Clinical and molecular evaluation of SHOX/PAR1 duplications in Léri–Weill dyschondrosteosis (LWD) and Idiopathic Short Stature (ISS)

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DOI: 10.1210/jc.2010-1689 · Source: PubMed
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Abstract
Léri-Weill dyschondrosteosis (LWD) is a skeletal dysplasia characterized by disproportionate short stature and the Madelung deformity of the forearm. SHOX mutations and pseudoautosomal region 1 deletions encompassing SHOX or its enhancers have been identified in approximately 60% of LWD and approximately 15% of idiopathic short stature (ISS) individuals. Recently SHOX duplications have been described in LWD/ISS but also in individuals with other clinical manifestations, thus questioning their pathogenicity. The objective of the study was to investigate the pathogenicity of SHOX duplications in LWD and ISS. Multiplex ligation-dependent probe amplification is routinely used in our unit to analyze for SHOX/pseudoautosomal region 1 copy number changes in LWD/ISS referrals. Quantitative PCR, microsatellite marker, and fluorescence in situ hybridization analysis were undertaken to confirm all identified duplications. During the routine analysis of 122 LWD and 613 ISS referrals, a total of four complete and 10 partial SHOX duplications or multiple copy number (n > 3) as well as one duplication of the SHOX 5' flanking region were identified in nine LWD and six ISS cases. Partial SHOX duplications appeared to have a more deleterious effect on skeletal dysplasia and height gain than complete SHOX duplications. Importantly, no increase in SHOX copy number was identified in 340 individuals with normal stature or 104 overgrowth referrals. MLPA analysis of SHOX/PAR1 led to the identification of partial and complete SHOX duplications or multiple copies associated with LWD or ISS, suggesting that they may represent an additional class of mutations implicated in the molecular etiology of these clinical entities.
J. Clin. Endocrinol. Metab. 2011 96:E404-E412 originally published online Dec 8, 2010; , doi: 10.1210/jc.2010-1689
Argente, J. L. Ross, A. R. Zinn, R. Gracia, P. Lapunzina, A. Campos-Barros and K. E. Heath
S. Benito-Sanz, E. Barroso, D. Heine-Suñer, A. Hisado-Oliva, V. Romanelli, J. Rosell, A. Aragones, M. Caimari, J.
Dyschondrosteosis (LWD) and Idiopathic Short Stature (ISS)
/PAR1 Duplications in Léri-WeillSHOXClinical and Molecular Evaluation of
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Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online
Clinical and Molecular Evaluation of SHOX/PAR1
Duplications in Le´ ri-Weill Dyschondrosteosis (LWD)
and Idiopathic Short Stature (ISS)
S. Benito-Sanz, E. Barroso, D. Heine-Sun˜ er, A. Hisado-Oliva, V. Romanelli,
J. Rosell, A. Aragones, M. Caimari, J. Argente, J. L. Ross, A. R. Zinn, R. Gracia,
P. Lapunzina, A. Campos-Barros, and K. E. Heath
Institute of Medical and Molecular Genetics (S.B.-S., E.B., A.H.-O., V.R., P.L., A.C.-B., K.E.H.), Hospital
Universitario La Paz, Universidad Auto´ noma de Madrid, IdiPAZ, and Centro de Investigacio´ n Biome´ dica
en Red de Enfermedades Raras, Instituto de Salud Carlos III, 28046 Madrid, Spain; Department of
Genetics (D.H.-S., J.R.) and Pediatric Endocrinology Unit (M.C.), Department of Pediatrics, Hospital
Universitario Son Dureta, 07014 Palma de Mallorca, Spain; Department of Pediatrics (A.A.), Hospital
Virgen de la Salud, 45004 Toledo, Spain; Department of Endocrinology (J.A.), Hospital Infantil
Universitario Nin˜ o Jesu´ s, Instituto de Investigacio´ n La Princesa and Department of Pediatrics, Universidad
Auto´ noma de Madrid, and Centro de Investigacio´ n Biome´ dica en Red de Fisiopatología, Obesidad y
Nutricio´ n, Instituto de Salud Carlos III, 28009 Madrid, Spain; Department of Pediatric Endocrinology (R.G.),
Hospital Universitario La Paz, 28046 Madrid, Spain; Department of Pediatrics (J.L.R.), Thomas Jefferson
University, Philadelphia, Pennsylvania 19107; and Dermott Center for Human Growth and Development and
Department of Internal Medicine (A.R.Z.), University of Texas Southwestern Medical School, Dallas, Texas 75235
Context: Le´ ri-Weill dyschondrosteosis (LWD) is a skeletal dysplasia characterized by disproportion-
ate short stature and the Madelung deformity of the forearm. SHOX mutations and pseudoau-
tosomal region 1 deletions encompassing SHOX or its enhancers have been identified in approx-
imately 60% of LWD and approximately 15% of idiopathic short stature (ISS) individuals. Recently
SHOX duplications have been described in LWD/ISS but also in individuals with other clinical man-
ifestations, thus questioning their pathogenicity.
