice. To explore the potential association of the HELQ variant with POI, we used CRISPR/Cas9 to create a knock-in mouse model harbouring the equivalent of the human HELQ variant identified in the POI patient. Surprisingly, Helq knock-in mice showed no discernible phenotype, with fertility levels, histological features, and follicle development similar to wild-type mice. Despite the lack of observable effects in mice, the potential role of HELQ in human fertility, especially in the context of POI, should not be dismissed. Larger studies encompassing diverse ethnic populations and alternative functional approaches will be necessary to further examine the role of HELQ in POI. Our results underscore the potential uncertainties associated with genomic variants and the limitations of in vivo animal modelling.

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis.

RESEARCH QUESTION: Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1-3.7% women. Genetic factors explain 20-30% of POI cases, but most causes remain unknown despite genomic advancements. DESIGN: We used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity. RESULTS: Despite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea - the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO. CONCLUSIONS: This study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.

Operative vs Nonoperative Management of Unstable Medial Malleolus Fractures: A Randomized Clinical Trial.

Importance: Unstable ankle fractures are routinely managed operatively. However, because of soft tissue and implant-related complications, recent literature has reported on the nonoperative management of well-reduced medial malleolus fractures after fibular stabilization, but with limited evidence supporting the routine application. Objective: To assess the superiority of internal fixation of well-reduced (displacement ≤2 mm) medial malleolus fractures compared with nonfixation after fibular stabilization. Design, Setting, and Participants: This superiority, pragmatic, parallel, prospective randomized clinical trial was conducted from October 1, 2017, to August 31, 2021. A total of 154 adult participants (≥16 years) with a closed, unstable bimalleolar or trimalleolar ankle fracture requiring surgery at an academic major trauma center in the UK were assessed. Exclusion criteria included injuries with no medial-sided fracture, open fractures, neurovascular injury, and the inability to comply with follow-up. Data analysis was performed in July 2022 and confirmed in September 2023. Interventions: Once the lateral (and where appropriate, posterior) malleolus had been fixed and satisfactory intraoperative reduction of the medial malleolus fracture was confirmed by the operating surgeon, participants were randomly allocated to fixation (n = 78) or nonfixation (n = 76) of the medial malleolus. Main Outcome and Measure: Olerud-Molander Ankle Score (OMAS) 1 year after randomization (range, 0-100 points, with 0 indicating worst possible outcome and 100 indicating best possible outcome). Results: Among 154 randomized participants (mean [SD] age, 56.5 [16.7] years; 119 [77%] female), 144 (94%) completed the trial. At 1 year, the median OMAS was 80.0 (IQR, 60.0-90.0) in the fixation group compared with 72.5 (IQR, 55.0-90.0) in the nonfixation group (P = .17). Complication rates were comparable. Significantly more patients in the nonfixation group developed a radiographic nonunion (20% vs 0%; P < .001), with 8 of 13 clinically asymptomatic; 1 patient required surgical reintervention for this. Fracture type and reduction quality appeared to influence fracture union and patient outcome. Conclusions and Relevance: In this randomized clinical trial comparing internal fixation of well-reduced medial malleolus fractures with nonfixation, after fibular stabilization, fixation was not superior according to the primary outcome. However, 1 in 5 patients developed a radiographic nonunion after nonfixation, and although the reintervention rate to manage this was low, the future implications are unknown. These results support selective nonfixation of anatomically reduced medial malleolar fractures after fibular stabilization. Trial Registration: ClinicalTrials.gov Identifier: NCT03362229.

Ultrasound assessment of humeral shaft nonunion risk: a feasibility and proof of concept study.

