Characterization of novel rare genetic variants identified by next generation sequencing

Transcription

1 Characterization of novel rare genetic variants identified by next generation sequencing Maja Stojiljković, PhD Institute of Molecular Genetics and Genetic Engineering University of Belgrade

2 RARE DISEASES Definition is based on prevalence Europe - rarer than 5 : USA - < Numerous: more than 7000 rare diseases Very heterogeneous: >80% has genetic origin (genetic testing is important) 20% account for infections, allergies etc

3 NEXT GENERATION SEQUENCING TRANSFORMED GENETIC TESTING OF RDs Sequencing High power sequencing is high capacity : billion bp/day Cost has rapidly decreased CHALLENGING DIAGNOSTICS GENETICALLY HETEROGENEOUS DISEASES (defects in multiple genes) WITH OVERLAPING BIOCHEMICAL AND CLINICAL PRESENTATION DISEASES WITH WIDE PHENOTYPIC VARIATION - THE DISEASE MAY TAKE DIFFERENT CLINICAL COURSES EVEN THOUGH THE SAME GENE IS INVOLVED KNOWING WHAT TO TEST, BUT GENETIC TEST IS NOT AVAILABLE LARGE GENES

4 STRATEGIES FOR GENETIC DIAGNOSIS OF RARE DISEASES CUSTOM GENE PANELS Interrogates for known disease associated genes Depth coverage High sensitivity and specificity Data can be examinated for CNV Many patients should be analyzed to be cost effective Limited number of genes (<100) CLINICAL EXOME SEQUENCING Sequencing all exons and flanking intron regions of 4813 clinicaly relevant genes (OMIM) Depth coverage (minimum 20X) Limited number of genes WHOLE EXOME SEQUENCING Interrogates for the protein coding exons (1-2% genome) Is used for detecting known genes and also new disease associated genes Depth coverage is not uniform Detects too many SNV with unclear phenotypic significance WHOLE GENOME SEQUENCING Sequencing all coding and noncoding genomic regions Depth coverage is not uniform Detects too many SNV with unclear phenotypic significance

5 STRATEGIES FOR GENETIC DIAGNOSIS OF RARE DISEASES CUSTOM GENE PANELS Interrogates for known disease associated genes Depth coverage High sensitivity and specificity Data can be examinated for CNV Many patients should be analyzed to be cost effective Limited number of genes (<100) CLINICAL EXOME SEQUENCING Sequencing all exons and flanking intron regions of 4813 clinicaly relevant genes (OMIM) Depth coverage (minimum 20X) Limited number of genes WHOLE EXOME SEQUENCING Interrogates for the protein coding exons (1-2% genome) Is used for detecting known genes and also new disease associated genes Depth coverage is not uniform Detects too many SNV with unclear phenotypic significance WHOLE GENOME SEQUENCING Sequencing all coding and noncoding genomic regions Depth coverage is not uniform Detects too many SNV with unclear phenotypic significance

6 RARE VARIANT - A NEW VARIANT? 1. Use valid HGVS nomenclature 2. Search scientific and medical literature 3. Search databases Population specific to obtain frequencies of variants (no info on functional effect) ExAc, EVS, 1000GP, dbsnp, dbvar Diseases specific contain variants found in patients with disease and assessment of the variants pathogenicity OMIM, HGMD, ClinVar, LSDBs

7 Genetic base of rare metabolic and endocrinological diseases in Serbia Preliminary diagnosis Final diagnosis Analysis Genes All/NEW variants Phenylketonuria PKU Sanger PAH 28/1 (4%) Congenital adrenal hyperplasia Glycogen storage diseases Organic acidurias CAH Sanger CYP21A2 18/2 (11%) GSD Ia GSD Ib GSD III GSD VI MMA MSUD PA Sanger & NGS Sanger & NGS G6PC SLC37A4 AGL PYGL MUT MMAA PCCB BCKDHA BCKDHB 10/3 (30%) 16/8 (50%) AVERAGE 72/14 (20%)

8 KLINIČKI ZNAČAJ DNK VARIJANTE Knowing the effect of the variant is crucial Benign or 100% 0% Pathogenic? *ACMG guidelines Variants of uncertain significance (VUS) should NOT BE USED in clinical decision making and genetic counselling Computational predictions should NOT BE USED as the only evidence to make a clinical assertion. For frequent variants clinical phenotypisation and statistical approach are possible For rare/unique variants, in vitro assay could be the only way to confirm the pathogenic effect In vitro analyses test the drug responsiveness of the variant

9 1. DETERMINE RESPONS TO THE DRUG IN USE 2. NEW VARIANT AS A NEW THERAPEUTICAL TARGET ACCELERATS DRUG DEVELOPMENT BASED ON THE MOLECULAR MECHANISM OF SPECIFIC VARIANTS SPLICING VARIANTS ANTISENSE THERAPY SPLICING FACTORS NONSENSE VARIANTS READTHROUGH DRUGS MISSENSE VARIANTS PHARMACOLOGICAL CHAPERONES VITAMINS/COFACTORS O N O P N O O O N P O Base 2 N

10 For in vitro functional analysis: chose a good cellular model Wellestablished, validated and reproducible functional assays are needed. Transient/stable expression of the aberrant protein using a vector in the cell culture Immortalized cells from a patient biopsied tissue (invasive!) Genome editing of human cell lines with CRISPR/Cas9 technology

11 Splice site variants RT-PCR and subsequent sequencing of cdna confirms splicing defect qrt-pcr informs about future protein quality and quantity truncated protein inactive protein of different size (smaller or longer) inactive or aberrant function

12 Missense variants Investigate its localization in cellular compartments Different biological functions of proteins - is there a partial rescue of the function? Enzymes residual activity Membrane transporters - channel conductance Structure and movement partial function Extracelular communication decreased signaling Histochem Cell Biol 2016

13 Missense variants Investigate its localization in cellular compartments Compare stability and activity of the aberrant protein with the wt protein

14 Missense variants Compare stability and activity of the aberrant protein with the wt protein Investigate its localization in cellular compartments Stability study check degradation of the aberrant protein in time

15 Missense variants Compare stability and activity of the aberrant protein with the wt protein Investigate its localization in cellular compartments Stability study check degradation of the aberrant protein in time Try rescuing the aberrant protein at the lower temperature or with a chaperone

16 Missense variants Compare stability and activity of the aberrant protein with the wt protein Investigate its localization in cellular compartments Stability study check degradation of the aberrant protein in time Try rescuing the aberrant protein at the lower temperature or with a chaperone Try to rescue the aberrant protein with pharmacological chaperone approved in the treatment of patients PKU Tetrahydrobiopterin treatment

17 Why functional characterization is CHALLENGING Each protein is specific, has a specific structure, function, localization, it is engaged in the net of interactions; Therefore, a good knowledge of a diseases and their molecular pathology is required when choosing tools and strategy for variant characterization and when interpreting results phenotypic effect of the variant. There are different types of variants which exert different kind of effects (truncated, unstable, aggregated, dislocated, nonfunctional protein etc) Why functional characterization is IMPORTANT Functional characterization of the novel variant in order to be used in clinical decision making and prenatal diagnostics Analysis of a drug effect on a specific mutation Identification of potential new drugs

18 IMGGE, UNIVERSITY OF BELGRADE Laboratory for Molecular Biomedicine Head: dr Sonja Pavlović Rare Diseases: Molecular Pathophysiology, Diagnostic and Therapeutic Modalities and Social, Ethical and Legal Aspects, research project funded by Ministry of Education and Science, Republic of Serbia (No. III 41004)