Extending the new era of genomic testing into pregnancy management: A proposed model for Australian prenatal services.

BACKGROUND: Trio exome sequencing can be used to investigate congenital abnormalities identified on pregnancy ultrasound, but its use in an Australian context has not been assessed. AIMS: Assess clinical outcomes and changes in management after expedited genomic testing in the prenatal period to guide the development of a model for widespread implementation. MATERIALS AND METHODS: Forty-three prospective referrals for whole exome sequencing, including 40 trios (parents and pregnancy), two singletons and one duo were assessed in a tertiary hospital setting with access to a state-wide pathology laboratory. Diagnostic yield, turn-around time (TAT), gestational age at reporting, pregnancy outcome, change in management and future pregnancy status were assessed for each family. RESULTS: A clinically significant genomic diagnosis was made in 15/43 pregnancies (35%), with an average TAT of 12 days. Gestational age at time of report ranged from 16 + 5 to 31 + 6 weeks (median 21 + 3 weeks). Molecular diagnoses included neuromuscular and skeletal disorders, RASopathies and a range of other rare Mendelian disorders. The majority of families actively used the results in pregnancy decision making as well as in management of future pregnancies. CONCLUSIONS: Rapid second trimester prenatal genomic testing can be successfully delivered to investigate structural abnormalities in pregnancy, providing crucial guidance for current and future pregnancy management. The time-sensitive nature of this testing requires close laboratory and clinical collaboration to ensure appropriate referral and result communication. We found the establishment of a prenatal coordinator role and dedicated reporting team to be important facilitators. We propose this as a model for genomic testing in other prenatal services.

Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

RNF43 pathogenic Germline variant in a family with colorectal cancer.

The role of RNF43 as a cause of an inherited predisposition to colorectal cancer (CRC) is yet to be fully explored. This report presents our findings of two individuals with CRC from a single family carrying a likely-pathogenic inherited germline variant in RNF43. The proband (III:1) and the proband's mother (II:2) were diagnosed with mismatch repair proficient CRCs at the age of 50 years and 65 years, respectively. Both patients had BRAFV600E mutated colon tumours, indicating that the CRCs arose in sessile serrated lesions. The germline variant RNF43:c.375+1G>A was identified in both patients. RNA studies showed that this variant resulted in an aberrantly spliced transcript, which was predicted to encode RNF43:p.Ala126Ilefs*50 resulting in premature termination of protein synthesis and was classified as a likely-pathogenic variant. Our report adds further evidence to the hereditary role of RNF43 as a tumour suppressor gene in colorectal tumorigenesis and supports the inclusion of RNF43 as a gene of interest in the investigation of CRC predispositions outside the setting of serrated polyposis.

Recurrent pneumothorax in a case of tenascin-X deficient Ehlers-Danlos syndrome: Broadening the phenotypic spectrum.

The genomic region surrounding the Tenascin-XB gene (TNXB) is a complex and duplicated region, with several pseudogenes that predispose to high rates of homologous recombination. Classical-like Ehlers-Danlos syndrome (clEDS) is the result of tenascin-X deficiency due to biallelic loss of function variants in the TNXB gene. Here we present a patient with clEDS and spontaneous pneumothorax, a feature not previously reported to be associated with this condition. Two inherited pathogenic/likely pathogenic variants were identified; a previously reported deletion resulting in a TNXA/TNXB chimeric gene and a novel frameshift variant. The Tenascin-XB gene is well described in the literature to be associated with collagen metabolism, stabilization of the fibrillar-collagen matrix and is expressed abundantly in the extracellular matrix. We propose that tenascin-X deficiency is directly related to pneumothorax predisposition. This case expands the phenotypic spectrum of clEDS and highlights the challenges with molecular analysis and diagnosis.

GATA2 deficiency syndrome: A decade of discovery.

GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms, of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 480 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 240 of these confirmed to be familial and 24 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0-78 years) and myelodysplastic syndrome is the first diagnosis in 75% of these cases with acute myeloid leukemia in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss-of-function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing, and expression. In this review, we provide the first expert-curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

New Pathogenic Mutations Associated with Diacylglycerol O-Acyltransferase 1 Deficiency.

