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Human Molecular Genetics Advance Access originally published online on August 11, 2006
Human Molecular Genetics 2006 15(18):2772-2783; doi:10.1093/hmg/ddl218
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Evidence for involvement of the vitamin D receptor gene in idiopathic short stature via a genome-wide linkage study and subsequent association studies

Astrid Dempfle1,*, Stefan A. Wudy2, Kathrin Saar3, Sandra Hagemann2, Susann Friedel4, André Scherag1, Lars D. Berthold5, Gerhard Alzen5, Ludwig Gortner6, Werner F. Blum2,7, Anke Hinney4, Peter Nürnberg8, Helmut Schäfer1 and Johannes Hebebrand4

1 Institute of Medical Biometry and Epidemiology, Philipps-University Marburg, Germany, 2 Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus Liebig-University Gießen, Germany, 3 Molecular Genetics and Gene Mapping Center, Max Delbrück Center, Berlin, Germany, 4 Department of Child and Adolescent Psychiatry and Psychotherapy, University Duisburg-Essen, Germany, 5 Pediatric Radiology, Center of Radiology, Justus Liebig-University Gießen, Germany, 6 Department of General Pediatrics and Neonatology, University of Saarland, Homburg, Germany, 7 Eli Lilly & Company, Bad Homburg, Germany and 8 Cologne Center for Genomics and Institute for Genetics, University of Cologne, Germany

* To whom correspondence should be addressed at: Institute of Medical Biometry and Epidemiology, Philipps-University Marburg, Bunsenstr. 3, 35037 Marburg, Germany. Tel: +49 6421/2866504; Fax: +49 6421/2868921; Email: dempfle{at}med.uni-marburg.de

Received June 24, 2006; Accepted August 2, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Stature is a highly heritable trait under both polygenic and major gene control. We aimed to identify genetic regions linked to idiopathic short stature (ISS) in childhood, through a whole genome scan in 92 families each with two affected children with ISS, including constitutional delay of growth and puberty and familial short stature. Linkage analysis was performed for ISS, height and bone age retardation. Chromosome 12q11 showed significant evidence of linkage to ISS and height (maximum non-parametric multipoint LOD scores 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. The region on chromosome 12q11 had previously shown significant linkage to adult stature in several genome scans and harbors the vitamin D receptor gene, which has been associated with variation in height. A single nucleotide polymorphism (SNP) (rs10735810, FokI), which leads to a functionally relevant alteration at the protein level, showed preferential transmission of the transcriptionally more active G-allele to affected children (P=0.04) and seems to be responsible for the observed linkage (P=0.05, GIST test). Bone age retardation showed moderate linkage to chromosomes 19p11–q11 and 7p14 (LOD scores 1.69 at 57 cM and 1.42 at 50 cM), but there was no clear overlap with linkage regions for stature. In conclusion, we identified significant linkage, which might be due to a functional SNP in the vitamin D receptor (VDR) gene and could be responsible for up to 34% of ISS cases in the population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Short stature in a child is the most common cause for referrals to pediatric endocrinologists. Many of these patients have no identifiable medical abnormality but represent the extremes of normal variation in growth and maturation. They are classified with diagnoses such as constitutional delay of growth and puberty (CDGP), familial short stature (FSS) or idiopathic short stature (ISS) in its narrow meaning. In young children, CDGP is typically defined by short stature (height below the 3rd or 5th age- and sex-specific percentile), bone age retardation of at least 1 year and a positive family history of delayed growth and pubertal development (1); however, in a strict sense the diagnosis cannot be made before the time of puberty, when a delay in sexual maturation becomes evident. Patients with FSS reveal a family history of short stature, while osseous development and sexual maturation are appropriate for chronological age. A diagnosis of ISS in its narrow meaning is made when both bone age and parental height are within the normal range (though often at the lower end). In clinical terms however, these individual diagnostic categories often cannot be distinguished, since both parental height and bone age delay show a continuous distribution and combinations of both lead to more severe short stature in childhood (2). The clinical cutoffs used for diagnosis often result in combined diagnoses (3,4). Therefore, we chose to summarize all these subtypes under the general term and broader definition of ISS following the recommendations of a consensus meeting (2,4). In most patients, the aetiology of their short stature is currently unknown, although it is believed that genetic variations are among the underlying causes (5,6). The genetic factors contributing to short stature in childhood could affect either height per se or timing of growth and pleiotropic effects on both are possible.

