SDHB and SDHD Mutation
Analysis in Renal Oncocytomas
and Chromophobe Renal Cell
Carcinomas
Joanne Ramsay
CMGS Spring Conference 2010
Renal Cell Cancer
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Renal cell cancer (RCC) is a heterogeneous
disease that can be classified into several
subtypes
Around 2-3% of all RCC cases are familial
There are several genes reported to be
associated with familial RCCs including
 VHL
 FLCN
(Birt Hogg Dube)
 FH (HLRCC)
 and more recently SDHB (no specific subtype)
Renal Oncocytoma
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Oncocytoma can occur in the thyroid gland, salivary
gland, adrenal gland and the kidney
Renal oncocytomas (ROs) are benign epithelial tumours
that arise from the intercalated cells of the renal collecting
duct
The main characteristics of ROs is the accumulation of
mitochondria in the cell cytoplasm and their deficiency in
electron transport chain complex I
Loss of chromosomes 1 and 14 common but ROs are not
associated with mutations in any one gene
Chromophobe RCC
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Renal Oncocytoma
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Chromophobe RCC
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Chromophobe RCC (CRCC)
and RO have overlapping
morphologies
CRCC can be malignant but
low mortality
Hybrid tumours have been
described
More significant chromosome
loses
Both CRCC and RO have been
reported in patients with SDHB
mutations
Succinate Dehydrogenase
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SDH is a nuclear encoded mitochondrial enzyme
4 subunits, A B C D
SDH acts in Kreb’s cycle to convert succinate to
fumarate
Also acts as complex II in the electron transport chain
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SDHB/SDHC/SDHD have tumour suppressor properties
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Biallelic SDHA mutations cause the genetically
heterogeneous Leigh syndrome
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SDH Associated Phenotypes
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Phaeochromocytoma (PC) and Paraganglioma (PGL)
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Renal Cancer – often with PGL
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No genotype/phenotype correlation to explain why some get
RCC
GIST
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PGL extra adrenal neuroendocrine tumours
PCs are essentially PGLs of the adrenal medulla
Mutations observed in sporadic and familial cases
SDHD associated PGLs/PCs show paternal inheritance
usually in combination with PGL
Cowden Syndrome – PTEN mutations
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Renal/thyroid cancer in ~5% SDH mutation carriers
Aims
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Patient with germline SDHB mutation – later developed a
RO
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LOH of normal allele observed in RO tissue
 SDHB associated ROs described in literature have not been confirmed
by LOH analysis of tumour tissue
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This is the largest cohort of sporadic RO samples
screened for SDHB/SDHD mutations
Hoped to establish genotype/phenotype correlations
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Could then offer SDHB/SDHD mutation screening to patients with
a certain RCC type
Offer predictive testing to at-risk family members
Methods
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DNA from 28 FFPE RO samples and 4 CRCC samples
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From sporadic cases – no FH
Normal tissue supplied for comparison
Use of these tissues for our research was approved by the
Ethical Committee of the Tayside Tissue Bank
Extraction by Pathology (lysis and column clean-up)
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Screened SDHB and SDHD in 13 PCR fragments using
direct bi-directional sequencing
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3 markers at each locus to identify LOH – compared
tumour tissue to normal tissue
Results - Sequencing
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No mutations identified in SDHB or SDHD
Several novel “polymorphisms” identified
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Had problems with DNA quality and quantity
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All deeply intronic and unlikely to be disease causing
SDHD p.His50Arg unclassified variant detected in 2 samples
PCR and sequencing failures
Increased cycles and amount of template DNA
Scored some fragments as ?No mutation detected
Identified skewed polymorphism ratios that indicated
LOH
SDHB c.-37T>C
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Mutation Surveyor DNA
Quantification tool detected
the C peak at 71.34%
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Sample was later comfirmed
to have LOH
SDHD – p.His50Arg
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Identified in both tumour and normal tissues
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Unclassified variant – based on literature and in silico evidence (PolyPhen
predicts damaging, others suggest benign)
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Recent study (Ni et al 2008) suggests Cowden Syndrome
association & did not observe variant in 700 control chromosomes
Family studies would provide best info
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Maternal inheritance goes against pathogenicity
Not possible in the context of this project
Freq. of around 1% in normal controls (Spanish population)
We aim to screen 500 Scottish controls (from Generation
Scotland DNA bank)
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Ethnic origin of the patients in our cohort are unknown
Summary of Variants Detected
Change
Novel
Alamut / Other evidence
c.-37T>C
Yes
No effect on splicing
c.287-26A>G
Yes
No effect on splicing (variant allele
at lower level – LOH)
c.642+17T>C
Reported once
1 algorithm predicted an effect on
splicing (seen in 1% controls)
(Burnichon et al 2009)
c.-139G>T
Reported once
(Burnichon et al 2009)
Seen in patient with pathogenic
VHL mutation
c.200+35G>A
No
Common poly
c.18A>C / p.A6A
No
Common poly
c.201-36T>G
No
Common poly
c.200+33G>A
No
Common poly
c.149A>G /
p.His50Arg (SDHD)
No but known UV
(in 2 RO samples)
Grantham dist: 29, conserved
nucleotide
Results - LOH
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No LOH observed at SDHD
locus
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Chromosome 11 loss not
common in ROs
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About 50% samples had
LOH at SDHB locus
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Marker D1S170 was
unstable in 4 samples
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In MFAP2 gene (expressed in
kidney)
Instability may contribute to
tumourigenesis
Additional work
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To confirm LOH, aCGH analysis was
carried out on 2 samples
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To characterise loss of chromosome 1
To show that the technique works well on FFPE tissue
Identified contamination in one sample –
introduced by pathology!
 Loss of 1p36 confirmed in the other sample
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In process of screening Generation
Scotland DNA controls for the p.H50R
SDHD variant
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Using TaqMan allelic discrimination protocol
Looked at 92 samples so far – p.H50R not detected
Plan to examine at least another 350 samples
Conclusions
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This is the largest cohort of ROs that have been
screened for mutations in SDHB and SDHD
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No mutations identified indicating that mutations in
SDHB and SDHD are not associated with sporadic ROs
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Confirmed that the SDHB region 1p36 is lost in about
50% of ROs
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Should examine all RCC tumour from SDH mutation
carriers for LOH
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Requires good communication between clinicians, pathology and
genetics
Acknowledgements
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Thanks to Michelle, Dave and everyone else in
molecular
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Also to the Stewart Fleming, Leslie Christie and the rest
of the pathology departments in Ninewells and Kirkcaldy
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And to Cytogenetics especially Gordon and Jasbani for
help with aCGH work