Diacylglycerol O-acyltransferase 1 deficiency is a recently discovered, rare congenital diarrheal disorder. We report 2 patients with newly described pathogenic mutations in diacylglycerol O-acyltransferase 1 with compound heterozygous inheritance and unusual phenotypes. This included a macrophage activation syndrome-like response seen in one patient, ameliorated with low dietary fat.

CDH1-related blepharocheilodontic syndrome is associated with diffuse gastric cancer risk.

We report the first case of diffuse gastric cancer in an individual with familial blepharocheilodontic syndrome (BCD) due to a germline CDH1 likely pathogenic variant. To date, other BCD affected relatives are nonpenetrant for diffuse gastric cancer posing challenges to counseling regarding gastric and breast cancer surveillance, and preventative total gastrectomy.

Identification of monogenic diabetes in an Australian cohort using the Exeter maturity-onset diabetes of the young (MODY) probability calculator and next-generation sequencing gene panel testing.

AIMS: This study aims to describe the prevalence of monogenic diabetes in an Australian referral cohort, in relation to Exeter maturity-onset diabetes of the young (MODY) probability calculator (EMPC) scores and next-generation sequencing with updated testing where relevant. METHODS: State-wide 5-year retrospective cohort study of individuals referred for monogenic diabetes genetic testing. RESULTS: After excluding individuals who had cascade testing for a familial variant (21) or declined research involvement (1), the final cohort comprised 40 probands. Incorporating updated testing, the final genetic result was positive (likely pathogenic/pathogenic variant) in 11/40 (27.5%), uncertain (variant of uncertain significance) in 8/40 (20%) and negative in 21/40 (52.5%) participants. Causative variants were found in GCK, HNF1A, MT-TL1 and HNF4A. Variants of uncertain significance included a novel multi-exonic GCK duplication. Amongst participants with EMPC scores ≥ 25%, a causative variant was identified in 37%. Cascade testing was positive in 9/10 tested relatives with diabetes and 0/6 tested relatives with no history of diabetes. CONCLUSIONS: Contemporary genetic testing produces a high yield of positive results in individuals with clinically suspected monogenic diabetes and their relatives with diabetes, highlighting the value of genetic testing for this condition. An EMPC score cutoff of ≥ 25% correctly yielded a positive predictive value of ≥ 25% in this multiethnic demographic. This is the first Australian study to describe EMPC scores in the Australian clinic setting, albeit a biased referral cohort. Larger studies may help characterise EMPC performance between ethnic subsets, noting differences in the expected probability of monogenic diabetes relative to type 2 diabetes.

BAP1 Tumour Predisposition Syndrome Due to Whole BAP1 Gene Deletion.

BRCA-1-associated protein-1 (BAP1) tumour predisposition syndrome (BAP1-TPDS) is a dominant hereditary cancer syndrome. The full spectrum of associated malignancies is yet to be fully characterised. We detail the phenotypic features of the first reported family with a whole BAP1 gene deletion. This report also adds to the emerging evidence that the rhabdoid subtype of meningioma is a part of the clinical spectrum of this tumour predisposition syndrome.

The VHL-dependent regulation of microRNAs in renal cancer.

