Pediatr Blood Cancer 2011;56:379–383

Isocitrate Dehydrogenase 1/2 Mutational Analyses and 2-Hydroxyglutarate

Measurements in Wilms Tumors

Dinesh Rakheja,

MD

,

1,

* Midori Mitui,

PhD

,

2

Richard L. Boriack,

MS

,

2

and Ralph J. DeBerardinis,

MD,PhD

3

Background. L-2-Hydroxyglutaric aciduria (L-2-HGA) is an

uncommon inborn error of metabolism, in which the patients
are predisposed to develop brain tumors.Elevated levels of D-2-
hydroxyglutarate have been demonstrated with malignant gliomas
and myeloid leukemias associated with somatic mutations of
the genes encoding NADP(

+)-dependent isocitrate dehydrogenases

(IDH1 and IDH2, respectively).Recently, we noted a Wilms tumor
in a child with L-2-HGA.Given the accumulating evidence that
both enantiomers of 2-hydroxyglutarate are associated with cellu-
lar transformation, we investigated if sporadic Wilms tumors are
associated with IDH1 or IDH2 mutations or with elevated levels
of 2-hydroxyglutarate. Procedure. We retrieved 21 frozen Wilms
tumor tissues.In 20 cases, we sequenced exon 4 and flanking
intronic regions of IDH1 and IDH2.In all 21 cases, we measured

2-hydroxyglutarate levels by liquid chromatography-tandem mass
spectrometry. Results. We did not find mutations at the hot spots
IDH1 codon 132 or IDH2 codon 172.Two cases (1 with favorable
histology and 1 with unfavorable histology) showed heterozy-
gous change c.211G

>A (p.Val71Ile) in IDH1, a change previously

reported as a mutation but listed as a single nucleotide polymor-
phism in the NCBI SNP database.We did not find increased levels of
2-hydroxygluatric acid in any sample. Conclusions. Our results sug-
gest that IDH1 codon 132 or IDH2 codon 172 mutations or elevated
2-hydroxyglutarate levels do not play a role in the biology of sporadic
Wilms tumors.The significance of heterozygous change c.211G

>A

(p.Val71Ile) in IDH1, seen in two tumors, is not clear.Pediatr Blood
Cancer 2011;56:379–383.

© 2010 Wiley-Liss, Inc.

Key words: 2-hydroxyglutarate; isocitrate dehydrogenase; Wilms tumor

INTRODUCTION

L-2-hydroxyglutaric aciduria (L-2-HGA, OMIM #236792) and

D-2-hydroxyglutaric aciduria (D-2-HGA, OMIM #600721) are
uncommon inborn errors of metabolism that are biochemically
characterized by elevated tissue and body fluid levels of L-2-
hydroxyglutarate (L-2-HG) and D-2-hydroxyglutarate (D-2-HG),
respectively [1,2]. A third related disorder, distinguished by ele-
vated levels of both L-2-HG and D-2-HG, has also been described,
although the molecular etiology is unknown [3]. At least nine
patients with L-2-HGA have been reported to have brain tumors
[4–9], and a right frontal bone osteoma has been described in one
patient [10]. Further, elevated levels of D-2-HG have been demon-
strated with malignant gliomas and myeloid leukemias associated
with acquired mutations of the genes encoding the cytoplasmic and
mitochondrial isoforms of NADP(

+)-dependent isocitrate dehy-

drogenases (IDH1 and IDH2, respectively). These mutations have
almost exclusively been observed in codon 132 of IDH1 or codon
172 of IDH2 and produce an enzyme with decreased ability to
convert isocitrate to alpha-ketoglutarate (

␣-KG) but with increased

ability to convert

␣-KG to D-2-HG [11–14].

We identified a patient with L-2-HGA who unexpectedly devel-

oped Wilms tumor [15]. Given the accumulating evidence that both
enantiomers of 2-HG are associated with malignant cellular trans-
formation, we investigated if sporadic Wilms tumors are associated
with elevated levels of 2-HG or with IDH1 or IDH2 mutations.

METHODS

Wilms Tumors

After approval by the UT Southwestern Institutional Review

Board and the Children’s Medical Center Tissue Resource Uti-
lization Committee, we retrieved 21 freshly frozen Wilms tumor
tissues from the Children’s Medical Center Pediatric Biospecimen
Repository.

