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The Journal of Clinical Endocrinology & Metabolism 90(9):5386 –5392 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-2520

A Novel EXT1 Splice Site Mutation in a Kindred with Hereditary Multiple Exostosis and Osteoporosis Manuel C. Lemos, Peter Kotanko, Paul T. Christie, Brian Harding, Theodora Javor, Christine Smith, Richard Eastell, and Rajesh V. Thakker Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Center for Diabetes, Endocrinology, and Metabolism, Churchill Hospital (M.C.L., P.T.C., B.H., R.V.T.), Headington, Oxford OX3 7LJ, United Kingdom; Krankenhaus der Barmherzigen Bru¨der, Department of Internal Medicine, Teaching Hospital Medical University Graz (P.K., T.J.), A-8020 Graz, Austria; and Bone Metabolism Group, Division of Clinical Sciences, University of Sheffield, Northern General Hospital (C.S., R.E.), Sheffield S5 7AU, United Kingdom Context: Hereditary multiple exostosis (HME) is an autosomal dominant disorder characterized by the development of benign cartilagecapped tumors at the juxta-epiphyseal regions of long bones. HME is usually caused by mutations of EXT1 or EXT2. Objective: The objective of this study was to investigate a threegeneration Austrian kindred with HME for EXT1 and EXT2 mutations and for abnormalities of bone mineral density (BMD). Methods: DNA sequence and mRNA analyses were used to identify the mutation and its associated consequences. Serum biochemical and radiological investigations assessed bone metabolism and BMD. Results: HME-affected members had a lower femoral neck BMD compared with nonaffected members (z-scores, ⫺2.98 vs. ⫺1.30; P ⫽ 0.011), and in those less than 30 yr of age, the lumbar spine BMD was

H

EREDITARY MULTIPLE EXOSTOSIS (HME; MIM 133700 and 133701) is an autosomal dominant disorder characterized by the occurrence of multiple benign cartilage-capped tumors, or exostoses, that are typically located at the juxta-epiphyseal regions of long bones and associated with disproportionately short stature (1). In addition, exostoses may occur at other sites, such as the ribs, scapula, and pelvis. The exostoses (EXT) may be asymptomatic or associated with complications due to compression of adjacent nerves, blood vessels, and tendons or occasionally to the development of chondrosarcomas or osteosarcomas (1). HME is a genetically heterogeneous disorder, and three different loci, designated EXT1, EXT2, and EXT3, have been mapped to chromosomes 8q24.1 (2), 11p11-p12 (3, 4), and 19p (5), respectively. The human EXT1 and EXT2 genes have been identified (6 – 8), and germline mutations in these are found in the majority of HME patients (9). The EXT1 and EXT2 proteins, which consist of 746 and 718 amino acids, respectively, are endoplasmic reticulum-localized, type II First Published Online June 28, 2005 Abbreviations: BMAD, Bone mineral apparent density; BMD, bone mineral density; DXA, dual-energy x-ray absorptiometry; EBV, EpsteinBarr virus; EXT, exostoses; HME, hereditary multiple exostosis; OPG, osteoprotegerin. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

also low (z-scores, ⫺2.68 vs. ⫺1.42; P ⫽ 0.005). However, they had normal mobility and normal serum concentrations of calcium, phosphate, alkaline phosphatase activity, creatinine, PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, and ␤-crosslaps. DNA sequence analysis of EXT1 revealed a heterozygous g3c transversion that altered the invariant ag dinucleotide of the intron 8 acceptor splice site. RT-PCR analysis using lymphoblastoid RNA showed that the mutation resulted in skipping of exon 9 with a premature termination at codon 599. DNA sequence abnormalities of the osteoprotegerin gene, which is in close proximity to the EXT1 gene, were not detected. Conclusions: A novel heterozygous acceptor splice site mutation of EXT1 results in HME that is associated with a low peak bone mass, indicating a possible additional role for EXT1 in bone biology and in regulating BMD. (J Clin Endocrinol Metab 90: 5386 –5392, 2005)