Objective: The objective of the study was to investigate the pathogenicity of SHOX duplications in
LWD and ISS.
Design and Methods: Multiplex ligation-dependent probe amplification is routinely used in our
unit to analyze for SHOX/pseudoautosomal region 1 copy number changes in LWD/ISS referrals.
Quantitative PCR, microsatellite marker, and fluorescence in situ hybridization analysis were un-
dertaken to confirm all identified duplications.
Results: During the routine analysis of 122 LWD and 613 ISS referrals, a total of four complete and
10 partial SHOX duplications or multiple copy number (n 3) as well as one duplication of the SHOX
5 flanking region were identified in nine LWD and six ISS cases. Partial SHOX duplications appeared
to have a more deleterious effect on skeletal dysplasia and height gain than complete SHOX
duplications. Importantly, no increase in SHOX copy number was identified in 340 individuals with
normal stature or 104 overgrowth referrals.
Conclusion: MLPA analysis of SHOX/PAR1 led to the identification of partial and complete SHOX
duplications or multiple copies associated with LWD or ISS, suggesting that they may represent an
additional class of mutations implicated in the molecular etiology of these clinical entities. (J Clin
Endocrinol Metab 96: E404 –E412, 2011)
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2010-1689 Received July 22, 2010. Accepted October 22, 2010.
First Published Online December 8, 2010
Abbreviations: FISH, Fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phos-
phate dehydrogenase; indel, insertions or deletions; ISS, idiopathic short stature; LWD,
Le´ ri-Weill dyschondrosteosis; MIM, Mendelian Inheritance in Man; MLPA, multiplex liga-
tion-dependent probe amplification;PAR1, pseudoautosomal region 1; qPCR, quantitative
real-time PCR; SDS,
SD score; SHOX, short-stature homeobox-containing gene.
JCEM ONLINE
Advances in Genetics—Endocrine Research
E404 jcem.endojournals.org J Clin Endocrinol Metab, February 2011, 96(2):E404–E412
L
e´ri-Weill dyschondrosteosis (LWD) [Mendelian Inher-
itance in Man (MIM) 127300] is a dominantly in-
herited skeletal dysplasia characterized by disproportion-
ate short stature, mesomelic limb shortening, and the
Madelung deformity of the forearm: the bowing of the
radius and the distal dislocation of the ulna (1, 2). Idio-
pathic short stature (ISS; MIM 300582) is a condition
defined as a height below 2
SD score (SDS) in the absence
of known specific causative disorders (3). To date, het-
erozygous deletions of two distinct regions of the pseudo-
autosomal region 1 (PAR1), the short-stature homeobox-
containing gene (SHOX; MIM 313865) (4, 5) and the
downstream enhancer region (6 8) or point and small
insertions or deletions (indel) mutations within SHOX,
have been identified in up to 60% of LWD and approx-
imately 15% ISS patients (9 –11). Homozygous or com-
pound heterozygous SHOX mutations as well as bial-
lelic deletions of SHOX and/or the downstream PAR1
region result in a more severe phenotype, known as
Langer mesomelic dysplasia (MIM 249700) (12–15).
Conversely, SHOX overdosage, caused by either struc-
tural rearrangements or numerical abnormalities of the
sex chromosomes, has been shown to be associated with
normal to tall stature (16 –18). Although the underlying
pathological mechanism remains unknown, it has been
proposed that the association of an extra copy of
SHOX, especially with estrogen deficiency, results in
tall stature through an abnormally prolonged growth
period in the late teenage years and early adulthood
(17). In contrast, SHOX duplications have been re-
cently described in three LWD/ISS patients as well as in
individuals with diverse clinical features, leading to un-
certainty of their involvement in LWD/ISS etiology
(19–22).
Until recently, the molecular diagnosis of LWD, Langer
mesomelic dysplasia, and ISS patients was commonly un-
dertaken by microsatellite and/or fluorescence in situ hy-
bridization (FISH) of the SHOX encompassing PAR1 se-
quence (9, 10, 23). We and others (6, 9, 24–27) have
shown the advantages of using multiplex ligation-de-
pendent probe amplification (MLPA) for the identifi-
cation of PAR1 deletions encompassing SHOX and/or
the downstream enhancer region. This technique has
permitted us now to identify 14 complete or partial
SHOX duplications or multiple copies (n 3) and one
duplication upstream of SHOX in nine LWD and six ISS
probands. We report our clinical and molecular find-
ings on the largest number of SHOX duplications re-
ported to date, with the aim to increase our knowledge
of whether such alterations are involved in the etiology
of LWD and ISS.
