Transcription of SHOX is dependent upon the interaction of the gene with a complex array of flanking regulatory elements. Duplications that contain flanking regulatory elements but not the SHOX gene have been reported in individuals with SHOX haploinsufficiency syndromes, suggesting that alterations to the physical organisation or genomic architecture may affect SHOX transcription. Individuals with tall stature and an additional X or Y chromosome have an extra copy of both the SHOX gene and the entire SHOX regulatory region, so all three copies of SHOX can be expressed fully. However, for a duplication of the SHOX gene that does not include all of the flanking regulatory elements, the potential effect on SHOX expression is difficult to predict. We present nine unpublished individuals with a SHOX whole gene duplication in whom the duplication contains variable amounts of the SHOX regulatory region, and we review 29 similar cases from the literature where phenotypic data were clearly stated. While tall stature was present in a proportion of these cases, we present evidence that SHOX whole gene duplications can also result in a phenotype more typically associated with SHOX haploinsufficiency and are significantly overrepresented in Leri-Weill dyschondrosteosis and idiopathic short stature probands compared to population controls. Although similar-looking duplications do not always produce a consistent phenotype, there may be potential genotype-phenotype correlations regarding the duplication size, regulatory element content, and the breakpoint proximity to the SHOX gene. Although ClinGen does not currently consider SHOX whole gene duplications to be clinically significant, the ClinGen triplosensitivity score does not take into account the context of the duplication, and more is now known about SHOX duplications and the role of flanking elements in SHOX regulation. The evidence presented here suggests that these duplications should not be discounted without considering the extent of the duplication and the patient phenotype, and should be included in diagnostic laboratory reports as variants of uncertain significance. Given the uncertain pathogenicity of these duplications, any reports should encourage the exclusion of all other causes of short stature where possible.

Haploinsufficiency of SHOX results in phenotypes ranging from Leri-Weill dyschondrosteosis (LWD; MIM #127300) [Leri and Weill, 1929] to short stature [Marchini et al., 2016]. Idiopathic short stature (ISS; MIM #300582) is defined as a height below the 3rd centile in the absence of a known specific causative disorder [Wit et al., 2008], and although SHOX loss-of-function variants are highly penetrant, clinical expression is extremely variable, even within the same family [Binder et al., 2003; Stuppia et al., 2003; Huber et al., 2006; Jorge et al., 2007; Rappold et al., 2007].

SHOX regulation is highly complex, with long-range enhancers both 5′ and 3′ of the gene. Comparative genomic studies identified multiple conserved noncoding DNA elements (CNEs) downstream of SHOX, four of which have transcriptional activity: CNE4 (X:714,085–714,740 [hg19]), CNE5 (X:750,825–751,850), CNE7 (ECR1; X:780,700–781,220), and CNE9 (ECS4; X:834,746–835,567) [Fukami et al., 2006; Sabherwal et al., 2007; Chen et al., 2009; Benito-Sanz et al., 2012]. The ZED element (zeugopodal enhancer downstream of SHOX) [Skuplik et al., 2018] was shown to be the critical functional element within the recurrent 47.5-kb X:780,550–828,092 downstream deletion [Benito-Sanz et al., 2012; Bunyan et al., 2013] and an additional cis-regulatory element was proposed around X:970,000 [Bunyan et al., 2014] that would potentially further extend the downstream regulatory region. Three active SHOX upstream CNEs have also been demonstrated, namely CNE-5 (X:398,357–398,906), CNE-3 (X:460,279–460,664), and CNE-2 (X:516,610–517,229) [Durand et al., 2010]. Duplications that contain only flanking SHOX regulatory elements have been reported in individuals with SHOX haploinsufficiency syndromes [Benito-Sanz et al., 2011; Fukami et al., 2015; Bunyan et al., 2016, 2021; Hirschfeldova and Solc, 2017; Sadler et al., 2020], suggesting that the physical organisation of the SHOX regulatory region is critical, so alterations to the physical separation or to the intervening genomic architecture may affect SHOX transcription.

An additional copy of the entire SHOX gene and all associated regulatory elements would be expected to cause overexpression, and tall stature is typically observed in individuals with an additional sex chromosome (47,XXX, 47,XXY or 47,XYY) [Ottesen et al., 2010]. However, the consequences of duplications which include the entire SHOX gene but only part of the regulatory “cassette” are hard to predict, even though many cases have been described in the literature [Thomas et al., 2009; Benito-Sanz et al., 2011; Brosens et al., 2014; Fukami et al., 2015; Donze et al., 2015; Tropeano et al., 2016; Hirschfeldova and Solc, 2017; Upners et al., 2017; Sadler et al., 2020]. SHOX whole gene duplications are associated with a wide range of phenotypes with no established genotype-phenotype correlations, making genetic counselling and clinical management difficult. One hypothesis is that the phenotypic variability may be directly related to the regulatory element content of the duplicated interval, and/or to the location of the duplicated fragment. We can test this hypothesis by studying individuals with SHOX whole gene duplications that contain a variable number of enhancers, i.e., where at least one breakpoint of the duplication maps within the X:398,357–970,000 interval which contains all currently known or proposed cis-regulatory elements.