In adults, stature is a highly heritable trait, with heritability estimates around 0.8 and higher (7–10). Height is a trait that follows a normal distribution in the whole population, as noted by Pearson and Lee (11) over 100 years ago, who suggested how this could be the result of many genes, each with a small, additive effect independent of the others, termed a polygenic model. More recent segregation analyses additionally showed evidence for major genes on top of polygenic inheritance (10,12). Studies on the genetics of onset of puberty, age at maximal growth spurt and skeletal maturation during childhood showed that these traits are also largely under genetic control, with heritabilities of 50–80% (13–15).

Currently, linkage genome scans of adult height have been performed in at least 22 separate samples, summarized in 12 publications (10,16–26). Most were carried out in samples ascertained for specific diseases unrelated to body height, such as diabetes (25) or asthma (10), while a few were performed in population samples, such as the Framingham Heart Study (17) or The Netherlands Twin Register (24). Not surprisingly, this large number of individual small studies yielded divergent results with suggestive or significant linkage on many chromosomes. Although no region was highlighted consistently across all scans, for some regions, the overall evidence for linkage from the majority of studies seems convincing. This is the case for regions on chromosomes 6, 7, 9 and 12, whereas regions on chromosomes 3, 5, 13, 14, 15 and 20 were also linked in at least two scans but failed to be replicated in other studies.

Many candidate genes have been proposed and studied for association with (final adult) stature, and also for timing of growth and delayed puberty (27). These include genes of the growth 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 pituitary development (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 monogenic forms of growth disorders have been extensively reviewed (14).

The genetic analysis of variation in stature can be supplemented by the study of genetic syndromes that include short or tall stature among their cardinal features, such as Noonan Syndrome (OMIM 163950 [OMIM] , http://www.ncbi.nlm.nih.gov/Omim), Prader–Willi Syndrome (PWS, OMIM 176270 [OMIM] ) and many others. In some cases, genes responsible for these syndromes might also have alleles that influence normal growth variation or the whole syndrome is caused by microdeletions which include dozens of genes (e.g. PWS), just one of which might be involved in growth regulation. Similarly, the short stature seen in Léri–Weill dyschondrosteosis (LWD, OMIM 127300 [OMIM] ) and Ullrich–Turner Syndrome is caused by haploinsufficiency due to heterozygous deletions or mutations of the SHOX gene or its regulatory regions (29–31). Such mutations or deletions in the SHOX gene also seem to be responsible for a proportion of patients diagnosed with ISS, with estimates ranging from 1–22% (32–36). This wide range may be due to rather small samples, the use of different ascertainment criteria (the typical skeletal abnormalities of LWD are less pronounced in younger children and males) and different sensitivities of molecular methods for mutation/deletion detection.

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


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Linkage results for ISS and height standard deviation scores
We investigated linkage of ISS and the quantitative phenotypes height [standard deviation scores (SDS)] and bone age retardation in a sample of 92 families with two affected children with ISS each, using 511 short tandem repeat (STR) markers. Table 1 gives the maximum and Figures 1 and 2 show the genome-wide LOD scores of the linkage analysis for ISS and height SDS. The main results for these traits were very similar, with the highest LOD scores on chromosome 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 out of 1000 simulations yielded a maximum LOD score greater than 3.18). This LOD score was reached at 12q11 at 58 cM. The second region that might harbor genes responsible for height was on chromosome 1q12, with maximum LOD scores of 2.02 (for height SDS) and 1.36 (for ISS) at 140–154 cM. Other LOD scores between one and two were on chromosomes 2q36, 13q12, 15q14 and 19q13 (Table 1), none of these reached genome-wide significance.