BACKGROUND: The commonest histological type of renal cancer, clear cell renal cell carcinoma (cc RCC), is associated with genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumour suppressor. VHL inactivation leads to induction of hypoxia-inducible factors (HIFs) and a hypoxic pattern of gene expression. Differential levels of specific microRNAs (miRNAs) are observed in several tumours when compared to normal tissue. Given the central role of VHL in renal cancer formation, we examined the VHL-dependent regulation of miRNAs in renal cancer. METHODS: VHL-dependent miRNA expression in cc RCC was determined by microarray analysis of renal cell line RCC4 with mutated VHL (RCC4-VHL) and reintroduced wild-type VHL (RCC4 + VHL). Five miRNAs highly upregulated in RCC4 + VHL and five miRNAs highly downregulated in RCC4 + VHL were studied further, in addition to miR-210, which is regulated by the HIF-VHL system. miRNA expression was also measured in 31 cc RCC tumours compared to adjacent normal tissue. RESULTS: A significant increase in miR-210, miR-155 and miR-21 expression was observed in the tumour tissue. miR-210 levels also showed a correlation with a HIF-regulated mRNA, carbonic anhydrase IX (CAIX), and with VHL mutation or promoter methylation. An inverse correlation was observed between miR-210 expression and patient survival, and a putative target of miR-210, iron-sulfur cluster assembly protein (ISCU1/2), shows reciprocal levels of mRNA expression in the tumours. CONCLUSIONS: We have identified VHL-regulated miRNAs and found that for some the regulation is HIF-dependent and for others it is HIF-independent. This pattern of regulation was also seen in renal cancer tissue for several of these miRNAs (miR-210, miR-155, let-7i and members of the miR-17-92 cluster) when compared with normal tissue. miR-210 showed marked increases in expression in renal cancer and levels correlated with patient survival. The inverse correlation between miR-210 levels and ISCU1/2 provides support for the hypothesis that ISCU1/2 is a target of miR-210 and that it may contribute to the anaerobic respiration seen in renal (and other) tumours.See Commentary: http://www.biomedcentral.com/1741-7015/8/65.

Aberrant Splicing of SDHC in Families With Unexplained Succinate Dehydrogenase-Deficient Paragangliomas.

CONTEXT: Germline mutations in the succinate dehydrogenase genes (SDHA/B/C/D, SDHAF2-collectively, "SDHx") have been implicated in paraganglioma (PGL), renal cell carcinoma (RCC), gastrointestinal stromal tumor (GIST), and pituitary adenoma (PA). Negative SDHB tumor staining is indicative of SDH-deficient tumors, usually reflecting an underlying germline SDHx mutation. However, approximately 20% of individuals with SDH-deficient tumors lack an identifiable germline SDHx mutation. METHODS: We performed whole-exome sequencing (WES) of germline and tumor DNA followed by Sanger sequencing validation, transcriptome analysis, metabolomic studies, and haplotype analysis in 2 Italian-Australian families with SDH-deficient PGLs and various neoplasms, including RCC, GIST, and PA. RESULTS: Germline WES revealed a novel SDHC intronic variant, which had been missed during previous routine testing, in 4 affected siblings of the index family. Transcriptome analysis demonstrated aberrant SDHC splicing, with the retained intronic segment introducing a premature stop codon. WES of available tumors in this family showed chromosome 1 deletion with loss of wild-type SDHC in a PGL and a somatic gain-of-function KIT mutation in a GIST. The SDHC intronic variant identified was subsequently detected in the second family, with haplotype analysis indicating a founder effect. CONCLUSIONS: This is the deepest intronic variant to be reported among the SDHx genes. Intronic variants beyond the limits of standard gene sequencing analysis should be considered in patients with SDH-deficient tumors but negative genetic test results.

RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

ClinGen Myeloid Malignancy Variant Curation Expert Panel recommendations for germline RUNX1 variants.

Standardized variant curation is essential for clinical care recommendations for patients with inherited disorders. Clinical Genome Resource (ClinGen) variant curation expert panels are developing disease-associated gene specifications using the 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines to reduce curation discrepancies. The ClinGen Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) was created collaboratively between the American Society of Hematology and ClinGen to perform gene- and disease-specific modifications for inherited myeloid malignancies. The MM-VCEP began optimizing ACMG/AMP rules for RUNX1 because many germline variants have been described in patients with familial platelet disorder with a predisposition to acute myeloid leukemia, characterized by thrombocytopenia, platelet functional/ultrastructural defects, and a predisposition to hematologic malignancies. The 28 ACMG/AMP codes were tailored for RUNX1 variants by modifying gene/disease specifications, incorporating strength adjustments of existing rules, or both. Key specifications included calculation of minor allele frequency thresholds, formulating a semi-quantitative approach to counting multiple independent variant occurrences, identifying functional domains and mutational hotspots, establishing functional assay thresholds, and characterizing phenotype-specific guidelines. Preliminary rules were tested by using a pilot set of 52 variants; among these, 50 were previously classified as benign/likely benign, pathogenic/likely pathogenic, variant of unknown significance (VUS), or conflicting interpretations (CONF) in ClinVar. The application of RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the benefit of gene-specific criteria and sharing internal laboratory data.

