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Human MutationRESEARCH ARTICLE
Alu-Alu Recombination Underlies the Vast Majority
of Large VHL Germline Deletions: Molecular
Characterization and Genotype–Phenotype Correlations
in VHL Patients
Gerlind Franke,1,2 Birke Bausch,1,5 Michael M. Hoffmann,3 Markus Cybulla,1 Christian Wilhelm,4 Ju¨rgen Kohlhase,4
Gerd Scherer,5 and Hartmut P.H. Neumann1�
1Department of Nephrology, University Medical Center Freiburg, Freiburg, Germany; 2Faculty for Biology, University of Freiburg, Freiburg,
Germany; 3Department of Laboratory Medicine, University of Freiburg, Freiburg, Germany; 4Center for Human Genetics, Freiburg, Germany;
5Institute of Human Genetics and Anthropology, University of Freiburg, Freiburg, Germany
Communicated by Haig H. Kazazian
Received 24 July 2008; accepted revised manuscript 5 November 2008.
Published online 11 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.20948
ABSTRACT: Von Hippel-Lindau disease (VHL) is an
autosomal dominant cancer syndrome. Affected indivi-
duals are predisposed to multiple tumors, primarily of the
central nervous system (CNS), eyes, adrenals, and
kidneys. The VHL tumor suppressor gene on chromo-
some 3p26–25 is partially or completely deleted in 20 to
30% of families with VHL. We identified deletions
ranging from 0.5 kb to 250 kb affecting part of or the
entire VHL and flanking genes in 54 families. In 33 of the
index patients, the breakpoints were precisely character-
ized by DNA sequencing. Of the 66 breakpoints, 90%
were located in Alu elements, revealing Alu-mediated
recombination as the major mechanism for large germ-
line deletions of the VHL gene, which lies in a region of
high Alu density. Interestingly, an AluYa5 element in
VHL intron 2, the evolutionarily youngest Alu element
and the only such element in the entire region, was found
to be the most recombinogenic, involved in 7 out of the
33 deletions. In comparison to VHL patients in general,
the 54 index cases and their affected relatives showed a
higher occurrence of renal cell carcinomas (RCC) and of
CNS hemangioblastomas. We not only noted the
association of RCC with retention of the HSPC300
gene, but also observed a significant correlation between
retention of HSPC300 and the development of retinal
angiomas (AR). This study reveals that germline VHL
deletions provide a particularly rich source for the study
of Alu-mediated unequal crossover events, and provides
evidence for a protective role of the loss of the
actin-regulator gene HSPC300 for the development of
both RCC and AR.
Hum Mutat 30:776–786, 2009. & 2009 Wiley-Liss, Inc.
KEY WORDS: Von Hippel-Lindau; AluYa5; FANCD2;
HSPC300; IRAK2; PRRT3; TMEM111; clear cell renal
cell carcinoma; angiomatosis retinae
Introduction
Von Hippel-Lindau disease (VHL; MIM 193300;
www.ncbi.nlm.nih.gov/omim) is an autosomal dominant familial
cancer syndrome caused by germline mutations in the VHL gene.
The disease occurs with an incidence of approximately 1 in 36,000
live births per year [Maher et al., 1991]. The penetrance is
estimated to be 80 to 90% by the age of 65 years [Couch et al.,
2000]. VHL disease is characterized by the presence of benign and
malignant neoplasias. The most frequent tumors are retinal and
central nervous system (CNS) hemangioblastomas, renal cell
carcinomas (RCCs), pheochromocytomas, pancreatic endocrine
tumors, and endolymphatic sac tumors (ELSTs) [Neumann and
Wiestler, 1991; Maher et al., 2004]. In addition, renal, pancreatic,
and epididymal cysts are common [Kaelin, 2007; Ong et al., 2007].
The manifestation and severity are highly variable both within and
between families [Webster et al., 1998].
The VHL gene is a tumor suppressor gene on chromosome
3p26–25. It spans a 10-kb region and consists of three exons. The
VHL mRNA encodes a protein (pVHL) of 213 amino acids with a
molecular weight of 30 kDa [Iliopoulos et al., 1995]. A second
pVHL isoform of approximately 19 kDa is produced as a result of
internal translation initiation at an in-frame start codon (ATG) at
codon 54 [Schoenfeld et al., 1998]. Both isoforms appear to retain
tumor suppressor activity [Kim and Kaelin, 2004]. The VHL gene
is widely expressed in both fetal and adult tissues, so its expression
is not restricted to the organs affected in VHL disease [Kim and
Kaelin, 2004]. pVHL, together with Elongin B, Elongin C, and
Cul2, interacts and modifies hypoxia inducible factor 1 (HIF 1)
and thus plays an important role in cellular response to hypoxia
with the regulation of angiogenesis and apoptosis [Carmeliet
et al., 1998; Maxwell et al., 1999].
OFFICIAL JOURNAL
www.hgvs.org
& 2009 WILEY-LISS, INC.
Additional Supporting Information can be found in the online version of this article.
Contract grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: NE
571/5-3.
Birke Bausch’s current address: Department of Internal Medicine, University
Hospital Zurich, Zurich, Switzerland.
�Correspondence to: Hartmut P.H. Neumann, MD, Medizinische Universita¨tsklinik,
Abteilung Innere Medizin 4, Hugstetter Str. 55, D 79106 Freiburg, Germany.
