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

Clinical Genetics

Leading the way in genetic issues

Sequence change: pathogenic or just a neutral variant?

Ans van den Ouweland

• Dept. Clinical Genetics

Erasmus MC

Indication for request molecular analysis

• Genetic confirmation of clinical diagnosis

Better treatment

• Carrier detection

Better risk calculation and / or treatment options

• Prenatal analysis

In the Netherlands: >99% affected fetus the pregnancy is terminated

Diagnostic setting

Scanning

• Analysis of genes proven to be the underlying cause of the

clinical symptoms (Sanger / WES/WGS (Whole exome sequencing / Whole genome sequencing) data and analyse with a filter of panel of genes). Filters need frequent update

• Analysis of WES/WGS complete data (in diagnostic setting most of

the time trio analysis: index and his/her parents)

Sequence analysis

Whole exome sequenced and analysed

What about findings not correlated to the clinical phenotype present in the patient / family? Informed consent needed!!! Patient / family wants to know:

• 1. Only sequence changes correlated to the disease in the family
• 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

Precounseling is essential!

Sequence analysis

Analysis of genes proven to cause the clinical symptoms: * Sanger sequencing * NGS: whole exome and analysed with a filter Advantage: no unexpected results with respect to other diseases Examples of NGS in the Netherlands: * Deafness and blindness * Intelectual disability * Cardiomyopathy * Neuronal migration defects * Neurodegenerative and movement disorders * Ciliopathy * Oncogenetics * Craniosynostosis

RNA Splicing

DONOR 5’ SPLICE SITE ACCEPTOR 3’ SPLICE SITE

EXON 2 EXON 3 Intron2 EXON 1 ATG TGA EXON 3 TGA ATG EXON 2 EXON 1 BRANCH POINT (between: - 50 and -10) PROTEIN

Genomic DNA mRNA Protein

Promoter

Transcription Translation

Spliceosome assembly and RNA splicing

Query, Nature (2009) 458:418-419

U1 – U6: snRNPs snRNP: is a complex of a small RNA and up to dozens of proteins

RNA Splicing Consensus sequences

A(38) A(62) G(77) C(31)

EXON INTRON A(71)

G(100) T(100) G(24) C(55) PY(84) PY(85) PY(58) X A(100) G(100) T(37)

INTRON EXON T(41)

G(50) A(24) EXON 1 EXON 2

DONOR ACCEPTOR INTRON INTRON +1 +2

• 2 -1

Zhang, Hum Mol Genet (1998) 7:919-932 Roca et al, Genome research (2008) 18:77-87

HGVS nomenclature (http://www.hgvs.org) Exon:

Nonsense c.3826C>T p.(Arg1276*) Frameshift c.3525_3526delAA p.(Arg1175fs) In-frame c.4312_4314delGAA p.(Glu1438del) Missense c.4418A>G p.(Gln1472Arg) Silent c.3468C>T p.(=)

Intron:

Splice site c.3113+1G>A p.? ATG translation codon: A is nucleotide number 1 That is not the case in the NM number!

Sequence analysis

Nonsense and Frameshift: Almost always pathogenic in genes proven to be the underlying cause of the disease Nonsense mutation at the N-terminal end of the protein: alternative ATG translation codon usage possible? At the C-terminal end of the protein: p.Lys3326* (BRCA2; 3418 aa): known neutral variant and what about all nonsense and frameshifts after this position? Splice site changes:

• 2, -1 and +1, +2 changes are almost always pathogenic

In-frame, missense, silent nucleotide changes and the other intron changes: How to decide whether they are pathogenic or a neutral variant?

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

Classification of sequence changes

Lindor et al. (2012) Hum Mut 33:8-21

Classification of sequence changes

Lindor et al. (2012) Hum Mut 33:8-21

Align-GVGD

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

• 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

Classification of sequence changes

All sequences changes except already classified as class 1, 2 or 5

• r 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 Further analysis can result in a reclassification (eg RNA studies, functional protein studies, LOH)

Classification of sequence changes

Alamut c.1235A>T (p.Glu412Val; TSC2) Protein level

probably damaging

Classification of sequence changes

Consensus sequence splice donor (5’ site): A(62) G(77) / g(100) t(100) a(71)

mutant normal

Alamut c.1235A>T (p.Glu412Val; TSC2) Splice site prediction

Classification of sequence changes

RNA (isolated from skin fibroblasts) analysis performed : RNA showed an abnormal pattern in agreement with the predictions. 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 Nomenclature: c.1235A>T p.(Glu412fs)

c.1235A>T p.(Glu412Val) in TSC2

Classification of sequence changes

TSC1: missense changes in same codon. Pathogenicity?

Functional analysis: p.(Arg190Cys): same as wildtype p.(Arg190Pro): pathogenic

p.(Arg190Cys) p.(Arg190Pro)

probably damaging probably damaging

Hereditary Breast/Ovarian Cancer

1:8 women develops breast cancer 5%: a genetic factor involved 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
• Functional data

Problem: Most VUS are very rare and therefore the likelihood ratios will not give the ultimate result

Hereditary Breast/Ovarian Cancer

c.5309G>T p.(Gly1770Val) (BRCA1)

Several small families; not enough for linkage analysis All families of Northern African origin aCGH of tumours: BRCA1 profile Functional analysis:

Pathogenic

Possibly damaging

Bloopers

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 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

Pitfall

• Collecting all classified sequence variants in the Netherlands from

Clinical Genetics DNA laboratories

• Example: SERPINA1 (Emphysema due to alpha1-antitrypsine

deficiency; Autosomal recessive mode of inheritance).

• c.1096G>A, p.(Glu366Lys): GoNL: 1.70%; ExAc: 1.12%.
• Sequence abnormality heterozygous identified in “open” WES

with the knowledge that the patient has breast cancer.

• Question: how would you classify this sequence change?

Pitfall

• c.1096G>A, p.(Glu366Lys) in SERPINA1 is frequent pathogenic

mutation, despite the clinical phenotype of the patient in which the pathogenic mutation is identified.

• Conclusion:
• The patient is a carrier of the pathogenic mutation c.1096G>A,

p.(Glu366Lys). This is an incidental finding.

Problem

Gout et al (2007) Nat Genet 39:427-428 Murphy et al (2004) Hum Mutat 24:296-304: Resp, 5% and 43% of all published mutations in PKD1 and CDKN2A are not reported correct Recommendation: Locus specific database: curators analyse the sequence changes which have been deposited http://chromium.liacs.nl/LOVD2/home.php or http://www.LOVD.nl More databases become available, but validation of the results (necessary for diagnostic use) is very poor.

Classification of sequence changes

Frequent misinterpretations

The abnormality is a pathogenic mutation because: 1) cosegregates with the disease 2) is the only abnormality identified 3) less frequent than 1%

Classification of sequence changes

Guidelines for finding genetic variants underlying human disease Posted in Genomes Unzipped: 24 Apr 2014 06:00 AM PDT Authors: Daniel MacArthur and Chris Gunter. New DNA sequencing technologies are rapidly transforming the diagnosis of rare genetic diseases, but they also carry a risk: by allowing us to see all of the hundreds of “interesting-looking” variants in a patient’s genome, they make it potentially easy for researchers to spin a causal narrative around genetic changes that have nothing to do with disease status. Such false positive reports can have serious consequences: incorrect diagnoses, unnecessary or ineffective treatment, and reproductive decisions (such as embryo termination) based on spurious test results. In order to minimize such outcomes the field needs to decide on clear statistical guidelines for deciding whether or not a variant is truly causally linked with disease.

Thank you for your attention