Transcript one gene, two inheritance patterns and two or three diseases

Fumarate Hydratase; One Gene,
Two Inheritance Patterns and Two
or Three Diseases
Oliver Ridgway
West Midlands Regional
Genetics Laboratory
Fumarate hydratase (FH)
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1q42.1
10 exons
~22 kb of DNA
Transcript length of 1,790bp
510 amino acids
Cytosolic and mitochondrial forms
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Transcribed from the same locus (posttranslational processing)
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Different electrophoresis mobility
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Presence of a mitochondrial signal peptide
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Brain – mitochondrial only
FH enzyme formed from a homotetramer
Two substrate binding sites (A and B)
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Site A – catalytic
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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)
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Functions in the Krebs cycle
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Fumarate is hydrated to form
malate by fumarate hydratase
(fumarase)
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The WMRGL already offers
testing for mutations in the
subunits of succinate
dehydrogenase
(SDHB, C and D)
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Therefore FH analysis is a
logical addition to our repertoire
www.iturrate.com
2 or 3 conditions?
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Different patterns of inheritance lead to
different diseases
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Autosomal recessive
 Fumarate Hydratase Deficiency
(OMIN: 606812)
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Autosomal dominant
 Hereditary Leiomyomatosis and Renal
Cell Cancer (OMIN: 605839)
 Multiple Cutaneous and Uterine
Leiomyomata (OMIN: 150800)
Fumarate Hydratase Deficiency (FHD)
- condition 1
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FHD
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Autosomal recessive
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Homozygous / compound heterozygous FH mutations
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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
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HLRCC
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Autosomal dominant
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Germline mutation followed by a second hit
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Cutaneous leiomyomata
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Skin coloured / brown papules / nodes
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Trunk, extremities and occasionally face
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Mean age of 25 years
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Increase in size and No. with age
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Uterine leiomyomata (fibroids)
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Almost all females
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18-52 years
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Renal tumour
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10-16% of HLRCC
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Usually unilateral
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Aggressive
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Type 2 papillary / collecting duct morphology
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Mean age of 44 years
Jorge et al, 2003
Leiomyoma – a benign tumour (as a
fibroid) consisting of smooth muscle
fibres
Multiple Cutaneous and Uterine
Leiomyomata (MCUL) - ? condition 3
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MCUL
 Same phenotype as HLRCC but -ve
for renal tumours
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However renal tumours only present
in 10-16% of HLRCC cases
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A case can be made for two
conditions being considered as one
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MCUL individuals are merely HLRCC
affected patients in whom renal cell
cancer has not developed
Mechanism of disease in HLRCC / MCUL,
the pseudo-hypoxic drive
VGEF = angiogenesis
PDGF / TGFα = growth stimulation
Loss of 2nd FH allele
Excess Succinate /
Fumarate inhibit HIF
prolyl hydroxylase
Adapted from
Sudarshan et al, 2007
GLUT1 = glucose transport
Over expression of
HIF1α
Up-regulation of;
VGEF, PDGF, TGFα
and GLUT1
Primers
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Supplied by Chris Ricketts (Institute
of Cancer Studies, Bham Uni)
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Worked first time but
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Not M13 tagged
SNP found under exon 6
reverse primer
Ordered M13 tagged primers
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Exon 2 stopped working
Used combinations of forward
and reverse primers
Supplied with an empty tube for
the forward primer
Exon 9 unidirectional
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Poly CT tract before exon 9
Moving the primer inward would result in the omission of the first
portion of the exon
Positive control
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Positive control sample was obtained
Confirmation a mutation previously identified in a research
setting
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Exon 8, missense
c.1189G>A
p.Gly397Arg
Referred patients
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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)
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Leiden Open Variation Database
 2 x in HLRCC / MCUL
 3 x in FHD
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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
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‘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
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A further 5 patients have been screened
All so far tested negative, expected for some but:
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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)
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Deletions account for ~ 4%
Duplications account for ~ 2%
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Need for a positive control
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Bayley et al, 2008
Conclusion
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The WMRGL is now offering screening for mutations in
fumarate hydratase
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Currently sequencing only
MLPA analysis to be validated
Potential to test samples which have tested negative
for other conditions with a clinical overlap
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Von Hippel-Lindau syndrome (VHL)
Hereditary papillary renal cancer (HPRC)
Birt-Hogg-Dube syndrome (BHD)
Acknowledgments
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Dr Chris Ricketts – Cancer Research Studies, University
of Birmingham
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Dr Fiona Macdonald – WMRGL
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Jennie Bell – WMRGL
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St George’s Hospital, London