Figure 2181
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  		Figure 1. Genome-wide non-parametric multipoint LOD scores for ISS.

 


Figure 2182
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  		Figure 2. Genome-wide non-parametric multipoint LOD scores for height SDS.

 


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

 
Sex-specific analyses for the linked region on chromosome 12q11 showed that the linkage peak was mostly due to sister–sister pairs, with a LOD score of 1.90 at 60 cM in only 22 pairs, whereas the 33 brother–brother pairs contributed a LOD of 0.27 and 37 opposite-sex pairs gave a LOD of 1.01. Chromosome 15 also showed a strong female-specific 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-specific LOD scores between 1.5 and 2 were found on chromosomes 2, 14 and 18 (empirical genome-wide adjusted P-values 0.72 to 0.82). The most prominent male-specific linkage was detected on 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 on chromosomes 3, 4 and 16 (empirical genome-wide adjusted P-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 relative bone age retardation. The linkage analysis of this trait yielded only moderate LOD scores, none of which reach genome-wide significance; there was some evidence of linkage of this phenotype to chromosomes 19p11–q11 and 7p14 (with maximum LOD scores of 1.69 and 1.42). There was no clear overlap of linkage regions for bone age retardation with linkage regions for ISS and height SDS.


Figure 2183
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  		Figure 3. Genome-wide non-parametric multipoint LOD scores for maximum relative bone age retardation.

 
Linkage analysis with imprinting on chromosome 15
For chromosome 15, which is known to harbor both maternally and paternally imprinted genes, the separate linkage analyses for maternal and paternal allele-sharing showed strong evidence for imprinting neither in the region of the Imprinting Center of PWS (15q11, around 0–1 cM), nor in the region relevant to Angelman Syndrome (15q11–q13, especially the UBE3A gene at 6 cM). The parent-of-origin specific LOD scores for our most distal marker on chromosome 15 (D15S822 at 12 cM) were 0.48 for maternal sharing and 0.10 for paternal sharing. However, we found evidence of a more proximal locus that was linked to ISS but only contributed 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 marker D15S1012 (36 cM, cytogenetic region 15q14), whereas the paternal-only LOD score was zero in this region. This was consistent with our overall maximum LOD score on chromosome 15 of 1.25 at the same marker. The test for imprinting showed that the difference between maternal and paternal LOD scores was significant (P=0.04).

Meta-analysis of chromosome 12 results of genome scans for adult stature
Linkage genome scans of final adult height have been performed previously by other groups for 22 samples unselected for height. Supplementary Material, Table S1 shows the maximum LOD scores on chromosome 12 (18–98 cM) obtained in these scans. The strongest linkage to adult height was obtained in a Finnish sample (18) with a LOD score of 3.35 at 56 cM, between markers D12S1090 and D12S398. Three other groups reported LOD scores above 1.5 for this region (10,22,26) and another three genome scans had LOD scores between 0.97 and 1.5 (17–19). Most other scans showed weak linkage to this region, with LOD scores below one, but remarkably, only six of the 22 samples had LOD scores below 0.2 while the maximum LOD scores in this region were between 0.2 and one in nine genome scans or sub-samples. Our meta-analysis of these results [using the multiple scan probability (MSP) method (39), but not including our own significant results for short stature in children] gave a total adjusted P-value of 0.03 for the adult height genome scans for this region on chromosome 12.