transmembrane glycosyltransferases that are involved in the chain elongation step of heparan sulfate biosynthesis (10, 11), and the expression of these has been found to be significantly reduced in HME-derived chondrocytes (12, 13). Loss of heterozygosity involving these EXT loci has been reported in chondrosarcomas associated with exostoses (14, 15), and EXT1 epigenetic inactivation has been found in other sporadic cancers (16), suggesting that the EXT genes may also have roles as tumor suppressors. We recently identified a large Austrian kindred with HME and undertook studies to further characterize the clinical features and identify the underlying genetic etiology. Patients and Methods Patients A three-generation Austrian kindred in which HME had affected 14 members (five males and nine females), with an age range of 19 –53 yr, was studied (Fig. 1). These affected members had multiple bone exostoses that were located at the juxta-epiphyseal regions of long bones as well as at other sites (Fig. 2). The radiology in some patients revealed evidence of loss of trabecular bone, which was also assessed by bone mineral density (BMD) measurements. In addition, 11 unaffected (seven males and four females) members, with an age range of 17– 47 yr, were included in the study, which was approved by the local research ethical committee.

Serum biochemistry and BMD measurements Serum calcium, phosphate, alkaline phosphatase activity, and creatinine were measured using standard methods, as previously reported

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Lemos et al. • EXT1 Mutation in HME and Osteoporosis

J Clin Endocrinol Metab, September 2005, 90(9):5386 –5392

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FIG. 1. Pedigree of the family segregating for HME and the EXT1 intragenic polymorphisms, C/T and G/A, at nucleotides c.1431 and c.1761, respectively. These polymorphisms cosegregated with HME, with LOD scores of 2.04 and 1.42, respectively, at 0% recombination, indicating the occurrence of a causative EXT1 mutation. The absence or presence of the g3c acceptor splice mutation (see Fig. 3), occurring at intron 8 of the EXT1 gene, is represented by ⫹ and ⫺, respectively. The peak LOD score between this heterozygous mutation and HME was 5.40, at 0% recombination. Individuals are represented as males (f and 䡺), females (F and E), unaffected (䡺 and E), affected (f and F), and deceased (oblique line through symbol). The paternal haplotypes are shown on the left, and the maternal haplotypes are shown on the right. Deduced haplotypes are within brackets. HME in this family is associated with haplotype [-, T, G], which is inherited from the affected male I-1. The proband (II-1) is indicated by the arrow. The BMD results for individuals available for study (see Table 1) are indicated. F⫹ and F⫺ indicate normal and low (i.e. t-score less than ⫺2.5) femoral neck BMD, respectively, and L⫹ and L⫺ indicate normal and low (i.e. t-score less than ⫺2.5) lumbar spine BMD, respectively. Nine of the 10 individuals affected with HME had t-scores below ⫺2.5, in contrast to two of the six HME-unaffected individuals (P ⬍ 0.05, by Fisher’s exact test).

(17). Serum intact PTH was measured using an electrochemiluminescence immunoassay (Roche, Indianapolis, IN), and serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured using 125I RIAs (Immunodiagnostic Systems, Boldon, UK). Serum osteocalcin and ␤-crosslaps, which is a collagen degradation product and marker for bone resorption, were measured using an electrochemiluminescence immunoassay (Roche). Dual-energy x-ray absorptiometry (DXA) of the femoral neck and lumbar spine was performed on 16 (10 affected and six unaffected) available family members. Bone area, bone mineral content, BMD, BMD t-scores and z-scores were obtained from DXA measurements using the manufacturer’s software (QDR 4500 Elite, Hologic, Inc., Bedford, MA). The Hologic reference database was used to define osteoporosis and osteopenia for the spine, and the National Health and Nutrition Examination Survey III reference database was used for the hip. Osteoporosis was defined according to World Health Organization criteria (i.e. BMD t-score less than ⫺2.5). The relative importance of bone size and volumetric bone density was evaluated by estimating lumbar spine bone volume (from bone area to the power of 1.5) and then calculating bone mineral apparent density (BMAD) by dividing the bone mineral content by the estimated volume (18). This analysis was not performed on the femoral neck, which, unlike vertebrae, often showed the presence of local exostoses that would interfere with interpretation of the results. Two patients (II-1 and II-2; Fig. 1) also had an evaluation by quantitative computed tomography of the lumbar spine using a Toshiba computed tomography scanner and the University of California normative database. BMD values, obtained by DXA and adjusted for age and gender (z-scores), were compared between HME-affected and unaffected relatives using a two-tailed Student’s t test, and the level of statistical significance was set at 0.05. Causes of secondary osteoporosis were excluded in patients using a previously published protocol (17).