E-mail: hartmut.neumann@uniklinik-freiburg.de
The risk for different tumors is influenced by the VHL mutation
type [Kaelin, 2007]. Clinically, VHL families can be subdivided
into different types, based on the likelihood of developing
pheochromocytoma or RCC [Zbar et al., 1996]. Type 1 is
characterized by a low risk for pheochromocytomas and by the
presence of RCC, whereas Type 2 is characterized by a high risk for
pheochromocytomas. Type 2 can be further subdivided into Type
2A (pheochromocytomas accompanied by a low risk for RCC),
Type 2B (pheochromocytomas and a high risk for RCC), and Type
2C (pheochromocytomas only) [Plate et al., 2007]. Whereas Type
1 is frequently caused by VHL deletions or truncating mutations
and missense mutations, Type 2 cases frequently harbor VHL
missense mutations [Chen et al., 1995; Zbar et al., 1996; Ong et al.,
2007]. Except for Type 2C families, all other VHL types develop
hemangioblastomas. A large number of families with VHL Type
2A are found in Southwest Germany and North America due to a
founder effect of the p.Y98H (c.292T4C; previously c.505T4C)
missense mutation [Brauch et al., 1995; Bender et al., 2001].
Individuals with familial VHL disease carry one wild-type and
one inherited mutant VHL allele. Approximately 20 to 37% of VHL
patients have large or partial germline deletions, 30 to 38% have
missense mutations, and 23 to 27% have nonsense or frameshift
mutations [Maher and Kaelin, 1997; Stolle et al., 1998]. For the
tumor to develop, a second hit must occur [Knudson, 1971] that
inactivates the wild-type VHL allele. In the majority of tumors, this
second hit consists of a large VHL deletion or a VHL point mutation
[Vortmeyer et al., 2002; Wait et al., 2004; Wong et al., 2007].
Not much is known regarding the relationship between particular
symptoms seen in VHL and the specific size of germline VHL
deletions, and the molecular mechanism behind these deletions. In
two previous VHL deletion studies, a reduced risk for renal cell
carcinoma has been noted when the actin regulator gene HSPC300
was codeleted together with the VHL gene [Maranchie et al., 2004;
Casco´n et al., 2007]. While a number of studies have characterized
partial and complete germline deletions of the VHL gene by
quantitative Southern blot, multiplex ligation-dependent probe
amplification (MLPA), and/or quantitative real-time polymerase
chain analyses (qPCR) [Stolle et al., 1998; Hes et al., 2000; Cybulski
et al., 2002; Gallou et al., 2004; Maranchie et al., 2004; Hoebeeck
et al., 2005; Casarin et al., 2006; Hattori et al., 2006; Casco´n et al.,
2007; Hes et al., 2007; Huang et al., 2007; Ong et al., 2007] and while
some of the breakpoints have been narrowed down more precisely,
the exact breakpoint sequence has only been determinedfor one
case, which was shown to result from Alu-Alu recombination
[Casarin et al., 2006]. Alu-Alu recombination-mediated deletions
have been described for various inherited disorders such as familial
hypercholesterolemia and a-thalassemia [for reviews see Deininger
and Batzer, 1999; Batzer and Deininger, 2002].
In this study, we identified and analyzed 54 VHL deletion
families, with the aim to characterize the size of the deletion and
the underlying mechanism causing deletion formation, and to see
if deletion size and loss of particular VHL flanking genes can be
correlated with clinical phenotype.
Materials and Methods
Patient Samples
This study is based on the Freiburg VHL registry of 308
unrelated familial or sporadic VHL index cases, fulfilling either
distinct clinical criteria of VHL or having a clear history of VHL in
their family. This study has been approved by the Ethics
Committee of the University of Freiburg. All patients provided
informed consent. DNA from patients has been collected since
1996. EDTA-anticoagulated blood samples were obtained from the
patients and their genomic DNA was extracted using standard
methods. In 254 out of the 308 index cases, point mutations or
small deletions/insertions (o20 bp) were identified by DNA
sequencing following complete mutation screening using PCR/
single strand conformation polymorphism (SSCP) and denaturing
high-performance liquid chromatography (DHPLC) (WAVE
analysis system; Transgenomics, Paris, France). A total of 144
patients from 42 unrelated families and 12 sporadic cases were
included in this study (54 index cases and 90 relatives). DNA
samples were available from 95 of the patients (54 index cases and
41 relatives).
Clinical Data
The Preventive Medicine Center of the University Medical Center
Freiburg, Germany, serves as the major VHL clinic for Germany. All
patients are registered with demographic data and detailed clinical
data. For this study an update was performed in 2007.
MLPA
Screening for large alterations was performed for all 95 DNA
samples by means of MLPA, following the manufacturer’s
instructions (MRC-Holland, Amsterdam, The Netherlands). The
commercial VHL-MLPA assay included eight pairs of probes
designed to amplify the three VHL exons, five probe pairs for
neighboring genes (three for FANCD2, one for IRAK2, and one for
GHRL), two additional probe pairs from the short arm of
chromosome 3 (9.6 Mb telomeric and 26 Mb centromeric from
the VHL gene), one probe pair from the long arm of chromosome
3, and 13 probe pairs for loci at other chromosomes. The size of
MLPA products was estimated by using a MegaBACE 500 DNA
Analysis System (Amersham Biosciences, Amersham, UK).