We describe nine previously unreported individuals with a SHOX whole gene duplication that includes only part of the X:398,357–970,000 region and have identified 29 further cases from the literature with a similar duplication where the individual’s phenotype was clearly stated. We have determined the frequencies of these duplications within various study cohorts and compared them to control cohorts. To limit ascertainment bias, our results include data from in-house and published array comparative genome hybridisation (aCGH) cohorts to determine how frequently probands with a SHOX whole gene duplication, detected incidentally, have an ISS or LWD phenotype.

The novel probands presented in this study were tested at the Wessex Regional Genetics Laboratory as part of the National Health Service (NHS). Purified genomic DNA obtained from an EDTA blood sample was extracted according to standard protocols.

This is a retrospective study. Within the NHS in England, probands with isolated short stature are only eligible for SHOX analysis and have no access to whole genome sequencing (WGS) and are not consented for research. However, where patients have had additional testing, we have listed this information in Table 1.

Table 1.

The ascertainment and phenotypes of the individuals with SHOX whole gene duplications (duplicating only part of the SHOX regulatory region) and the minimum duplication sizes

Patient numberSexAgeTest methodology/clinical details/other testingInheritancePhenotype designationDuplication (chrX)
P1 SHOX cohort testing. Height on the 0.4th centile, no skeletal abnormalities. Additional testing unknown Unknown ISS 307,417–618,170 
P2 16 SHOX cohort testing. Height on the 0.4th centile, brachydactyly. No dysmorphism or Madelung deformity. Additional testing unknown Unknown ISS 580,427–618,170 
P3 10 aCGH testing for height <3rd centile, craniosynostosis, moderate developmental delay, large head, learning difficulties. Also tested for Noonan syndrome and Russell-Silver syndrome but no other abnormality was detected. FISH analysis of the duplication gave a single signal at Xp22.3 Paternal ISS 185,483–850,643 
P4 aCGH testing for neonatal oedema, over-riding toes, deep palmar creases. Height on the 75th centile. Additional testing unknown Unknown 4th–96th centile 581,707–917,693 
P5 0.1 aCGH testing for faltering growth, height <0.4th centile with relative preservation of head circumference, almond-shaped eyes, mild bilateral hip immaturity. Not dysmorphic but has a small, blind-ending sacral dimple. Also tested for Russell-Silver syndrome, spinal muscular atrophy, congenital generalised lipodystrophy gene panel, multi-locus imprinting disorders and pseudohypoparathyroidism but no other abnormality detected Unknown ISS 387,593–621,873 
P6 aCGH and epilepsy gene panel testing for pharmaco-resistant epilepsy with previous generalized tonic-clonic seizures and frequent absence seizures with myoclonus, learning difficulties, attention deficit hyperactive disorder, behavioural difficulties, sleep difficulties. Height on the 3rd centile. Also tested by whole genome sequencing but no other abnormality detected Maternal ISS 452,148–1,233,875 
P7 aCGH testing for autism and developmental delay except gross motor skills, height on the 50th centile. Additional testing unknown Maternal 4th–96th centile 1–624,771 
P8 aCGH testing for heart defects and an imperforate anus. Height on the 50th centile. Additional testing unknown Paternal 4th–96th centile 561,527–1,259,089 
P9 Originally referred for achondroplasia/hypochondroplasia testing because of rhizomelia, a short neck and height <0.4th centile. aCGH subsequently requested following a normal FGFR3 result Maternal (height on the 17th centile) ISS 581,707–620,291 
Th1 aCGH testing for Asperger syndrome. Height on the 91st–98th centile Maternal (height on the 75th–91st centile) Tall stature 420,000–758,000 
Th2 68 SHOX cohort testing. Height on the 0.4th centile, Madelung deformity Unknown LWD 570,000–618,000 
Th3 aCGH testing for cleft palate. Height on the 97th centile Paternal (height on the 75th–90th centile) Tall stature 500,000–706,000 
BS1 13 SHOX cohort testing. Height <3rd centile, slightly shortened neck Maternal (height <3rd centile) ISS 460,000–706,200 
BS2 >18 SHOX cohort testing. Height <3rd centile Maternal (height <3rd centile) ISS 420,000–618,170 