Genetic and DNA sequence analysis Venous blood samples were obtained after obtaining informed consent, using guidelines approved by the local research ethical committee, from 28 family members (12 affected, 11 unaffected, and five spouses). Leukocyte DNA was extracted and used with appropriate PCR primers (19, 20) and restriction enzymes to detect previously reported intragenic polymorphisms in the EXT1 and EXT2 genes (21). The EXT1 polymorphisms consist of a C3 T transition of the third base of codon 477, which encodes a Pro residue in exon 6, and a G3 A transition of the third base of codon 587, which encodes a Glu residue in exon 9. The C3 T and G3 A transitions, which occur at nucleotides c.1431 and c.1761, respectively (nucleotide numbers based on cDNA sequence), both result in the loss of an MnlI restriction enzyme site. The previously reported EXT2 polymorphism consists of a C3 A transition of the first base of codon 10, which encodes an Arg residue in exon 2, and occurs at nucleotide c.28 (21). The resultant genotypes were used to calculate conventional two point LOD scores (22). DNA sequence analysis of the EXT1 gene, which consists of 11 exons that encode a 746-amino-acid protein, was performed using leukocyte DNA and EXT1-specific primers (details available on request) for PCR amplification, using methods previously described (19, 20). DNA sequence analysis of the five exons encoding the osteoprotegerin (OPG) gene, which is also referred to as TNF receptor superfamily member 11B (TNFRSF11B), was performed with leukocyte DNA and OPG-specific primers (details available on request) for PCR amplification, using methods previously described (23). The DNA sequences of the PCR products were determined by Taq polymerase cycle sequencing performed using a semiautomated detection system (ABI 373XL sequencer, Applied Biosystems, Foster City, CA) (24). DNA sequence abnormalities were confirmed by restriction enzyme analysis and were demonstrated to cosegregate with the disorder and to be absent as common sequence polymorphisms by studying 110 alleles using DNA obtained from 55 (26 males and 29 females) unrelated normal individuals.

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FIG. 2. Radiographs from patients with HME. A–D, Radiological appearance of exostoses in the proband (individual II-1, Fig. 1). Exostoses are indicated by arrows: A, proximal left humerus; B, distal right ulna; C, lower right ribs; and D, right calcaneum. The presence of these multiple bony outgrowths (exostoses) in individual II-1 as well as in other members of the family (not shown) led to the diagnosis of HME. E, Lateral radiograph of lumbar spine (individual II-2, Fig. 1), showing relative osteopenia with prominence of the vertebral trabeculae.

RT-PCR analysis RT-PCR was used to investigate mRNA splicing abnormalities, using total RNA extracted from an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line from the proband (II-1; Fig. 1) and two unrelated normal individuals, as previously described (25). RT-PCR was performed using EXT1-specific primers (forward, 5⬘-GTCGTCATTGAAGGAGAGAG-3⬘; reverse, 5⬘-GCCCAACGGGAAGCCCGAGA-3⬘) and 35 cycles with an annealing temperature of 58 C. The forward primer is complementary to a part of exon 7 (nucleotides c.1609_1628), and the reverse primer is complementary to a part of exon 11 (nucleotides c.2059_2078). The DNA sequences of the RT-PCR products were determined using methods previously reported (24).

Results

Analysis of the EXT1 and EXT2 intragenic polymorphisms in the kindred revealed that the EXT1 polymorphisms cosegregated with HME. The peak LOD scores between HME and the C3 T and G3 A EXT1 polymorphisms were 2.04 and 1.42, respectively, at 0% recombination. DNA sequence analysis of the 2241 bp of the EXT1 coding region and the 20 splice sites was therefore undertaken. This revealed a heterozygous guanine to cytosine (g3c) transversion at position ⫺1 of the acceptor splice site of intron 8 (Fig. 3). This DNA sequence change, which altered the invariant ag dinucleotide of the acceptor splice consensus site, resulted in the loss of a PstI restriction endonuclease site, and this was used to confirm the presence of the heterozygous mutation and its cosegregation with HME in the family (Fig. 3). The peak LOD score between HME and the heterozygous mutation of EXT1, which is consistent with the autosomal dominant phenotype in this kindred, was 5.40, at 0% recombination. In addition, an analysis of the DNA from 55 unrelated normal individuals

