Sequence change: Better risk calculation and / or treatment options - PowerPoint PPT Presentation

Indication for request molecular analysis Clinical Genetics Leading the way in genetic issues • Genetic confirmation of clinical diagnosis Better treatment • Carrier detection Sequence change: Better risk calculation and / or treatment options pathogenic or just a • Prenatal analysis neutral variant? In the Netherlands: >99% affected fetus the pregnancy is terminated Ans van den Ouweland Dept. Clinical Genetics Erasmus MC Diagnostic setting Sequence analysis Whole exome sequenced and analysed Scanning What about findings not correlated to the clinical phenotype present in the patient / family? Informed consent needed!!! Analysis of genes proven to be the underlying cause of the • clinical symptoms (Sanger / WES/WGS (Whole exome sequencing Patient / family wants to know: / Whole genome sequencing) data and analyse with a filter of 1. Only sequence changes correlated to the disease in the family panel of genes). Filters need frequent update 2. Only sequence changes correlated to the disease in the family and other treatable disorders 3. All sequence changes and the impact of the changes • Analysis of WES/WGS complete data (in diagnostic setting most of the time trio analysis: index and his/her parents) Precounseling is essential!

Sequence analysis RNA Splicing BRANCH POINT (between: - 50 and -10) Analysis of genes proven to cause the clinical symptoms: ATG TGA Genomic DNA * Sanger sequencing Promoter Intron2 EXON 1 EXON 2 EXON 3 * NGS: whole exome and analysed with a filter DONOR Advantage: 5’ SPLICE SITE no unexpected results with respect to other diseases ACCEPTOR 3’ SPLICE SITE Examples of NGS in the Netherlands: Transcription * Deafness and blindness * Intelectual disability ATG TGA * Cardiomyopathy * Neuronal migration defects EXON 1 EXON 2 EXON 3 mRNA * Neurodegenerative and movement disorders * Ciliopathy * Oncogenetics Translation * Craniosynostosis PROTEIN Protein RNA Splicing Consensus sequences Spliceosome assembly and RNA splicing +1 +2 A(38) A(71) INTRON EXON 1 A(62) G(77) G(100) T(100) C(31) G(24) DONOR INTRON EXON INTRON -2 -1 EXON 2 C(55) T(41) U1 – U6: snRNPs PY(84) PY(85) PY(58) X A(100) G(100) G(50) ACCEPTOR T(37) A(24) snRNP: is a complex of a small RNA and up to dozens of proteins INTRON EXON Query, Nature (2009) 458:418-419 Zhang, Hum Mol Genet (1998) 7:919-932 Roca et al, Genome research (2008) 18:77-87

HGVS nomenclature Sequence analysis (http://www.hgvs.org) Nonsense and Frameshift: ATG translation codon: A is nucleotide number 1 Almost always pathogenic in genes proven to be the underlying That is not the case in the NM number! cause of the disease Exon: Nonsense mutation at the N-terminal end of the protein: alternative ATG translation codon usage possible? Nonsense c.3826C>T p.(Arg1276*) Frameshift c.3525_3526delAA p.(Arg1175fs) At the C-terminal end of the protein: In-frame c.4312_4314delGAA p.(Glu1438del) p.Lys3326* (BRCA2; 3418 aa): known neutral variant and what Missense c.4418A>G p.(Gln1472Arg) about all nonsense and frameshifts after this position? Silent c.3468C>T p.(=) Splice site changes: -2, -1 and +1, +2 changes are almost always pathogenic Intron: In-frame, missense, silent nucleotide changes and the other Splice site c.3113+1G>A p.? intron changes: How to decide whether they are pathogenic or a neutral variant? Classification of sequence changes Classification of sequence changes 5 Class system (Plon et al. Hum Mutat (2008) 29: 1282 – 1291) Class Description Probability of being pathogenic 5 Definitely pathogenic * > 0.99 4 Likely pathogenic** 0.95 - 0.99 3 Uncertain 0.05 - 0.949 2 Likely not pathogenic or of little clinical significance 0.001 - 0.049 1 Not pathogenic or of no clinical significance <0.001 *: prenatal and carrier detection is offered **: segregation analysis is offered Lindor et al. (2012) Hum Mut 33:8-21