IDH1/IDH2 Mutational Analyses

Twenty-five milligrams of frozen Wilms tumor tissue was

homogenized in 600

␮l Buffer RLT Plus (Qiagen, Valencia, CA)

using Omni TH tissue homogenizer fitted with disposable plas-
tic soft tissue generator probes. Twenty-five microliters of the
homogenate was used for measurement of 2-HG (see below). From
the remainder of the homogenate, DNA was extracted using AllPrep
DNA/RNA Mini Kit (Qiagen) in 100

␮l of elution volume. Con-

centration of the extracted DNA was measured using NanoDrop
2000c (Thermo Scientific, Wilmington, DE). PCR amplification
was performed with HotStarTaq Plus Master Mix (Qiagen), using
primers targeting exon 4 and flanking intronic regions of IDH1
and IDH2. The primer sequences for PCR and sequencing were:
IDH1-Exon 4 (F): GCCAGTGCTAAAACTTGGCAG, IDH1-Exon
4 (R): TCAATTTCATACCTTGCTTAATGGG, IDH2-Exon 4 (F):
GCAGACTCCAGAGCCCACAC, and IDH2-Exon 4 (R): TGC-
CATCTTTTGGGGTGAAG. The PCR reaction contained 400 nM
of each primer, 0.2 mM of dNTPs, 1.5 mM MgCl

2

, 2 U of Hot Start

Taq DNA polymerase, and 50 ng of DNA template. The cycling
condition was: 95

◦

C/5 min; 35 cycles 94

◦

C/10 sec, 50

◦

C/30 sec,

72

◦

C/30 sec; final extension 72

◦

C/7 min. After PCR amplification,

amplicons were purified using QIAquick PCR Purification Kit
(Qiagen). Sequencing reaction and capillary electrophoresis were

1

Department of Pathology, Children’s Medical Center and UT South-

western Medical Center, Dallas, Texas;

2

Department of Pathology,

Children’s Medical Center, Dallas, Texas;

3

Department of Pediatrics,

Children’s Medical Center and UT Southwestern Medical Center, Dal-
las, Texas

Conflict of interest: nothing to declare.

*Correspondence to: Dinesh Rakheja, Department of Pathology, MC
9073, UT Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, TX 75390, USA. E-mail: [e-mail]

Received 10 March 2010; Accepted 18 May 2010

© 2010 Wiley-Liss,Inc.
DOI 10.1002/pbc.22697
Published online 22 November 2010 in Wiley Online Library
(wileyonlinelibrary.com).

380

Rakheja et al.

performed at UT Southwestern Sequencing Core facility and com-
pared to NCBI reference sequences NM 005896.2 (IDH1) and
NM 002168.2 (IDH2). Position 1 corresponds to the first A of the
ATG translation-initiating codon.

Measurement of 2-Hydroxyglutaric Acid

Chemicals and reagents. Solvents were all Optima LC:MS

grade (Fisher Scientific, Fair Lawn, NJ) where available. Zinc
powder was from Riedel-de-Ha¨en (Seelze, Switzerland); ammo-
nium formate and formic acid from Fluka (Steinheim, Germany);
2-ketopentanedioic acid-[

2

D

6

] from Isotec (St. Louis, MO); (

+)-

Di-O-acetyl-L-tartaric anhydride (DATAN), L-2-HG, and D-2-HG
were from Sigma–Aldrich (St. Louis, MO); acetic acid was from
JT Baker (Phillipsburg, NJ); and 6 N hydrochloric acid was from
Ricca (Arlington, TX). The internal standard (IS) D,L-[1,3,3,4,4,5-

2

D

6

]-2-hydroxyglutaric acid–zinc salt was prepared in-house as

previously described [16]. Briefly, 150 mg 2-ketopentanedioic acid-
[

2

D

6

] diluted in 0.5 ml water was slowly added to 120 mg zinc

powder in 1 ml ice-cold water. The mixture was stirred on ice for
30 min and excess zinc removed by centrifugation. The zinc pel-
let was washed two times with ice-cold water and the washes were
combined with the original supernatant. D,L-[1,3,3,4,4,5-

2

D

6

]-2-

hydroxyglutaric acid–zinc salt was precipitated with the addition of
three volumes of acetone followed by centrifugation. The pellet was
washed two times with 10 ml acetone, dried under a gentle stream
of nitrogen at 37

◦

C, and stored at

−20

◦

C.

