Clinical utility of the functional mrna evaluation of rare genetic variants in diagnostic practice Celia Duff-Farrier: STP MSc project 2017 Funding provided by the Showering Fund
mrna functional evaluation requirement Genomic analysis generates many variants requiring interpretation A proportion of these affect mrna splicing ACGS practice guidelines for the evaluation of pathogenicity of sequence variants: (UK Clinical Molecular Genetics Society, Dutch Society of Clinical Genetic Laboratory Specialists. 2013) In silico tools stand only as predictions-confirmation of impact requires functional analysis Functional analysis is the only way to confirm pathogenic mechanism Scotti, M.M. & Swanson, M.S., 2015. RNA mis-splicing in disease. Nature Reviews Genetics, 17(1), pp.19 32.
MSc project Can mrna splicing effects be demonstrated in the diagnostic setting of Bristol Genetics Laboratory? What is the clinical utility of mrna functional analysis?
Assessment of lab databases Inter-lab collaboration Sample acquisition Clinical teams -cases where mrna analysis helpful to clarify a diagnosis Recruited from a range of testing pathways: NGS panel, exome sequencing and DDD study Consent for resampling; study inclusion request letters Sampling instructions; PAXgene and EDTA requested Familial controls used, randomised control patient when unavailable 10 variants analysed (18 patients in total)
Assay details SAMPLE COLLECTION RNA EXTRACTED cdna SYNTHESISED VARIANT SPECIFIC AMPLICONS GENERATED AGAROSE GEL ELECTROPHORESIS EXPERIMENTAL REPORTS ISSUED SEQUENCE ANALYSIS SANGER SEQUENCING
MSc patient samples Gene Genetic variant Affected phenotype In silico predictions KIF1B c.[3121+1g>t];[=] Neuropathy Alteration of intron 29 donor site MFN2 c.[2230g>a];[=] Neuropathy Novel splice donor site within exon 19 NEXN c.[490-9a>g];[=] Hypertrophic cardiomyopathy Undefined TNNI3 c.[24+5g>a];[(24+5g> A)] End-stage dilated cardiomyopathy Intron 2 donor site abolished PRKCD c.[788-2a>g];[=] Autoimmune Intron 9 acceptor site abolished lymphoproliferative syndrome MYBPC3 c.[1224-52g>a];[=] Hypertrophic cardiomyopathy Activation of cryptic splice site intron 13 TAZ c.[646+1del];[=] Barth syndrome Alteration splice donor site exon 8 WDR45 c.[236-18a>g];[=] NBIA Activation of a cryptic splice site in intron 6 ANKRD11 c.[7471-1g>c];[=] KBG syndrome Intron 10 splice acceptor abolished KAT6A c.[1364-2a>t];[=] KAT6A syndrome Novel splice acceptor within exon 8
KBG syndrome; ANKRD11 c.[7471-1g>c ];[=] BGL specialist KBG syndrome service: Low K, et al 2016.Clinical and genetic aspects of KBG syndrome. Am J Med Genet Part A. 2016;170: 2835 2846. mild intellectual disability, Asperger traits, ADHD, short stature, marked speech delay, delayed bone age, brachydactyly and 5th finger clinodactyly, macrodontia and typical facial features. Non-consanguineous healthy parents ANKRD11 c.7471-1g>c splice acceptor site of intron 9 Absent in dbsnp, ExAC,gnomAD or EVS datasets Patient at 10 years (left) and 19 years (right) One parent available for testing variant negative
KBG syndrome; ANKRD11 c.[7471-1g>c ];[=] Exon 10 Exon 11 (C-terminus of ANKRD11, consensus D-box sequences underlined, exon 10 region removed by the variant) ANKRD11 protein influences genes involved in neural development 2 critical D-box consensus sequences in highly conserved C-terminus >95% ANKRD11 truncating variants lacking D-box sequence 1 missense variant p.(arg2512gln) located within D-box 1 splice variant c.7570-1g>c 2 amino acid deletion in D-box p.(glu2524_lys2525del) ANKRD11 c.[7471-1g>c ];[=] splice acceptor site of intron 9 Does ANKRD11 c.7471-1g>c fit the disease mechanism?