qPCR
The qPCR with SYBR Green I detection (SYBR Green PCR
Master Mix; Qiagen, Hilden, Germany) was carried out on an ABI
Prism 7900 Sequence Detection System (PE Applied Biosystems
[ABI], Foster City, CA). The Sequence Detection System software
(SDS version 2.2.1; ABI) was used to analyze the data. About 300
different amplicons were used for the analysis of the 453.5-kb
region on chromosome 3, spanning the region from 9,843,500 to
10,297,000 (sequence coordinates according to Ensembl Human
Genome Browser, Database version 48.36j; www.ensembl.org/
Homo_sapiens/index.html). All amplicons were 100 to 350 bp in
length. Primers were designed using the Primer3 software (http://
frodo.wi.mit.edu/primer3/input.htm) and the Primer3 human
Mispriming Library (repeat library) to avoid primer design in any
repeat region [Rozen and Skaletsky, 2000]. (Data for primers, their
positions, and amplicon sizes are available in Supporting Table S4;
available online at http://www.interscience.wiley.com/jpages/1059-
7794/suppmat). PCR reactions and quantification of PCR
products were performed essentially as described [Boehm et al.,
2004; Borozdin et al., 2004; Bausch et al., 2007]. All measurements
were carried out in duplicate and repeated twice for critical
amplicons. Ratio values of 0.85–1.15 were accepted as a diploid
situation, and values of 0.40–0.70 as a haploid situation.
GenBank accession and RefSeq numbers for the eight genes
studied are as follows: 1) von Hippel-Lindau (VHL), AF010238,
RefSeq NM_000551.2; 2) proline-rich transmembrane protein 3
HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009 777
(PRRT3), AC018809, RefSeq NM_207351.1; 3) transmembrane
protein 111 (TMEM111), AF157321, RefSeq NM_018447.1; 4)
Fanconi anemia complementation group D2 (FANCD2),
DQ341263, RefSeq NM_033084.3; 5) HSPC300, currently desig-
nated C3orf10 by HUGO, AF161418, RefSeq NM_018462.4; 6)
interleukin-1 receptor-associated kinase 2 (IRAK2), AJ496794,
RefSeq NM_001570.3; 7) TatD DNase domain containing 2
(TATDN2), D86972, NM_014760.2; and 8) ghrelin/obestatin
preprohormone (GHRL), EU072083, RefSeq NM_016362.2.
Long-Range PCR (LR-PCR)
Either Taq DNA polymerase (GE Healthcare, Freiburg,
Germany) or Phusion Hot Start high-fidelity DNA polymerase
(Finnzymes/New England Biolabs, Ipswich, MA) were used
according to the manufacturer’s recommendations. In some cases,
betain (at 5 mM) was included. The primers of the nearest
nondeleted amplicons, already designed for qPCR, were used. In
some cases, nested PCRs were performed on gel extracted
products (QIAquick Gel Extraction Kit; Qiagen) to obtain clean
products in amounts suitable for sequence analysis. For this,
additional primers were designed as described above. (Primers are
available in Supp. Table S4).
Cloning and Sequencing
PCR products were directly sequenced in both orientations on
an ABI PRISM 3100 Genetic Analyzer (ABI, Darmstadt, Germany)
using the BigDye Terminator Cycle Sequencing-Kit (ABI). For
Families 10 and 14, the special structure at the junction sites made
cloning of the PCR products into the pGEMs-T Easy vector
(Promega, Madison, WI) necessary before sequencing. Compar-
ison of the sequences obtained with the human genomic reference
sequence (Ensembl Human Genome Browser, version 48.36)
revealed the breakpoints within the limits shown in Table 3,
column F.
Statistical Analysis
Fisher’s Exact Test was used to test for possible associations
between certain classes of deletions vs. specific aspects of the
phenotype (P values). All tests were two-sided and Pr0.05 was
considered statistically significant.
Results
Gross Deletion Mapping by MLPA
Using MLPA, we identified partial or complete germline
deletions of the VHL gene with or without deletion of flanking
genes in 42 unrelated VHL families and 12 sporadic VHL
cases (subsequently designated 54 VHL families for ease of
presentation). The MLPA results can be subdivided into 10 groups
(Table 1; Fig. 1). A partial or complete deletion of only the VHL
gene (groups A–F) is detected in 40 families, whereas in 14
families the deletion extends towards the IRAK2 (groups G and H)
plus the FANCD2 gene (groups I and K), the only VHL flanking
genes monitored by the MLPA kit used. As to be expected, the
MLPA results were consistent within families.
Fine Deletion Mapping by qPCR
For fine-mapping of the respective deletion site, at least one
patient sample from each family was subjected to qPCR. The
extent of the deletions is shown in Figs. 1 and 2. For 21 of the 54
families, the minimal and maximal size estimates for the deletion
could be determined (Table 2). For 33 families, the exact size of
the deletion could be obtained by sequencing across the deletion
breakpoints (Table 3).
The deletions range in size from 568 bp (Family 8) to about
250 kb (Family 51). They include at least one other gene apart
from the VHL gene in 28 out of the 54 families. In the region
telomeric to the VHL gene, the deletions extend into and beyond
the FANCD2 gene in seven families (Families 48–54), and into and
beyond the HSPC300 gene in two families (Families 39 and 45),
while only the VHL flanking gene ENST 197804 is deleted,partially or completely, in another seven families (Families 2, 6, 7,
10, 11, 35, and 38). On the centromeric side of VHL, deletions
disrupt or eliminate the flanking IRAK2 gene in 17 families
(Families 23, 24, 27, 33, 34, 39–44, 46, 48, and 51–53) and extend
beyond the putative transcript LOC728426 and into the TATDN2
gene in four families (Families 45, 47, 50, and 54).
