Fumarate Hydratase; One Gene, Two Inheritance Patterns and Two or Three Diseases

1. Fumarate Hydratase; One Gene, Two Inheritance Patterns and Two or Three Diseases Oliver Ridgway West Midlands Regional Genetics Laboratory

2. Fumarate hydratase (FH) 1q42.1 10 exons ~22 kb of DNA Transcript length of 1,790bp 510 amino acids Cytosolic and mitochondrial forms Transcribed from the same locus (post-translational processing) Different electrophoresis mobility Presence of a mitochondrial signal peptide Brain – mitochondrial only FH enzyme formed from a homotetramer Two substrate binding sites (A and B) Site A – catalytic Site B – substrate binding-activation To date (18/02/09), 111 independent FH mutations reported on the ‘Leiden Open Variation Database’ Alam et al, 2005

3. Fumarate hydratase (FH) Functions in the Krebs cycle Fumarate is hydrated to form malate by fumarate hydratase (fumarase) The WMRGL already offers testing for mutations in the subunits of succinate dehydrogenase (SDHB, C and D) Therefore FH analysis is a logical addition to our repertoire www.iturrate.com

4. 2 or 3 conditions? • Different patterns of inheritance lead to different diseases • Autosomal recessive • Fumarate Hydratase Deficiency (OMIN: 606812) • Autosomal dominant • Hereditary Leiomyomatosis and Renal Cell Cancer (OMIN: 605839) • Multiple Cutaneous and Uterine Leiomyomata (OMIN: 150800)

5. Fumarate Hydratase Deficiency (FHD) - condition 1 FHD Autosomal recessive Homozygous / compound heterozygous FH mutations Early-onset, failure to thrive, hypotonia, cerebral atrophy, mental retardation Death in the first decade of life OMIM – Phoenix New Times, 12/29/2005 ‘Forbidden fruit: inbreeding among polygamists along the Arizona-Utah border is producing a cast of severely retarded and deformed children’ An unusually high incidence of FHD in the south-western United states among member of the fundamentalist Church of Jesus Christ of Latter Day Saints, a religions community that practices inbreeding and polygamy. The genetic defect was traced to one of the communities founder and the first of his plural wives (14 children) Disease mechanism somewhat overlooked in the literature ATP deficiency Backlog of metabolites

6. Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) – condition 2 HLRCC Autosomal dominant Germline mutation followed by a second hit Cutaneous leiomyomata Skin coloured / brown papules / nodes Trunk, extremities and occasionally face Mean age of 25 years Increase in size and No. with age Uterine leiomyomata (fibroids) Almost all females 18-52 years Renal tumour 10-16% of HLRCC Usually unilateral Aggressive Type 2 papillary / collecting duct morphology Mean age of 44 years Jorge et al, 2003 Leiomyoma – a benign tumour (as a fibroid) consisting of smooth muscle fibres

7. Multiple Cutaneous and Uterine Leiomyomata (MCUL) - ? condition 3 MCUL Same phenotype as HLRCC but -ve for renal tumours However renal tumours only present in 10-16% of HLRCC cases A case can be made for two conditions being considered as one MCUL individuals are merely HLRCC affected patients in whom renal cell cancer has not developed

8. Mechanism of disease in HLRCC / MCUL, the pseudo-hypoxic drive VGEF = angiogenesis PDGF / TGFα = growth stimulation GLUT1 = glucose transport Loss of 2nd FH allele Excess Succinate / Fumarate inhibit HIF prolyl hydroxylase Over expression of HIF1α Up-regulation of; VGEF, PDGF, TGFα and GLUT1 Adapted from Sudarshan et al, 2007

9. Primers Supplied by Chris Ricketts (Institute of Cancer Studies, Bham Uni) Worked first time but Not M13 tagged SNP found under exon 6 reverse primer Ordered M13 tagged primers Exon 2 stopped working Used combinations of forward and reverse primers Supplied with an empty tube for the forward primer

10. Exon 9 unidirectional Poly CT tract before exon 9 Moving the primer inward would result in the omission of the first portion of the exon

11. Positive control Positive control sample was obtained Confirmation a mutation previously identified in a research setting Exon 8, missense c.1189G>A p.Gly397Arg

12. Referred patients Male, 42 years (A) – Histologically proven cutaneous leiomyomata Female, 47 (B) (sister of A) - Histologically proven cutaneous leiomyomata and possible uterine fibroids Many other family members with skin lesions and aunt recently died of cancer (no details or sample available) A: c.698G>A (p.Arg233His) -heterozygous B: c.698G>A (p.Arg233His) - heterozygous

13. c.698G>A (p.Arg233His) Leiden Open Variation Database 2 x in HLRCC / MCUL 3 x in FHD Literature search Alam et al, 2005 – Arg233 found to be conserved across species Tomlinson et al, 2002 – p.Arg233His found in a woman with 15 cutaneous leiomyomata Toro et al, 2003 – p.Arg233His found in 11/35 families with cutaneous leiomyomata ‘This result is consistent with a clinical diagnosis of HLRCC/MCUL in this patient. Based on this result it is possible to offer testing to other family members’

14. A further 5 patients have been screened All so far tested negative, expected for some but: 45 year old female – uterine fibroids and a strong family history of cancer 47 year old female – multiple cutaneous leiomyomas and uterine fibroids, mother also has uterine fibroids Next – MLPA (P198) Deletions account for ~ 4% Duplications account for ~ 2% Need for a positive control Referred patients Bayley et al, 2008

15. Conclusion • The WMRGL is now offering screening for mutations in fumarate hydratase • Currently sequencing only • MLPA analysis to be validated • Potential to test samples which have tested negative for other conditions with a clinical overlap • Von Hippel-Lindau syndrome (VHL) • Hereditary papillary renal cancer (HPRC) • Birt-Hogg-Dube syndrome (BHD)

16. Acknowledgments Dr Chris Ricketts – Cancer Research Studies, University of Birmingham Dr Fiona Macdonald – WMRGL Jennie Bell – WMRGL St George’s Hospital, London