Preparation of standards. For preparing standard curves,

10

␩mol, 1 ␩mol, 100 ρmol, and 10 ρmol of L-2-HG and D-2-HG (in

aqueous solutions) were added to separate 16

× 100 mm screw cap

tubes. 1

␩mol IS was added to each. The standards were vortexed,

dried under a gentle stream of nitrogen at 37

◦

C, and derivatized with

DATAN.

Metabolite extraction from tissues. Methanol extraction was

performed using a modification of a previously described method
[17]. Briefly, 25

␮l of homogenized tissue (see above), 1 ␩mol IS,

and 300

␮l methanol:water (80:20) pre-cooled on dry ice were added

to a 1.5 ml polypropylene microcentrifuge tube, vortexed, held on
dry ice for 30 min, and centrifuged at 13,300g for 5 min at 4

◦

C.

The supernatant was transferred to a 16 mm

× 100 mm screw cap

tube and the pellet extracted with 200

␮l methanol:water (80:20)

as before. The supernatants were combined, dried under a gentle
stream of nitrogen at 37

◦

C, and derivatized with DATAN.

DATAN derivatization. To each tube of dried standards and

tissue extracts was added 50

␮l of freshly made DATAN solution

(50 mg/ml in methylene chloride:acetic acid, 80:20). Derivatization
proceeded at 75

◦

C for 30 min, after which the tubes were cooled

to room temperature and the solutions dried under a gentle stream
of nitrogen. The dried residue was dissolved in 0.5 ml water and
centrifuged to remove any precipitate.

Measurement of 2-HG enantiomers by liquid chromato-

graphy-tandem mass spectrometry (LC-MS/MS). LC-MS/MS
analyses was adapted from a previously described method [18].
Twenty microliters aliquot of the derivatized samples were injected
for chromatographic separation on an Agilent Hypersil ODS 4.0

×

250 mm, 5

␮m column (Santa Clara, CA). The column oven was set

at 30

◦

C. Solvent A was 125 mg/L ammonium formate, pH

= 3.6,

solvent B was acetonitrile. The enantiomers were eluted using
the program: 0–0.5 min—100% A, 0.5–3.5 min—linear gradient to
96.5% A, 3.5–25 min—96.5% A and 3.5% B. The flow rate was

TABLE I. Results of Sequencing of Exon 4 and Flanking Intronic
Regions of IDH1 and IDH2 in Wilms Tumor Tissues

Case no. Gene Exon/intron Nucleotide change Amino acid change

2

IDH1

4

c.315C

>T het

p.Gly105Gly

4

IDH1

4

c.315C

>T het

p.Gly105Gly

5

IDH1

4

c.211G

>A het

p.Val71Ile

IDH1

4

c.315C

>T het

p.Gly105Gly

8

a

IDH1

4

c.211G

>A het

p.Val71Ile

IDH1

4

c.315C

>T het

p.Gly105Gly

15

IDH1

4

c.315C

>T het

p.Gly105Gly

17

IDH1

4

c.315C

>T het

p.Gly105Gly

16

IDH2

IVS 4

c.535-40G

>A het

NA

a

Wilms tumor with unfavorable histology (diffuse anaplasia).

set at 0.5 ml/min, 50% of the postcolumn eluate was diverted to
waste, and the remainder injected into API 3000 triple-quadrupole
mass spectrometer equipped with an electrospray ionization source
(Applied Biosystems, Foster City, CA) and operating in negative ion
mode. Nebulizer gas was set at 8 L/min, the temperature at 500

◦

C,

and the ionspray voltage at

−4,200 V. Product ion transitions were

monitored at 363.2 to 147.2 for L-2-HG and D-2-HG and at 367.1
to 151.1 for the IS.

RESULTS

Wilms Tumors

The 21 cases of Wilms tumors were received between 2004

and 2008. The patient age at the time of surgical procedure ranged
from 9 months to 8 years/5 months (mean 3 years/1 month, median
3 years/11 months). There were 15 males and 6 females. His-
tologically, 18 tumors showed favorable histology and 3 showed
unfavorable histology (diffuse anaplasia). The surgical procedure
was nephrectomy in 18 cases, biopsy in 2 cases, and resection of
lung metastasis in 1 case. Of the 18 resections, 1 was staged at local
stage I, 9 at local stage II, and 8 at local stage III. Of the 20 primary
tumors, 17 were unifocal, 1 was multifocal (unilateral), and 2 were
bilateral.

