Fumarate Hydratase; One Gene, Two Inheritance Patterns and Two

or Three Diseases

Oliver Ridgway

West Midlands Regional Genetics Laboratory

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

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

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)

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

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

Leiomyoma – a benign tumour (as a fibroid) consisting of smooth muscle

fibres

Jorge et al, 2003

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

Excess Succinate / Fumarate inhibit HIF prolyl hydroxylase

Loss of 2nd FH allele

Over expression of HIF1α

Up-regulation of; VGEF, PDGF, TGFα

and GLUT1

Mechanism of disease in HLRCC / MCUL, the pseudo-hypoxic drive

VGEF = angiogenesis

PDGF / TGFα = growth stimulation

GLUT1 = glucose transport

Adapted from Sudarshan et al, 2007

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

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

Positive control

Positive control sample was obtained Confirmation a mutation previously identified in a research

setting

Exon 8, missense c.1189G>A p.Gly397Arg

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

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’

Referred patients

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

Bayley et al, 2008

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)

Acknowledgments

Dr Chris Ricketts – Cancer Research Studies, University of Birmingham

Dr Fiona Macdonald – WMRGL

Jennie Bell – WMRGL

St George’s Hospital, London