BACKGROUND:Unexplained stillbirth may occur owing to premature placental aging, with an unexpected deterioration of placental function for the gestational age. Circular RNAs are enzyme-resistant RNA molecules that accumulate in aging tissues. Furthermore, circular RNAs bind to genomic DNA directly, forming complexes that can induce DNA breaks and genomic instability. OBJECTIVE:This study investigated tissue aging and circular RNA accumulation with gestational age in healthy and stillbirth placentae and determined whether circular RNAs directly interact with placental DNA, causing DNA damage and cellular senescence. STUDY DESIGN:Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, at 23, 26, 31, and 34 weeks' gestation) were assessed. The abundance of 7 candidate circular RNAs and their linear transcripts was quantified. The physical interaction of candidate circular RNAs with DNA was confirmed. Telomere length, relative abundance of senescence-associated genes, and DNA damage were assessed. Patient-derived trophoblast stem cell differentiation into syncytiotrophoblasts or extravillous trophoblasts was confirmed before circ_0000284 knockdown. The abundance of circular RNAs in maternal blood sampled between 15 and 16 weeks' gestation (n=12 control, n=6 women who went on to have a stillbirth) was determined using quantitative polymerase chain reaction. Appropriate statistical analyses were undertaken (SPSS). RESULTS:Placental DNA damage, senescence, and expression of 7 candidate circular RNAs, but not their linear transcripts, were increased in 40 and 41+ weeks' gestation samples and in stillbirth compared with earlier gestations (37-39 weeks). DNA:RNA immunoprecipitation-quantitative polymerase chain reaction signal size confirmed that all candidate circular RNA loci bind to placental DNA. The abundance of circular RNA was significantly decreased with the addition of RNase H1 compared with all healthy gestation samples, indicating that stillbirth placentae may lack RNase H1. Telomere length is shorter in placentae from stillbirths than in healthy 37 weeks' gestation placentae. Depletion of circ_0000284 by specific small interfering RNA in primary cells significantly reduced DNA damage and increased expression of senescence-associated genes compared with the control. The abundance of candidate circular RNAs is increased in maternal blood at 16 weeks' gestation for women who went on to have a stillbirth compared with women who had live births. CONCLUSION:Stillbirth placentae show accelerated aging with shortened telomeres, premature DNA breaks, increased cellular senescence, and accumulation of candidate circular RNAs at levels consistent with older gestation tissue. These circular RNAs bind to DNA in the placenta, and circ_0000284 knockdown reduces DNA breaks and senescence in primary placental cells. Therefore, circular RNAs play a role in placental aging and are associated with stillbirth, likely via decreased RNase H1 abundance, preventing circular RNA degradation and facilitating circular RNA accumulation and subsequent circular RNA:DNA complex formation. Circular RNAs may present a viable method of stillbirth risk screening.

Prenatal presentation of polycystic kidney disease (PKD), characterized by bilateral renal cysts and enlarged echogenic kidneys on ultrasound, often results in perinatal death. Prenatal manifestations of PKD are generally associated with autosomal recessive PKD, most commonly a result of pathogenic variants in PKHD1, but in rare cases can also be driven by bi-allelic inheritance of pathogenic variants in genes more commonly associated with autosomal dominant PKD such as PKD1. Diagnosing the underlying cause of prenatal PKD can be complicated by atypical histology, and/or a prenatal phenotype that does not align with family history. In this study, five cases of prenatal PKD with atypical or inconclusive features identified during post-mortem investigations underwent trio exome or genome sequencing, termed a genomic autopsy. Genomic autopsy was able to delineate the genetic basis of prenatal PKD in all five families. Our findings demonstrate the diagnostic utility of a genomic autopsy in providing a genetic diagnosis for fetal PKD cases post-mortem, particularly in atypical presentations. A genetic diagnosis is highly beneficial for future family planning, including the use of reproductive technologies, as well as identifying presymptomatic parents who are likely to develop PKD in the future.

