Evidence for involvement of the vitamin D receptor gene in idiopathic short stature via a...

Evidence for involvement of the vitamin Dreceptor gene in idiopathic short stature viaa genome-wide linkage study and subsequentassociation studies

Astrid Dempfle1,*, Stefan A. Wudy2, Kathrin Saar3, Sandra Hagemann2, Susann Friedel4,

Andre Scherag1, Lars D. Berthold5, Gerhard Alzen5, Ludwig Gortner6, Werner F. Blum2,7,

Anke Hinney4, Peter Nurnberg8, Helmut Schafer1 and Johannes Hebebrand4

1Institute of Medical Biometry and Epidemiology, Philipps-University Marburg, Germany, 2Pediatric Endocrinology and

Diabetology, Center of Child and Adolescent Medicine, Justus Liebig-University Gießen, Germany, 3Molecular

Genetics and Gene Mapping Center, Max Delbruck Center, Berlin, Germany, 4Department of Child and Adolescent

Psychiatry and Psychotherapy, University Duisburg-Essen, Germany, 5Pediatric Radiology, Center of Radiology,

Justus Liebig-University Gießen, Germany, 6Department of General Pediatrics and Neonatology, University of

Saarland, Homburg, Germany, 7Eli Lilly & Company, Bad Homburg, Germany and 8Cologne Center for Genomics and

Institute for Genetics, University of Cologne, Germany

Received June 24, 2006; Revised and Accepted August 2, 2006

Stature is a highly heritable trait under both polygenic and major gene control. We aimed to identify geneticregions linked to idiopathic short stature (ISS) in childhood, through a whole genome scan in 92 familieseach with two affected children with ISS, including constitutional delay of growth and puberty and familialshort stature. Linkage analysis was performed for ISS, height and bone age retardation. Chromosome12q11 showed significant evidence of linkage to ISS and height (maximum non-parametric multipoint LODscores 3.18 and 2.31 at 55–58 cM, between D12S1301 and D12S1048), especially in sister–sister pairs(LOD score of 1.9 for ISS in 22 pairs). These traits were also linked to chromosomes 1q12 and 2q36. Theregion on chromosome 12q11 had previously shown significant linkage to adult stature in several genomescans and harbors the vitamin D receptor gene, which has been associated with variation in height. Asingle nucleotide polymorphism (SNP) (rs10735810, FokI), which leads to a functionally relevant alterationat the protein level, showed preferential transmission of the transcriptionally more active G-allele to affectedchildren (P 5 0.04) and seems to be responsible for the observed linkage (P 5 0.05, GIST test). Bone ageretardation showed moderate linkage to chromosomes 19p11–q11 and 7p14 (LOD scores 1.69 at 57 cMand 1.42 at 50 cM), but there was no clear overlap with linkage regions for stature. In conclusion, we identifiedsignificant linkage, which might be due to a functional SNP in the vitamin D receptor (VDR) gene and could beresponsible for up to 34% of ISS cases in the population.

INTRODUCTION

Short stature in a child is the most common cause for referralsto pediatric endocrinologists. Many of these patients have noidentifiable medical abnormality but represent the extremes

of normal variation in growth and maturation. They are classi-fied with diagnoses such as constitutional delay of growth andpuberty (CDGP), familial short stature (FSS) or idiopathicshort stature (ISS) in its narrow meaning. In young children,CDGP is typically defined by short stature (height below the

# The Author 2006. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

*To whom correspondence should be addressed at: Institute of Medical Biometry and Epidemiology, Philipps-University Marburg, Bunsenstr. 3, 35037Marburg, Germany. Tel: þ49 6421/2866504; Fax: þ49 6421/2868921; Email: [email protected]

Human Molecular Genetics, 2006, Vol. 15, No. 18 2772–2783doi:10.1093/hmg/ddl218Advance Access published on August 11, 2006

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3rd or 5th age- and sex-specific percentile), bone age retar-dation of at least 1 year and a positive family history ofdelayed growth and pubertal development (1); however, in astrict sense the diagnosis cannot be made before the time ofpuberty, when a delay in sexual maturation becomesevident. Patients with FSS reveal a family history of shortstature, while osseous development and sexual maturationare appropriate for chronological age. A diagnosis of ISS inits narrow meaning is made when both bone age and parentalheight are within the normal range (though often at the lowerend). In clinical terms however, these individual diagnosticcategories often cannot be distinguished, since both parentalheight and bone age delay show a continuous distributionand combinations of both lead to more severe short staturein childhood (2). The clinical cutoffs used for diagnosisoften result in combined diagnoses (3,4). Therefore, wechose to summarize all these subtypes under the generalterm and broader definition of ISS following the recommen-dations of a consensus meeting (2,4). In most patients, theaetiology of their short stature is currently unknown, althoughit is believed that genetic variations are among the underlyingcauses (5,6). The genetic factors contributing to short staturein childhood could affect either height per se or timing ofgrowth and pleiotropic effects on both are possible.