Patients and Methods
Clinical patients
The study was approved by the local ethical committees
and all participants provided informed consent. The LWD and
ISS patient samples were referred from endocrinology and
genetic clinics. LWD patients were ascertained using the in-
clusion criteria of the presence of the Madelung deformity and
mesomelic shortening of the limbs in the proband or a direct
family member. Stature was recorded and SDS were deter-
mined according to the population standards for age and gen-
der (28) (http://www.aepap.org/crecorbegozo.ht; http://www.
magicfoundation.org/). ISS patients with stature less than 2 SDS
were ascertained using the current consensus criteria (3).
Two control cohorts were also obtained and analyzed. A co-
hort of 340 normal controls, obtained from the Spanish DNA
bank (University of Salamanca, Salamanca, Spain), with heights
within the normal range for the Spanish population for age and
gender (2 SDS ⬍⫹2). The second control cohort consisted
of 104 overgrowth referrals with heights greater than 3 SDS.
Peripheral blood was drawn from probands and, when pos-
sible, from relevant family members. All samples were reported
to have a normal G-banding karyotype. Genomic DNA was iso-
lated from whole blood using the salt precipitation method
(blood kit; QIAGEN, Valencia, CA).
MLPA assay
MLPA analysis was carried out using the commercial SHOX/
PAR1 MLPA kit (Salsas P018B-D1; MRC Holland, Amsterdam,
The Netherlands) in accordance to the manufacturer’s recom-
mendations. In particular cases, SHOX duplications were fur-
ther delimited using an additional MLPA assay including six
novel probes, located between the Xp telomere and SHOX
(kindly donated by Dr. Simon Thomas, Wessex Regional Ge-
netics Service, Wiltshire, UK). The MLPA assays were analyzed
with help of GeneMapper software (Applied Biosystems, Foster
City, CA). Subsequently the ratios of the proband’ peak areas vs.
controls’ samples were determined using an Excel data sheet
(Microsoft Corp., Richmond, CA). Normal peaks were classified
as showing a ratio of 0.65–1.35, whereas deletions and dupli-
cations were classified as having a ratio less than 0.65 or greater
than 1.35, respectively. Each alteration was confirmed in an in-
dependent MLPA replicate.
Validation of SHOX copy number
Microsatellite analysis
Samples were analyzed for the presence of a triallelic pattern
with the help of a panel of previously described PAR1 mic-
rosatellite markers: DXYS10137, DXYS10138, DXYS201,
DXYS10092, DYS290, DXYS10093, and DXYS10083 (6, 24).
Quantitative real-time PCR (qPCR)
Three qPCR assays targeting sequences within SHOX and
flanking regions were designed to confirm the SHOX copy num-
ber. Primer sequences and probes are described in Supplemental
Table 1, published on The Endocrine Society’s Journals Online
web site at http://jcem.endojournals.org. The qPCR was per-
formed in a final volume of 10
l. The reactions, in duplicate,
contained 1 LightCycler 480 probes master mix (Roche,
Mannheim, Germany), 10
M of each primer, 10
M of both
J Clin Endocrinol Metab, February 2011, 96(2):E404 –E412 jcem.endojournals.org E405
target (labeled with FAM) and calibrator [glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), labeled with HEX] probes,
and 10 ng of genomic DNA. The cycling conditions were as
follows: a prerun at 95 C for 10 min, 45 cycles of 10 sec at 95 C,
30 sec at 60 C and 1 sec at 72 C, and finally a 40 C cooling step
for 30 sec. A no-template negative control was included in each
assay. The samples were amplified in a Lightcycler 480 system
(Roche). Test samples and normal controls were first normalized
to the GAPDH control and subsequently to the mean Ct value
determined for five normal controls. Normal peaks were classi-
fied as showing a ratio of 0.65–1.35, whereas duplications were
classified as having a ratio greater than 1.35.
FISH analysis
To determine if the detected extra copy or copies were the
consequence of chromosomal translocations, we undertook
FISH analysis on metaphase chromosomes prepared from pe-
ripheral blood lymphocytes by standard techniques (29). FISH
was performed with cosmids from the distal PAR1, including
two spanning the entire SHOX, LLNOYCO3M15D10 and
LLNOYCO3M34F5 (30). Centromeric probes DXZ2 and
DYZ3 were used as controls.
Exclusion of point mutations, small deletions, or
insertions in SHOX
All LWD and ISS probands were excluded for SHOX muta-
tions by denaturing HPLC, high resolution melting analysis,
and/or DNA sequencing, as previously reported (9).