confirmed the absence of this g3c transversion at position ⫺1 of intron 8 in 110 alleles, indicating that it was not a common polymorphism that would be expected to occur in over 1% of the population. The g nucleotide at position ⫺1 of the acceptor splice consensus sequence is invariant in eukaryotic sequences (26), and mutations of this ⫺1 bp g nucleotide have been previously reported in patients with endocrine disorders, such as multiple endocrine neoplasia type 1 (27) and hypoparathyroidism, deafness, and renal dysplasia syndrome (28). These and other studies have also revealed that mutations in the acceptor splice site regions may be associated with an accumulation of unspliced precursor mRNA, retention of incompletely spliced precursors, complete absence of transcripts, or the appearance of aberrantly processed mRNA from the use of alternative normally occurring splice sites or cryptic splice sites. To investigate these possibilities, we examined EXT1 mRNA processing by the detection of its transcription in EBV-transformed lymphoblastoid cell lines (Fig. 4). This revealed, in addition to the wild-type product, the presence of an aberrantly processed mRNA that was 161 bp smaller than normal. DNA sequence analysis of the mutant EXT1 product revealed exon skipping in which exon 9, which is 161 bp in size, was lost, and exon 8 was spliced to exon 10. The loss of this 161 bp of exon 9 is predicted to cause a frameshift, starting at codon 575, followed by 24 missense amino acids and a premature termination at codon 599. During the course of radiological investigations to assess the extent of exostosis in the HME patients, evidence of osteopenia was found (Fig. 2). This was also assessed by


FIG. 3. Detection of mutation in intron 8 in the HME family by restriction enzyme analysis. DNA sequence analysis of an individual (II-6) affected with HME revealed a g3c transversion of the invariant ag dinucleotide of the acceptor splice site consensus sequence of intron 8 (A). The g3c transversion resulted in the loss of a PstI restriction enzyme site (ctgca/g) from the wild-type (WT) sequence (A), and this facilitated detection of the mutation in other affected members (III-3 and III-4 are shown) of this family (B). After PCR amplification and PstI digestion, one product of 235 bp was obtained from the mutant (m) sequence, but two products of 197 and 38 bp (not shown in B) were obtained from the wild-type normal sequence (C). The affected individuals (II-6, III-3, and III-4 are shown) are heterozygous (WT/m), and the unaffected individuals (I-2, I-5, and III-2 are shown) are homozygous (WT/WT; B). The symbols representing the individuals are described in Fig. 1. The positions of the size markers (S; 100-bp ladder) at 200 and 300 bp are shown. Cosegregation of this EXT1 mutation with HME in the family was demonstrated (Fig. 1), and its absence from 110 alleles of 55 unrelated normal individuals (not shown) indicated that it is not a common DNA sequence polymorphism.

BMD measurements in 16 available family members (Table 1). Nine (three males and six females; age range, 19 –53 yr) of 10 (three males and seven females; age range, 19 –53 yr) HME-affected relatives and two males, aged 46 and 47 yr, of six nonaffected relatives (five males and one female; age range, 17– 47 yr), for whom data were available, were found to have osteoporosis (i.e. t-score less than ⫺2.5; Table 1). As a group, HME-affected relatives, compared with unaffected relatives, had significantly lower z-scores at the femoral neck (⫺2.98 vs. ⫺1.30; P ⫽ 0.011), but not at the lumbar spine (⫺2.47 vs. ⫺1.80; P ⫽ 0.133). Differences in BMD were larger when considering only individuals under the age of 30 yr, represented by the third generation in Fig. 1 (lumbar spine z-scores, ⫺2.68 vs. ⫺1.42; P ⫽ 0.005). Separate analysis for males and females did not reveal a gender effect on z-score differences between affected and nonaffected relatives. Correction for bone size (29, 30) showed that the low BMD in HME patients was associated with low BMAD and normal or high bone volume (Fig. 5). In addition, lumbar spine quantitative computed tomography carried out on two individuals (II-1 and II-2; Fig. 1) showed BMD of 56.3 mg/cm3 (z-score, ⫺4.75) and 100.2 mg/cm3 (z-score, ⫺1.80), respec-