In adults, stature is a highly heritable trait, with heritabilityestimates around 0.8 and higher (7–10). Height is a trait thatfollows a normal distribution in the whole population, as notedby Pearson and Lee (11) over 100 years ago, who suggestedhow this could be the result of many genes, each with asmall, additive effect independent of the others, termed a poly-genic model. More recent segregation analyses additionallyshowed evidence for major genes on top of polygenic inheri-tance (10,12). Studies on the genetics of onset of puberty, ageat maximal growth spurt and skeletal maturation during child-hood showed that these traits are also largely under geneticcontrol, with heritabilities of 50–80% (13–15).

Currently, linkage genome scans of adult height have beenperformed in at least 22 separate samples, summarized in 12publications (10,16–26). Most were carried out in samplesascertained for specific diseases unrelated to body height,such as diabetes (25) or asthma (10), while a few were per-formed in population samples, such as the FraminghamHeart Study (17) or The Netherlands Twin Register (24).Not surprisingly, this large number of individual smallstudies yielded divergent results with suggestive or significantlinkage on many chromosomes. Although no region was high-lighted consistently across all scans, for some regions, theoverall evidence for linkage from the majority of studiesseems convincing. This is the case for regions on chromo-somes 6, 7, 9 and 12, whereas regions on chromosomes 3, 5,13, 14, 15 and 20 were also linked in at least two scans butfailed to be replicated in other studies.

Many candidate genes have been proposed and studied forassociation with (final adult) stature, and also for timing ofgrowth and delayed puberty (27). These include genes of thegrowth hormone-IGF-system (e.g. GH1, GHR, GHRHR,GHSR, IGF-1, IR, STAT5b), genes regulating bone formation(e.g. COL1A1, BMP2, FGFR3), genes involved in pituitarydevelopment (e.g. POU1F1, PROP1, LHX3, LHX4, HESX1)and genes such as the VDR or estrogen receptor alpha (28).

Studies relating to these candidate genes and monogenicforms of growth disorders have been extensively reviewed (14).

The genetic analysis of variation in stature can be sup-plemented by the study of genetic syndromes that includeshort or tall stature among their cardinal features, such asNoonan Syndrome (OMIM 163950, http://www.ncbi.nlm.nih.gov/Omim), Prader–Willi Syndrome (PWS, OMIM 176270)andmany others. In some cases, genes responsible for these syn-dromes might also have alleles that influence normal growthvariation or the whole syndrome is caused by microdeletionswhich include dozens of genes (e.g. PWS), just one of whichmight be involved in growth regulation. Similarly, the shortstature seen in Leri–Weill dyschondrosteosis (LWD, OMIM127300) and Ullrich–Turner Syndrome is caused by haploin-sufficiency due to heterozygous deletions or mutations of theSHOX gene or its regulatory regions (29–31). Such mutationsor deletions in the SHOX gene also seem to be responsible fora proportion of patients diagnosed with ISS, with estimatesranging from 1–22% (32–36). This wide range may be due torather small samples, the use of different ascertainment criteria(the typical skeletal abnormalities of LWD are less pronouncedin younger children and males) and different sensitivities ofmolecular methods for mutation/deletion detection.

Here, we present data from a genome scan in children withISS using affected sibling pairs and both their biologicalparents. Such a selected sample of children with an extremephenotype should have more power to identify linkage toheight than samples of unselected probands (37,38). Inaddition, many of our probands have delayed bone age andwe used this as a quantitative phenotype for linkage analysis.

RESULTS

Linkage results for ISS and height standarddeviation scores

We investigated linkage of ISS and the quantitative pheno-types height [standard deviation scores (SDS)] and bone ageretardation in a sample of 92 families with two affected chil-dren with ISS each, using 511 short tandem repeat (STR)markers. Table 1 gives the maximum and Figures 1 and 2show the genome-wide LOD scores of the linkage analysisfor ISS and height SDS. The main results for these traitswere very similar, with the highest LOD scores on chromo-some 12 (3.18 and 2.31 for ISS and height SDS, respectively)with an adjusted empirical P-value of 0.02 for ISS (i.e. 20 outof 1000 simulations yielded a maximum LOD score greaterthan 3.18). This LOD score was reached at 12q11 at 58 cM.The second region that might harbor genes responsible forheight was on chromosome 1q12, with maximum LODscores of 2.02 (for height SDS) and 1.36 (for ISS) at 140–154 cM. Other LOD scores between one and two were onchromosomes 2q36, 13q12, 15q14 and 19q13 (Table 1),none of these reached genome-wide significance.

Sex-specific analyses for the linked region on chromosome12q11 showed that the linkage peak was mostly due to sister–sister pairs, with a LOD score of 1.90 at 60 cM in only 22pairs, whereas the 33 brother–brother pairs contributed aLOD of 0.27 and 37 opposite-sex pairs gave a LOD of 1.01.Chromosome 15 also showed a strong female-specific

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linkage signal, with a maximum LOD of 2.68 at 14 cM(empirical genome-wide adjusted P-value 0.136; in males,the maximum LOD was 0.65 at 38 cM). Other female-specificLOD scores between 1.5 and 2 were found on chromosomes 2,14 and 18 (empirical genome-wide adjusted P-values 0.72 to0.82). The most prominent male-specific linkage was detectedon chromosome 13 with a maximum LOD of 2.59 at 102 cM(empirical genome-wide adjusted P-value 0.156; in females,the maximum LOD was 0.06 in this region), other

male-specific LOD scores between 1.5 and 2 were found onchromosomes 3, 4 and 16 (empirical genome-wide adjustedP-values 0.47 to 0.83).