Deletion Breakpoint Sequencing Reveals a High
Frequency of Alu-Alu Recombinations
The exact positions of the breakpoints could be identified in 33
out of the 54 families by sequencing across the deletion junction
following breakpoint-spanning LR-PCR. The results are listed in
Table 3, which also provides information about the repeats
involved. Of the 33 deletions, 30 were clear-cut deletions (for
example, see Supporting Fig. S1), while more complex insertions/
deletions were observed in three cases (Families 6, 10, and 14;
Supporting Figs. S2–S4). Due to the sequence similarity between
the ends of the deletion, the sequence directly at the deletion
junction can derive from either the telomeric or centromeric end
of the deletion (underlined in Supporting Fig. S1 and shown in
Table 3, column F). Unequal homologous recombination between
Alu elements underlies the deletion in 29 families (Table 3). In two
families, an Alu element is located at one deletion breakpoint and
another repeat at the second breakpoint (Families 6 and 11). A
long terminal repeat (LTR) is located at one breakpoint but no
repeat at the other breakpoint in Family 47, while the deletion
breakpoints are not located in any repeat region in Family 8.
Surprisingly, Families 27 and 33, which to our knowledge are not
related, show the same breakpoints with an identical deletion
junction sequence. The 50 breakpoint in these two families is in the
Alu element most frequently involved in deletions in this study. In
the other 31 families the sequences are unique.
Table 1. MLPA Results, Distributed into 10 Groups
Groups Number of families
A Del VHL Ex 1 8
B Del VHL Ex 1, 2 3
C Del VHL Ex 2 8
D Del VHL Ex 2, 3 5
E Del VHL Ex 3 10
F Del VHL Ex 1, 2, 3 6
G Del VHL Ex 3, IRAK2 2
H Del VHL Ex 1, 2, 3, IRAK2 5
I Del FANCD2 partial, VHL Ex 1, 2, 3, IRAK2 3
K Del FANCD2, VHL Ex 1, 2, 3, IRAK2 4
Total 54
FANCD2, Fanconi anemia complementation group D2: NM_033084.3; VHL, von
Hippel-Lindau: NM_000551.2; IRAK2, interleukin-1 receptor-associated kinase 2:
NM_001570.3.
778 HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009
In the 29 families with Alu elements at each end of the deletion,
the recombination event occurred in each case between Alu
elements in the same orientation (columns M and N in Table 3).
The same is apparent from Fig. 2, which shows a 20-kb region
around the VHL gene with the Alu elements color-coded according
to the Alu subfamily to which they belong. While most
Alu elements are involved only once in deletion formation, several
nd at deletion junctions. These are summarized in Supporting Table
S1 and include repeats involved two and up to seven times (AluYa5;
located in VHL intron 2, Fig. 2 in deletion formation). Interestingly, in
Families 16, 18, and 19, the unequal homologous recombination event
occurred between the same telomeric AluY and centromeric AluYa5
repeat, but the exact breakpoints differ (Table 3). The same situation is
found in Families 26 and 32, where the recombination event affected
the same telomeric AluSc and centromeric AluSq repeat, but the exact
breakpoints again differ (Table 3). In Families 13 and 20, where the
recombination events affected the same telomeric AluSg but different
centromeric AluSx elements, remarkably the breakpoints occurred
almost at the same positions within the Alu elements involved, as
shown by the nearly identical junction sequences (Table 3).
VHL Germline Deletion Size and Genotype–Phenotype
Correlations
In total, clinical data were available for 144 deletion carriers (54
index cases and 90 relatives) (Supporting Table S2; extended
version available on request). Clinical characteristics of all patients
from one family were merged. The 54 families were represented on
average by 2.4 patients, including at least one index case and up to
12 affected relatives per family. In general, deletion carriers had a
mean age at diagnosis of VHL of 23.3 years (median 21.5 years)
with an age range of 5–48 years. The mean age of the patients was
34.1 years (median 36 years; range 5–70 years) at the time of
clinical update for this study. The male to female ratio was 1.4:1.
Hemangioblastomas of the brain and spinal cord were present in
93% and 79.1% of the patients, respectively, retinal angiomas
(AR) in 79.2%, RCCs in 58.5%, and pheochromocytomas in
17.9% (Supporting Table S2). An involvement of broad ligaments
was not reported for female VHL carriers, whereas cysts of the
epididymides were observed in 52.2% of male VHL carriers.
Seven families belong to MLPA groups I and K with the largest
detected deletions (Fig. 1). While these seven families show an
involvement of the brain/cerebellum, as do most of the other MLPA
groups (100% vs. 91.7%, P�1), only 3 out of the 7 families (42.9%)
show involvement of the spinal cord (Supporting Table S2). This is a
significantly lower rate than in all the other MLPA groups (86.1%;
P5 0.026). Particularly striking is the absence of AR in all seven
large-deletion families as compared to the remaining families
belonging to MLPA groups A–H (0% vs. 92.7%; Po0.00001).
It has previously been noted that codeletion of the HSPC300
gene together with the VHL gene correlates with a lower
prevalence of RCC [Maranchie et al., 2004; Casco´n et al., 2007].
Figure 1. Extent of the germline deletions detected by MLPA and qPCR analyses in 54 VHL families. A 350-kb region is shown at the top with
the sizes of the VHL gene and neighboring genes drawn to scale. Dashed vertical lines indicate gene borders. Family IDs (on the left) are
grouped according to the deletions detected by MLPA (MLPA Group at right). The numbers at the bottom of the diagram give the nucleotide
position on chromosome 3. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009 779
The 54 VHL deletion families were divided into two groups: one
group retaining the HSPC300 gene, comprised of 45 families
(Families 1–38, 40–44, 46, and 47), and a second group with
partial or complete deletion of the HSPC300 gene, comprised of
nine families (Families 39, 45, and 48–54). The patients of both
groups had comparable mean ages at the time of data collection
(HSPC300 retained: mean age 34.1 (median 34) years, range 5–70
years, N5 69; HSPC300 deleted: mean age 34.2 (median 41) years,
range 10–59 years, N5 20).