SNP analysis in the VDR gene
We performed a family-based association analysis in our linkage sample of seven common single nucleotide polymorphisms (SNPs) in the VDR (1,25-dihydroxyvitamin D3 receptor gene) and obtained evidence for association of the G-allele of SNP rs10735810 with stature (nominal P=0.04, Table 2). This effect was very similar in males and females with a transmission ratio of 58% for the G-allele in both sexes. The estimated genotype relative risk for the at-risk genotypes 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 fraction of 0.34 (95% CI: 0 to 0.83). The other six investigated SNPs showed no transmission disequilibrium in the whole sample (Table 2). If the analysis was restricted to affected children of one sex only, two SNPs (rs4516035 and rs7139166) showed over-transmission to affected females only, but these effects were not significant in the resulting smaller samples, with transmission ratios of 62% (C-allele of rs4516035, nominal P=0.07) and 61% (T-allele of rs7139166, nominal P=0.11) to affected females, while transmission was slightly in the opposite direction to affected males (49% each). In haplotype-based association analysis (for all children), a combination of all seven SNPs resulted in a P-value of 0.0046; after correction for multiple testing of all possible 127 combinations of markers, this remained close to significance (adjusted P=0.06, see Supplementary Material, Table S2 for detailed results of haplotype-based association analysis). There was strong linkage disequilibrium (LD) between the first three VDR SNPs and also between the last three SNPs in our sample, with little LD between these blocks (Supplementary Material, Fig. S1). This was in complete agreement with results of a recent high-resolution LD mapping of 68 common SNPs in the VDR gene in a large family sample (40) which indicated the presence of three blocks of high LD. Our first three and last three VDR SNPs map in two of these blocks, respectively, while rs10735810 was in a 1.3 kb LD breaking spot separating two of these LD blocks and showed no LD to any other common SNP.


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

 
We tested whether one of the SNPs might explain the linkage result at this locus, and found that rs10735810 showed the most promising results again, with P-values of 0.05 under both recessive and additive genetic models [GIST program (41)]. A P-value of 0.06 was also observed for rs731236 under an additive model, whereas all other SNPs had P-values >0.13 (most >0.3) for all three considered models.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
This genome scan yielded significant evidence for linkage of ISS in children to chromosome 12q11 (LOD score 3.18, empirical genome-wide P-value 0.02) with the maximum LOD score at D12S1048. This was supported by the quantitative linkage analysis of height SDS, which also gave the largest LOD score in this region. In this selected sample of children with height below the 5th or 15th age- and sex-specific percentile, it could be expected that linkage analyses of the qualitative affection status of short stature and the underlying quantitative phenotype would yield similar results.

A comparison of our linkage results in a sample of children with short stature with linkage genome scans for final adult height in samples not selected for height showed a concordance for the chromosome 12 region in several scans. Our meta-analysis of these results confirmed significant linkage. The observed pattern of moderate linkage in most studies would be expected for either a gene with a relatively small effect in small, unselected samples (42,43) or a gene with relatively rare alleles with moderate effect, which have different frequencies in the samples and might be most prevalent in selected samples, thus leading to higher power (37,38). This is consistent with our significant linkage finding in a much smaller (184 phenotyped children) but selected sample, compared to the larger unselected samples of adult stature. The quantitative phenotype stature in an unselected sample of adults and the affection status ISS in children are closely related but distinct phenotypes, especially because our sample includes a large proportion of children who have also a delay in maturation (evidenced by bone age retardation). Many of our children were pre-pubertal; some of these children may have a height within the normal range after their pubertal growth spurt.

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

Probably the strongest candidate within our linked region on chromosome 12 is the VDR gene on 12q13.11 (at ~62 cM). The VDR is a member of the steroid/thyroid hormone receptor superfamily and has an important role in the vitamin D endocrine system, which is involved in skeletal metabolism, cellular growth and differentiation in many target organs and a number of other relevant metabolic pathways, e.g. in the immune system. Consequently, the VDR gene has been associated with a large number of traits and diseases, primarily bone mineral density and osteodystrophy, but also stature, diabetes and diabetic retinopathy (45), obesity and vascular disease (46) or inflammatory bowel disease and asthma (47). The family-based association analysis indicated that rs10735810 (denoted FokI in several previous publications) was associated with short stature and may at least partly explain our linkage peak. This polymorphism is an A to G substitution in the start codon (Met1Thr), abolishing the first translation initiation site and resulting in a peptide lacking three amino acids (G-allele), which increases the transcriptional activity of VDR (45,48). This more active allele was over-transmitted to affected children in our sample giving estimates of moderate relative risks of 1.33 and 1.9 for heterozygous and homozygous carriers. On a population level, this variant might be responsible for ~34% of ISS cases, but the rather small sample size in the present analysis leads to imprecise estimates and large confidence intervals (49). Although over-transmission to male and female affected children was similar for rs10735810 (58% transmission ratio), two other SNPs in the VDR (rs4516035 and rs7139166) showed strong over-transmission only to females, which might explain the observation of stronger linkage in sister–sister pairs and is in line with previous investigations of an effect of VDR polymorphisms on height especially in females (50–54). Therefore, the effect of this putative susceptibility locus seems to be more pronounced in females.