BS3 13.8 SHOX cohort testing. Height <3rd centile Paternal (height on the 75th centile) ISS 420,000–618,170 
BS4 >18 SHOX cohort testing. Height on the 25th–50th centile, Madelung deformity, short ulnars Unknown LWD 580,500–618,170 
B1 Not given aCGH testing for esophageal atresia. Height on the 2nd centile, limb anomalies. FISH analysis of the duplication gave a single signal at Xp22.3 Maternal ISS 405,941–673,267 
F1 1.9 SHOX cohort testing. Height <3rd centile. Sequencing showed that the duplication is a direct tandem repeat Unknown ISS 486,700–757,437 
F2 3.2 SHOX cohort testing. Height <3rd centile. FISH analysis of the duplication gave a single signal at Xp22.3 Unknown ISS 521,908–1,262,229 
D1 5.6 SHOX cohort testing. Height <3rd centile Unknown ISS 307,417–611,726 
D2 3.3 SHOX cohort testing. Height <0.4th centile Paternal (height on the 75th centile) ISS 307,417–611,726 
Tr1 >40 aCGH testing for Asperger syndrome. Height on the 25th–50th centile Unknown 4th–96th centile 286,524–658,258 
Tr2 >50 aCGH testing for Asperger syndrome. Height on 50th–75th centile Unknown 4th–96th centile 286,524–774,548 
Tr3 <2 aCGH testing as part of a non-ASD control cohort. Also has talipes equinovarus Paternal Club foot 154,061–664,616 
Tr4 <2 aCGH testing for ASD. Also has bilateral talipes equinovarus Unknown Club foot 169,063–618,036 
Tr5 <2 aCGH testing as part of a non-ASD control cohort. Height >97th centile Maternal Tall stature 169,063–664,616 
Tr6 10 aCGH testing as part of a non-ASD control cohort. Height >97th centile Unknown Tall stature 342,016–664,616 
Tr7 <2 aCGH testing as part of a non-ASD control cohort. Height <3rd centile Paternal ISS 481,940–819,243 
Tr8 54 aCGH testing for ASD. Height <3rd centile Maternal ISS 509,393–624,771 
Tr9 aCGH testing for ASD. Height <3rd centile Unknown ISS 550,457–618,036 
HS1 Not given SHOX MLPA population control. Height on the 75th–90th centile Unknown 4th–96th centile 580,427–694,862 
HS2 Not given SHOX cohort testing. Height <3rd centile Unknown ISS 580,427–899,388 
U1 13 SHOX MLPA for tall stature. Height >99th centile. Normal karyotype Paternal (height on the 75th–90th centile) Tall stature 307,417–899,318 
U2 10 SHOX MLPA for tall stature. Height >99th centile. Normal karyotype Paternal (height on the 75th–90th centile) Tall stature 307,417–850,580 
S1 SHOX MLPA for club foot. Bilateral talipes equinovarus, amniotic band syndrome, bilateral symbrachydactly, sixth nerve palsy, height on the 8th centile Inherited Club foot 395,644–631,222 
S2 14 SHOX MLPA for club foot. Bilateral talipes equinovarus, developmental delay, height unknown. Also has a 16p13.11 duplication De novo Club foot 390,150–631,408 
S3 14 SHOX MLPA for club foot. Bilateral talipes equinovarus, height unknown Unknown Club foot 450,941–728,093 
S4 13 SHOX MLPA for club foot. Left talipes equinovarus, adolescent idiopathic scoliosis, height on the 48th centile Unknown Club foot 330,223–631,389 
Patient numberSexAgeTest methodology/clinical details/other testingInheritancePhenotype designationDuplication (chrX)
P1 SHOX cohort testing. Height on the 0.4th centile, no skeletal abnormalities. Additional testing unknown Unknown ISS 307,417–618,170 
P2 16 SHOX cohort testing. Height on the 0.4th centile, brachydactyly. No dysmorphism or Madelung deformity. Additional testing unknown Unknown ISS 580,427–618,170 
P3 10 aCGH testing for height <3rd centile, craniosynostosis, moderate developmental delay, large head, learning difficulties. Also tested for Noonan syndrome and Russell-Silver syndrome but no other abnormality was detected. FISH analysis of the duplication gave a single signal at Xp22.3 Paternal ISS 185,483–850,643 
P4 aCGH testing for neonatal oedema, over-riding toes, deep palmar creases. Height on the 75th centile. Additional testing unknown Unknown 4th–96th centile 581,707–917,693 
P5 0.1 aCGH testing for faltering growth, height <0.4th centile with relative preservation of head circumference, almond-shaped eyes, mild bilateral hip immaturity. Not dysmorphic but has a small, blind-ending sacral dimple. Also tested for Russell-Silver syndrome, spinal muscular atrophy, congenital generalised lipodystrophy gene panel, multi-locus imprinting disorders and pseudohypoparathyroidism but no other abnormality detected Unknown ISS 387,593–621,873 