IDH1/IDH2 Mutational Analyses

Adequate DNA was available in 20 out of the 21 Wilms

tumors. We sequenced exon 4 and flanking intronic regions of
IDH1 and IDH2 to look for the mutations that have previously
been noted in malignant gliomas and myeloid leukemias [19–
22]. Sequence analysis did not reveal mutations at IDH1 codon
132 or at IDH2 codon 172. Six cases (5 favorable histology, 1
unfavorable histology) showed heterozygous silent polymorphism
c.315C

>T (p.Gly105Gly) in IDH1, 2 cases (1 with favorable histol-

ogy and 1 with unfavorable histology) showed heterozygous change
c.211G

>A (p.Val71Ile) in IDH1, and 1 case (favorable histology)

showed heterozygous change c.535-40 G

>A in intron 4 of IDH2

(Table I).

Measurement of 2-Hydroxyglutaric Acid

Extracts of all 21 Wilms tumor samples gave quantifiable signals

on the LC-MS/MS (Table II). The total 2-HG ranged from 6.8 to
40.5

ρmol/mg tissue (mean 17.6 ρmol/mg, median 15.4 ρmol/mg).

Pediatr Blood Cancer DOI 10.1002/pbc

IDH1/2 and 2-Hydroxyglutarate in Wilms Tumors

381

TABLE II. 2-Hydroxyglutarate Levels in Wilms Tumor Tissues

Case no.

L-2-HG (

ρmol/mg tissue)

D-2-HG (

ρmol/mg tissue)

Total 2-HG (

ρmol/mg tissue)

L-2-HG/D-2-HG (ratio)

1

9

.8

5

.3

15

.1

1

.9

2

8

.2

4

.0

12

.2

2

.1

3

a

3

.0

3

.8

6

.8

0

.8

4

5

.4

4

.3

9

.7

1

.2

5

14

.6

7

.9

22

.5

1

.9

6

15

.5

8

.8

24

.3

1

.8

7

9

.8

5

.2

15

.0

1

.9

8

a

6

.7

4

.1

10

.8

1

.6

9

22

.0

18

.5

40

.5

1

.2

10

12

.7

17

.7

30

.4

0

.7

11

7

.6

7

.8

15

.4

1

.0

12

6

.6

7

.4

14

.1

0

.9

13

3

.4

5

.0

8

.5

0

.7

14

10

.5

6

.8

17

.4

1

.5

15

12

.4

14

.6

27

.0

0

.9

16

13

.1

10

.2

23

.3

1

.3

17

5

.7

3

.4

9

.1

1

.7

18

11

.8

4

.4

16

.2

2

.7

19

a

9

.7

6

.8

16

.5

1

.4

20

4

.9

6

.4

11

.4

0

.8

21

19

.0

4

.7

23

.6

4

.1

2-HG, 2-hydroxyglutarate; L-2-HG, L-2-hydroxyglutarate; D-2-HG, D-2-hydroxyglutarate;

a

Wilms tumor with unfavorable histology (diffuse

anaplasia).

L-2-HG ranged from 3 to 22

ρmol/mg tissue (mean 10.1 ρmol/mg,

median 9.8

ρmol/mg). D-2-HG ranged from 3.4 to 18.5 ρmol/mg

(mean 7.5

ρmol/mg, median 6.4 ρmol/mg). The ratio of L-2-HG to

D-2-HG ranged from 0.7:1 to 4.1:1 (mean 1.5, median 1.4). No data
are available for levels of 2-HG in normal kidney tissues. However,
for comparison, we also measured 2-HG in the Wilms tumor of
the patient with L-2-HGA [15]. In this tumor, the total 2-HG was
222.7

ρmol/mg tissue, of which L-2-HG was 215 ρmol/mg and D-

2-HG was 7.7

ρmol/mg, with L-2-HG to D-2-HG ratio of 28.1:1.