Following termination of pregnancy for fetal anomaly or unexplained perinatal death (PND), clinical geneticists advise on possible genetic causes and likelihood of recurrence, often with limited use of molecular analysis. In the Australian Genomic Autopsy Study (GAS) cases that were unresolved following standard-of-care investigations underwent exome and/or genome sequencing (ES/GS). This diagnostic before-and-after study measured the changes in clinical management, in terms of the effect on clinical counselling that was provided to parents following ES/GS. Clinicians were surveyed before and after receiving sequencing results about the likelihood of recurrence and the reproductive planning advice they would provide to families. 161 pairs of before-and-after surveys were completed. Clinician estimates regarding PND recurrence changed for 45% (73/161) of families after receiving test results, despite a genetic diagnosis being found in only 19%. Families with an 'unknown likelihood' of recurrence reduced from 26% to 15% (p = 0.01). The information provided to parents about recurrence and reproductive planning increased significantly, both with and without a diagnosis, and clinicians reported that most parents expressed value was obtained from the investigation. The utility of genomic autopsy for clinical management is not restricted to families with a genetic finding.

Unexplained stillbirth is hypothesised to occur due to premature placental ageing, with unexpected deterioration of placental function for gestational age. Circular RNAs (circRNAs) are enzyme resistant RNA molecules that accumulate in ageing tissues. Furthermore, circRNAs bind gDNA directly, forming circRNA:DNA complexes which can induce DNA breaks. This study investigated circRNA accumulation with gestational age in healthy and stillbirth placentae and determined whether circRNAs directly interact with placental DNA causing DNA damage. Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, 23, 26, 31, 34 weeks' gestation) were assessed for DNA damage using an alkaline Comet Assay. Expression levels of 6 candidate circRNAs (circ\_0009000, circ\_0024157, circ\_0061017, circ\_0036877, circ\_0054624 and circ\_0111277), and their linear transcripts, were quantified using qPCR. Physical interaction of candidate circRNAs with DNA was confirmed by DNA:RNA ImmunoPrecipitation (DRIP). The effect of circ\_0009000 knockdown in HEK293T cells was assessed following transfection with either a siRNA (designed to knockdown circ\_0009000) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. DNA damage was assessed by Comet Assay. Compared with earlier gestations (37, 38, 39 and 40 weeks'), placental DNA damage and expression of all 6 candidate circRNAs, but not their linear transcripts, were increased in 40 and 41+ weeks' gestation samples, and in stillbirth. DRIP-qPCR signal size was significantly larger in term placentae than in enzyme-treated controls, confirming that all candidate circRNA loci bind to placental DNA. Depletion of circ\_0009000 by specific siRNA in HEK293T cells, significantly reduced DNA damage compared to control. Stillbirth placentae show accelerated ageing with premature accumulation of candidate circRNAs (first evidence in humans) at levels consistent with older gestation tissue. Importantly, these circRNAs bind to DNA and circ\_0009000 causes DNA breaks in placenta. Therefore, circRNAs (circ\_0009000, circ\_0024157, circ\_0061017, circ\_0036877, circ\_0054624 and circ\_0111277) play a role in placental ageing and associate with stillbirth, likely via DNA damage. ### Competing Interest Statement Provisional patent #1297095, filed 2nd September 2024.