Different polymorphisms in this gene have been studied repeatedly for linkage and/or association with stature, bone mineral density and related traits (18,50–64). Most studies found an association with height, though not all (56,57,60,61,63). The linkage genome scan for adult height with the largest LOD score in this region (18), found no association with markers near the VDR, but did not report details of markers tested. Our most strongly associated marker rs10735810 (FokI) has been tested for association with stature in seven previous studies: the GG-genotype was associated with shorter stature in two studies (50,51), association with other VDR polymorphisms but not the FokI marker was identified in two studies (53,54) and the final four studies found no association with any of the investigated VDR markers (including FokI) (55,57,61,63), although one of these studies was restricted to male subjects only (61). It is possible that rs10735810 is not the actual causal variant itself but rather in LD with it, and such inconsistencies might be due to different degrees of LD between FokI and a putative causal variant in the investigated populations. However, high resolution LD analysis revealed that FokI was not in significant LD with any of 68 common SNPs (minor allele frequency >10%) in the VDR in four Caucasian populations (40). Alternatively, low power due to small sample sizes, differences in the investigated phenotype (adult versus childhood stature) or a differential effect depending on sex might be responsible for inconsistent results.

A comparison of our linkage results with genetic syndromes that include short stature as a main symptom pointed particularly to PWS and Noonan Syndrome, which map to chromosomes 15q11–q13 and 12q24.1, respectively. The PWS region has been shown to be under the influence of maternal imprinting and PWS is caused by a deletion on the paternal chromosome or uniparental maternal disomy (65–68). For this region on chromosome 15, we obtained a maximum LOD score of 1.25 in the non-parametric linkage analysis of all children and a maximum LOD of 2.68 at 14 cM in the 22 sister–sister pairs. The parent-of-origin specific linkage analysis gave a maximum LOD score for maternal sharing of 2.48 at 36 cM and a LOD score for paternal sharing of zero. This was contrary to what would be expected from PWS, where only the paternally-derived alleles are expressed. However, the imprinted locus appeared to be more proximal than the PWS locus and our imprinted locus seems to be distinct from that responsible for the short stature of PWS. It is known that the same region on chromosome 15q11–q13 also contains maternally expressed (paternally imprinted) genes, as seen in Angelman Syndrome (OMIM 105830 [OMIM] ). Patients with Angelman Syndrome also generally have sub-average height (69), although not as pronounced as patients with PWS. This seems to be the case for patients with 15q11–q13 deletions and imprinting defects, not for patients with mutations in the UBE3A gene (69), indicating another imprinted gene (besides UBE3A) in the 15q11–q13 region that is related to short stature.