As shown in Figure 3, statistically significant differences
between the groups at the P5 0.05 level exist for two organs:
the kidney and the eye. As for the kidney, RCCs developed in 22
out of 33 (67%) HSPC300 nondeletion families and in only 2 out
of 8 (25%) HSPC300 deletion families (P5 0.048), while cyst
development occurred in 24 out of 32 (75%) and 2 out of 9 (22%)
HSPC300 nondeletion and deletion families, respectively
(P5 0.0065). A significant difference between the two groups is
observed for the development of AR, which occurred in 37 out of
39 (95%) HSPC300 nondeletion families but in only 1 out of the 9
(11%) HSPC300 deletion families (Po0.00001). In contrast,
hemangioblastomas of the spinal cord showed only a marginally
significant difference (P5 0.074) between the HSPC300 nondele-
tion group (29/34; 85%) and the deletion group (5/9; 56%).
Retention or loss of HSPC300 had no significant effect on the
incidence of pheochromocytomas, cerebellar hemangioblastomas,
ELSTs, pancreatic cysts, islet cell tumors, epididymal cysts, or
other tumors and cysts (Fig. 3).
We alsoanalyzed the clinical data for potential genotype–phe-
notype correlations with respect to deletion or retention of the
ENST197804 gene or the IRAK2 gene, and the deletion of only the
VHL gene vs. the codeletion of the VHL gene with other genes.
The nine families with partial or complete deletion of the
HSPC300 gene were excluded from these calculations. No
correlations below the P5 0.05 level were observed.
Discussion
Alu-Mediated Recombination Is a Major Mechanism for
VHL Germline Deletions
We have characterized germline deletions of the VHL gene and
its flanking genes of up to 250 kb in size in 54 VHL families and
have successfully identified the precise deletion breakpoints in 33
families. This is to our knowledge the most extensive character-
ization of deletion breakpoints at the sequence level in VHL
disease reported so far. We found that 18 out of 54 families (33%)
harbor deletions of at least all three exons of the VHL gene. This is
a similar frequency to the findings in other studies [Cybulski et al.,
Figure 2. Involvement of Alu and other repeats in germline deletions in 33 VHL families. A region of 20 kb is shown, extending 5 kb either side
of the VHL gene. All LINE, LTR, and SINE (Alu) repeats in this region are shown. Broken lines indicate deletions with breakpoints located beyond
the limits chosen for this figure. The direction of the arrowheads denotes the orientation of the repeats (complete repeat elements in filled,
incomplete in open arrowheads). Each subfamily is represented by a different color (see legend on the figure). The region shown includes 38 out
of the 66 breakpoints identified.
780 HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009
2002; Rocha et al., 2003; Maranchie et al., 2004; Casarin et al.,
2006; Casco´n et al., 2007].
Of the 66 sequenced breakpoints, 60 (90%) were located in Alu
elements, revealing unequal homologous Alu-mediated recombi-
nation as a major mechanism for germline VHL deletions. In a
previous study, the exact breakpoint sequence has been
determined in a single VHL deletion patient and was also shown
to result from Alu-Alu recombination [Casarin et al., 2006].
Unequal crossover between Alu elements is a frequent cause of
inherited disorders such as hypercholesterolemia, a-thalassemia
and lissencephaly [Batzer and Deininger, 2002; Mei et al., 2008].
Genes showing high levels of Alu-Alu recombination tend to have
a high density of Alu sequences, but not every Alu-rich gene is
prone to this type of deletion formation, such as the thymidine
kinase or b-tubulin genes [Deininger and Batzer, 1999; Batzer and
Deininger, 2002; and references therein]. Analysis of the VHL
locus using the software CENSOR (www.girinst.org/censor/
index.php) [Kohany et al., 2006] revealed an Alu density of 30%
for the 322-kb region that includes all deletion breakpoints from
this study, while the 20-kb region extending 5 kb either side of the
VHL gene (Fig. 2) has an Alu density of 37%. Introns 1 and 2 of
the VHL gene have an even higher Alu density of 50% and 44%,
respectively. This is a much higher ratio than the average of the
human genome where Alu elements account for about 10%,
occurring on average once every 3 kb [Lander et al., 2001]. The
very high density of Alu elements in and around the VHL gene
thus appears to predispose the VHL gene to a high frequency of
Alu-mediated deletions. A recent study of more than 20 genes also
found evidence that a high content of transposable elements such
as Alu elements results in increased frequency of gene disruption
by gross deletions in human disease [van Zelm et al., 2008].
An analysis of the positions of the deletion breakpoints within
the respective Alu elements reveals that the breakpoints are
distributed more or less evenly over the entire element. This is
summarized in Fig. 4, where the positions of the breakpoints with
their respective family IDs are shown relative to a consensus
Alu element. This contrasts with the deletion breakpoints in
the LDL receptor and globin genes, which cluster in the left
arm between the A and B boxes that function as promoter
elements for RNA polymerase III. This has been interpreted as
indicating that an unusual configuration during transcription may
render the Alu element prone to recombination [Lehrman et al.,
1987b]. A recombination hotspot between the A and B boxes
has also been described for Alu/Alu deletions in the human vs.
the chimpanzee genome [Sen et al., 2006]. The fact that we do
not observe such a hotspot could be due to our limited
sample size.
