Genetic Testing in Common Disorders of Coagulation Tahnee Causey, ms, lcgc Genetic counselor Division of clinical genetics, department of human & Molecular genetics November 1, 2017

Evolution of DNA to Protein

F8 and F9 genes F8 gene is at the telomeric end of X (Xq28) F9 is more proximal at Xq27.1 F8 has 26 exons F9 has 8 exons, with exon 8 coding for ~50% of gene

Factor VIII gene (F8) 2 notable introns in F8 gene—intron 1 and intron 22 These introns have nucleotide sequences that are repeated outside of the F8 gene at telomeric end of X X chromosome can bend and align in these regions of homology If crossing over occurs, results in a disrupted F8 gene

Inversion in intron 22 F8

Inversion Intron 22 Result of inversion is complete disruption of F8 gene No FVIII is made as the exons are completely separated—exons 1-22 are at one end of X chromosome and exons 23-26 stay in position Inversion results in severe FVIII deficiency, phenotype, genotype correlation Inversion in intron 22 accounts for ~45-48% of severe factor VIII deficiency

Inversion in Intron 1 of F8 Similar issues develop with intron 1 inversion Region of homology near telomere and within intron 1 of F8

Inversion in intron 1 of F8 Result of inversion is a gene that is completely disrupted No factor VIII is made as the exons are completely separated—exon 1 is at one end of X chromosome and exons 2-26 stay in position All inversion intron 1 pts will be severe FVIII def. Inversion in intron 1 accounts for ~2% of severe factor VIII deficiency

Additional Mutations in F8 Outside of common inversions, there are no other common mutations that account for a large percentage of F8 mutations Missense, nonsense, deletion, splice site mutations have all been documented

Molecular Genetics of FVIII Deficiency CDC database of mutations within the F8 contains ~3000 mutations

Mutations within F8 gene For severe hemophilia A, inversions account for up to 50% cases, remainder have many different types of mutations For mild and moderate hemophilia A, almost all cases are due to missense mutations Mutations can be identified in up to 98% of affected individuals Unidentified mutations may be in introns

What genetic tests to order in FVIII Deficiency? In pt with severe factor VIII, start with inversion testing for intron 22/1, reflex to full gene sequencing For mild-moderate factor VIII, start with sequencing Can reflex to deletion/duplication if needed

What does F8 genetic testing cost? Depends upon many factors-lab, pt insurance, institution, etc. For inversion testing only (intron 22 or 1 or both), ~$200-$400 Full gene sequencing additional $700-$1000 Deletion/duplication additional $600-$1000 Targeted variant analysis $250-$400 Institutional vs direct insurance vs self pay Insurance preauth and out of pocket expense

F9 gene F9 gene is smaller than F8 No notable introns/exons and no issues with external sequence homology in F9 No common mutations or mechanisms that account for a large percentage of variants as in F8 There is no mutational hot spot within F9

Molecular Genetics of FIX Deficiency >1100 mutations identified in F9 in CDC database

Mutations in F9 Majority of mutations are point mutations Mutations leading to severe disease are often nonsense, promoter mutations, splice site defects Deletions and insertions have also been seen in F9 Mutation detection up to 98-99%

What genetic tests to order? For factor IX, sequencing is primary test May reflex to deletion/duplication if sequencing is negative

How much does F9 genetic testing cost? Full gene sequencing $700-$1000 Deletion/duplication additional $600-$1000 Targeted variant analysis $250-$400 Institutional vs direct insurance vs self pay Insurance preauth and out of pocket expense

Why pursue genetic testing? WHO recommends identification of disease-causing mutation in individuals with FVIII/IX deficiencies Factor levels in females are less reliable for carrier testing than molecular testing Genotype-phenotype correlations Emerging correlations in specific mutations and inhibitor development risk Targeted molecular medicine

How to determine hemophilia carrier status A woman who has a son and a brother with hemophilia is an obligate hemophilia carrier A woman who has two sons with hemophilia is assumed to be a hemophilia carrier (germline mosaicism) A woman whose father has hemophilia is a known carrier

How to determine carrier status 1/3 to 1/2 of males have no family hx of hemophilia When a woman has a son with hemophilia, there is an 80% (4/5) chance that a woman will be a hemophilia carrier When a son has an intron 22 inversion, there is a 98% chance that the mother is a carrier For index cases, either proband has de novo mutation, mom has a de novo mutation, or mom has a mutation she inherited that occurred in her father’s sperm

How to determine carrier status Genetic testing for known hemophilia mutation is the only reliable method of determining carrier status Testing an affected male first is optimal

Genotype-Phenotype Correlations Males within same family will generally have same approximate disease severity as they all carry the same mutation, some variability Unrelated males with the same mutation will also usually have a comparable disease severity (e.g. inversion 22/1)

Genotype-Phenotype Correlations Null mutations are usually severe in phenotype

Genotype-Phenotype Correlations Missense mutations (amino acid is replaced by another—often a single nucleotide base substitution) Some missense mutations recur frequently, most infrequent/rare