Linkage results for bone age retardation

Table 1 gives the maximum and Figure 3 shows the genome-wide LOD scores of the linkage analysis for maximum relativebone age retardation. The linkage analysis of this trait yielded

Figure 1. Genome-wide non-parametric multipoint LOD scores for ISS.

Table 1. Non-parametric multipoint LOD scores above one and adjacent markers for the investigated traits

Trait Chromosome Adjacent marker Position of maximum LODscorea (Kosambi cM)

Multipoint LOD score Empirical genome-widesignificanceb

ISS1 D1S534 154 1.36 0.782 M02HT01Ac 222 1.81 0.4512 D12S1048 58 3.18 0.0215 D15S1012 36 1.25 0.8419 D19S880 95 1.10 0.91

Height SDS1 D1S3723 140 2.02 0.282 D2S9999 222 1.64 0.5912 D12S1301 55 2.31 0.1813 D13S1242 14 1.37 0.74

Maximum relative bone age retardation7 D7S817 50 1.42 0.7611 D11S1999 17 1.07 0.9514 D14S1280 23 1.27 0.8519 D19S208 57 1.69 0.5321 D21S1994 16 1.14 0.92

aAccording to Marshfield map (http://research.marshfieldclinic.org).bDerived by simulating 1000 genome-wide replicates under the null hypothesis of no linkage.cLocally established marker, primers CCTCCAGTGGCAGTAAGC and CCAGGGAGATGGCAGTT.

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only moderate LOD scores, none of which reach genome-widesignificance; there was some evidence of linkage of this phe-notype to chromosomes 19p11–q11 and 7p14 (with maximumLOD scores of 1.69 and 1.42). There was no clear overlap oflinkage regions for bone age retardation with linkage regionsfor ISS and height SDS.

Linkage analysis with imprinting on chromosome 15

For chromosome 15, which is known to harbor both mater-nally and paternally imprinted genes, the separate linkage ana-lyses for maternal and paternal allele-sharing showed strongevidence for imprinting neither in the region of the Imprinting

Center of PWS (15q11, around 0–1 cM), nor in the regionrelevant to Angelman Syndrome (15q11–q13, especially theUBE3A gene at 6 cM). The parent-of-origin specific LODscores for our most distal marker on chromosome 15(D15S822 at 12 cM) were 0.48 for maternal sharing and0.10 for paternal sharing. However, we found evidence of amore proximal locus that was linked to ISS but only contrib-uted if the relevant allele(s) were transmitted from the mother(which would be consistent with a paternally imprinted gene).The maximum maternal-only LOD score was 2.48 at markerD15S1012 (36 cM, cytogenetic region 15q14), whereasthe paternal-only LOD score was zero in this region. Thiswas consistent with our overall maximum LOD score on

Figure 2. Genome-wide non-parametric multipoint LOD scores for height SDS.

Figure 3. Genome-wide non-parametric multipoint LOD scores for maximum relative bone age retardation.




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chromosome 15 of 1.25 at the same marker. The test forimprinting showed that the difference between maternal andpaternal LOD scores was significant (P ¼ 0.04).

Meta-analysis of chromosome 12 results of genomescans for adult stature

Linkage genome scans of final adult height have been per-formed previously by other groups for 22 samples unselectedfor height. Supplementary Material, Table S1 shows themaximum LOD scores on chromosome 12 (18–98 cM)obtained in these scans. The strongest linkage to adultheight was obtained in a Finnish sample (18) with a LODscore of 3.35 at 56 cM, between markers D12S1090 andD12S398. Three other groups reported LOD scores above1.5 for this region (10,22,26) and another three genomescans had LOD scores between 0.97 and 1.5 (17–19). Mostother scans showed weak linkage to this region, with LODscores below one, but remarkably, only six of the 22samples had LOD scores below 0.2 while the maximumLOD scores in this region were between 0.2 and one innine genome scans or sub-samples. Our meta-analysis ofthese results [using the multiple scan probability (MSP)method (39), but not including our own significant resultsfor short stature in children] gave a total adjusted P-valueof 0.03 for the adult height genome scans for this region onchromosome 12.