All Alu-Alu recombination events in the VHL deletions have
their breakpoints in the same arm in both Alu elements involved
(Table 3; Fig. 4). As listed in Table 3, the unequal crossover events
always occurred between Alu elements in the same plus or minus
strand orientation except for Family 10, where one breakpoint lies
in an AluSq element on the plus strand and where the other
breakpoint is located at the junction between an AluSx element
and a truncated MLT1H2 element on the minus strand (Table 3;
dark-gray arrow-boxes in Fig. 4). This unique crossover event
resulted in a complex 14.5-kb deletion with concomitant insertion
of a 131–134-bp sequence (Supporting Fig. S4). BLAST-like
Table 2. Approximately Defined Breakpoints and Deletion Sizes in 21 VHL Families Determined by qPCR Analysis�
1 2 3 4 5 6 7 8 9
MLPA
Family
ID
Limits of telomeric
deletion breakpoint
Limits of centromeric
deletion breakpoint
Minimal size of
deletion (bp)
Maximal size of
deletion (bp) VHL exons and neighboring genes deleted
A 1a 10,155,000 10,158,460 10,158,920 10,163,170 461 8,171 VHL Ex 1
2 10,150,693 10,151,062 10,161,696 10,162,051 10,634 11,359 VHL Ex 11ENST197804 (PD)
5 10,153,219 10,154,621 10,160,419 10,162,051 5,799 8,333 VHL Ex 1
B 9 10,153,219 10,154,621 10,165,291 10,165,897 10,671 12,679 VHL Ex 1, 2
D 21 10,159,746 10,160,570 10,169,214 10,169,507 8,645 9,762 VHL Ex 2, 3
24 10,162,208 10,162,618 10,187,714 10,193,945 25,097 31,738 VHL Ex 2, 31IRAK2 (PD)
E 25 10,164,491 10,165,291 10,174,355 10,175,487 9,065 10,997 VHL Ex 3
28 10,164,491 10,165,291 10,174,525 10,174,926 9,235 10,436 VHL Ex 3
29 10,164,491 10,165,291 10,174,926 10,175,487 9,636 10,997 VHL Ex 3
31 10,164,491 10,165,291 10,174,926 10,175,662 9,636 11,172 VHL Ex 3
34 10,164,491 10,165,291 10,184,077 10,184,484 18,787 19,994 VHL Ex 31IRAK2 (PD)
F 35 10,150,713 10,151,062 10,168,537 10,169,507 17,476 18,795 VHL Ex 1, 2, 31ENST197804 (PD)
36 10,157,305 10,157,660 10,168,537 10,169,507 10,878 12,203 VHL Ex 1, 2, 3
38 10,143,152 10,146,442 10,174,525 10,174,926 28,084 31,775 VHL Ex 1, 2, 31putatively HSPC300 (PD)1ENST197804
G 41 10,164,491 10,165,291 10,198,312 10,199,259 33,022 34,769 VHL Ex 31IRAK2 (PD)
42 10,164,491 10,165,291 10,226,205 10,227,036 60,915 62,546 VHL Ex 31IRAK2 (PD)
H 43 10,150,713 10,151,062 10,256,699 10,257,398 105,638 106,686 VHL Ex 1, 2, 31ENST197804 (PD)1IRAK2 (PD)
45 10,139,901 10,140,729 10,288,355 10,291,979 147,627 152,079 VHL Ex 1, 2, 31HSPC300 (PD)1ENST1978041IRAK21
TATDN2 (PD)
I 48 10,106,203 10,106,593 10,264,465 10,264,835 157,873 158,633 FANCD2 (PD)1VHL Ex 1, 2, 31IRAK2 (incl. HSPC3001
ENST197804)
50 10,090,490 10,096,345 10,274,262 10,275,246 177,918 184,757 FANCD2 (PD)1VHL Ex 1, 2, 31IRAK21TATDN2 (PD)
(incl. HSPC3001ENST197804)
K 52 9,991,786 9,993,333 10,237,722 10,238,646 244,390 246,861 oFANCD2, VHL Ex 1, 2, 31IRAK2 (PD) (incl. HSPC3001
ENST197804)
�The MLPA group and family IDs are given in columns 1 and 2, respectively. The end position of the last undeleted amplicon and the start position of the first deleted
amplicon flanking the telomeric deletion breakpoint are given in columns 3 and 4, respectively. Likewise, the end position of the last deleted amplicon and the start position
of the first undeleted amplicon flanking the centromericdeletion breakpoint are given in columns 5 and 6, respectively. The size of the deletion lies anywhere between the
minimal size given in column 7 (difference between values in columns 4 and 5) and the maximal size given in column 8 (difference between values in columns 3 and 6).
Columns 3–6: sequence coordinates are according to Ensembl Human Genome Browser, Release 48, GeneBuild Ensembl, December 2006, Database version 48.36j;
www.ensembl.org/Homo_sapiens/index.html. Column 9: GenBank accession and RefSeq numbers for the genes analyzed are listed in Materials and Methods (qPCR).
aDeletion size for Family 1 is based on MLPA analysis only, due to sample limitation.
PD, partial deletion.
HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009 781
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782 HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009
alignment tool (BLAT; http://genome.ucsc.edu/cgi-bin/hgBlat?or-
g5 human) searches failed to identify a perfect match to the
complete insert, but different parts of the insert sequence showed
imperfect matches to different Alu elements on different
chromosomes.
