1. How do we interpret the variants?

2. Overview How do we prioritize the filtered variants? What filters can be used to identify the causative variants? What databases do we use to interpret filtered variants? How do we interpret the primary variants in relation to the phenotype?

3. How many variants? 40,000 Filtered variants from the exome ~1000 Good quality variants Variants with a HGMD and Clinvar entry Remove variants >10 bp intronic <5% MAF for all variants 1-2 Reportable variants

4. What information is available for the filtered variants? Information regarding the variant Reference Allele Alternative Allele Genomic Location Coding HGVS Protein Change Population Frequencies 1000 Genomes Insilco Programs Location of the variant Segmental duplication Depth of coverage Transcript ID Zygosity OMIM HGMD Type of Variant ExAC ESP Internal Frequency Polyphen Sift Mutation Taster Low Coverage Exon Region of Homology ACMG Incidental Gene Effect

5. Now we are ready for the analysis of the variants within the exome What information can I use to start filtering? – Look at the pedigree (pattern of inheritance)

6. What clinical information do I have…… Pedigree

7. Filtering the variants 892 Filtered variants for the patient 237 Autosomal recessive variants

8. Now we are ready to analyze the variants within the exome What information can I use to start filtering? Look at the pedigree (pattern of inheritance) – Filter the common variants (>1%)

9. How many variants? 892 Filtered variants for the patient 236 Autosomal recessive variants 132 Filter the common variants (>1%)

10. Now we are ready to analyze the variants within the exome What information can I use to start filtering? Look at the pedigree (pattern of inheritance) Filter the common variants (>1%) Look at only the variants with an OMIM entry

11. What is OMIM? Online Mendelian Inheritance in Man (OMIM) Updated catalog of human genes and genetic disorders and traits 8,062/ 23,000 entries in OMIM represented phenotypes

12. OMIM: Cystic Fibrosis

14. How many variants? 892 Filtered variants for the patient 236 Autosomal recessive variants 132 Filter the common variants (>1%) 131 Variants with an OMIM entry

15. Now we are ready to analyze the variants within the exome What information can I use to start filtering? Look at the pedigree (pattern of inheritance) Filter the common variants (>1%) Look at only the variants with an OMIM entry Look at only the variants with a DM entry in HGMD

16. What is HGMD? The Human Gene Mutation Database (HGMD ® ) is a database of gene mutations responsible for causing disease that have been published in the peer-reviewed literature. The database contains over 141,000 different mutations detected in over 5,700 different genes.

17. HGMD screenshot for LRRK2

20. Categories of mutations and polymorphsims

21. How many variants? 892 Filtered variants for the patient 236 Autosomal recessive variants 132 Filter the common variants (>1%) 131 Variants with an OMIM entry 2 Variants with a DM entry in HGMD

22. What do we know about Norum Disease Clinical Features of our patient Global developmental delay Delayed speech and language development Rigidity Poor muscle coordination Hypertelorism Delayed fine motor development Delayed gross motor development Blue Sclerae Clinical Features of Norum Disease Renal failure Hemolytic anemia Corneal lipid deposits Increased plasma free cholesterol Increased plasma triglycerides Increased plasma phospholipids ✘ ✘ ✘ ✘ ✘ ✘

23. What do we know about Pantothenate kinase-associated neurodegeneration (PKAN)? PKAN is a disorder of the nervous system. Characterized by an abnormal buildup of iron in certain areas of the brain. PANK2 is the only gene in which mutation is known to cause PKAN. PKAN is inherited in an autosomal recessive manner

24. Comparison of clinical features of PKAN and our patient Clinical Features of our patient Global developmental delay Delayed speech and language development Rigidity Poor muscle coordination Hypertelorism Delayed fine motor development Delayed gross motor development Blue Sclerae Clinical Features of PKAN Cognitive decline Speech abnormalities Stiffness Decreased muscle mass Ataxia Dementia, progressive Decreased muscle mass MRI shows decreased signal intensity in the pallidal nuclei with central hyperintensity ('eye of the tiger' sign) ✔ ✔ ✔ ✔ ✔

25. ‘Eye of the tiger' Arrow indicates the 'eye of the tiger' change characteristic of PKAN.

26. Comparison of clinical features of PKAN and our patient Clinical Features of our patient Global developmental delay Delayed speech and language development Rigidity Poor muscle coordination Hypertelorism Delayed fine motor development Delayed gross motor development Blue Sclerae Clinical Features of PKAN Cognitive decline Speech abnormalities Stiffness Decreased muscle mass Ataxia Dementia, progressive Decreased muscle mass MRI shows decreased signal intensity in the pallidal nuclei with central hyperintensity ('eye of the tiger' sign) ✔ ✔ ✔ ✔ ✔ ✔

27. How many variants? 892 Filtered variants for the patient 236 Autosomal recessive variants 132 Filter the common variants (>1%) 131 Variants with an OMIM entry 2 Variants with a DM entry in HGMD 1 Reportable variant

28. ACMG guidelines used to classify the PANK2 variant

29. Very strong evidence of a Pathogenic variant (PVS) – Any variant that is truncating in a gene where loss of function is a known mechanism of disease – PVS1 Moderate evidence of pathogenicity (PM) – Absent from control databases (ExAC, ESP) – PM2

30. Research Report

31. Sanger confirmation in a clinical laboraotry c.828_829del (p.Cys276fs*)/ c.828_829del (p.Cys276fs*)

32. Report issued to the family

33. Summary Discussed the different filters and databases that can be used to assess the variants in an exome Reviewed how the laboratory classifies and interprets the primary variants in relation to the phenotype Reviewed how variants are reported through a research and clinical reports.