DISCUSSION

L-2-HG (equivalent to S-2-HG using the R/S nomenclature)

and D-2-HG (equivalent to R-2-HG using the R/S nomenclature)
are enantiomers with identical physicochemical properties such as
melting point and solubility. However, they have different three-
dimensional spatial configurations that are mirror images of each
other and they rotate plane-polarized light either clockwise or coun-
terclockwise. Thus, L-2-HG rotates plane-polarized light clockwise
(

+) and the D-2-HG rotates plane-polarized light counterclockwise

(

−) [1,14]. The different three-dimensional spatial configurations

have biologic significance because the two compounds are rec-
ognized by different enzymes and are therefore associated with
distinct metabolic disorders. L-2-HG appears to be formed from
␣-KG by the side activity of L-malate dehydrogenase, which nor-
mally catalyzes the interconversion of L-malate and oxaloacetate in
the tricarboxylic acid (TCA) cycle. L-2-HG, which has no known
metabolic function in eukaryotes, is converted back to

␣-KG by

the catalytic action of L-2-hydroxyglutarate dehydrogenase (L-
2-HGDH), a mitochondrial FAD-linked dehydrogenase [2,23,24].
A deficiency of L-2-HGDH, caused by mutations in the gene
L2HGDH located on chromosome region 14q22.1, is responsible
for the metabolic disorder L-2-HGA [25–27]. L-2-HGA is clinically
characterized by childhood-onset neurodevelopmental delay and

subsequent variably progressive neurodegeneration with pyrami-
dal and extrapyramidal findings, seizures, and ataxia. The ultimate
developmental outcome is poor, with average intelligence quotients
in the 40–50 range. Typical magnetic resonance imaging (MRI)
findings include cortical atrophy, subcortical leukoencephalopathy,
and high-signal intensity in dentate nucleus and putamen [28–29].
D-2-HG participates in the succinic acid–glycine cycle, where it
is formed by the oxidation of gamma-hydroxybutyrate catalyzed
by hydroxyacid–oxoacid transhydrogenase (HOT). D-2-HG is con-
verted to

␣-KG by the catalytic action of D-2-HGDH, which is

also likely FAD-linked [1]. A deficiency of D-2-HGDH, caused by
mutations in the gene D2HGDH located on chromosome 2q37.3,
give rise to the metabolic disorder D-2-HGA [30–32]. D-2-HGA
may present with a severe phenotype of neonatal- or early infantile-
onset epileptic encephalopathy and severe developmental delay,
along with cardiomyopathy and facial dysmorphism. MRI findings
include delayed cerebral maturation, ventricular white matter abnor-
malities, and subependymal cyst formation. D-2-HGA may also be
asymptomatic or present with a milder phenotype of variable hypo-
tonia and developmental delay along with milder MRI changes [1].
A third disorder involving 2-HG has been described in four patients
severe neonatal-onset encephalopathy, in whom there were elevated
levels of both L-2-HG and D-2-HG (combined D,L-2-HGA) and of
␣-KG. Three of the infants with combined D,L-2-HGA died in the
first year of life and the fourth died at 3.5 years of age. The molec-
ular basis of this disorder is not known [3,33]. Elevated levels of
2-HG also occur in multiple acyl-coenzyme A dehydrogenase defi-
ciency (MADD, OMIM #231680), also known as glutaric aciduria
II (GA-II). MADD results from defects of electron transfer from
primary flavoprotein dehydrogenases to coenzyme Q10 in the mito-
chondrial electron transport chain and gives rise to neonatal onset of
hypoglycemic encephalopathy with (type I) or without (type II) con-
genital anomalies, or a later onset, milder disease with progressive
muscle weakness (type III) [34,35].

Pediatr Blood Cancer DOI 10.1002/pbc

382

Rakheja et al.

In addition to giving rise to metabolic complications, there is

accumulating evidence that elevated levels of either of the two
enantiomers of 2-HG are associated with malignant cellular trans-
formation. Thus, at least nine patients with L-2-HGA have been
reported to have brain tumors [4–9], and a right frontal bone osteoma
has been described in another patient [10]. Yazici et al. [36] reported
the occurrence of a medulloblastoma in a patient with apparent L-
2-HGA, which may have been a misdiagnosis for MADD [37].
Elevated levels of D-2-HG have been demonstrated with brain
tumors and myeloid leukemias associated with somatic mutations
of IDH1 and IDH2. Oncogenic mutations of IDH1 and IDH2 almost
exclusively occur at the same respective codon and give rise to amino
acid change R132C in IDH1 and R172K in IDH2. These mutations
reduce the enzymes’ affinity for isocitrate and increase the affinity
for

␣-KG and NADPH. This prevents the oxidative decarboxyla-

tion of isocitrate to

␣-KG and facilitates the reduction of ␣-KG to

D-2-HG [11–14].