Genetic testing of blood relatives of individuals at high risk of dominant conditions has significant preventive health benefits. However, cascade testing uptake is <50%. Research shows increased testing uptake when health professionals (HPs) contact at-risk relatives directly, with patient consent. Despite international support, this is not standard practice in Australia. We aimed to gather perspectives of genetic testing patients about direct-contact methods. Using an online survey, we surveyed Australian adults with genetic results of relevance for relatives, including patients who (i) self-categorised as being directly contacted by a clinical service, (ii) self-categorised as being referred by a HP, and (iii) received genetic results through a research study. Overall, 442 patients responded (clinical n = 363; research n = 79). Clinical patients self-categorised as 49.0% directly-contacted and 51.0% referred. Overall, the majority of patients had no privacy concerns about direct-contact methods (direct-contact 97%; referred 77%; research 76%). Less than 5% of the combined cohort (n = 19/442) reported significant concerns. The most prevalent concerns were the need for consent to provide HPs with relatives' contact details, and a patient preference to notify relatives before HP contact. Other key findings include preferences about contact methods, including that most patients who received a letter from a genetics service preferred a letter with specific information about the familial genetic condition (n = 141/149; 94.6%) than one with general information about genetic risk. Our findings indicate Australian patients support HPs using direct-contact methods to assist with risk communication to relatives. Findings also identify concerns to be addressed in the design of direct-contact programs.

Pregnancy loss and perinatal death are devastating events for families. We assessed 'genomic autopsy' as an adjunct to standard autopsy for 200 families who had experienced fetal or newborn death, providing a definitive or candidate genetic diagnosis in 105 families. Our cohort provides evidence of severe atypical in utero presentations of known genetic disorders and identifies novel phenotypes and disease genes. Inheritance of 42% of definitive diagnoses were either autosomal recessive (30.8%), X-linked recessive (3.8%) or autosomal dominant (excluding de novos, 7.7%), with risk of recurrence in future pregnancies. We report that at least ten families (5%) used their diagnosis for preimplantation (5) or prenatal diagnosis (5) of 12 pregnancies. We emphasize the clinical importance of genomic investigations of pregnancy loss and perinatal death, with short turnaround times for diagnostic reporting and followed by systematic research follow-up investigations. This approach has the potential to enable accurate counseling for future pregnancies.

Purpose Families living with mitochondrial diseases (MD) often endure prolonged diagnostic journeys and invasive testing, yet many remain without a molecular diagnosis. Through a national team of clinicians, diagnostic, and research scientists, the Australian Genomics Mitochondrial disease flagship conducted a prospective study to identify the diagnostic utility of singleton genomic sequencing using blood samples as a first step to diagnose MD. Methods 140 children and adults living with suspected MD were recruited using modified Nijmegen criteria (MNC) and randomised to either exome + mtDNA sequencing (ES+mtDNAseq) or genome sequencing (GS). Results Diagnostic yield was 55% (n=77) with variants in nuclear (n=37) and mtDNA (n=18) MD genes, as well as phenocopy genes (n=22). A nuclear gene aetiology was identified in 77% of diagnoses, irrespective of disease onset. Diagnostic rate was higher in paediatric-onset (71%) than adult-onset (31%) cases. For children, higher MNC scores correlated with increased diagnostic yield and fewer diagnoses in phenocopy genes. Additionally, three adult patients had a mtDNA deletion discovered in skeletal muscle that was not initially detected in blood. Conclusion Genomic sequencing from blood can simplify the diagnostic pathway for individuals living with suspected MD, especially those with childhood onset diseases and high MNC scores.

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.

Lethal short-limb skeletal dysplasia Al-Gazali type (OMIM %601356), also called dysplastic cortical hyperostosis, Al-Gazali type, is an ultra-rare disorder previously reported in only three unrelated individuals. The genetic etiology for Al-Gazali skeletal dysplasia has up until now been unknown. Through international collaborative efforts involving seven clinical centers worldwide, a cohort of nine patients with clinical and radiographic features consistent with short-limb skeletal dysplasia Al-Gazali type was collected. The affected individuals presented with moderate intrauterine growth restriction, relative macrocephaly, hypertrichosis, large anterior fontanelle, short neck, short and stiff limbs with small hands and feet, severe brachydactyly, and generalized bone sclerosis with mild platyspondyly. Biallelic disease-causing variants in ADAMTSL2 were detected using massively parallel sequencing (MPS) and Sanger sequencing techniques. Six individuals were compound heterozygous and one individual was homozygous for pathogenic variants in ADAMTSL2. In one of the families, pathogenic variants were detected in parental samples only. Overall, this study sheds light on the genetic cause of Al-Gazali skeletal dysplasia and identifies it as a semi-lethal part of the spectrum of ADAMTSL2-related disorders. Furthermore, we highlight the importance of meticulous analysis of the pseudogene region of ADAMTSL2 where disease-causing variants might be located. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .

Perinatal death, of a fetus or newborn, is a devastating event for families. Following nationwide multicentre recruitment, we assessed ‘genomic autopsy’ as an adjunct to standard autopsy for 200 families who experienced perinatal death, and provided a definite or candidate genetic diagnosis in 105 families. From this understudied cohort, half of the (candidate) diagnoses were phenotype expansions or novel disease genes, revealing previously unknown in-utero presentations of existing developmental disorders, and genomic disorders that are likely incompatible with life. Among the definite diagnoses, 43% were recessively or dominantly inherited, posing a 25% or 50% recurrence risk for future pregnancies. Ten families used their diagnosis for preimplantation or prenatal diagnosis of 12 pregnancies, facilitating the delivery of ten healthy newborns and management of two affected pregnancies. We emphasize the clinical importance of genomic investigations of perinatal death, with short turn-around times, enabling accurate counselling and options for families to prevent recurrence.

Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17β-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures. Treated PA2 mice had an earlier onset of seizures coupled with a subsequent reduction in seizures later in postnatal life, with a complete absence of any seizures during the analysis at 7 weeks of age. Despite the reduction in seizures, 17β-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (Log2FC > ± 0.5, P-value<0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17β-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. We conclude that 17β-estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome nor improve mortality or behavioural and cognitive deficits.

Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH-SCKS) include a heterogeneous group of autosomal recessive inherited diseases characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Here, we report biallelic variants in NUP85 in two pedigrees with an MCPH-SCKS phenotype spectrum without SRNS, thereby expanding the phenotypic spectrum of NUP85-linked diseases. Structural analysis predicts the identified NUP85 variants cause conformational changes that could have an effect on NPC architecture or on its interaction with other NUPs. We show that mutant NUP85 is, however, associated with a reduced number of NPCs but unaltered nucleocytoplasmic compartmentalization, abnormal mitotic spindle morphology, and decreased cell viability and proliferation in one patient's cells. Our results also indicate the link of common cellular mechanisms involved in MCPH-SCKS spectrum disorders and NUP85-associated diseases. In addition to the previous studies, our results broaden the phenotypic spectrum of NUP85-linked human disease and propose a role for NUP85 in nervous system development.

BackgroundThis sub-study of the Australian Genomics Cardiovascular Genetic Disorders Flagship sought to conduct the first nation-wide audit in Australia to establish the current practices across cardiac genetics clinics.MethodAn audit of records of patients with a suspected genetic heart disease (cardiomyopathy, primary arrhythmia, autosomal dominant congenital heart disease) who had a cardiac genetics consultation between 1st January 2016 and 31 July 2018 and were offered a diagnostic genetic test.ResultsThis audit included 536 records at multidisciplinary cardiac genetics clinics from 11 public tertiary hospitals across five Australian states. Most genetic consultations occurred in a clinic setting (90%), followed by inpatient (6%) and Telehealth (4%). Queensland had the highest proportion of Telehealth consultations (9% of state total). Sixty-six percent of patients had a clinical diagnosis of a cardiomyopathy, 28% a primary arrhythmia, and 0.7% congenital heart disease. The reason for diagnosis was most commonly as a result of investigations of symptoms (73%). Most patients were referred by a cardiologist (85%), followed by a general practitioner (9%) and most genetic tests were funded by the state Genetic Health Service (73%). Nationally, 29% of genetic tests identified a pathogenic or likely pathogenic gene variant; 32% of cardiomyopathies, 26% of primary arrhythmia syndromes, and 25% of congenital heart disease.ConclusionWe provide important information describing the current models of care for genetic heart diseases throughout Australia. These baseline data will inform the implementation and impact of whole genome sequencing in the Australian healthcare landscape.