PURPOSE: To determine the feasibility and reliability of ultrasound in the assessment of humeral shaft fracture healing and estimate the accuracy of 6wk ultrasound in predicting nonunion. METHODS: Twelve adults with a non-operatively managed humeral shaft fracture were prospectively recruited and underwent ultrasound scanning at 6wks and 12wks post-injury. Seven blinded observers evaluated sonographic callus appearance to determine intra- and inter-observer reliability. Nonunion prediction accuracy was estimated by comparing images for patients that united (n = 10/12) with those that developed a nonunion (n = 2/12). RESULTS: The mean scan duration was 8 min (5-12) and all patients tolerated the procedure. At 6wks and 12wks, sonographic callus (SC) was present in 11 patients (10 united, one nonunion) and sonographic bridging callus (SBC) in seven (all united). Ultrasound had substantial intra- (weighted kappa: 6wk 0.75; 12wk 0.75) and inter-observer reliability (intraclass correlation coefficient: 6wk 0.60; 12wk 0.76). At 6wks, the absence of SC demonstrated sensitivity 50%, specificity 100%, positive predictive value (PPV) 100% and negative predictive value (NPV) 91% in nonunion prediction (overall accuracy 92%). The absence of SBC demonstrated sensitivity 100%, specificity 70%, PPV 40% and NPV 100% in nonunion prediction (overall accuracy 75%). Of three patients at risk of nonunion (Radiographic Union Score for HUmeral fractures < 8), one had SBC on 6wk ultrasound (that subsequently united) and the others had non-bridging/absent SC (both developed nonunion). CONCLUSIONS: Ultrasound assessment of humeral shaft fracture healing was feasible, reliable and may predict nonunion. Ultrasound could be useful in defining nonunion risk among patients with reduced radiographic callus formation.

Fifteen-year prospective longitudinal cohort study of outcomes following single radius total knee arthroplasty.

Aims: This prospective study reports longitudinal, within-patient, patient-reported outcome measures (PROMs) over a 15-year period following cemented single radius total knee arthroplasty (TKA). Secondary aims included reporting PROMs trajectory, 15-year implant survival, and patient attrition from follow-up. Methods: From 2006 to 2007, 462 consecutive cemented cruciate-retaining Triathlon TKAs were implanted in 426 patients (mean age 69 years (21 to 89); 290 (62.7%) female). PROMs (12-item Short Form Survey (SF-12), Oxford Knee Score (OKS), and satisfaction) were assessed preoperatively and at one, five, ten, and 15 years. Kaplan-Meier survival and univariate analysis were performed. Results: At 15 years, 28 patients were lost to follow-up (6.1%) and 221 patients (51.9%) had died, with the mean age of the remaining cohort reducing by four years. PROMs response rates among surviving patients were: one-year 63%; five-year 72%; ten-year 94%; and 15-year 59%. OKS and SF-12 scores changed significantly over 15 years (p < 0.001). The mean improvement in OKS was 18.8 (95% confidence (CI) 16.7 to 19.0) at one year. OKS peaked at five years (median 43 years) declining thereafter (p < 0.001), though at 15 years it remained 17.5 better than preoperatively. Age and sex did not alter this trajectory. A quarter of patients experienced a clinically significant decline (≥ 7) in OKS from five to ten years and from ten to 15 years. The SF-12 physical component score displayed a similar trajectory, peaking at one year (p < 0.001). Patient satisfaction was 88% at one, five, and ten years, and 94% at 15 years. In all, 15-year Kaplan-Meier survival was 97.6% (95% CI 96.0% to 99.2%) for any revision, and 98.9% (95% CI 97.9% to 99.9%) for aseptic revision. Conclusion: Improvements in PROMs were significant and maintained following single radius TKA, with OKS peaking at five years, and generic physical health peaking at one year. Patient satisfaction remained high at 15 years, at which point 2.4% had been revised.

QuickDASH and PRWE Are Not Optimal Patient-Reported Outcome Measures After Distal Radial Fracture Due to Ceiling Effect: Potential Implications for Future Research.