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FIG. 4. Exon skipping due to an intron 8 acceptor splice consensus sequence mutation. The transcription of exons 7–11 of the EXT1 gene was detected by RT-PCR using RNA obtained from EBV-transformed lymphoblastoids of normal subjects (N1 and N2) and the proband (II-1) with HME who had the intron 8 acceptor splice site consensus sequence mutation (see Fig. 3). In the normal individuals, correctly spliced EXT1 cDNA was observed (A) at the expected size of 470 bp (B). This was confirmed by DNA sequence analysis. However, the proband with HME and the g3c transversion of the intron 8 acceptor splice site was found to have two products. One of these is the normal EXT1 cDNA of 470 bp, and the other is an abnormal EXT1 cDNA of 309 bp (A). This mutant (m) cDNA differed from the wild-type (WT) cDNA by 161 bp, which corresponds to the size of exon 9 (B). DNA sequence analysis of the mutant cDNA confirmed exon 9 skipping, with splicing of exon 8 to exon 10. This loss of exon 9 would, if translated, result in a frameshift from codon 575 with 24 missense amino acids, followed by a premature stop signal (TGA) at codon 599. DNA size standard (S) markers, which were a 100-bp ladder, are indicated.

tively, thus supporting the initial findings of low BMD, by an independent method. The HME patients had normal mobility and did not have a history of limited weight-bearing exercise during adolescence; hence, the low BMD in these affected individuals could not be accounted for by diminished physical activity. Furthermore, the low BMD in the patients affected with HME was not associated with abnormalities of serum calcium, phosphate, alkaline phosphatase activity, creatinine, intact PTH, 25-hydroxyvitamin D, 1,25dihydroxyvitamin D, osteocalcin, or ␤-crosslaps. In addition, DNA sequence analysis of the OPG gene, which is within 1 megabase of the EXT1 gene, did not detect any abnormalities, indicating that an OPG mutation is unlikely to be the cause of the low BMD in the HME patients. Discussion

Our study of this large HME kindred has identified a novel heterozygous acceptor splice site mutation in intron 8 of the EXT1 gene. This mutation leads to skipping of exon 9, which results in a frameshift from codon 575 with 24 missense amino acids and a premature termination at codon 599. This would lead to loss of the highly conserved C terminal of the protein, which appears to be involved in protein-protein interactions (31). The importance of this domain has been previously shown by showing that deletion of the histidine residue at codon 627 in exon 9 results in a loss of enzyme activity (32). Thus, this heterozygous mutation, which is

M M F M M M

M F F F F M F F F M

Sex

47 46 25 19 17 22

53 51 50 49 45 44 42 24 19 19

165 165 165

162 164

146 144 141 139 145 148 141 150 150 157

20 48

59 15 49 14 43 64 30

Age Ht No. of (yr) (cm) EXT

0.821 0.730 0.927 0.910 0.826 0.953

0.580 0.687 0.776 0.702 0.914 0.722 0.802 0.765 0.713 0.767

⫺2.45 ⫺3.24 ⫺1.09 ⫺1.64 ⫺2.41 ⫺1.26

⫺4.65 ⫺3.28 ⫺2.47 ⫺3.14 ⫺1.21 ⫺3.36 ⫺2.23 ⫺2.57 ⫺3.04 ⫺2.95

BMD T score (g/cm2)

⫺2.16 ⫺2.99 ⫺1.00 ⫺1.42 ⫺1.98 ⫺1.26 ⫺1.80 ⫾ 0.73

⫺4.22 ⫺2.43 ⫺1.71 ⫺2.47 ⫺0.76 ⫺3.17 ⫺1.89 ⫺2.47 ⫺2.77 ⫺2.79 ⫺2.47* ⫾ 0.92

Z score

Lumbar spine (L1–L4)

0.677 0.640 0.768 0.901 0.705 0.941

0.417 0.497 0.582 0.443 0.716 0.466 0.538 0.533 0.605 0.644

⫺2.75 ⫺3.06 ⫺1.26 ⫺0.71 ⫺2.49 ⫺0.35

⫺5.10 ⫺3.98 ⫺3.13 ⫺4.52 ⫺1.79 ⫺4.66 ⫺3.57 ⫺3.62 ⫺2.89 ⫺3.05

BMD T score (g/cm2)