SNP analysis in the VDR gene

We performed a family-based association analysis in ourlinkage sample of seven common single nucleotide poly-morphisms (SNPs) in the VDR (1,25-dihydroxyvitamin D3

receptor gene) and obtained evidence for association of theG-allele of SNP rs10735810 with stature (nominal P ¼ 0.04,Table 2). This effect was very similar in males and femaleswith a transmission ratio of 58% for the G-allele in bothsexes. The estimated genotype relative risk for the at-riskgenotypes AG and GG compared to genotype AA are 1.33(95% CI: 0.672 to 2.63) and 1.90 (95% CI: 0.903 to 3.98),respectively. Considering the high allele frequency of G(63%), this corresponds to a population attributable fractionof 0.34 (95% CI: 0 to 0.83). The other six investigatedSNPs showed no transmission disequilibrium in the wholesample (Table 2). If the analysis was restricted to affected chil-dren of one sex only, two SNPs (rs4516035 and rs7139166)

showed over-transmission to affected females only, but theseeffects were not significant in the resulting smaller samples,with transmission ratios of 62% (C-allele of rs4516035,nominal P ¼ 0.07) and 61% (T-allele of rs7139166, nominalP ¼ 0.11) to affected females, while transmission was slightlyin the opposite direction to affected males (49% each). Inhaplotype-based association analysis (for all children), a com-bination of all seven SNPs resulted in a P-value of 0.0046;after correction for multiple testing of all possible 127 combi-nations of markers, this remained close to significance(adjusted P ¼ 0.06, see Supplementary Material, Table S2for detailed results of haplotype-based association analysis).There was strong linkage disequilibrium (LD) between thefirst three VDR SNPs and also between the last three SNPsin our sample, with little LD between these blocks (Sup-plementary Material, Fig. S1). This was in complete agree-ment with results of a recent high-resolution LD mapping of68 common SNPs in the VDR gene in a large family sample(40) which indicated the presence of three blocks of highLD. Our first three and last three VDR SNPs map in two ofthese blocks, respectively, while rs10735810 was in a 1.3 kbLD breaking spot separating two of these LD blocks andshowed no LD to any other common SNP.

We tested whether one of the SNPs might explain thelinkage result at this locus, and found that rs10735810showed the most promising results again, with P-values of0.05 under both recessive and additive genetic models[GIST program (41)]. A P-value of 0.06 was also observedfor rs731236 under an additive model, whereas all otherSNPs had P-values .0.13 (most .0.3) for all three con-sidered models.

DISCUSSION

This genome scan yielded significant evidence for linkage ofISS in children to chromosome 12q11 (LOD score 3.18,empirical genome-wide P-value 0.02) with the maximumLOD score at D12S1048. This was supported by the quantitat-ive linkage analysis of height SDS, which also gave the largestLOD score in this region. In this selected sample of childrenwith height below the 5th or 15th age- and sex-specific percen-tile, it could be expected that linkage analyses of the qualitat-ive affection status of short stature and the underlyingquantitative phenotype would yield similar results.

A comparison of our linkage results in a sample of childrenwith short stature with linkage genome scans for final adult

Table 2. Single marker association analysis of VDR gene SNPs (previously used names are given in parentheses). Numbers of transmissions from heterozygousparents are given

Marker Over-transmited allele Frequency ofover-transmitted allele

Transmitted Non-Transmitted Transmission ratio P-value(FAMHAP)

rs7139166 C 0.54 80 66 0.55 0.27rs4516035 T 0.55 76 64 0.54 0.35rs2238136 A 0.26 66 60 0.52 0.66rs10735810 (FokI) G 0.63 106 76 0.58 0.04rs1544410 (BsmI) A 0.39 86 80 0.52 0.70rs7975232 (ApaI) A 0.54 97 83 0.54 0.33rs731236 (TaqI) — 0.38 (C allele) 85 85 0.50 1




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height in samples not selected for height showed a concor-dance for the chromosome 12 region in several scans. Ourmeta-analysis of these results confirmed significant linkage.The observed pattern of moderate linkage in most studieswould be expected for either a gene with a relatively smalleffect in small, unselected samples (42,43) or a gene with rela-tively rare alleles with moderate effect, which have differentfrequencies in the samples and might be most prevalent inselected samples, thus leading to higher power (37,38). Thisis consistent with our significant linkage finding in a muchsmaller (184 phenotyped children) but selected sample, com-pared to the larger unselected samples of adult stature. Thequantitative phenotype stature in an unselected sample ofadults and the affection status ISS in children are closelyrelated but distinct phenotypes, especially because oursample includes a large proportion of children who havealso a delay in maturation (evidenced by bone age retar-dation). Many of our children were pre-pubertal; some ofthese children may have a height within the normal rangeafter their pubertal growth spurt.

Other regions that showed linkage to adult stature in severalgenome scans were on chromosomes 6 (10,17–19,24,26), 7(18–20,25) and 9 (10,17,19,20,22). These regions were notlinked to short stature in children in our sample and mightbe more specific to normal variation in stature. Genes withcommon alleles that have a small effect each (polygenicmodel) and together account for much of the normal variationin stature may not be identifiable with our small, selectedsample (43,44). However, the extremes of this trait, such asshort stature defined by height below the 5th or 15th age-and sex-specific percentiles, may be caused by few geneswith infrequent alleles that have larger effects; evidence formajor genes from segregation analyses suggests this (10,12).Therefore, alleles in one or more genes that have a somewhatlarger effect on height may be present in our selected sample.These might be infrequent in the general population andmostly found in the extremes of the trait distribution, whichcould be the case for our linkage on chromosome 12.