BACKGROUND: The aim of this study was to determine the floor and ceiling effects for both the QuickDASH (shortened version of the Disabilities of the Arm, Shoulder and Hand [DASH] questionnaire) and the PRWE (Patient-Rated Wrist Evaluation) following a distal radial fracture (DRF). Secondary aims were to determine the degree to which patients with a floor or ceiling effect felt that their wrist was "normal" according to the Normal Wrist Score (NWS) and if there were patient factors associated with achieving a floor or ceiling effect. METHODS: A retrospective cohort study of patients in whom a DRF was managed at the study center during a single year was undertaken. Outcome measures included the QuickDASH, PRWE, EuroQol-5 Dimensions-3 Levels (EQ-5D-3L), and NWS. RESULTS: There were 526 patients with a mean age of 65 years (range, 20 to 95 years), and 421 (80%) were female. Most patients were managed nonsurgically (73%, n = 385). The mean follow-up was 4.8 years (range, 4.3 to 5.5 years). A ceiling effect was observed for both the QuickDASH (22.3% of patients with the best possible score) and the PRWE (28.5%). When defined as a score that differed from the best available score by less than the minimum clinically important difference (MCID) for the scoring system, the ceiling effect increased to 62.8% for the QuickDASH and 60% for the PRWE. Patients who had a ceiling score on the QuickDASH and the PWRE had a median NWS of 96 and 98, respectively, and those who had a score within 1 MCID of the ceiling score reported a median NWS of 91 and 92, respectively. On logistic regression analysis, a dominant-hand injury and better health-related quality of life were the factors associated with both QuickDASH and PRWE ceiling scores (all p < 0.05). CONCLUSIONS: The QuickDASH and PRWE demonstrate ceiling effects when used to assess the outcome of DRF management. Some patients achieving ceiling scores did not consider their wrist to be "normal." Future research on patient-reported outcome assessment tools for DRFs should aim to limit the ceiling effect, especially for individuals or groups that are more likely to achieve a ceiling score. LEVEL OF EVIDENCE: Prognostic Level III . See Instructions for Authors for a complete description of levels of evidence.

Integrated multi-omics for rapid rare disease diagnosis on a national scale.

Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner.

Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

Modeling human skeletal development using human pluripotent stem cells.

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

The Radiographic Union Score for ulnar fractures (RUSU) predicts ulnar shaft nonunion.

AIMS: To develop a reliable and effective radiological score to assess the healing of isolated ulnar shaft fractures (IUSF), the Radiographic Union Score for Ulna fractures (RUSU). METHODS: Initially, 20 patients with radiographs six weeks following a non-operatively managed ulnar shaft fracture were selected and scored by three blinded observers. After intraclass correlation (ICC) analysis, a second group of 54 patients with radiographs six weeks after injury (18 who developed a nonunion and 36 who united) were scored by the same observers. RESULTS: In the initial study, interobserver and intraobserver ICC were 0.89 and 0.93, respectively. In the validation study, the interobserver ICC was 0.85. The median score for patients who united was significantly higher than those who developed a nonunion (11 vs. 7, p < 0.001). A ROC curve demonstrated that a RUSU ≤ 8 had a sensitivity of 88.9% and specificity of 86.1% in identifying patients at risk of nonunion. Patients with a RUSU ≤ 8 (n = 21) were more likely to develop a nonunion (n = 16/21) than those with a RUSU ≥ 9 (n = 2/33; OR 49.6, 95% CI 8.6-284.7). Based on a PPV of 76%, if all patients with a RUSU ≤ 8 underwent fixation at 6 weeks, the number of procedures needed to avoid one nonunion would be 1.3. CONCLUSION: The RUSU shows good interobserver and intraobserver reliability and is effective in identifying patients at risk of nonunion six weeks after fracture. This tool requires external validation but may enhance the management of patients with isolated ulnar shaft fractures.

Surgical Versus Nonsurgical Management of Humeral Shaft Fractures: A Systematic Review and Meta-Analysis of Randomized Trials.

INTRODUCTION: The aim was to compare surgical and nonsurgical management for adults with humeral shaft fractures in terms of patient-reported upper limb function, health-related quality of life, radiographic outcomes, and complications. METHODS: MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature, PubMed, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, International Clinical Trials Registry, and OpenGrey (Repository for Grey Literature in Europe) were searched in September 2021. All published prospective randomized trials comparing surgical and nonsurgical management of humeral shaft fractures in adults were included. Of 715 studies identified, five were included in the systematic review and four in the meta-analysis. Data were extracted by two independent reviewers according to the Preferred Reporting Items for Systematic Review and Meta-Analysis statement. Methodological quality was assessed using the revised Cochrane risk-of-bias tool for randomized trials. Pooled data were analyzed using a random-effects model. RESULTS: The meta-analysis comprised 292 patients (mean age 41 [18 to 83] years, 67% male). Surgery was associated with superior Disabilities of the Arm, Shoulder and Hand (DASH) and Constant-Murley scores at 6 months (mean DASH difference 7.6, P = 0.01; mean Constant-Murley difference 8.0, P = 0.003), but there was no difference at 1 year (DASH, P = 0.30; Constant-Murley, P = 0.33). No differences in health-related quality of life or pain scores were found. Surgery was associated with a lower risk of nonunion (0.7% versus 15.7%; odds ratio [OR] 0.13, P = 0.004). The number needed to treat with surgery to avoid one nonunion was 7. Surgery was associated with a higher risk of transient radial nerve palsy (17.4% versus 0.7%; OR 8.23, P = 0.01) but not infection (OR 3.57, P = 0.13). Surgery was also associated with a lower risk of reintervention (1.4% versus 19.3%; OR 0.14, P = 0.04). CONCLUSIONS: Surgery may confer an early functional advantage to adults with humeral shaft fractures, but this is not sustained beyond 6 months. The lower risk of nonunion should be balanced against the higher risk of transient radial nerve palsy. LEVEL OF EVIDENCE: Level I.