Classification of sequence changes Classification of sequence changes 1. rs data: ExAc or ESP or GoNL > 1% (more than 200 chromosomes analysed Class 1 2. Nonsense, frameshift and consensus sequences of intron Class 5 / 4 Align-GVGD 3. In silico prediction programs (protein and RNA splicing) We use Alamut software program: 4 in silico protein prediction programs (based on homology) 5 in silico RNA splicing programs (10% difference in at least 2 programs) 4. Functional studies 5. Locus specific databases 6. Literature Lindor et al. (2012) Hum Mut 33:8-21 Classification of sequence changes Classification of sequence changes All sequences changes except already classified as class 1, 2 or 5 Alamut c.1235A>T (p.Glu412Val; TSC2) Protein level or in-frame del/ins (always Class 3): run all predition programs in Alamut Silent changes and intron changes outside consensus: no effect on RNA splicing Class 2 • effect on RNA splicing Class 3 • Missense changes and no further data (eg functional) Effect on RNA splicing Class 3 • 1 out of 4 protein in silico deleterious Class 2 • Remaining Class 3 • probably damaging Further analysis can result in a reclassification (eg RNA studies, functional protein studies, LOH)

Classification of sequence changes Classification of sequence changes Alamut c.1235A>T (p.Glu412Val; TSC2) Splice site prediction c.1235A>T p.(Glu412Val) in TSC2 RNA (isolated from skin fibroblasts) analysis performed : RNA showed an abnormal pattern in agreement with the predictions. normal If possible: use an intragenic heterozygous SNP to rule out the possibility that the abnormal spliced RNA is a product of the normal allele. Use enough (about 5) controls to rule out leaky transcription artefacts Sequence change c.1235A>T is a pathogenic mutation; it influences RNA splicing of TSC2 and therefore it is not a missense mutation but a splice site mutation mutant Nomenclature: c.1235A>T p.(Glu412fs) Consensus sequence splice donor (5’ site): A(62) G(77) / g(100) t(100) a(71) Classification of sequence changes Hereditary Breast/Ovarian Cancer TSC1: missense changes in same codon. Pathogenicity? 1:8 women develops breast cancer 5%: a genetic factor involved p.(Arg190Cys) p.(Arg190Pro) 10-15% a pathogenic mutation in either BRCA1 or BRCA2 A lot of VUS identified Co-occurrence of 2 deleterious mutations: • In BRCA1 not possible; BRCA2: other phenotype (Fanconi Anemia D1) Cosegregation • Pathology: e.g. array CGH profiles; Loss of Heterozygosity of VUS • probably damaging probably damaging Functional data • Problem: Most VUS are very rare and therefore the likelihood ratios will not give the ultimate result Functional analysis: p.(Arg190Cys): same as wildtype p.(Arg190Pro): pathogenic

Hereditary Breast/Ovarian Cancer Bloopers c.5309G>T p.(Gly1770Val) (BRCA1) p.(Met1628Val) variant in BRCA1 (first classified as neutral/low risk variant) Phelan et al (2005) J. Med. Genet. 42: 138-146: functional test: pathogenic mutation Possibly damaging Carvalho and Monteiro (2007) J. Med. Genet. 44: 78: mistake in construct; not only 1628V variant, but also a deletion of 7 nucleotides. p.(Met1628Val) is a neutral variant Several small families; not enough for linkage analysis All families of Northern African origin aCGH of tumours: BRCA1 profile Functional analysis: Pathogenic Pitfall Pitfall Collecting all classified sequence variants in the Netherlands from c.1096G>A, p.(Glu366Lys) in SERPINA1 is frequent pathogenic • • Clinical Genetics DNA laboratories mutation, despite the clinical phenotype of the patient in which the pathogenic mutation is identified. Example: SERPINA1 (Emphysema due to alpha1-antitrypsine • deficiency; Autosomal recessive mode of inheritance). Conclusion: • The patient is a carrier of the pathogenic mutation c.1096G>A, • c.1096G>A, p.(Glu366Lys): GoNL: 1.70%; ExAc: 1.12%. p.(Glu366Lys). This is an incidental finding. • Sequence abnormality heterozygous identified in “open” WES • with the knowledge that the patient has breast cancer. Question: how would you classify this sequence change? •