Jessica M Clarke¹, Melanie Galea¹, Luke Hesson¹.

¹Department of Molecular Genetics, Douglass Hanly Moir Pathology, Macquarie Park, 2113

The introduction of Medicare Benefits Schedule (MBS) item 73451 in late 2023, has facilitated more equitable access to reproductive carrier screening for the Australian population.¹ Since the introduction of this MBS item, the uptake of reproductive carrier screening has increased markedly, highlighting the important role federal funding plays on accessibility of critical healthcare services. 

Included in this MBS item is screening of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.^2,3 Many testing methods are available and utilised by genomic laboratories for CFTR screening, including both fragment analysis and sequencing based methods. A challenge faced by genomic laboratories today is in providing a service that is clinically responsible, sustainable and high throughput to maintain quality equitable access.

Here we outline a strategy for increasing the diagnostic yield of a commercially available multiplex PCR assay by identifying atypical results for orthogonal testing using both Sanger sequencing and MLPA. We show that this represents an efficient strategy for maximising the yield of reportable variants to meet the requirements of the MBS rebate. This ultimately provides a comprehensive and sustainable service for patients and genomic laboratories.

References:

1.       Australian Government Department of Health. (2024). Medicare Benefits Schedule: Item 73451. Available at: https://www.mbsonline.gov.au

2.       Cystic Fibrosis Australia. (2023). Cystic fibrosis standards of care: Australia. Available at: https://www.cysticfibrosis.org.au.

3.       CFTR2 Consortium. (2024). CFTR2 database. Cystic Fibrosis Foundation. Available at https://cftr2.org/

 

Krystle Standen¹, Rebecca Walsh¹, George Elaksis¹, Bianca Rodgrigues¹

¹ Department of Genomics, NSW Health Pathology, Randwick, NSW, 2031

 

Background: Gonadal mosaicism can present diagnostic challenges. Deep sequencing approaches have proven useful in identifying mosaic pathogenic variants in conditions such as Tuberous sclerosis complex and Neurofibromatosis 1 where gonadal/gonosomal mosaicism has been reported. Identification of familial variants is necessary for access to therapy and reproductive options.

 

Methodology and results: Samples were processed using a TWIST 79-gene custom probe set kit with deep massively parallel sequencing. Libraries were sequenced on an Illumina NextSeq 500 and data processed using the Dragen server and an in house pipeline.

Case 1 showed a maternally inherited mosaic variant in TSC2. The variant was not detected in peripheral blood but was detected in an angiomyolipoma biopsy at 57% VAF.

Case 2 showed a paternally inherited mosaic variant in TSC2 in peripheral blood at 4.2% VAF. On review, they had clinical features consistent with Tuberous sclerosis.

Case 3 showed two pathogenic variants in NF1; variant 1 was detected in both left (21% VAF) and right (26% VAF) cutaneous neurofibromas biopsies, as well as in peripheral blood (2.5% VAF).

 

Conclusion: Deep sequencing can identify mosaic pathogenic variants below the limit of detection of standard sequencing assays. This, along with testing an appropriate tissue type can be used to infer gonadal mosaicism and guide appropriate genetic counselling.

 

1. Chung CWT, Bournazos AM, Chan LCD, et al. Deep Sequencing and Phenotyping in an Australian Tuberous Sclerosis Complex "No Mutations Identified" Cohort. Mol Genet Genomic Med. 2024;12(10):e70017. doi:10.1002/mgg3.70017

2. Chen JL, Miller DT, Schmidt LS, et al. Mosaicism in Tumor Suppressor Gene Syndromes: Prevalence, Diagnostic Strategies, and Transmission Risk. Annu Rev Genomics Hum Genet. 2022;23:331-361. doi:10.1146/annurev-genom-120121-105450

3. Klonowska K, Giannikou K, Grevelink JM, et al. Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex. Am J Hum Genet. 2023;110(6):979-988. doi:10.1016/j.ajhg.2023.04.002

Jessica Wright¹, Tamaryn J Knezovitch¹, Mark G Williams¹, Ritesh Chatrapati², Pauline Higgins², Amanda Goh^2,3, Abhijit Kulkarni¹, Edward Chew¹

¹Genomic Diagnostics, 460 Lower Heidelberg Road, Heidelberg, Victoria, 3084; ²QML Pathology, 11 Riverview Place, Murarrie, Queensland, 4172; ³Faculty of Medicine, The University of Queensland, 20 Weightman St, Herston QLD 4006

The introduction of Medicare-funded Next Generation Sequencing (NGS) panel testing for haematological malignancies has transformed diagnostic workflows, offering unparalleled insights into the genetic basis of these disorders. The extensive data generated by NGS significantly increases the analytical workload which raises critical questions about the clinical utility of this additional information: does it genuinely assist clinicians, and does it translate into better patient outcomes? To explore these questions, we focused on one key gene from the expanded NGS panel: SF3B1.