Probably the strongest candidate within our linked region onchromosome 12 is the VDR gene on 12q13.11 (at ~62 cM).The VDR is a member of the steroid/thyroid hormone receptorsuperfamily and has an important role in the vitamin D endo-crine system, which is involved in skeletal metabolism, cellu-lar growth and differentiation in many target organs and anumber of other relevant metabolic pathways, e.g. in theimmune system. Consequently, the VDR gene has been associ-ated with a large number of traits and diseases, primarily bonemineral density and osteodystrophy, but also stature, diabetesand diabetic retinopathy (45), obesity and vascular disease(46) or inflammatory bowel disease and asthma (47). Thefamily-based association analysis indicated that rs10735810(denoted FokI in several previous publications) was associatedwith short stature and may at least partly explain our linkagepeak. This polymorphism is an A to G substitution in thestart codon (Met1Thr), abolishing the first translation initiationsite and resulting in a peptide lacking three amino acids(G-allele), which increases the transcriptional activity ofVDR (45,48). This more active allele was over-transmittedto affected children in our sample giving estimates of moder-ate relative risks of 1.33 and 1.9 for heterozygous and

homozygous carriers. On a population level, this variantmight be responsible for ~34% of ISS cases, but the rathersmall sample size in the present analysis leads to impreciseestimates and large confidence intervals (49). Although over-transmission to male and female affected children wassimilar for rs10735810 (58% transmission ratio), two otherSNPs in the VDR (rs4516035 and rs7139166) showed strongover-transmission only to females, which might explain theobservation of stronger linkage in sister–sister pairs and isin line with previous investigations of an effect of VDR poly-morphisms on height especially in females (50–54). There-fore, the effect of this putative susceptibility locus seems tobe more pronounced in females.

Different polymorphisms in this gene have been studiedrepeatedly for linkage and/or association with stature, bonemineral density and related traits (18,50–64). Most studiesfound an association with height, though not all(56,57,60,61,63). The linkage genome scan for adult heightwith the largest LOD score in this region (18), found noassociation with markers near the VDR, but did not reportdetails of markers tested. Our most strongly associatedmarker rs10735810 (FokI) has been tested for associationwith stature in seven previous studies: the GG-genotype wasassociated with shorter stature in two studies (50,51), associ-ation with other VDR polymorphisms but not the FokImarker was identified in two studies (53,54) and the finalfour studies found no association with any of the investigatedVDR markers (including FokI) (55,57,61,63), although one ofthese studies was restricted to male subjects only (61). It ispossible that rs10735810 is not the actual causal variantitself but rather in LD with it, and such inconsistenciesmight be due to different degrees of LD between FokI and aputative causal variant in the investigated populations.However, high resolution LD analysis revealed that FokIwas not in significant LD with any of 68 common SNPs(minor allele frequency .10%) in the VDR in four Caucasianpopulations (40). Alternatively, low power due to smallsample sizes, differences in the investigated phenotype(adult versus childhood stature) or a differential effect depend-ing on sex might be responsible for inconsistent results.

A comparison of our linkage results with genetic syndromesthat include short stature as a main symptom pointed parti-cularly to PWS and Noonan Syndrome, which map to chromo-somes 15q11–q13 and 12q24.1, respectively. The PWS regionhas been shown to be under the influence of maternal imprint-ing and PWS is caused by a deletion on the paternal chromo-some or uniparental maternal disomy (65–68). For this regionon chromosome 15, we obtained a maximum LOD score of1.25 in the non-parametric linkage analysis of all childrenand a maximum LOD of 2.68 at 14 cM in the 22 sister–sister pairs. The parent-of-origin specific linkage analysisgave a maximum LOD score for maternal sharing of 2.48 at36 cM and a LOD score for paternal sharing of zero. Thiswas contrary to what would be expected from PWS, whereonly the paternally-derived alleles are expressed. However,the imprinted locus appeared to be more proximal than thePWS locus and our imprinted locus seems to be distinctfrom that responsible for the short stature of PWS. It isknown that the same region on chromosome 15q11–q13also contains maternally expressed (paternally imprinted)




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genes, as seen in Angelman Syndrome (OMIM 105830).Patients with Angelman Syndrome also generally have sub-average height (69), although not as pronounced as patientswith PWS. This seems to be the case for patients with15q11–q13 deletions and imprinting defects, not for patientswith mutations in the UBE3A gene (69), indicating anotherimprinted gene (besides UBE3A) in the 15q11–q13 regionthat is related to short stature.