We reported the occurrence of Wilms tumor in a child with L-2-

HGA [15]. Therefore, we investigated sporadic Wilms tumors for
association with elevated levels of 2-HG or with IDH1 or IDH2
mutations. In our study of 21 Wilms tumors, we did not find any
with significantly elevated levels of 2-HG. In 20 cases, we sequenced
exon 4 and flanking intronic regions of IDH1 and IDH2 and did not
find mutations at IDH1 codon 132 or at IDH2 codon 172. Two
Wilms tumors showed heterozygous change c.211G

>A that results

in an amino acid change at codon 71 of IDH1 (p.Val71Ile). This
change has previously been reported as a mutation in one case of
follicular thyroid carcinoma, one case of anaplastic thyroid carci-
noma, and in the myeloma cell line RPMI-8226 [38,39]. However,
c.211G

>A (p.Val71Ile) is listed as a single nucleotide polymor-

phism in the NCBI SNP database [40]. The population frequency
of IDH1 c.211G

>A allele is not known. It is interesting that in

our study, 1 of 3 (33.3%) Wilms tumors with unfavorable his-
tology carried the c.211G

>A (p.Val71Ile) allele, while only 1 of

18 (5.5%) tumors with favorable histology harbored this allele.
We do not know if this change leads to a loss of IDH1 func-
tion, but based on our data, it is not associated with increased
D-2-HG. It is possible that a compensatory mechanism, such as
increased activity of D-2-HGDH, may contribute to normaliza-
tion of D-2-HG levels. However, no functional studies of variant
IDH1 with c.211G

>A (p.Val71Ile) change have been reported in the

literature.

It should be noted that other aberrations of the TCA cycle

metabolism are also associated with cancer. Mutations in nuclear
genes encoding subunits of succinate dehydrogenase (SDH) have
been found in pheochromocytoma and paraganglioma [41–43].
Fumarate hydratase (FH) mutations are responsible for a familial
syndrome of uterine fibroids, skin leiomyomata, and renal papil-
lary cell cancer [44]. This is pertinent to our hypothesis, because it
shows that renal carcinogenesis may be associated with abnormali-
ties of the TCA cycle. Together, the involvement of aberrant alleles
of L-2-HGDH, IDH1, IDH2, FH, and genes encoding subunits
of the SDH complex strongly suggest a relationship between the
intermediary metabolism, particularly the TCA cycle, and tumorige-
nesis. It is interesting to speculate that abnormalities in intermediary
metabolism could be at the root of a subset of Wilms tumors, most
of which lack a clear genetic cause [45]. Because our sample set
of Wilms tumors is small, it is still possible that a small sub-
set of Wilms tumors do have these or other abnormalities of the
TCA cycle.

REFERENCES

1. Struys EA. D-2-Hydroxyglutaric aciduria: Unravelling the bio-

chemical pathway and the genetic defect. J Inherit Metab Dis
2006;29:21–29.

2. Van

Schaftingen

E,

Rzem

R,

Veiga-da-Cunha

M.

L-2-

Hydroxyglutaric aciduria, a disorder of metabolite repair. J Inherit
Metab Dis 2009;32:135–142.

3. Muntau AC, Roschinger W, Merkenschlager A, et al. Com-

bined D-2- and L-2-hydroxyglutaric aciduria with neonatal onset
encephalopathy: A third biochemical variant of 2-hydroxyglutaric
aciduria? Neuropediatrics 2000;31:137–140.

4. Moroni I, Bugiani M, D’Incerti L, et al. L-2-Hydroxyglutaric

aciduria and brain malignant tumors: A predisposing condition?
Neurology 2004;62:1882–1884.

5. Wilcken B, Pitt J, Heath D, et al. L-2-Hydroxyglutaric aciduria:

Three Australian cases. J Inherit Metab Dis 1993;16:501–504.

6. Barbot C, Fineza I, Diogo L, et al. L-2-Hydroxyglutaric aciduria:

Clinical, biochemical and magnetic resonance imaging in six Por-
tuguese pediatric patients. Brain Dev 1997;19:268–273.