KBG syndrome; ANKRD11 c.[7471-1g>c ];[=] Exon 10
KBG syndrome; ANKRD11 c.[7471-1g>c ];[=] 1: Affected patient 2: WT familial control -: negative water control Exon 11 Exon 9 In support of the disease mechanism proposed by Walz et al., 2015 A splice-site variant in ANKRD11 associated with classical KBG syndrome. Karen Low, Alison Hills, Maggie Williams, Celia Duff- Farrier, Shane McKee, Sarah Smithson Am J Med Genet Submitted
Barth syndrome; TAZ c.[646+1del];[=] Exon 8
Barth syndrome; TAZ c.[646+1del];[=] 1: Twin 1 severe phenotype TAZ c.[646+1del];[=] 2: Twin 2 mild phenotype TAZ c.[646+1del];[=] 3-5: Unaffected family members +: K562 cell line -: H 2 0 Exon 7 Exon 9
WDR45 c.[236-18a>g];[=] Neurodegeneration with brain iron accumulation (NBIA) WDR45 c.[236-18a>g];[=] variant detected as part of the DDD study Associated with Neurodegeneration with Brain Iron Accumulation Disorders (NBIA) Second sample type processed as part of this study-fibroblasts received
NBIA; WDR45 c.[236-18a>g];[=] Healthy non-consanguineous parents. No significant family history, dev delay with craniosynostosis of metopic suture, epileptic encephalopathy with late-onset West syndrome. Non progressive white matter changes on MRI-brain imaging WDR45 c.236-18a>g absent from population databases Classified Class 3 VUS by Sheffield and specialist lab in the USA Consideration of NBIA panel testing and 100,000 genomes recruitment Functional mrna analysis is the only way to confirm pathogenicity
NBIA; WDR45 c.[236-18a>g];[=] Exon 7
NBIA; WDR45 c.[236-18a>g];[=] 1: Affected c.[236-18a>g];[=] patient 2: Positive K562 control - negative H 2 0 Exon 5 Intron 6 17bp insertion Exon 7
NBIA; WDR45 c.[236-18a>g];[=] 11 intronic splicing variants reported in WDR45 in association with a neurodegenerative phenotype (HGMD pro 2017) First report of a clearly pathogenic variant located into the intron(-18) displaying a functional splicing effect Prevented needless further testing: NBIA panel testing and 100,000 genomes recruitment MYBPC3 c.[1224-52g>a];[=] Hypertrophic cardiomyopathy-splicing effect demonstrated
KAT6A syndrome; KAT6A c.[1364-2a>t];[=] DDD patient Likely pathogenic variant but requiring mrna confirmation Mild features: autism, speech delay, absent seizures, subtle dysmorphism, recurrent ear infections, submucous cleft palate. Sister also has a cleft palate
KAT6A syndrome; KAT6A c.[1364-2a>t];[=] Newly reported syndrome 2015 DDD study results indicate KAT6A mutations may be causal in 1% of patients with an undiagnosed developmental delay (Wright et al., 2015) KAT6A regulates a multitude of genes including the developmental Hox genes and is important for homeotic regulation (Tham et al. 2015) Heterozygous truncating mutations in all patients to date Haploinsufficiency postulated as cause of the phenotype mrna analysis important to demonstrate pathogenicity
KAT6A syndrome; KAT6A c.[1364-2a>t];[=] Exon 8
KAT6A syndrome; KAT6A c.[1364-2a>t];[=] 1:WT familial control 2:Affected c.[1364-2a>t];[=] patient - negative H 2 0 Exon 7 Shifted into exon 8
KAT6A syndrome; KAT6A c.[1364-2a>t];[=] First report of a splicing mutation causing KAT6A syndrome (HGMD professional 2017.1 database) Family felt it was Very helpful to have diagnosis mrna analysis important to demonstrate pathogenicity
Clinical utility of mrna functional analysis Gene Genetic variant Phenotype Result NEXN c.[490-9a>g];[=] Hypertrophic TNNI3 c.[24+5g>a];[(24+5 G>A)] cardiomyopathy Dilated cardiomyopathy Undefined Not expressed to sufficient level in peripheral blood MFN2 c.[2230g>a];[=] Neuropathy No effect demonstrated, splicing effect predicted KIF1B c.[3121+1g>t];[=] Neuropathy Exon skipping. Influenced treatment option PRKCD c.[788-2a>g];[=] Autoimmune lymphoproliferativ e syndrome MYBPC3 c.[1224-52g>a];[=] Hypertrophic cardiomyopathy Splicing effect demonstrated, complex picture, work ongoing Splicing effect demonstrated, complex picture, work ongoing TAZ c.[646+1del];[=] Barth syndrome Exon skipping demonstrated. Class 4>5 based on findings. New mechanism for disease WDR45 c.[236-18a>g];[=] NBIA Splicing effect demonstrated; Pathogenicity demonstrated Class 3>? based on findings. Implications for specialist centre and NBIA protocols ANKRD11 c.[7471-1g>c];[=] KBG syndrome Splicing effect demonstrated; exon partial deletion. Conformation of pathogenicity and further support for proposed disease mechanism KAT6A c.[1364-2a>t];[=] KAT6A Syndrome Splicing effect demonstrated, exon skipping. First report of a splicing mutation causing KAT6A syndrome
Clinical utility of mrna functional analysis High diagnostic yield Further evidence for the significance of intronic variants Decreased need for further testing Confirmation of variant pathogenicity and mechanism Influences treatment options Increases scientific understanding Useful technique for demonstrating specific variants are pathogenic
Acknowledgements Mark Greenslade Maggie Williams Dave Bunyan Extraction team BGL Joanna Davies Jennifer Glauert And extraction team MRD team BGL Paula Waits Paul Archer Amy White Leanne Grimes Stuart Cook Dr Francis Sansbury, University Hospitals Bristol Prof Ruth Newbury-Ecob, University Hospitals Bristol Dr Joanna Kennedy, University Hospitals Bristol Dr Marion Roderick, University Hospitals Bristol Dr Melissa Lees, Great Ormond Street Hospital Dr Meena Balasubramanian, Sheffield Clinical Genetics Service Prof Patrick Morrison, Belfast Regional Medical Genetics Center Dr. Liz Househam, Plymouth Hospitals NHS Trust Dr Ian Berry, Leeds Teaching Hospitals Dr Sinead Murphy Tallaght Hospital, Dublin Professor David Bennett, John Radcliffe Hospital, Oxford Dr Karen Low, University Hospitals Bristol Showering fund for funding the project