⫺1.67 ⫺2.00 ⫺1.26 n/a n/a ⫺0.25 ⫺1.30 ⫾ 0.76

⫺3.80 ⫺2.90 ⫺2.13 ⫺3.60 ⫺1.08 ⫺3.74 ⫺2.99 ⫺3.61 n/a n/a ⫺2.98** ⫾ 0.95

Z score

Femoral neck

2.38 2.39 2.38 2.39 ⫾ 0.03 2.0 –2.6

2.44 2.37

2.29 2.61 2.38 2.78 2.32 2.23 2.28 2.57 2.39 2.25 2.41 ⫾ 0.18

Ca (mmol/liter)

94 95

127 98 ⫾ 32

147 108 75 82 101 124 79 43

ALP (U/liter)

1.27 1.08

0.96 1.02 0.85 0.81 0.86 0.86 0.90 0.75 0.76 0.95 0.87 ⫾ 0.09

Cr (mg/dl)

13.5 16.8

39.6 38.8 43.8 41.6 27.2 30.5 57.8 21.2 13.9 51.2 36.6 ⫾ 13.5

PTH (pg/ml)

3.5 150 0.88 3.8 190 0.94 4.2 80 0.95 3.6 122 1.02 15.2 ⫾ 0.5 ⫾ 47 ⫾ 0.16 ⫾ 2.3 2.3–5.0 50 –170 0.5–1.2b 15– 65

2.8 3.9

3.5 3.4 3.1 3.4 3.8 3.0 3.5 4.0 2.8 3.8 3.4 ⫾ 0.4

Pi (mg/dl)

Serum

17.1 27.4 25.7 33.2 ⫾ 20.2 23–113

62.7

34.2 91.9 27.5 25.2 25.0 41.5 46.3 42.7 21.5 39.5 ⫾ 21.6

127.9 33.4 23.4 57.3 ⫾ 47.8 20 – 67

44.5

14.4 43.7 36.6 15.6 32.6 35.5 29.6 48.7 18.2 30.5 ⫾ 12.3

25-OHD 1,25-OHD (nmol/liter) (pg/ml)

0.45 ⫾ 0.08

0.42

0.57 0.43 0.39

␤CTX (ng/ml)

80.0 80.0 25.0 53.7 ⫾ 30.5 11–70c 0.00 –1.56d

29.6

52.3 31.0 28.6 10.0 18.9 15.5 30.8 30.3 81.3 33.2 ⫾ 21.7

OC (ng/ml)


M, Male; F, female; Ht, height; n/a, not available due to lack of appropriate age-matched reference population; Ca, calcium; Pi, phosphate; ALP, alkaline phosphatase activity; Cr, creatinine; 25-OHD, 25-hydroxyvitamin D; 1,25-OHD, 1,25-dihydroxyvitamin D; OC, osteocalcin; ␤CTX, beta-crosslaps. Asterisks indicate significant Z-score differences between affected and unaffected relatives. *, t test P value 0.13 (all members analyzed) and 0.005 [only third-generation members (III-1 to III-12) analyzed]. **, t test P value 0.011 (all members analyzed); third generation not separately analyzed due to lack of appropriate age-matched reference population. a Member identification indicates position in pedigree (Fig. 1). b Normal: male, 0.7–1.2; female, 0.5– 0.9. c Normal: male 18 –30 yr, 24 –70; male 30 –50 yr, 14 – 42; male 50 –70 yr, 14 – 46; female premenopausal, 11– 43; postmenopausal, 15– 46. d Normal: female premenopausal, 0 – 0.87; postmenopausal, 0 –1.56.

Unaffected II-7 II-8 III-1 III-10 III-11 III-12 Mean ⫾ SD Normal range

Affected II-1 II-2 II-4 II-6 II-10 II-11 II-14 III-3 III-4 III-5 Mean ⫾ SD

Membera

TABLE 1. Clinical, biochemical, and BMD data obtained from HME affected and unaffected relatives

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FIG. 5. Comparison of bone size in HME patients and unaffected (normal) individuals. The 10 HME patients for whom data were available (Table 1) were found to have normal to high bone volume at the lumbar spine, despite the short stature, and low BMAD compared with their six normal (unaffected) relatives (Table 1) and with the reference range (RR), which has been previously established (29) from a study of healthy individuals from Sheffield, UK. This reference range, from a British population, does not differ from that established in a German population as part of the Osteoporosis and Ultrasound Study (30) (Glu¨er, C., unpublished observation). These results indicate that BMD is low in the HME patients because of a true decrease in volumetric BMD and not because of a decrease in bone size. AP LS, Anteroposterior lumbar spine.