One other gene that has been shown to be responsible for both non-syndromic and syndromic short stature is the SHOX gene in the pseudoautosomal region on the X chromosome (31). In isolated short stature, mutations in this gene are rather 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 through genetic association studies, but are not expected to lead to significant linkage in a rather small sample (70). It is, therefore, not surprising that we obtained a LOD score of zero for both of our makers in the pseudoautosomal region on the X chromosome. Another candidate gene, which has recently been suggested as responsible for a considerable fraction of ISS cases, is NPR2 (71); this lies at chromosome 9p21–p12 where we had an LOD score below 0.5. Therefore, mutations in NPR2 did not seem to contribute substantially to the short stature in our sample. Similarly, mutations in the Ghrelin receptor 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 retardation or delayed puberty) is often found in children with short stature (4) and in our sample, 121 of 184 children had a bone age delay of at least 1 year, which is usually taken as a diagnostic criterion for CDGP. The linkage analysis of this quantitative trait revealed only moderate linkage. This approach of analyzing bone age delay as a quantitative trait has the advantage that all children of our sample contributed to the linkage analysis while an analysis of the affection status CDGP would have diminished the eligible sample and thus have lower power. We standardized bone age retardation for chronological age; nevertheless, this phenotype might be more heterogeneous than stature (for which we had age- and sex-adjusted SDS values), as we had probands with a large age range before and after the onset of puberty. The bone age retardation at a specific age might represent a more homogeneous and more heritable phenotype, but we are not aware of any conclusive formal genetic studies regarding this phenotype. To our knowledge, no other linkage genome scan of skeletal maturation during childhood, or related phenotypes such as delayed puberty, pubertal onset or age of growth spurt or menarche, has been performed. Several genetic association studies of candidate genes for such traits have been performed, especially with genes of the hypothalamic–pituitary–gonadal axis. Recent studies imply that the G-protein-coupled receptor gene GPR54, which maps to chromosome 19p13.3 (0 cM), together with its ligand KiSS-1 is a regulator of initiation of puberty (73–75). Our maximum LOD score of 1.69 on chromosome 19 is at 57 cM, which probably is not due to variation in GPR54. Although it is possible that common polymorphisms in GPR54 exist and regulate normal variation in pubertal onset, the mutations identified up to now are very rare and have severe phenotypic consequences (hypogonadotropic hypogonadism) (75). Another strong candidate gene for timing of maturation is the estrogen receptor alpha gene (ESR1) on chromosome 6q25, which was linked to and associated with age at menarche in different ethnic groups (76) and also with height (77). Again, in our sample, we did not find evidence for linkage of either height or bone age retardation to this region on chromosome 6.

In our sample, it seems that often both factors governing height per se (evident from short target height based on average parental height) and factors responsible for the timing of growth (bone age delay) contribute to the final diagnosis of short stature in children. In some cases, one of these factors is predominantly evident, whereas in other cases, a combination of both may lead to an especially extreme phenotype. There may be both shared and separate genetic factors for bone age retardation and stature. In our sample, there was no apparent overlap of linked regions for these two quantitative traits; all identified regions seemed to be specific for only one of the traits. A formal multivariate linkage analysis, e.g. using variance component methods, was not possible, as these traits are not normally distributed in our highly selected sample.

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


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 References
 
Study sample
For this study, 92 families, all including two children with ISS, were ascertained, examined and phenotypically characterized between November 2001 and August 2003 at the endocrine outpatient clinic of the Center of Child and Adolescent Medicine of the Justus Liebig University of Gießen, Germany. The study protocol was approved by the Ethics Committee of the Justus Liebig University of Gießen and written informed consent was obtained from the parents. The study was conducted in accordance with the guidelines of The Declaration of Helsinki.

Families were included if the height of one child was below the 5th and the height of a sibling was below the 15th sex- and age-adjusted percentile of the most current German reference data from more than 34 000 children and adolescents (78). This less stringent threshold for affected siblings was chosen to allow the ascertainment of at least a moderately sized sample, and both the quantitative phenotype stature and the affection status ISS were used for genetic analysis (see below). Only Caucasian families were considered; all parents were of German origin with the exception of two fathers from France and Croatia. In eight families, DNA of only one parent was available, whereas in all other families, DNA of both parents could be obtained. Medical histories of children and parents were recorded using structured and standardized interviews. All interviews and clinical examinations were performed by the same investigator (S.H.). As has been described in full previously (4), pathological reasons for short stature were excluded. Children with dysmorphic features or chronic disease were excluded. Absence of chronic disease was confirmed by tests for chronic inflammatory, coeliac, hepatic or renal disease and hypothyroidism. Growth hormone deficiency was considered to be unlikely based on serum insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) levels.