In a subset of patients investigated for haematological malignancies, we observed that SF3B1 mutations frequently co-occurred with JAK2, CALR, and MPL driver variants. Through a series of case studies, we illustrate how the detection of SF3B1 mutations added significant diagnostic value, refining disease classification and informing clinical decision-making.

Our findings emphasize that while the complexity of NGS data can be challenging, its capacity to reveal the genetic heterogeneity of myeloproliferative neoplasms / myelodysplastic syndrome (MPN/MDS) markedly enhances diagnostic accuracy and patient care. We conclude that the investment in comprehensive NGS testing is not only justifiable but essential for delivering precise and impactful diagnoses.

Dr Melissa Robinson^1
1Mater Pathology, Brisbane, Australia

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that leads to impaired corticosteroid synthesis. Three overlapping phenotypes can be seen - salt-wasting, simple virilising and non-classic; the phenotype can be attributed to the amount of functional enzyme produced. The phenotype can often be predicted from the least severe genetic variant. 

 

Next Generation Sequencing (NGS) is a cost effective testing methodology, commonly used in large reproductive carrier screening panels. However, there are caveats to the information available from short-read NGS. Certain genes have low depth of coverage and/or may be difficult to map to the reference genome. This can be due to highly repetitive sequence regions, or significant homology with a pseudogene. CYP21A2 is one such gene, due to the presence of the CYP21A2P pseudogene. Testing of this gene is further complicated by variable numbers of the active gene and pseudogene per allele. 

 

This talk will use clinical cases to demonstrate some of the complex alleles that can be identified in CYP21A2. The limitations of performing this testing on NGS and when to consider full CYP21A2 screening.

Jennifer Phan^{1, 2}, Ellie Wu¹, Kasey Peachy¹, Renata Bird¹, Niamh Hewitt¹, Jillian Nicholl¹, Kathryn Friend¹, Carol Siu¹, Sui Yu¹

¹Department of Genetics and Molecular Pathology, SA Pathology, Adelaide, SA, Australia; ² Genomics, Pathology Queensland, Herston, QLD, Australia.

Our case involves a 10-year-old female who was referred for SNP microarray and Fragile X testing as part of routine autism spectrum disorder (ASD) work up. No clinically significant copy number variants were detected on SNP microarray, and Fragile X repeat expansion testing detected a single peak in the normal repeat range with no evidence of an expanded allele. However, SNP microarray detected an unexpected XY sex chromosome complement.

Sex mismatches can be the result of laboratory technical errors or biological causes. A thorough laboratory investigation was performed, including additional testing and a sample recollection, to rule out a laboratory technical error. Discussion with the referring paediatrician did not reveal any significant clinical history that provided a biological cause for the sex mismatch. The patient was referred to paediatric endocrinology for further work up, and a disorders of sex development (DSD) gene panel was performed. A hemizygous Likely Pathogenic variant in the androgen receptor (AR) gene was detected, which confirmed a diagnosis of complete androgen insensitivity syndrome. This is a rare case of an incidental finding of DSD in a pre-pubertal patient and highlights the potential complexity that can occur with genome-wide testing.

Crystle Lee¹, Sebastian Hollizeck¹, Paul de Fazio¹, Ain Roesley¹, Belinda Chong¹, Simon Sadedin^1,2, Sebastian Lunke^1,2,3

¹ Victorian Clinical Genetics Services, Melbourne, Victoria

² Murdoch Children’s Research Institute, Melbourne, Victoria

³ University of Melbourne, Melbourne, Victoria

Background: Expanded carrier screening (ECS) aims to identify couples at risk of having children with a severe, early-onset recessive condition. The benefits of ECS have been widely reported, driving the uptake of this test. Performing ECS at scale presents a major challenge for diagnostic laboratories. Consequently, improving efficiencies through automated workflows is crucial.

Aim: We developed a workflow that minimises manual interaction to enable large-scale, high-throughput, couple-based expanded carrier screening. 

Method: The Illumina Emedgene platform, combined with custom in-silico proband variant calling, was used to develop a prioritisation strategy which uses annotations from various databases to identify candidate variants of interest. Couples with no variants of interest are automatically progressed to a low-chance report. To validate the algorithm, we used 34 known “high chance” and 40 known “low chance” couples. 

Results: All known “high chance” couples were correctly identified, and 55% of known “low chance” couples were progressed to automatic reporting. The remaining 45% returned variants for manual curation, none of which were reportable.

Conclusion: Workflows utilizing automated processes and optimised variant calling strategies facilitate the delivery of ECS at scale. Various improvements have been identified which will further improve the workflow.