consistent with the autosomal dominant phenotype, is likely to be of significance in the etiology of HME in this family. Our study is the first to report an association between HME and osteoporosis. Although benign tumors and skeletal deformities are hallmarks of HME, no effect on overall BMD has been previously reported. Presently, it is unknown whether this is a mutation-specific phenotype or whether other published mutations, with similar effects on the EXT1 protein, are also associated with osteoporosis. A reevaluation to assess osteoporosis in these other families with EXT1 mutations is warranted to clarify this issue. The BMD data from our study of HME patients with EXT1 mutations and any future studies will need to be interpreted with caution, because there is evidence that values are related to skeletal size. This is because smaller bones may exhibit lower BMD when analyzed by the two-dimensional projection obtained by DXA scanning compared with the true volumetric BMD (29). Because short stature was a common feature in our patients, height may have contributed to the low BMD values. However, correction for bone size showed that the low BMD in patients was associated with low BMAD, but normal or high bone volume. Furthermore, the BMD data obtained by quantitative computed tomography, which is independent of bone size, in two of the HME patients confirmed that results were not significantly distorted by short stature alone. Although several unaffected relatives showed variable degrees of osteopenia, as a group, the HME patients had significantly lower z-scores than unaffected relatives. This difference was more striking when considering only individuals less than 30 yr of age, pointing toward an inherited, rather than acquired, characteristic. The EXT1 mutation may be the underlying cause of osteoporosis in this kindred by interfering with the role of the EXT1 protein in bone metabolism or by another unidentified pleiotropic effect. The EXT1 protein has been shown to be an endoplasmic reticulum-localized, type II transmembrane glycoprotein that forms a Golgi-localized complex with the EXT2 protein that appears to be involved in heparan sulfate polymerization (33, 34). It is interesting to note that transgenic expres-

sion of the EXT2 gene in mice up-regulates the expression of EXT1 and the formation of trabeculae, although the effects of this on BMD remain to be assessed (35). EXT1 haplo-insufficient mice failed to develop exostoses, but had a decline in heparan sulfate formation and a slight shortening of bone length (36) together with mild reductions in humeral and femoral BMD (37). Thus, our findings of low BMD in the HME patients with an EXT1 mutation may be consistent with the BMD phenotype of the EXT1 haplo-insufficient mice (37). An alternative explanation for the observed association with osteoporosis in the HME family is represented by the possibility that there may be linkage disequilibrium of the EXT1 locus with another neighboring gene involved in bone metabolism. Genes that are located in 8q24 and would represent strong candidates for having an etiological role in osteoporosis include hyaluronic acid synthase 2 (HAS2; MIM 601636), syndecan 2 or heparan sulfate proteoglycan (SDC2; MIM 142460), and OPG (TNFRSF11B; MIM 602643), which has been reported to have roles in the regulation of BMD (38), osteoporosis (39), and juvenile Paget’s disease (23). However, polymorphisms in these genes are unlikely to have the extreme effect on BMD that is observed in HME patients. The low BMD in osteoporosis is thought to be caused by a combined large number of risk alleles or polymorphisms, each having a subtle effect on BMD, but together resulting in substantially decreased BMD (40). Given this, we reasoned that it would have to be a severe mutation in one of these genes to cause the low BMD in the HME patients. We sought such a mutation in OPG because of its role in regulating BMD and osteoporosis (38, 39). Our analysis did not reveal OPG mutations in the HME patients, and this indicates that the EXT1 mutation is likely to be responsible for the low BMD phenotype in this family. In summary, our study of an Austrian HME kindred has identified a novel acceptor splice site mutation of the EXT1 gene that leads to exon skipping and a truncated EXT1 protein that would result in a loss of function. Furthermore, our study revealed a previously unrecognized association of osteoporosis with HME due to an EXT1 mutation, and this may help to elucidate the role of this gene in bone metabolism.

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Acknowledgments We thank Dr. R. Stacher for providing the radiographs together with help in their interpretation. Received December 22, 2004. Accepted June 21, 2005. Address all correspondence and requests for reprints to: Dr. Rajesh V. Thakker, Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Center for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, United Kingdom. E-mail: [email protected]. This work was supported by the Medical Research Council, United Kingdom (to M.C.L., P.T.C., B.H., and R.V.T.), and the Portuguese Foundation for Science and Technology (SFRH/BD/12415/2003; to M.C.L.).

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