One other gene that has been shown to be responsible forboth non-syndromic and syndromic short stature is theSHOX gene in the pseudoautosomal region on the X chromo-some (31). In isolated short stature, mutations in this gene arerather rare and have been implicated in ~1–22% of cases (32–36), with the largest study to date reporting only 2.4% (34).Such rare causal mutations are more identifiable throughgenetic association studies, but are not expected to lead to sig-nificant linkage in a rather small sample (70). It is, therefore,not surprising that we obtained a LOD score of zero for bothof our makers in the pseudoautosomal region on the Xchromosome. Another candidate gene, which has recentlybeen suggested as responsible for a considerable fraction ofISS cases, is NPR2 (71); this lies at chromosome 9p21–p12where we had an LOD score below 0.5. Therefore, mutationsin NPR2 did not seem to contribute substantially to the shortstature in our sample. Similarly, mutations in the Ghrelinreceptor gene (GHSR, at 3q26.31), identified in FSS (72),did not seem to give rise to a linkage signal.

Delayed maturation (manifested through bone age retar-dation or delayed puberty) is often found in children withshort stature (4) and in our sample, 121 of 184 children hada bone age delay of at least 1 year, which is usually takenas a diagnostic criterion for CDGP. The linkage analysis ofthis quantitative trait revealed only moderate linkage. Thisapproach of analyzing bone age delay as a quantitative traithas the advantage that all children of our sample contributedto the linkage analysis while an analysis of the affectionstatus CDGP would have diminished the eligible sample andthus have lower power. We standardized bone age retardationfor chronological age; nevertheless, this phenotype might bemore heterogeneous than stature (for which we had age- andsex-adjusted SDS values), as we had probands with a largeage range before and after the onset of puberty. The boneage retardation at a specific age might represent a more homo-geneous and more heritable phenotype, but we are not awareof any conclusive formal genetic studies regarding this pheno-type. To our knowledge, no other linkage genome scan ofskeletal maturation during childhood, or related phenotypessuch as delayed puberty, pubertal onset or age of growthspurt or menarche, has been performed. Several geneticassociation studies of candidate genes for such traits havebeen performed, especially with genes of the hypothalamic–pituitary–gonadal axis. Recent studies imply that theG-protein-coupled receptor gene GPR54, which maps tochromosome 19p13.3 (0 cM), together with its ligandKiSS-1 is a regulator of initiation of puberty (73–75). Ourmaximum LOD score of 1.69 on chromosome 19 is at57 cM, which probably is not due to variation in GPR54.Although it is possible that common polymorphisms inGPR54 exist and regulate normal variation in pubertal onset,the mutations identified up to now are very rare and have

severe phenotypic consequences (hypogonadotropic hypogo-nadism) (75). Another strong candidate gene for timing ofmaturation is the estrogen receptor alpha gene (ESR1) onchromosome 6q25, which was linked to and associated withage at menarche in different ethnic groups (76) and alsowith height (77). Again, in our sample, we did not find evi-dence for linkage of either height or bone age retardation tothis region on chromosome 6.

In our sample, it seems that often both factors governingheight per se (evident from short target height based onaverage parental height) and factors responsible for thetiming of growth (bone age delay) contribute to the final diag-nosis of short stature in children. In some cases, one of thesefactors is predominantly evident, whereas in other cases, acombination of both may lead to an especially extreme pheno-type. There may be both shared and separate genetic factorsfor bone age retardation and stature. In our sample, therewas no apparent overlap of linked regions for these two quan-titative traits; all identified regions seemed to be specific foronly one of the traits. A formal multivariate linkage analysis,e.g. using variance component methods, was not possible, asthese traits are not normally distributed in our highly selectedsample.

In conclusion, we identified significant linkage of ISS inchildren to chromosome 12q11, which harbors the VDRgene as a candidate gene previously associated with staturein several studies. The transcriptionally more active allele ofthe functional FokI SNP (rs10735810) was over-transmittedto children affected with ISS and may be responsible for ourlinkage finding. Moderate relative risks of 1.33 and 1.9 wereassociated with heterozygous and homozygous presence ofthe risk allele, leading to an estimate of population attributablerisk of 34%. Haplotype analysis indicated the possibility thatother functionally relevant variants within the VDR genealso contributed to the short stature in our sample.

MATERIALS AND METHODS

Study sample

For this study, 92 families, all including two children with ISS,were ascertained, examined and phenotypically characterizedbetween November 2001 and August 2003 at the endocrineoutpatient clinic of the Center of Child and Adolescent Medi-cine of the Justus Liebig University of Gießen, Germany. Thestudy protocol was approved by the Ethics Committee of theJustus Liebig University of Gießen and written informedconsent was obtained from the parents. The study was con-ducted in accordance with the guidelines of The Declarationof Helsinki.

Families were included if the height of one child was belowthe 5th and the height of a sibling was below the 15th sex- andage-adjusted percentile of the most current German referencedata from more than 34 000 children and adolescents (78).This less stringent threshold for affected siblings was chosento allow the ascertainment of at least a moderately sizedsample, and both the quantitative phenotype stature and theaffection status ISS were used for genetic analysis (seebelow). Only Caucasian families were considered; allparents were of German origin with the exception of two




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fathers from France and Croatia. In eight families, DNA ofonly one parent was available, whereas in all other families,DNA of both parents could be obtained. Medical histories ofchildren and parents were recorded using structured and stan-dardized interviews. All interviews and clinical examinationswere performed by the same investigator (S.H.). As hasbeen described in full previously (4), pathological reasonsfor short stature were excluded. Children with dysmorphic fea-tures or chronic disease were excluded. Absence of chronicdisease was confirmed by tests for chronic inflammatory,coeliac, hepatic or renal disease and hypothyroidism.Growth hormone deficiency was considered to be unlikelybased on serum insulin-like growth factor 1 (IGF-1) andinsulin-like growth factor binding protein 3 (IGFBP-3) levels.