As noted above, several Alu elements are repeatedly found at
deletion junctions. One particular Alu element stands out, an
AluYa5 element located in VHL intron 2, which is involved in
seven deletions (Table 3; Fig. 2; Supporting Table S1). Interest-
ingly, the same AluYa5 element has recombined with the same
AluY element as in our Families 16, 18, and 19 in the 2,142-bp
VHL exon 2 deletion described by Casarin et al. [2006]. (The AluY
element was erroneously denoted as AluSx element by these
authors.) This AluYa5 is the evolutionarily youngest of all Alu
elements that we found involved in deletion formation, with an
age of 2.6 million years (Supporting Table S3), and is the only
AluYa5 element not only within the 20-kb region around the VHL
gene (Fig. 2) but also within the entire 322-kb region spanning all
deletion breakpoints studied. This low density is not surprising, as
there are only 2,640 copies of AluYa5 elements in the human
genome [Batzer and Deininger, 2002], averaging less than one
such element per megabase (Mb). The AluYa5 subfamily is
human-specific [Kehrer-Sawatzki and Cooper, 2007], and accord-
ingly, there is no AluYa5 element at the homologous position in
intron 2 of VHL in the chimpanzee. Why a young Alu element
should be more recombinogenic than older Alu elements is not
readily apparent. Apart from the AluYa5 element, AluY elements
are involved in 33% of the VHL deletions (Supporting Table S3),
but account for only 14% of all Alu elements (57/392) in the 322-
kb region (CENSOR analysis), which is similar to their frequency
of 18% of all Alu elements in the human genome [Batzer and
Deininger, 2002]. Interestingly, by comparing the reference human
and chimpanzee genomes, it has been noted that young AluY
elements are also overrepresented at loci of Alu recombination-
mediated human-specific and chimpanzee-specific deletions [Sen
et al., 2006; Han et al., 2007].
It has been argued that the level of recombination between Alu
elements from different subfamilies should vary as a function of
Figure 3. Frequency of organ and/or tumor involvement in VHL families without (45 families) or with (9 families) deletion of HSPC300. N
gives the number of families affected by the respective lesion of all families with clinical data for the lesion. P values at right are two-sided
probabilities calculated by the Fisher’s Exact Test. The data are from Supporting Table S2. RCC, clear cell renal cell carcinoma; AR, angiomatosis
retinae; ELST, endolymphatic sac tumor; ICT, islet cell tumor.
Figure 4. Positions of VHL deletion breakpoints relative to a consensus Alu element. A consensus Alu element is shown [Deininger et al.,
1981] with left and right direct repeats (arms) and the A, A0, B, and B0 boxes of the internal polymerase III promoter [Paolella et al., 1983]. The
positions of the breakpoints are shown by arrow-boxes with the respective family number. The direction of the arrowheads denotes the
orientation of the affected repeats (arrowheads to the left indicate orientation on the reverse strand (–) and to the right on the forward (1)
strand). All detected breakpoints which affect at least one Alu element are shown. Alu-Alu recombinations in all families but one (Family 10)
occur between elements in the same orientation. Except for Family 10, all breakpoints in both involved Alu elements are in the same arm. The
rearrangement in Family 10 (marked by two dark-gray arrow-boxes) is more complex (Supporting Fig. S4) (Figure adapted from Lehrman et al.
[1987a]).
HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009 783
pairwise sequence divergence between elements, with older Alu
elements that have higher pairwise divergence (�15–20%) being
much less likely to recombine than younger Alu insertions that
have lower pairwise divergence [Batzer and Deininger, 2002]. This
assumption is born out by the analysis shown in Table 4. Of the 29
Alu-Alu recombinations, 11 occurred between elements belonging
to the younger AluY subfamily, with pairwise divergence from 10.4
to 14.5% for AluY-AluY (mean 12.7%) and from 8.6 to 10.3% for
AluYa5-AluY (mean 9.6%). In contrast, only eight recombination
events occurred between complete Alu elements of the more
abundant and evolutionary older AluS subfamily, with pairwise
divergence from 14.2 to 25.4% (mean 20.5%); the exception are
the two half-AluSg/x elements of Family 39, with only 13.4%
divergence. The remaining nine are inter-Alu subfamily recombi-
nations: four AluY-AluS with 17.5 to 20.7% divergence (mean
18.7%); two AluYa5-AluS with 10.4% and 16.4% divergence
(mean 13.4%); two AluSx-AluJo with 20.2% and 25.8% divergence
(mean 23.0%); and one AluY-AluJb with 25% divergence. Clearly,
the frequency of intra-Alu subfamily recombinations correlates
with both the lower pairwise sequence divergence between the
recombining Alu elements and the age of the subfamily, whereas
inter-Alu subfamily recombinations are less frequent and have on
average higher pairwise sequence divergences between the
recombining Alu elements.
Deletion of HSPC300 : Protection from Development of RCC
and AR?
Compared to VHL patients in general, regardless of their type
of germline mutation, the analyzed VHL families with deletions
show a higher frequency of RCC, of AR, and of CNS
hemangioblastomas, both cerebellar and spinal (Supporting Table
S2). This finding was not related to deletion size and is in general
agreement with previous studies of VHL deletion cases [Cybulski
et al., 2002; Gallou et al., 2004; Maranchie et al., 2004; Huang
et al., 2007]. The high frequency in particular of CNS
hemangioblastomas in VHL deletion cases has been stressed by
different authors [Hes et al., 2000; Cybulski et al., 2002; Huang
et al., 2007], a finding we can confirm.