P6 aCGH and epilepsy gene panel testing for pharmaco-resistant epilepsy with previous generalized tonic-clonic seizures and frequent absence seizures with myoclonus, learning difficulties, attention deficit hyperactive disorder, behavioural difficulties, sleep difficulties. Height on the 3rd centile. Also tested by whole genome sequencing but no other abnormality detected Maternal ISS 452,148–1,233,875 
P7 aCGH testing for autism and developmental delay except gross motor skills, height on the 50th centile. Additional testing unknown Maternal 4th–96th centile 1–624,771 
P8 aCGH testing for heart defects and an imperforate anus. Height on the 50th centile. Additional testing unknown Paternal 4th–96th centile 561,527–1,259,089 
P9 Originally referred for achondroplasia/hypochondroplasia testing because of rhizomelia, a short neck and height <0.4th centile. aCGH subsequently requested following a normal FGFR3 result Maternal (height on the 17th centile) ISS 581,707–620,291 
Th1 aCGH testing for Asperger syndrome. Height on the 91st–98th centile Maternal (height on the 75th–91st centile) Tall stature 420,000–758,000 
Th2 68 SHOX cohort testing. Height on the 0.4th centile, Madelung deformity Unknown LWD 570,000–618,000 
Th3 aCGH testing for cleft palate. Height on the 97th centile Paternal (height on the 75th–90th centile) Tall stature 500,000–706,000 
BS1 13 SHOX cohort testing. Height <3rd centile, slightly shortened neck Maternal (height <3rd centile) ISS 460,000–706,200 
BS2 >18 SHOX cohort testing. Height <3rd centile Maternal (height <3rd centile) ISS 420,000–618,170 
BS3 13.8 SHOX cohort testing. Height <3rd centile Paternal (height on the 75th centile) ISS 420,000–618,170 
BS4 >18 SHOX cohort testing. Height on the 25th–50th centile, Madelung deformity, short ulnars Unknown LWD 580,500–618,170 
B1 Not given aCGH testing for esophageal atresia. Height on the 2nd centile, limb anomalies. FISH analysis of the duplication gave a single signal at Xp22.3 Maternal ISS 405,941–673,267 
F1 1.9 SHOX cohort testing. Height <3rd centile. Sequencing showed that the duplication is a direct tandem repeat Unknown ISS 486,700–757,437 
F2 3.2 SHOX cohort testing. Height <3rd centile. FISH analysis of the duplication gave a single signal at Xp22.3 Unknown ISS 521,908–1,262,229 
D1 5.6 SHOX cohort testing. Height <3rd centile Unknown ISS 307,417–611,726 
D2 3.3 SHOX cohort testing. Height <0.4th centile Paternal (height on the 75th centile) ISS 307,417–611,726 
Tr1 >40 aCGH testing for Asperger syndrome. Height on the 25th–50th centile Unknown 4th–96th centile 286,524–658,258 
Tr2 >50 aCGH testing for Asperger syndrome. Height on 50th–75th centile Unknown 4th–96th centile 286,524–774,548 
Tr3 <2 aCGH testing as part of a non-ASD control cohort. Also has talipes equinovarus Paternal Club foot 154,061–664,616 
Tr4 <2 aCGH testing for ASD. Also has bilateral talipes equinovarus Unknown Club foot 169,063–618,036 
Tr5 <2 aCGH testing as part of a non-ASD control cohort. Height >97th centile Maternal Tall stature 169,063–664,616 
Tr6 10 aCGH testing as part of a non-ASD control cohort. Height >97th centile Unknown Tall stature 342,016–664,616 
Tr7 <2 aCGH testing as part of a non-ASD control cohort. Height <3rd centile Paternal ISS 481,940–819,243 
Tr8 54 aCGH testing for ASD. Height <3rd centile Maternal ISS 509,393–624,771 
Tr9 aCGH testing for ASD. Height <3rd centile Unknown ISS 550,457–618,036 
HS1 Not given SHOX MLPA population control. Height on the 75th–90th centile Unknown 4th–96th centile 580,427–694,862 
HS2 Not given SHOX cohort testing. Height <3rd centile Unknown ISS 580,427–899,388 
U1 13 SHOX MLPA for tall stature. Height >99th centile. Normal karyotype Paternal (height on the 75th–90th centile) Tall stature 307,417–899,318 
U2 10 SHOX MLPA for tall stature. Height >99th centile. Normal karyotype Paternal (height on the 75th–90th centile) Tall stature 307,417–850,580 
S1 SHOX MLPA for club foot. Bilateral talipes equinovarus, amniotic band syndrome, bilateral symbrachydactly, sixth nerve palsy, height on the 8th centile Inherited Club foot 395,644–631,222 
S2 14 SHOX MLPA for club foot. Bilateral talipes equinovarus, developmental delay, height unknown. Also has a 16p13.11 duplication De novo Club foot 390,150–631,408 
S3 14 SHOX MLPA for club foot. Bilateral talipes equinovarus, height unknown Unknown Club foot 450,941–728,093 
S4 13 SHOX MLPA for club foot. Left talipes equinovarus, adolescent idiopathic scoliosis, height on the 48th centile Unknown Club foot 330,223–631,389 