The sample included 103 boys and 81 girls, with a mean age of 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 (standard deviation 4.1 years, range 3.5–21.3 years). Among the 184 children included in this study, 96 (52.2%) were pre-pubertal (Tanner stage 1 for pubic hair, breast and genital development), this applies to 58 (65.3%) of the boys, and 38 (46.9%) of the girls. This distribution is typical for ISS which usually becomes manifest early in childhood. A bone age retardation of at least 1 year in at least one of the available X-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%) boys and 36 (44.4%) girls had a combination of both conditions, whereas only 10 (9.7%) boys and seven (8.6%) girls of the 184 patients had no pronounced bone age delay and normal mid-parental height.

Phenotypes for genetic analysis
We performed non-parametric linkage analysis of the affection status, ISS, and quantitative trait linkage analysis on the phenotypes of height SDS and bone age retardation. These were measured and defined as follows: current height was measured to the nearest 0.1 cm using an Ulm Stadiometer (Busse, Ulm, Germany) and converted to SDS using the most current German reference data (78). The mean height SDS for the 103 boys in this study was –1.99 (standard deviation 0.6, range –3.67 to –1.05); for the 81 girls, the mean was –1.87 (standard deviation 0.6, range –3.29 to –1.09). Bone age was independently assessed by three pediatric radiologists according to the method of Greulich and Pyle (79) from X-ray films of the left hand. Bone age determination was performed blinded for the patients' birth date and the mean of three ratings for each radiograph was used. The inter-observer agreement was good as judged from pair-wise Bland-Altman bias plots (80), where mean differences between two raters were between 0.5 and 2.3 months and 95% limits of agreement were between 14 and 24 months. Bone age retardation was defined as bone age–chronological age. If available, X-ray films from earlier presentations at our outpatient clinic were also considered (one to six X-ray films were available per child) and the mean and maximal bone age retardations at different time points were calculated. Relative bone age retardation was defined as (bone age–chronological age)/chronological age. The phenotype used for genetic linkage analysis was the maximum of the child's relative bone age retardations calculated from all available X-rays (analyses with maximum or current absolute bone age retardation were similar—results not shown). For boys, this phenotype had a mean of –0.15 (standard deviation 0.13, range –0.50 to –0.29), whereas the mean for girls was –0.14 (standard deviation 0.14, range –0.52 to –0.08).

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

Genotyping of SNPs in the VDR gene was performed by PCR with subsequent diagnostic restriction fragment length polymorphism analyses for SNPs rs7139166 (=–1521 C>G), rs4516035 (=–1012 T>C), rs2238136, rs10735810 (previously termed FokI-polymorphism, also denoted as rs2228570, rs8179174 or rs17881966), rs1544410 (previously termed BsmI-polymorphism), rs7975232 (previously termed ApaI-polymorphism) and rs731236 (previously termed TaqI-polymorphism). See Supplementary Material, Table S3 for primers and restriction enzymes. Positive controls for the variant alleles were run on each gel. To validate the genotypes, allele determinations were rated independently by at least two experienced individuals. Discrepancies were resolved unambiguously either by reaching consensus or by retyping.

Statistical analysis
Familial relationships were verified using the GRR program (82) on the genome scan markers, which confirmed that all sibling-pairs were full siblings as reported. We checked all markers for Mendelian inconsistencies using PedCheck (83) and in the event of inconsistencies set the genotypes of all family members for the respective marker and family as missing. Hardy–Weinberg equilibrium was checked for all genetic markers on parental genotypes by {chi}2 tests as implemented in Mega2 version 3.0 (84) and Merlin version 1.0 alpha/Pedstats (85,86). We checked for likely genotyping errors (e.g. manifesting as close double recombinants) using Merlin (85) and set unlikely genotypes as missing for the respective individuals. Allele frequencies for linkage analysis (relevant only for the eight missing parents) were estimated from all parental alleles.