Maranchie et al. [2004] were the first to note the surprising
association between deletion of the HSPC300 gene and a lower
incidence of RCC in VHL deletion cases, which was confirmed in a
recent study [Casco´n et al., 2007]. We also observed a significantly
lower frequency of RCC in the group with deletion of HSPC300
relative to the group with retention of this gene. This apparent
paradox, that codeletion of the VHL-flanking gene HSPC300
seems to protect against the development of RCC, has been
explained by suggesting that tumor cell proliferation is compro-
mised in the absence of HSPC300 [Casco´n et al., 2007]. In fact,
HSPC300 has been shown to play a role in regulation of the actin
cytoskeleton [Frank and Smith, 2002], and depletion of HSPC300
in RCC cell lines resulted in cytokinesis arrest and reduced
motility, thus reducing the invasive potential [Casco´n et al., 2007].
We also observed a highly significant correlation between
deletion of HSPC300 and a lower incidence of retinal angiomas.
This contrasts with the results of another study, where no such
effect was apparent [Casco´n et al., 2007]. On the other hand, an
association between complete VHL deletions and a relatively low
risk for retinal angiomas has been noted before, but the extent of
the deletions beyond the VHL gene was not determined [Cybulski
et al., 2002; Chew, 2005; Wong et al., 2007]. We also observed a
significant correlation between deletion of HSPC300 and a lower
incidence of kidney cysts. This correlation was not significant in
the study by Casco´n et al. [2007], but renal cystic volume was
significantly reduced (P5 0.05) in the HSPC300 deletion group. It
appears from our study that loss ofthe actin regulator HSPC300
may have a protective effect regarding not only the development
of RCCs, but also of kidney cysts and of retinal angiomas. It
remains to be seen whether future VHL germline deletion studies
can replicate our findings.
A low risk for pheochromocytomas is the determining factor
for classification of VHL patients as Type 1, the class commonly
associated with VHL deletions and frameshift or nonsense
mutations, but not with VHL missense mutations. Some previous
reports noted that families with VHL germline deletions have a
low risk for pheochromocytomas [Crossey et al., 1994; Maher
et al., 1996; Zbar et al., 1996; Hes et al., 2000; Huang et al., 2007;
Ong et al., 2007]. Our data do not support this observation.
Pheochromocytomas developed in 18% of our VHL deletion
families, which is in the range of the reported frequency of 7 to
20% for pheochromocytomas in VHL patients in general
(Supporting Table S2).
The symptoms outside the VHL spectrum that the VHL
deletion families developed show no correlation with the size of
the deletion. The exception is sporadic case 51, who at age 42 years
presented with multiple lesions outside the VHL spectrum, such
as a congenital heart defect, emphysema of the lung, chronic
obstructive pulmonary disease (COPD), anemia and reduced
hemoglobin level, hyperostosis (head) and unsuccessful surgery
after a fracture of the cheekbone, hiatal hernia, hemorrhoids, and
extremely dry skin. The 250-kb deletion in this patient, the largest
deletion in our sample, extends from the centromeric IRAK2 to
the telomeric PRRT3 gene, partially deleting both genes (Fig. 1).
Sporadic case 52 has the second-largest deletion (245 kb), which
partially deletes the centromeric IRAK2 and the telomeric
TMEM111 genes leaving the PRRT3 gene intact, but he developed
cerebellar hemangioblastoma as his only symptom at age 40 years.
It remains unclear whether or not this remarkable difference in
clinical manifestations can be attributed to retention or loss of the
PRRT3 gene, as the function of the encoded transmembrane
protein is not known and the gene has so far not been linked to
any disease.
Table 4. Recombination and Sequence Divergence between
Alu Subfamilies
Alu subfamily Family IDa Occurrence Sequence divergence (%)b
Y2Y 3, 23, 40, 46, 49, 53 6 10.4–14.5
Ya52Y 16118119, 27133 5 8.6–10.3
Sx2Sx 7, 51, 54 3 14.2–21.1
Sc2Sq 26132 2 25.1
Sg2Sx 13, 20 2 17.9118.6
Sx2Jo 12, 30 2 20.2126.0
Y2Sq 22, 44 2 17.8120.7
Y2Sc 17 1 17.5
Y2Sg 4 1 18.9
Ya52Sc 14 1 16.4
Ya52Sg 15 1 10.4
Sq2Sx 10 1 25.4
Sg/x2Sg/xc 39 1 13.4
Y2Jb 37 1 25.0
aA plus sign between Family IDs indicates that the recombinations occurred between
the same Alu elements (see Table 3).
bSequence divergence calculated from pairwise alignment of Alu elements involved,
excluding the poly A-tail. Gaps were counted as one mismatch, normalizing to the
shorter sequence.
cRecombination between two half Alu elements.
784 HUMAN MUTATION, Vol. 30, No. 5, 776–786, 2009
Acknowledgments
We thank the many clinical physicians for their cooperation, by kindly
providing DNA samples and/or by helping to make contact to the patients.
We thank all patients for information and for kindly answering our
questionnaire. We thank Dr. Wiktor Borozdin for supervising the first
steps of qPCR, and Alexander Craig for help with cloning. We thank
Godehard Hoexter for his kind support in generating statistical analyses,
Dr. Bernhard Schermer and Prof. Dr. Thomas Benzing for fruitful
discussions, and Prof. Dr. Werner Schempp and Dr. Deborah Morris-
Rosendahl for helpful comments on the manuscript.
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