The cause of neurodegeneration in MPS mouse models is the focus of much debate and what the underlying cause of disease pathology in MPS mice is. The timing of development of pathology and when this can be reversed or impacted is the key to developing suitable therapies in MPS. This study is the first of its kind to correlate the biochemical changes with the functional outcome as assessed using non-invasive behaviour testing across multiple mucopolysaccharidosis (MPS) mouse models. In the MPS brain, the primary lysosomal enzyme dysfunction leads to accumulation of primary glycosaminoglycans (GAGs) with gangliosides (GM2 and GM3) being the major secondary storage products. With a focus on the neuropathology, a time course experiment was conducted in MPS I, MPS IIIA, MPS VII (severe and attenuated models) in order to understand the relative timing and level of GAG and ganglioside accumulation and how this correlates to behaviour deficits. Time course analysis from 1 to 6 months of age was conducted on brain samples to assess primary GAG (uronic acid), β-hexosaminidase enzyme activity and levels of GM2 and GM3 gangliosides. This was compared to a battery of non-invasive behaviour tests including open field, inverted grid, rotarod and water cross maze were assessed to determine effects on motor function, activity and learning ability. The results show that the GAG and ganglioside accumulation begins prior to the onset of detectable changes in learning ability and behaviour. Interestingly, the highest levels of GAG and ganglioside accumulation was observed in the MPS IIIA mouse despite having 3% residual enzyme activity. Deficits in motor function were clearly observed in the severe Gusmps/mps, which were significantly delayed in the attenuated Gustm(L175F)Sly model despite their minimal increase in detectable enzyme activity. This suggests that genotype and residual enzyme activity are not indicative of severity of disease pathology in MPS disease and there exists a window when there are considerable storage products without detectable functional deficits which may allow an alteration to occur with therapy.

We show that the loss of Iqsec2 function in mice recapitulates key aspects of the human phenotype, irrespective of the X-inactivation status of the gene between species. Our understanding of the traditional X-chromosome inheritance with heterozygous female sparing needs revisiting. Clinical presentations of mutations in the IQSEC2 gene on the X-chromosome initially implicated to cause non-syndromic intellectual disability (ID) in males have expanded to include early onset seizures in males as well as in females. The molecular pathogenesis is not well understood, nor the mechanisms driving disease expression in heterozygous females. Using a CRISPR/Cas9–edited Iqsec2 KO mouse model, we confirm the loss of Iqsec2 mRNA expression and lack of Iqsec2 protein within the brain of both founder and progeny mice. Both male (52%) and female (46%) Iqsec2 KO mice present with frequent and recurrent seizures. Focusing on Iqsec2 KO heterozygous female mice, we demonstrate increased hyperactivity, altered anxiety and fear responses, decreased social interactions, delayed learning capacity and decreased memory retention/novel recognition, recapitulating psychiatric issues, autistic-like features, and cognitive deficits present in female patients with loss-of-function IQSEC2 variants. Despite Iqsec2 normally acting to activate Arf6 substrate, we demonstrate that mice modelling the loss of Iqsec2 function present with increased levels of activated Arf6. We contend that loss of Iqsec2 function leads to altered regulation of activated Arf6-mediated responses to synaptic signalling and immature synaptic networks. We highlight the importance of IQSEC2 function for females by reporting a novel nonsense variant c.566C > A, p.(S189*) in an elderly female patient with profound intellectual disability, generalised seizures, and behavioural disturbances. Our human and mouse data reaffirm IQSEC2 as another disease gene with an unexpected X-chromosome heterozygous female phenotype. Our Iqsec2 mouse model recapitulates the phenotypes observed in human patients despite the differences in the IQSEC2/Iqsec2 gene X-chromosome inactivation between the species.