7. Ozisik PA, Akalan N, Palaoglu S, et al. Medulloblastoma in a child

with the metabolic disease L-2-hydroxyglutaric aciduria. Pediatr
Neurosurg 2002;37:22–26.

8. Haliloglu G, Jobard F, Oguz KK, et al. L-2-Hydroxyglutaric

aciduria and brain tumors in children with mutations in
the L2HGDH gene: Neuroimaging findings. Neuropediatrics
2008;39:119–122.

9. Aghili M, Zahedi F, Rafiee E. Hydroxyglutaric aciduria and malig-

nant brain tumor: A case report and literature review. J Neurooncol
2009;91:233–236.

10. Larnaout A, Amouri R, Neji S, et al. Osteoma of the calvaria in

L-2-hydroxyglutaric aciduria. J Inherit Metab Dis 2007;30:980.

11. Ward PS, Patel J, Wise DR, et al. The common feature of leukemia-

associated IDH1 and IDH2 mutations is a neomorphic enzyme
activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Can-
cer Cell 2010;17:225–234.

12. Gross S, Cairns RA, Minden MD, et al. Cancer-associated

metabolite 2-hydroxyglutarate accumulates in acute myelogenous
leukemia with isocitrate dehydrogenase 1 and 2 mutations. J Exp
Med 2010;207:339–344.

13. Frezza C, Tennant DA, Gottlieb E. IDH1 mutations in gliomas:

When an enzyme loses its grip. Cancer Cell 2010;17:7–9.

14. Dang L, White DW, Gross S, et al. Cancer-associated IDH1 muta-

tions produce 2-hydroxyglutarate. Nature 2009;462:739–744.

15. Rogers RE, Deberardinis RJ, Klesse LJ, et al. Wilms tumor in a

child with L-2-hydroxyglutaric aciduria. Pediatr Dev Pathol 2010;
in press. It is available at www.pedpath.org/doi/pdf/10.2350/09-12-
0768-CR.1

16. Gibson KM, ten Brink HJ, Schor DS, et al. Stable-isotope dilution

analysis of D- and L-2-hydroxyglutaric acid: Application to the
detection and prenatal diagnosis of D- and L-2-hydroxyglutaric
acidemias. Pediatr Res 1993;34:277–280.

17. Lu W, Kimball E, Rabinowitz JD. A high-performance liquid

chromatography-tandem mass spectrometry method for quantita-
tion of nitrogen-containing intracellular metabolites. J Am Soc
Mass Spectrom 2006;17:37–50.

18. Struys EA, Jansen EE, Verhoeven NM, et al. Measurement

of urinary D- and L-2-hydroxyglutarate enantiomers by stable-
isotope-dilution liquid chromatography-tandem mass spectrometry
after derivatization with diacetyl-L-tartaric anhydride. Clin Chem
2004;50:1391–1395.

19. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in

gliomas. N Engl J Med 2009;360:765–773.

20. Green A, Beer P. Somatic mutations of IDH1 and IDH2 in the

leukemic transformation of myeloproliferative neoplasms. N Engl
J Med 2010;362:369–370.

Pediatr Blood Cancer DOI 10.1002/pbc

IDH1/2 and 2-Hydroxyglutarate in Wilms Tumors

383

21. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found

by sequencing an acute myeloid leukemia genome. N Engl J Med
2009;361:1058–1066.

22. Parsons DW, Jones S, Zhang X, et al. An integrated

genomic analysis of human glioblastoma multiforme. Science
2008;321:1807–1812.

23. Struys EA, Verhoeven NM, Jansen EE, et al. Metabolism of gamma-

hydroxybutyrate to D-2-hydroxyglutarate in mammals: Further
evidence for D-2-hydroxyglutarate transhydrogenase. Metabolism
2006;55:353–358.

24. Struys EA, Verhoeven NM, Ten Brink HJ, et al. Kinetic char-

acterization of human hydroxyacid–oxoacid transhydrogenase:
Relevance to D-2-hydroxyglutaric and gamma-hydroxybutyric
acidurias. J Inherit Metab Dis 2005;28:921–930.

25. Steenweg ME, Jakobs C, Errami A, et al. An overview of L-

2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: A
genotype–phenotype study. Hum Mutat 2010;31:380–390.