Results
Characterization of SHOX duplications
Four complete and 10 partial SHOX duplications or
multiple copies (n 3) as well as one duplication of the
SHOX 5 flanking region were identified in nine LWD and
six ISS probands (Table 1 and Fig. 1). Eight of nine LWD
probands presented with partial SHOX duplications or
multiple copies; one LWD proband had a complete SHOX
duplication, whereas three of six ISS referrals presented
with whole SHOX duplications; two presented with two
different partial SHOX duplications, and one ISS proband
presented with a duplication of the SHOX 5 flanking
region (Fig. 2). No pathogenic PAR1 deletions or point or
indel SHOX mutations were identified in the 15 probands.
Proband heights and clinical details are shown in Fig. 1 and
Table 1. No increases in SHOX copy number were detected
in 340 normal height controls or 104 overgrowth referrals.
The complete SHOX duplications ranged in size from
38 to 346 kb, extending from the SHOX 5 flanking region
through at least exon 6b in three ISS cases, and from the
SHOX 5 flanking region through to approximately 100
kb downstream of SHOX in the single LWD case present-
ing with a complete gene duplication (Fig. 2). Partial
SHOX duplications or multiple copies ranged from 13 to
294 kb, extending from the SHOX 5 flanking region
TABLE 1. Clinical features of the 15 LWD/ISS probands with complete or partial SHOX or SHOX 5 flanking
duplications or multiple copies
Proband Gender LWD/ISS
Duplication
type (extents)
Height SDS (adult
or age in years)
MD
(Y/N)
Other clinical
characteristics
1 F ISS Complete 2.57 (13.0) N Slightly shortened neck
2 F ISS Complete 2.19 (adult) N
3 F ISS Complete 2.29 (13.8) N Postponderal delay,
dermatitis atopica
4 F LWD Complete 0.30 (adult) Y Shortening of the ulnas,
cubitus valgus,
articular enlarging of
hands, articular pain
in wrists, hips, and
knees
5 F ISS Partial (5-Ex3) 2.01 (6.7) N
6 M LWD Partial (5-Ex3) 2.25 (13.0) Y
7 M LWD Partial (Ex2-6b) 1.80 (10.7) Y
8 M LWD Partial (Ex2-6a) 3.88 (15.5) Y
9 F LWD Partial (Ex2-6a) 4.13 (5.0) Y Piloric stenosis, vesicle
uretheral reflux
10 F LWD Partial (Ex2-6a) 2.24 (13.0) Y Minimal cubitus valgus
11 M LWD Partial (Ex2-6a) 2.06 (10.0) Y
12 F LWD Partial (Ex2-6a) 3.95 (13.5) Y Menarche at 8 yr
13 F LWD Partial (Ex2-6a) 1.88 (7.5) N Small for gestional age,
premature adrenarche
14 F ISS Partial (Ex4-3) 2.40 (14.0) N Mental retardation,
dysmorphic facies
15 F ISS 5 region 2.10 (adult) N
F, Female; M, male; MD, Madelung deformity present (Y) or absent (N).
E406 Benito-Sanz et al. SHOX Duplications in LWD/ISS J Clin Endocrinol Metab, February 2011, 96(2):E404–E412
through to exon 3, in two cases (one LWD and one ISS);
from exon 2 to exon 6a in six LWD cases; from exon 2
through to exon 6b in one LWD case; and from exon 4 to
approximately150 kb downstream of SHOX in one ISS
referral (Fig. 2). Proband 15 carried a duplication located
upstream of SHOX, which did not include any SHOX
coding sequence (Fig. 2). The duplication was at least 320
kb in size, extending from the most telomerically analyzed
probe, P1 (250 kb from telomere), to marker DYS201,
15 kb upstream of SHOX.
The height SDS of the eight LWD probands with partial
SHOX duplications or multiple copies ranged between
4.13 and 1.80, whereas the single LWD who had a
complete SHOX duplication (proband 4) had a height
within the normal range (0.3 SDS). She presented with
shortening of the ulnas, Madelung deformity, curving of
the lower limbs, and cubitus valgus and complained of
articular pain in the wrists, hips, and knees.
Six LWD referrals (probands 8 –13) carried a similar
duplication spanning SHOX exons 2–6a (Fig. 2). No
SHOX haplotype was shared between these six probands.
Interestingly, in one case (proband 8), both affected par-
ents presented with a similar alteration as the LWD diag-
nosed proband (Fig. 1), although no common SHOX hap-
lotype was shared between them (data not shown). PAR1
haplotype analysis confirmed that proband 8 had inher-
ited the allele with the extra copies from his mother and
shared the same nonduplicated paternal allele with his
healthy brother, who had inherited the nonduplicated al-
leles form both parents. Probands 5 (ISS) and 6 (LWD)
appear to share the same duplication extents, spanning
from probe P2 in the SHOX 5 flanking region to exon 3,
FIG. 1. Family pedigrees of the 15 LWD/ISS probands with duplications or multiple copy number of SHOX or the 5 flanking region. The probands
are indicated by the arrow. Black-filled symbols indicate the clinical diagnosis of