Our primary linkage analysis was a multipoint analysis on the qualitative affection status ISS using the non-parametric Sall statistic (87) which can be converted to a non-parametric LOD score (88) as implemented in Merlin (85). We also performed linkage analyses on the quantitative phenotypes of height SDS and (maximum) relative bone age retardation using Merlin-regress (89), assuming a heritability of 0.9 for height SDS and 0.7 for relative bone age retardation and a population mean (variance) of 0 (1) for height SDS and 0 (0.01) for relative bone age retardation. Setting heritability parameters too low might result in false positive linkage peaks, so we chose relatively high values. This analysis does not require that the phenotypes are normally distributed in the sample.

The empirical genome-wide significance of linkage results was assessed by simulating 1000 replicates of our data under the null hypothesis of no linkage with the same characteristics regarding family structures, phenotypes, marker spacing and informativity. These simulated scans were analyzed analogously to the real data and the highest non-parametric LOD score on each chromosome in each scan was recorded. We used a step-down maxT algorithm (90) to calculate adjusted P-values using these simulated replicates. This means that for the maximum score obtained in the real data, the empirical genome-wide adjusted P-value is the percentage of times that the simulated maximum score exceeds the real maximum score.

We performed sex-specific linkage analyses post hoc, because previous studies indicated that the VDR gene, in our most prominent linkage peak, might have a more pronounced effect in females. We used only brother–brother or sister–sister pairs for non-parametric linkage analysis. Because the non-parametric linkage test is based on the number of alleles shared identical by descent between relative pairs, this gives a sensible sex-specific non-parametric linkage analysis. However, the sample size is quite small for each sex and discordant pairs do not contribute to this analysis. Our sample included 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 families with an arbitrary number of affected children, (basically a generalization of the transmission disequilibrium test) was performed for seven SNPs in the VDR gene using a permutation test (91) as implemented in FAMHAP (92). Transmitted and non-transmitted alleles are permutated independently for the affected children in one family, thus making this a test for linkage in the presence of association. To consider all possible haplotype configurations, this test was performed for all 127 combinations of one to seven markers and a P-value that is corrected for this multiple testing is given (adjusted P-value) together with the nominal (uncorrected) P-values for single markers.

To provide quantitative information on the individual and epidemiological relevance of the associated variant, we calculated genotype relative risks and attributable risk (together with confidence intervals) on the basis of a conditional logistic regression 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 evidence for linkage on chromosome 12, we tested for a correlation between SNP genotypes of the affected children and the family-specific NPL scores, as implemented in the GIST program (41); three different genetic models (dominant, recessive and additive) were considered and nominal P-values are given.

Non-parametric linkage analysis with imprinting on chromosome 15 (esp. in the PWS region on 15q11) was performed using the imprinting functionality in Allegro version 2 (96,97). A weighted scoring function allows investigation of linkage based on separate maternal and paternal allele-sharing, which makes this a test for linkage that allows for imprinting. Additionally, we tested directly whether imprinting exists, i.e. whether maternal and paternal allele-sharing are significantly different, using a test proposed by Knapp and Strauch (98).

For a region on chromosome 12q, in which we obtained a significant LOD score, we performed a meta-analysis of published results from 22 linkage genome scans of adult height. We used a variation of Fisher's method for the combination of P-values, which was corrected for multiple testing by taking into account the size of the genetic region over which different studies reached their maximum LOD scores (called the MSP) (39). This results in one adjusted meta-analysis P-value for the genetic region considered. The published quantitative trait locus linkage analyses in the unselected adult samples had been done by variance components, which yields an LOD score that can easily be converted to P-values (99–101) and used in the meta-analysis.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
References
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors would like to thank the families for their participation in this study. They also thank Sieglinde Dürkopp, Jitka Andrä, Regina Pospiech and Inka Szangolies for excellent technical assistance, Gundula Huth for exceptional data management. 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 conflict of interest. Co-author W.F.B. is an employee and holds stock of Eli Lilly and Co., a maker of rhGH.


   References
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 

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