Patients P1–P9 are the novel cases from this study while patients Th1–Th3, BS1–BS4, B1, F1, F2, D1, D2, Tr1–Tr9, HS1, HS2, U1, U2, and S1–S4 are taken from the respective published manuscripts [Thomas et al., 2009; Benito-Sanz et al., 2011; Brosens et al., 2014; Fukami et al., 2015; Donze et al., 2015; Tropeano et al., 2016; Hirschfeldova and Solc, 2017; Upners et al., 2017; Sadler et al., 2020]. Parental heights are shown where available.

Novel Duplications in This Study

Patients 1 and 2 were identified from a cohort of 1,959 referrals from local, national, and international referrers between June 2003 and March 2020 for SHOX testing only. Analysis of SHOX and its flanking regions was carried out using multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. For patients 1 and 2, the duplication sizes were further defined using aCGH.

Patients 3–9 were identified from 22,018 individuals referred for aCGH (mostly investigated for developmental delay) from local, national, and international referrers between March 2009 and March 2020. Individuals in the aCGH cohort were not specifically referred for SHOX analysis. Probands with SHOX whole gene duplications from this cohort were identified solely to allow a comparison of their frequency versus the SHOX cohort, and also to determine the presence of SHOX-related phenotypes in independently ascertained individuals with SHOX whole gene duplications.

Methods

MLPA [Schouten et al., 2002] was performed using the current SHOX kit at the time of testing according to the manufacturer's protocol (P018; MRC-Holland, Amsterdam, The Netherlands). The current kit version (P018-G2) contains probes for every exon of the SHOX gene and every CNE shown in Figure 1. The proposed X:970,000 regulatory element does not contain an MLPA probe but is flanked by probes at approximately X:963,700 and X:1,029,700.

Fig. 1.

The minimum duplication sizes of the nine probands from this study and the 29 probands from the literature where phenotypic data were available.

Fig. 1.

The minimum duplication sizes of the nine probands from this study and the 29 probands from the literature where phenotypic data were available.

Close modal

Direct sequencing of all coding exons (isoform A, NM_000451.3, exons 2 to 6a) was used to exclude the presence of single nucleotide variants and small deletions/insertions in the SHOX coding sequence and intron/exon boundaries (primer sequences available upon request).

aCGH was performed using Oxford Gene Technologies (OGT, Oxford, UK) 60-mer oligo-array printed in 8 × 60 K International Standard Cytogenomic Array (ISCA) Consortium configuration, according to manufacturer’s instructions, using Kreatech’s pooled control DNA as a reference (Kreatech Diagnostics, Amsterdam, Holland). Slides were scanned using a G2539A Agilent microarray scanner (Agilent Technologies, Wokingham, UK) and analysed using OGT’s CytoSure Interpret (v3.6) microarray software.

Phenotypes

The phenotypes of the nine novel individuals with a SHOX duplication are given in Table 1, together with the phenotypes of the 29 probands from the literature with similar duplications. Parental samples were received for five of the nine novel probands identified in our laboratory. We do not have accurate heights for four of the parents who carry the same duplication as their offspring, and we have been unable to retrospectively obtain this information, although the original referral forms stated that the father of patient 3 is “not particularly short” and the father of patient 8 is “phenotypically normal.” The mother of patient 9 is 155 cm tall, putting her on the 17th centile.