26. Topcu M, Jobard F, Halliez S, et al. L-2-Hydroxyglutaric aciduria:

Identification of a mutant gene C14orf160, localized on chromo-
some 14q22. 1. Hum Mol Genet 2004;13:2803–2811.

27. Rzem R, Veiga-da-Cunha M, Noel G, et al. A gene encoding a

putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is
mutated in L-2-hydroxyglutaric aciduria. Proc Natl Acad Sci USA
2004;101:16849–16854.

28. Topcu M, Aydin OF, Yalcinkaya C, et al. L-2-Hydroxyglutaric

aciduria: A report of 29 patients. Turk J Pediatr 2005;47:1–7.

29. Barth PG, Hoffmann GF, Jaeken J, et al. L-2-Hydroxyglutaric

acidaemia: Clinical and biochemical findings in 12 patients and
preliminary report on L-2-hydroxyacid dehydrogenase. J Inherit
Metab Dis 1993;16:753–761.

30. Kranendijk M, Struys EA, Gibson KM, et al. Evidence for

genetic heterogeneity in D-2-hydroxyglutaric aciduria. Hum Mutat
2009;31:279–283.

31. Struys EA, Korman SH, Salomons GS, et al. Mutations in

phenotypically mild D-2-hydroxyglutaric aciduria. Ann Neurol
2005;58:626–630.

32. Struys EA, Salomons GS, Achouri Y, et al. Mutations in the D-2-

hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric
aciduria. Am J Hum Genet 2005;76:358–360.

33. Read MH, Bonamy C, Laloum D, et al. Clinical, biochemical,

magnetic resonance imaging (MRI) and proton magnetic res-

onance spectroscopy (1H MRS) findings in a fourth case of
combined D- and L-2 hydroxyglutaric aciduria. J Inherit Metab
Dis 2005;28:1149–1150.

34. Angle B, Burton BK. Risk of sudden death and acute life-

threatening events in patients with glutaric acidemia type II. Mol
Genet Metab 2008;93:36–39.

35. Yee WC. Two eminently treatable genetic metabolic myopathies.

Neurol India 2008;56:333–338.

36. Yazici N, Sarialioglu F, Alkan O, et al. L-2-Hydroxyglutaric

aciduria and brain tumors: A case report and review of the literature.
J Pediatr Hematol Oncol 2009;31:865–869.

37. Rakheja D. L-2-Hydroxyglutaric aciduria and multiple acyl-

coenzyme A dehydrogenase deficiency are distinct metabolic
disorders. J Pediatr Hematol Oncol 2010;32:169–170.

38. Murugan AK, Bojdani E, Xing M. Identification and functional

characterization of isocitrate dehydrogenase 1 (IDH1) mutations
in thyroid cancer. Biochem Biophys Res Commun 2010;393:555–
559.

39. Bleeker FE, Lamba S, Leenstra S, et al. IDH1 mutations at residue

p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but
not in other solid tumors. Hum Mutat 2009;30:7–11.

40. National Center for Biotechnology Information Single Nucleotide

Polymorphism Database. www.ncbi.nlm.nih.gov/projects/SNP/
snp ref.cgi?locusId

=3417.

41. Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in

SDHD, a mitochondrial complex II gene, in hereditary paragan-
glioma. Science 2000;287:848–851.

42. Niemann S, Muller U. Mutations in SDHC cause autosomal dom-

inant paraganglioma, type 3. Nat Genet 2000;26:268–270.

43. Astuti D, Latif F, Dallol A, et al. Gene mutations in the succi-

nate dehydrogenase subunit SDHB cause susceptibility to familial
pheochromocytoma and to familial paraganglioma. Am J Hum
Genet 2001;69:49–54.

44. Tomlinson IP, Alam NA, Rowan AJ, et al. Germline mutations in FH

predispose to dominantly inherited uterine fibroids, skin leiomy-
omata and papillary renal cell cancer. Nat Genet 2002;30:406–
410.

45. Ruteshouser EC, Robinson SM, Huff V. Wilms tumor genetics:

Mutations in WT1, WTX, and CTNNB1 account for only about
one-third of tumors. Genes Chromosomes Cancer 2008;47:461–
470.

Pediatr Blood Cancer DOI 10.1002/pbc