Outcome Following Acute Suture Anchor Repair of the Ulnar Collateral Ligament of the Thumb.

Background: The aim of this study was to evaluate the outcomes following acute repair of the ulnar collateral ligament of the thumb metacarpophalangeal joint (thumb UCL) using a suture anchor technique. Methods: From 2011 to 2019, we retrospectively identified 40 adult patients from a single centre who had undergone an acute thumb UCL repair (≤6 weeks post-injury). The mean age of the study cohort was 37 years (range 16-70) and 68% (n = 27/40) were male. The short-term outcomes included postoperative complications and failure of repair. The long-term outcomes were QuickDASH, the EuroQol 5-Dimension (EQ-5D), Visual Analogue Scale (EQ-VAS), return to sport and work and satisfaction with outcome. Results: The outcomes survey was completed at a mean of 4.3 years (range 1.0-9.2) for 33 patients (83%). Postoperative complications included self-limiting sensory disturbance (7.5%, n = 3/40), superficial infection (requiring oral antibiotics; 5%, n = 2/40) and wound dehiscence (requiring surgical debridement and re-closure; 2.5%, n = 1/40). No failures of repair were reported. The mean QuickDASH was 3.7 (range 0-27.3), EQ-5D 0.821 (range -0.041 to 1) and EQ-VAS 84 (range 60-100). Of the 32 employed patients, all returned to work at a median of 0.5 weeks (range 0-416) and the mean QuickDASH Work Module was 4.1 (range 0-50). Of the 24 patients playing sport prior to injury, 96% (n = 23/24) returned at a median of 16 weeks (range 5-52) and the mean QuickDASH Sport Module was 4.6 (range 0-25). All the patients were satisfied with their outcome (mean satisfaction score 9.8/10 [8-10O]). Conclusions: Thumb UCL repair using a suture anchor technique is safe and effective up to 6 weeks post injury. Pain and stiffness may persist in the longer term, but most patients report excellent upper limb function and health-related quality of life. The majority return to work and sport and are highly satisfied with their outcome. Level of Evidence: Level IV (Therapeutic).

Integral Role of the Mitochondrial Ribosome in Supporting Ovarian Function: MRPS7 Variants in Syndromic Premature Ovarian Insufficiency.

The mitochondrial ribosome is critical to mitochondrial protein synthesis. Defects in both the large and small subunits of the mitochondrial ribosome can cause human disease, including, but not limited to, cardiomyopathy, hypoglycaemia, neurological dysfunction, sensorineural hearing loss and premature ovarian insufficiency (POI). POI is a common cause of infertility, characterised by elevated follicle-stimulating hormone and amenorrhea in women under the age of 40. Here we describe a patient with POI, sensorineural hearing loss and Hashimoto's disease. The co-occurrence of POI with sensorineural hearing loss indicates Perrault syndrome. Whole exome sequencing identified two compound heterozygous variants in mitochondrial ribosomal protein 7 (MRPS7), c.373A>T/p.(Lys125*) and c.536G>A/p.(Arg179His). Both novel variants are predicted to be pathogenic via in-silico algorithms. Variants in MRPS7 have been described only once in the literature and were identified in sisters, one of whom presented with congenital sensorineural hearing loss and POI, consistent with our patient phenotype. The other affected sister had a more severe disease course and died in early adolescence due to liver and renal failure before the reproductive phenotype was known. This second independent report validates that variants in MRPS7 are a cause of syndromic POI/Perrault syndrome. We present this case and review the current evidence supporting the integral role of the mitochondrial ribosome in supporting ovarian function.

Premature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights.

CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.