Previously Reported Duplications

The first published collection of SHOX whole gene duplications contained four cases [Thomas et al., 2009]. One case was ascertained through screening a cohort of patients with Madelung deformity and had height on the 11th centile, suggestive of a possible diagnosis of LWD. Two cases were originally referred for aCGH analysis (because of Asperger syndrome and familial cleft palate, respectively). As the cohort sizes were not given, these three cases have not been included in any detection rate calculations. The fourth proband from this publication has been excluded from the genotype/phenotype component of this study as they also have a SHOX whole gene deletion, considered the explanation for their diagnosis of LWD. All individuals were negative for pathogenic SHOX sequence variants.

In a subsequenct study, MLPA analysis of 122 LWD and 613 ISS referrals identified SHOX whole gene duplications in three individuals with ISS and one with LWD [Benito-Sanz et al., 2011]. This study also included controls, and no SHOX whole gene duplication was identified in 340 individuals with normal stature (relative to age and gender) or 104 tall stature individuals with height above the 99th centile.

MLPA analysis also identified a duplication in a further four ISS individuals, two from an unspecified number of patients with short stature [Donze et al., 2015], one from a cohort of 245 patients with ISS or LWD [Fukami et al., 2015], and one from a Czech cohort of 352 ISS or LWD patients [Hirschfeldova and Solc, 2017]. The significance of the duplication in the latter Czech case was questioned in the manuscript because a different SHOX whole gene duplication was identified in one of the 250 population control individuals (whose height was on the 75th–90th centile).

Six other relevant cases in the literature came from cohorts screened specifically for SHOX dosage abnormalities (as SHOX was considered to be a likely cause of the phenotype under investigation): (1) The first study was a cohort of 81 girls with tall stature [Upners et al., 2017] which identified two relevant SHOX whole gene duplications in individuals with a height above the 99th centile and a normal karyotype; (2) Another four cases were identified in a cohort of 816 unrelated individuals with club foot (talipes equinovarus) [Sadler et al., 2020], three in probands with bilateral clubfoot and one with unilateral clubfoot. Heights were only available for two of the individuals with club foot; one was on the 8th centile and the other was on the 48th. In this latter manuscript, no similar duplications were detected in any of the 2,645 in-house controls which included 1,197 with adolescent idiopathic scoliosis, 334 with Chiari 1 malformation, 433 with male infertility, and 637 with amyotrophic lateral sclerosis.

Finally, two published manuscripts reported individuals with SHOX whole gene duplications, incidentally ascertained with regard to height, by aCGH genome-wide testing: (1) The first was a cohort of 180 patients with esophageal atresia that identified a SHOX duplication in a single patient with ISS [Brosens et al., 2014]. This male patient had a height on the 2nd centile and limb anomalies; (2) The second was a very large and detailed study that tested 26,664 individuals with autistic spectrum disorder (ASD) plus 12,594 controls [Tropeano et al., 2016]. This latter study identified 55 individuals with SHOX whole gene duplications (48 in individuals with a neurodevelopmental disorder and 7 in the non-ASD aCGH cohort), but sufficient clinical information for inclusion in this manuscript was only provided for nine of these cases – two where club foot is mentioned and seven where the height is listed (two >97th centile, three <3rd centile, one with a height on the 25th–50th centile, and one with a height on the 50th–75th centile). The 12,594 controls were selected on the basis that they did not have ASD but they had been referred for aCGH testing because of other phenotypes such as congenital malformations, physical dysmorphism, growth/skeletal abnormalities, and endocrine/metabolic conditions, so for results purposes we have treated these individuals as a mixed aCGH cohort rather than population-based normal controls.

Details of the nine novel and 29 published duplications are set out in Table 1 and Figure 1. All chromosomal location data are based on the hg19 GRCh37 build. For all probands, the minimum and maximum duplication sizes were sufficiently determined to allow the regulatory element content to be fully defined.

The overall incidence of SHOX whole gene duplications detected in patients referred to our laboratory for diagnostic SHOX testing was 4/1,959. Of these four cases, two were excluded from Table 1 and Figure 1 because of the presence of a second SHOX variant, so the effect of the SHOX whole gene duplication in those individuals could not be clearly determined. The overall incidence of SHOX whole gene duplications in individuals tested by aCGH in our laboratory was 9/22,018. Two of these nine cases were excluded from Table 1 and Figure 1: the first because we were unable to obtain any clinical information, and the second because, although their height was <2nd centile, they also had abnormal vertebral segmentation with fused vertebrae and several absent vertebral pedicles which was deemed likely to be the major cause of their height loss, so any compounding effect of the SHOX duplication could not be determined. No SHOX whole gene duplications were identified by MLPA in 471 anonymised normal controls in our laboratory (previously published in Bunyan et al. [2013]). These controls were variant-negative individuals who had undergone carrier-testing for autosomal recessive conditions, or were the parents of patients with a de novo structural abnormality. No height data were available on the control group, but all had been seen by a clinical geneticist prior to referral, so are unlikely to have a phenotype that would bring them to clinical attention and are expected to be representative of the general population.