The sample included 103 boys and 81 girls, with a mean ageof 11.8 years in boys (standard deviation 3.7 years, range 4.6–19.3 years) and a mean age of 12.6 years in girls (standarddeviation 4.1 years, range 3.5–21.3 years). Among the 184children included in this study, 96 (52.2%) were pre-pubertal(Tanner stage 1 for pubic hair, breast and genital develop-ment), this applies to 58 (65.3%) of the boys, and 38(46.9%) of the girls. This distribution is typical for ISSwhich usually becomes manifest early in childhood. A boneage retardation of at least 1 year in at least one of the availableX-rays was observed in 70 (68.0%) of the boys and 54 (66.7%)of the girls; 72 (69.9%) boys and 56 (69.1%) girls had FSS(based on mid-parental height). Of these, 49 (47.6%) boysand 36 (44.4%) girls had a combination of both conditions,whereas only 10 (9.7%) boys and seven (8.6%) girls of the184 patients had no pronounced bone age delay and normalmid-parental height.

Phenotypes for genetic analysis

We performed non-parametric linkage analysis of the affectionstatus, ISS, and quantitative trait linkage analysis on the phe-notypes of height SDS and bone age retardation. These weremeasured and defined as follows: current height was measuredto the nearest 0.1 cm using an Ulm Stadiometer (Busse, Ulm,Germany) and converted to SDS using the most currentGerman reference data (78). The mean height SDS for the103 boys in this study was 21.99 (standard deviation 0.6,range 23.67 to 21.05); for the 81 girls, the mean was21.87 (standard deviation 0.6, range 23.29 to 21.09).Bone age was independently assessed by three pediatric radi-ologists according to the method of Greulich and Pyle (79)from X-ray films of the left hand. Bone age determinationwas performed blinded for the patients’ birth date and themean of three ratings for each radiograph was used. The inter-observer agreement was good as judged from pair-wiseBland-Altman bias plots (80), where mean differencesbetween two raters were between 0.5 and 2.3 months and95% limits of agreement were between 14 and 24 months.Bone age retardation was defined as bone age2chronologicalage. If available, X-ray films from earlier presentations at ouroutpatient clinic were also considered (one to six X-ray filmswere available per child) and the mean and maximal bone ageretardations at different time points were calculated. Relativebone age retardation was defined as (bone age2chronologicalage)/chronological age. The phenotype used for genetic

linkage analysis was the maximum of the child’s relativebone age retardations calculated from all available X-rays(analyses with maximum or current absolute bone age retar-dation were similar—results not shown). For boys, this pheno-type had a mean of 20.15 (standard deviation 0.13, range20.50 to20.29), whereas the mean for girls was20.14 (stan-dard deviation 0.14, range 20.52 to 20.08).

Genotyping

We performed a genome-wide scan based on a total of 360individuals (in eight of the 92 families, only one parent wasavailable) with 485 autosomal, 24 X-chromosomal and twopseudoautosomal STR markers spanning the entire genomewith an average (maximum) distance of 7 cM (13 cM)(adapted from Saar et al. (81).). Briefly, markers were amplifiedin multiplex reactions in 384 well microtiter plates on ABIGeneAmp PCR 9700 machines (Applied Biosystems, Darm-stadt, Germany) and an aliquot of the PCR reaction was analyzedon ABI 3730 sequencers. Semi-automated genotyping was per-formed with the help of the GeneMapper software version 3.0(ABI). All genotypes were scored independently by an experi-enced lab technician and subsequently by one scientist.

Genotyping of SNPs in the VDR gene was performed byPCR with subsequent diagnostic restriction fragment lengthpolymorphism analyses for SNPs rs7139166(¼21521 C.G), rs4516035 (¼21012 T.C), rs2238136,rs10735810 (previously termed FokI-polymorphism, alsodenoted as rs2228570, rs8179174 or rs17881966), rs1544410(previously termed BsmI-polymorphism), rs7975232 (pre-viously termed ApaI-polymorphism) and rs731236 (previouslytermed TaqI-polymorphism). See Supplementary Material,Table S3 for primers and restriction enzymes. Positive con-trols for the variant alleles were run on each gel. To validatethe genotypes, allele determinations were rated independentlyby at least two experienced individuals. Discrepancies wereresolved unambiguously either by reaching consensus or byretyping.