Dominant TP63 missense variants lead to constitutive activation and premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a leading form of female infertility, characterised by menstrual disturbance and elevated follicle-stimulating hormone before age 40. It is highly heterogeneous with variants in over 80 genes potentially causative, but the majority of cases having no known cause. One gene implicated in POI pathology is TP63. TP63 encodes multiple p63 isoforms, one of which has been shown to have a role in the surveillance of genetic quality in oocytes. TP63 C-terminal truncation variants and N-terminal duplication have been described in association with POI, however, functional validation has been lacking. Here we identify three novel TP63 missense variants in women with nonsyndromic POI, including one in the N-terminal activation domain, one in the C-terminal inhibition domain, and one affecting a unique and poorly understood p63 isoform, TA*p63. Via blue-native page and luciferase reporter assays we demonstrate that two of these variants disrupt p63 dimerization, leading to constitutively active p63 tetramer that significantly increases the transcription of downstream targets. This is the first evidence that TP63 missense variants can cause isolated POI and provides mechanistic insight that TP63 variants cause POI due to constitutive p63 activation and accelerated oocyte loss in the absence of DNA damage.

Whole exome sequencing reveals copy number variants in individuals with disorders of sex development.

Complete androgen insensitivity syndrome (CAIS), where 46,XY individuals present as female, is caused by variants in the androgen receptor gene (AR). We analyzed the DNA of a patient with suspected CAIS using a targeted gene sequencing panel and whole exome sequencing (WES) but did not detect any small nucleotide variants in AR. Analysis of WES data using our bioinformatics pipeline designed to detect copy number variations (CNV) uncovered a rare duplication of exon 2 of AR. Using array comparative genomic hybridization, the duplication was found to span 43.6 kb and is predicted to cause a frameshift and loss of AR protein. We confirmed the power of our WES-CNV detection protocol by identifying pathogenic CNVs in FSHR and NR5A1 in previously undiagnosed patients with disorders of sex development. Our findings illustrate the usefulness of CNV analysis in WES data to detect pathogenic genomic changes that may go undetected using only standard analysis protocols.

Lessons learnt from multifaceted diagnostic approaches to the first 150 families in Victoria's Undiagnosed Diseases Program.

BACKGROUND: Clinical exome sequencing typically achieves diagnostic yields of 30%-57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals. AIM: We share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases. METHODS: We enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis. RESULTS: In 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis. CONCLUSION: We share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease-gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

Pathogenic variants in nucleoporin TPR (translocated promoter region, nuclear basket protein) cause severe intellectual disability in humans.

The nuclear pore complex (NPC) is a multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Genetic variants in components of the NPC have been shown to cause a range of neurological disorders, including intellectual disability and microcephaly. Translocated promoter region, nuclear basket protein (TPR) is a critical scaffolding element of the nuclear facing interior of the NPC. Here, we present two siblings with biallelic variants in TPR who present with a phenotype of microcephaly, ataxia and severe intellectual disability. The variants result in a premature truncation variant, and a splice variant leading to a 12-amino acid deletion respectively. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR levels, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, and decreased global RNA intensity in the nucleus. The discovery of variants that partly disable TPR function provide valuable insight into this essential protein in human disease, and our findings suggest that TPR variants are the cause of the siblings' neurological disorder.

Meiotic genes in premature ovarian insufficiency: variants in HROB and REC8 as likely genetic causes.

Premature ovarian insufficiency (POI), affecting 1 in 100 women, is characterised by loss of ovarian function associated with elevated gonadotropin, before the age of 40. In addition to infertility, patients face increased risk of comorbidities such as heart disease, osteoporosis, cancer and/or early mortality. We used whole exome sequencing to identify the genetic cause of POI in seven women. Each had biallelic candidate variants in genes with a primary role in DNA damage repair and/or meiosis. This includes two genes, REC8 and HROB, not previously associated with autosomal recessive POI. REC8 encodes a component of the cohesin complex and HROB encodes a factor that recruits MCM8/9 for DNA damage repair. In silico analyses, combined with concordant mouse model phenotypes support these as new genetic causes of POI. We also identified novel variants in MCM8, NUP107, STAG3 and HFM1 and a known variant in POF1B. Our study highlights the pivotal role of meiosis in ovarian function. We identify novel variants, consolidate the pathogenicity of variants previously considered of unknown significance, and propose HROB and REC8 variants as new genetic causes while exploring their link to pathogenesis.