Mutation type and Inhibitor Risk CDC-funded study (HIRS) looked at mutations in F8 and F9 genes and assessed risk of inhibitor development by type/location of mutation In hem A: deletions, inversion 22, nonsense mutations were all statistically more likely to develop inhibitors In hem B, only 2 in their study had inhibitors, but both of these had severe disease, one had a complete F9 gene deletion and one had large partial gene deletion

Mutation Type and Inhibitor Risk In mild/moderate hemophilia, most mutations are not null mutations, meaning some factor made 2013 article in Blood found a subset of 19 missense mutations that were associated with an increased risk for inhibitor development in non-severe hemophilia A (Blood, 12 Sept 2013 x Vol 122, # 11) Common unifying factor among these mutations was their location within the F8 gene

Hemophilia B Leyden Up to 3% of FIX patients have levels that increase in puberty due to Hemophilia B Leyden Individuals have mutations within the promoter upstream of F9 Promoters are regulatory elements that initiate transcription of a gene

Hemophilia B Leyden These promotor mutations have similar elements to the androgen receptor transcription factor (ARTF), which binds to DNA and initiates transcription of a gene in the presence of testosterone At puberty, testosterone levels inc, this mutation can lead to AR TF binding, inc F9 transcription, less bleeding

What happens once you decide to pursue molecular genetic testing? Identify a reputable, CLIA certified lab https://www.ncbi.nlm.nih.gov/gtr/ Blood Center of Wisconsin Ensure that lab can perform full gene sequencing and deletion/duplication analyses, not just inversion testing Call Genetics! Order the appropriate testing Start with inversion testing for severe FVIII Don’t perform full gene sequencing when you know the familial mutation Order appropriate gene

Variant Interpretation Once a sequence variant is identified, lab then determines whether it is likely disease causing Has it ever been reported in hemophilia? What does mutation do to protein? What type of mutation is it? Available mutation databases for hem A and B CDC CHAMP F8 mutation list (>2000 F8 mutations) CDC CHBMP F9 mutation list (>1000 F9 mutations) ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) F8 Variant database (http://www.factorviii-db.org/) F9 variant database (http://www.factorix.org/)

Variant Interpretation Pathogenic / Likely pathogenic Variant of uncertain clinical significance Likely benign / Benign Once a pathogenic mutation identified, other at risk carriers or males can be tested Female’s carrier status should not be determined based upon a VUS

Molecular Genetics of VWD VWF is located at 12p13.31 Mutations within VWF can result in qualitative or quantitative issues with VWF

Molecular Genetics of VWD VWF spans 178 Kb and 52 exons Partial pseudogene on chr 22 Benign variants are extremely common, with over 200 documented,10% of individuals with VWD type 1 have more than one variant identified, either in cis or trans Over 700 pathogenic mutations identified in VWF Most mutations in VWF are missense, although null mutations reported Genetic testing in vWD not as commonly performed as in Hem A and B

Molecular Genetics of VWD type 1 Variety of published studies identified a mutation in VWF in 2/3 pts with type 1 VWD, 1/10 may have more than one possible mutation In ~1/3 of pts with VWF:Ag levels >30% no mutations were identified If stringent criteria of VWF:Ag levels <30% used, detection rate of pathogenic variant is reported as high as 87%

Molecular Genetics of VWD type 2 Genetic testing can identify a mutation in >90% of individuals with type 2 VWD Type 2A: Mutations primarily located in exon 28, some in exons 11-16, 22, 25-27, and 52 Type 2B: Mutations primarily in exon 28

Molecular Genetics of VWD type 2 Type 2M: Mutations primarily in exon 28, some in exons 29-32 Type 2N: Most mutations in exons 18-20, lower proportion in exons 17 and 24-25; affected FVIII binding

Molecular Genetics of VWD type 3 Mutations are detected in >90% of individuals with type 3 VWD, entire coding region 82% of mutations were null mutations (7% large deletions) Offspring may have mild type 1 VWD

Molecular Genetics of VWD

Platelet type pseudo-VWD PT-VWD: Clinical presentation similar to type 2B, results from mutations in GP1BA Up to 15% of pts dx’d with 2B VWD may have mutation in GP1BA

Why pursue genetic testing for VWD? Accurate diagnosis and recurrence risk estimation Testing for types 2N and 2B may impact treatment Large deletion mutations in type 3 may indicate an increase risk for developing antibodies and anaphlylaxis

How much does VWF genetic testing cost? Full VWF sequencing $1300-$3000 Deletion/duplication an additional $600-1000 Single exon (28) $700 and up

Does insurance cover genetic testing? Depends upon insurance policy—very few insurance policies have direct policies that address F8, F9, VWD Biggest questions insurance companies ask How will this information affected medical management? Can you make the diagnosis biochemically? Will this information improve clinical outcomes?