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Additional file 3. Review History.

Additional file 2: Figure S1. Comparison of in silico splice-prediction tools on validated non-splice-altering variants. Using an UpSet plot, variants identified here by one or more tools are in silico false positives. The CADD and CADD-splice tools contributed the largest number of false positives. Figure S2. Novel Functions incorporated into Introme. A: Position Weight Matricesfor the exonic splicing enhancer motifs from ESEfinder [15] showing relative representationof nucleotides at each position of the motif. B: PWM for the hnRNP A1 exonic splicing silencer motif [50]. C: Normal splicing pattern with the blocks representing the location of the exons. Creation of an AG motif greater or equal to 15 nucleotides away from the branchpoint results in the use of the new splice site. Creation of an AG less than 15 nucleotides away from the branchpoint results in exon skipping due to the AG exclusion zone. The consensus motifs for exon definition of the minor spliceosomeare different than the major spliceosome. Intronic deletions which result in the branchpoint being less than 43 nucleotides away from the intron’s 5’SS result in the skipping of the preceding exon or intron retention. Figure S3. Importance of features included in Introme. Feature importance is an estimate of the contribution of each variable to the model. Boxes are coloured and labelled according to the source of the feature annotated. Figure S4. Overview of the methods and data sources for each section. Figure S5. Performance of in silico splice-prediction tools on three validation data sets. Precision recall curves and the corresponding positions of the splice-altering variants in the dataset for A-B: ncVar, C-D: MFASS and E-F: Shiraishi. Figure S6. Performance of in silico splice-prediction tools on three validation data sets, broken down by regions.

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.

Refereed/Peer-reviewed 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.

Refereed/Peer-reviewed 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.

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.

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 (G(M2) and G(M3)) 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), beta-hexosaminidase enzyme activity and levels of G(M2) and G(M3) 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 Gus(mps/mps), which were significantly delayed in the attenuated Gus(tm(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. usc Refereed/Peer-reviewed

Background: This 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. Method: An 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. Results: This 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. Conclusion: We 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 Refereed/Peer-reviewed

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.

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII. Refereed/Peer-reviewed

Mucopolysaccharidosis type I (MPS I) is the most common form of the MPS group of genetic diseases. MPS I results from a deficiency in the lysosomal enzyme α-l-iduronidase, leading to accumulation of undegraded heparan and dermatan sulphate glycosaminoglycan (GAG) chains in patient cells. MPS children suffer from multiple organ failure and die in their teens to early twenties. In particular, MPS I children also suffer from profound mental retardation and skeletal disease that restricts growth and movement. Neither brain nor skeletal disease is adequately treated by current therapy approaches. To overcome these barriers to effective therapy we have developed and tested a treatment called substrate deprivation therapy (SDT). MPS I knockout mice were treated with weekly intravenous injections of 1 mg/kg rhodamine B for six months to assess the efficacy of SDT. Mice were assessed using biochemistry, micro-CT and a battery of behaviour tests to determine the outcome of treatment. A reduction in female bodyweight gain was observed with the treatment as well as a decrease in lung GAG. Behavioural studies showed slight improvements in inverted grid and significant improvements in learning ability for female MPS I mice treated with rhodamine B. Skeletal disease also improved with a reduction in bone mineral volume observed. Overall, rhodamine B is safe to administer to MPS I knockout mice where it had an effect on improving aspects of neurological and skeletal disease symptoms and may therefore provide a potential therapy or adjunct therapy for MPS I patients. usc Refereed/Peer-reviewed