Germline variants in familial pituitary tumour syndrome genes are common in young patients and families with additional endocrine tumours.

OBJECTIVE: Familial pituitary tumour syndromes (FPTS) account for 5% of pituitary adenomas. Multi-gene analysis via next-generation sequencing (NGS) may unveil greater prevalence and inform clinical care. We aimed to identify germline variants in selected patients with pituitary adenomas using a targeted NGS panel. DESIGN: We undertook a nationwide cross-sectional study of patients with pituitary adenomas with onset ≤40 years of age and/or other personal/family history of endocrine neoplasia. A custom NGS panel was performed on germline DNA to interrogate eight FPTS genes. Genome data were analysed via a custom bioinformatic pipeline, and validation was performed by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) was performed in cases with heightened suspicion for MEN1, CDKN1B and AIP mutations. The main outcomes were frequency and pathogenicity of rare variants in AIP, CDKN1B, MEN1, PRKAR1A, SDHA, SDHB, SDHC and SDHD. RESULTS: Forty-four patients with pituitary tumours, 14 of whom had a personal history of other endocrine tumours and/or a family history of pituitary or other endocrine tumours, were referred from endocrine tertiary-referral centres across Australia. Eleven patients (25%) had a rare variant across the eight FPTS genes tested: AIP (p.A299V, p.R106C, p.F269F, p.R304X, p.K156K, p.R271W), MEN1 (p.R176Q), SDHB (p.A2V, p.S8S), SDHC (p.E110Q) and SDHD (p.G12S), with two patients harbouring dual variants. Variants were classified as pathogenic or of uncertain significance in 9/44 patients (20%). No deletions/duplications were identified in MEN1, CDKN1B or AIP. CONCLUSIONS: A high yield of rare variants in genes implicated in FPTS can be found in selected patients using an NGS panel. It may also identify individuals harbouring more than one rare variant.

Vestibular schwannoma in a patient with neurofibromatosis type 1: clinical report and literature review.

We describe a young patient with typical neurofibromatosis type 1 on the basis of a mutation in the NF1 gene, who was diagnosed with a unilateral vestibular schwannoma caused by a somatic mutation in the NF2 gene. This combination has not been described before. This report highlights the requirement for ongoing surveillance regarding other manifestations of neurofibromatosis type 2 in such patients, as mosaicism cannot be ruled out. In addition to the NF1 mutation, the NF2 mutation should be considered in such cases if pre-implantation genetic diagnosis in undertaken.

Phylogeographic analysis of the green python, Morelia viridis, reveals cryptic diversity.

Green pythons, which are regionally variable in colour patterns, are found throughout the lowland rainforest of New Guinea and adjacent far northeastern Australia. The species is popular in commercial trade and management of this trade and its impacts on natural populations could be assisted by molecular identification tools. We used mitochondrial nucleotide sequences and a limited allozyme data to test whether significantly differentiated populations occur within the species range. Phylogenetic analysis of mtDNA sequences revealed hierarchal phylogeographic structure both within New Guinea and between New Guinea and Australia. Strongly supported reciprocally monophyletic mitochondrial lineages, northern and southern, were found either side of the central mountain range that runs nearly the length of New Guinea. Limited allozyme data suggest that population differentiation is reflected in the nuclear as well as the mitochondrial genome. A previous morphological analysis did not find any phenotypic concordance with the pattern of differentiation observed in the molecular data. The southern mitochondrial lineage includes all of the Australian haplotypes, which form a single lineage, nested among the southern New Guinean haplotypes.