A summary of clinical information is provided for all 38 individuals in Table 1, taken from either the diagnostic referral, the relevant publication or retrospective information from the referring clinician. The clinical information has been used to assign a specific phenotype designation. We categorised 32 of the probands as either LWD (n = 2), ISS (n = 18), tall stature (n = 6) or club foot (n = 6). For the remaining six we have listed their heights in centiles and categorised them as “4th–96th centile”.

While some of the SHOX duplications identified in our laboratory or published in the literature were excluded from Table 1, either due to the lack of clinical information or the presence of a second SHOX variant, Table 2 includes the frequency of all SHOX whole gene duplications detected in every cohort where the total size of the test cohort was provided. Table 2 shows that SHOX whole gene duplications are present at a low level in anonymised control cohorts (1/3,721; 0.03%) and in mixed aCGH cohorts (16/34,612; 0.05%). In contrast, in the LWD/ISS group, whole gene duplications were seen in 0.33% of cases (11/3,291).

Table 2.

SHOX whole gene duplication detection rates in this study and in cohorts from the literature

PublicationDetection rate in the different cohort types, n/N (%)
LWD or ISSclub footesophageal atresiatall statureASDunselected aCGH cohortscontrols
This study; Thomas et al., 2009; Bunyan et al., 2013 4/1,959 (0.20)     9/22,018 (0.04) 0/471 (0) 
Benito-Sanz et al., 2011 4/735 (0.54)   0/104 (0)   0/340 (0) 
Brosens et al., 2014   1/180 (0.56)     
Fukami et al., 2015 2/245 (0.82)      0/15 (0) 
Tropeano et al., 2016     48/26,664 (0.18) 7/12,594 (0.06)  
Hirschfeldova and Solc, 2017 1/352 (0.28)      1/250 (0.4) 
Upners et al., 2017    2/81 (2.47)    
Sadler et al., 2020  4/816 (0.49)     0/2,645 (0) 
Totals 11/3,291 (0.33) 4/816 (0.49) 1/180 (0.56) 2/185 (1.08) 48/26,664 (0.18) 16/34,612 (0.05) 1/3,721 (0.03) 
PublicationDetection rate in the different cohort types, n/N (%)
LWD or ISSclub footesophageal atresiatall statureASDunselected aCGH cohortscontrols
This study; Thomas et al., 2009; Bunyan et al., 2013 4/1,959 (0.20)     9/22,018 (0.04) 0/471 (0) 
Benito-Sanz et al., 2011 4/735 (0.54)   0/104 (0)   0/340 (0) 
Brosens et al., 2014   1/180 (0.56)     
Fukami et al., 2015 2/245 (0.82)      0/15 (0) 
Tropeano et al., 2016     48/26,664 (0.18) 7/12,594 (0.06)  
Hirschfeldova and Solc, 2017 1/352 (0.28)      1/250 (0.4) 
Upners et al., 2017    2/81 (2.47)    
Sadler et al., 2020  4/816 (0.49)     0/2,645 (0) 
Totals 11/3,291 (0.33) 4/816 (0.49) 1/180 (0.56) 2/185 (1.08) 48/26,664 (0.18) 16/34,612 (0.05) 1/3,721 (0.03) 

In aCGH cohorts [Tropeano et al., 2016; our study] where SHOX whole gene duplications were detected incidentally (as opposed to targeted SHOX testing), a minimum of seven of the 64 probands have ISS (10.9%). As we do not have definitive clinical data for 46 of these 64 probands, the actual incidence of ISS in the mixed aCGH group may be higher.

Alterations in SHOX expression have significant clinical consequences and are associated with a wide range of phenotypic presentations. SHOX haploinsufficiency is a common cause of short stature and can also include additional skeletal features, such as Madelung deformity, in individuals with LWD. However, because the regulation of SHOX is so complex and subject to long-range position effects, it is difficult to precisely define which SHOX variants would result in loss of function. Establishing that specific microdeletions within the SHOX regulatory region are pathogenic has been very successful, even if phenotypic variability can confound segregation analysis. Investigating whether duplications within the SHOX regulatory region are pathogenic is much more challenging as the mode of pathogenicity is not obvious. The identification of duplications of flanking regulatory elements that do not include the SHOX gene in patients with various SHOX-related phenotypes [Benito-Sanz et al., 2011; Fukami et al., 2015; Bunyan et al., 2016, 2021; Hirschfeldova and Solc, 2017; Sadler et al., 2020] suggests that a general disruptive effect on genome architecture may explain the presence of a SHOX-related phenotype and that the maintenance of the SHOX region genomic architecture is critical to normal gene function.