Statistical analysis

Familial relationships were verified using the GRR program(82) on the genome scan markers, which confirmed that allsibling-pairs were full siblings as reported. We checked allmarkers for Mendelian inconsistencies using PedCheck (83)and in the event of inconsistencies set the genotypes ofall family members for the respective marker and family asmissing. Hardy–Weinberg equilibrium was checked for allgenetic markers on parental genotypes by x2 tests asimplemented in Mega2 version 3.0 (84) and Merlin version1.0 alpha/Pedstats (85,86). We checked for likely genotypingerrors (e.g. manifesting as close double recombinants) usingMerlin (85) and set unlikely genotypes as missing for therespective individuals. Allele frequencies for linkage analysis(relevant only for the eight missing parents) were estimatedfrom all parental alleles.

Our primary linkage analysis was a multipoint analysis onthe qualitative affection status ISS using the non-parametricSall statistic (87) which can be converted to a non-parametricLOD score (88) as implemented in Merlin (85). We also




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performed linkage analyses on the quantitative phenotypes ofheight SDS and (maximum) relative bone age retardationusing Merlin-regress (89), assuming a heritability of 0.9 forheight SDS and 0.7 for relative bone age retardation and apopulation mean (variance) of 0 (1) for height SDS and 0(0.01) for relative bone age retardation. Setting heritabilityparameters too low might result in false positive linkagepeaks, so we chose relatively high values. This analysis doesnot require that the phenotypes are normally distributed inthe sample.

The empirical genome-wide significance of linkage resultswas assessed by simulating 1000 replicates of our data underthe null hypothesis of no linkage with the same characteristicsregarding family structures, phenotypes, marker spacing andinformativity. These simulated scans were analyzed analo-gously to the real data and the highest non-parametric LODscore on each chromosome in each scan was recorded. Weused a step-down maxT algorithm (90) to calculate adjustedP-values using these simulated replicates. This means thatfor the maximum score obtained in the real data, the empiricalgenome-wide adjusted P-value is the percentage of times thatthe simulated maximum score exceeds the real maximumscore.

We performed sex-specific linkage analyses post hoc,because previous studies indicated that the VDR gene, in ourmost prominent linkage peak, might have a more pronouncedeffect in females. We used only brother–brother or sister–sister pairs for non-parametric linkage analysis. Because thenon-parametric linkage test is based on the number of allelesshared identical by descent between relative pairs, this givesa sensible sex-specific non-parametric linkage analysis.However, the sample size is quite small for each sex and dis-cordant pairs do not contribute to this analysis. Our sampleincluded 33 brother–brother, 22 sister–sister and 37 opposite-sex pairs. Adjusted P-values were obtained as above.

A family-based association test, which is valid for familieswith an arbitrary number of affected children, (basically ageneralization of the transmission disequilibrium test) wasperformed for seven SNPs in the VDR gene using a permu-tation test (91) as implemented in FAMHAP (92). Transmittedand non-transmitted alleles are permutated independently forthe affected children in one family, thus making this a testfor linkage in the presence of association. To consider all poss-ible haplotype configurations, this test was performed for all127 combinations of one to seven markers and a P-valuethat is corrected for this multiple testing is given (adjustedP-value) together with the nominal (uncorrected) P-valuesfor single markers.

To provide quantitative information on the individual andepidemiological relevance of the associated variant, we calcu-lated genotype relative risks and attributable risk (togetherwith confidence intervals) on the basis of a conditional logisticregression model (93,94). LD patterns, as measured by D’between the seven markers, are displayed using Haploview(95).

To evaluate whether one of the VDR SNPs explains the evi-dence for linkage on chromosome 12, we tested for a corre-lation between SNP genotypes of the affected children andthe family-specific NPL scores, as implemented in the GISTprogram (41); three different genetic models (dominant,

recessive and additive) were considered and nominalP-values are given.

Non-parametric linkage analysis with imprinting onchromosome 15 (esp. in the PWS region on 15q11) was per-formed using the imprinting functionality in Allegro version2 (96,97). A weighted scoring function allows investigationof linkage based on separate maternal and paternal allele-sharing, which makes this a test for linkage that allows forimprinting. Additionally, we tested directly whether imprint-ing exists, i.e. whether maternal and paternal allele-sharingare significantly different, using a test proposed by Knappand Strauch (98).

For a region on chromosome 12q, in which we obtained asignificant LOD score, we performed a meta-analysis of pub-lished results from 22 linkage genome scans of adult height.We used a variation of Fisher’s method for the combinationof P-values, which was corrected for multiple testing bytaking into account the size of the genetic region over whichdifferent studies reached their maximum LOD scores (calledthe MSP) (39). This results in one adjusted meta-analysisP-value for the genetic region considered. The publishedquantitative trait locus linkage analyses in the unselectedadult samples had been done by variance components, whichyields an LOD score that can easily be converted toP-values (99–101) and used in the meta-analysis.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

The authors would like to thank the families for their partici-pation in this study. They also thank Sieglinde Durkopp, JitkaAndra, Regina Pospiech and Inka Szangolies for excellenttechnical assistance, Gundula Huth for exceptional data man-agement. The German Ministry for Education and Research(National Genome Research Net2) and the European Union(Diet and Obesity) financially supported this study.

Conflict of Interest statement. The authors declare no conflictof interest. Co-author W.F.B. is an employee and holds stockof Eli Lilly and Co., a maker of rhGH.

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