There is strong evidence, including four de novo cases, that duplications of the SHOX gene are associated with club foot [Sadler et al., 2020], plus a statistically significant overrepresentation in individuals with ASD [Tropeano et al., 2016]. However, single reports of SHOX CNVs from a specific clinical cohort can lead to ascertainment bias, therefore in this study we have brought together individuals from multiple different cohorts. The duplications were identified in patients with a wide range of phenotypes, including 20 individuals with phenotypes more typically associated with SHOX haploinsufficiency (LWD and ISS) and six with tall stature suggestive of SHOX overexpression. We present nine individuals with unpublished duplications, three identified through targeted SHOX analysis and six detected through diagnostic aCGH testing. We also describe 29 previously reported individuals with partial duplications of the SHOX regulatory region (including the entire SHOX gene) where the clinical phenotype was clearly stated.

The prevalence of whole gene duplications in ISS/LWD cohorts provides evidence that they can cause SHOX haploinsufficiency (see Table 2): in this study, such duplications have a much higher prevalence in SHOX-specific LWD/ISS cohorts (11/3,291; 0.33%) than in population controls (1/3,721; 0.03%), a statistically significant increase (χ2 [1, N = 7,012] = 9.6, p < 0.05). In order to limit ascertainment bias, we have also looked at the frequency of SHOX whole gene duplications detected by aCGH where the referrals were unrelated to height. Although the overall incidence (16/34,612; 0.05%) was very similar to controls, the number of probands with ISS was higher than expected by chance. ISS has an incidence of 2.3% in the general population [Pedicelli et al., 2009], but in the aCGH cohorts a minimum of seven of the 64 probands (10.9%) have ISS. We do not have definitive clinical data for 46 of these 64 probands, so the actual incidence of ISS in the mixed aCGH group may be even higher. However, many of the mixed aCGH probands may have a secondary genetic diagnosis that includes short stature as part of a wider syndrome, and the same may be true of some members of the SHOX/ISS cohort. Although a secondary genetic diagnosis may provide an explanation for the high level of ISS in aCGH probands with SHOX whole gene duplications, it would not explain the increased prevalence of such duplications in the SHOX/ISS cohort.

The duplications presented in this study are extremely rare, and while some may share a common breakpoint, of the 38 duplications in Table 1 there are at least 36 different breakpoint combinations. However, it is clear from Figure 1 that similar duplications do not always produce a consistent phenotype. The duplications are frequently inherited from a phenotypically normal parent, so there are high levels of phenotypic variability even in individuals with the same variant, and some of the duplications may be co-incidental findings. This heterogeneity makes it difficult to extrapolate information from one duplication to another, and both laboratory reporting and genetic counselling are challenging. The data provide evidence that a subset of SHOX whole gene duplications can result in SHOX haploinsufficiency, but assigning causality to any individual duplication is very difficult, and excluding all other causes of ISS is not straightforward. The ACMG guidelines for interpreting copy number variants [Riggs et al., 2020] are not designed for genes with variable penetrance, and heavy weighting is given to the ClinGen (clinicalgenome.org) triplosensitivity score. For SHOX, this is 0, suggesting that SHOX whole gene duplications are not currently thought to be clinically important. However, this assessment is likely to be in the context of an intact SHOX regulatory region and an assumption that three copies of SHOX will result in overexpression.

For whole gene duplications that do not include all enhancers, several factors should to be taken into consideration. Firstly, is the location of the duplicated interval known? In order to disrupt SHOX regulation, the duplicated interval would be expected to reside within the SHOX regulatory region such that two copies of SHOX are competing for the same enhancers. Approximately 95% of large duplications genome-wide are reported to be tandem [Richardson et al., 2019]. The location of the duplicated fragment was only investigated in four of the ISS cases presented in this manuscript. In patient F1 the duplication was proven to be a direct tandem repeat by Sanger sequencing, and in patients F2, P3, and B1 fluorescence in situ hybridisation (FISH) analysis gave a single signal at Xp22.3. Where divergent phenotypes are seen in individuals with similar duplications, one explanation is that the duplicated fragments are in different genomic locations. Both individuals (U1 and U2) from the tall stature cohort [Upners et al., 2017] have a maximum duplication size consistent with a terminal rather than an interstitial duplication, and patient P7 (who has a height on the 50th centile) is known to have a terminal duplication, so for these individuals there is a higher likelihood that the extra copy may be translocated elsewhere in the genome [Qian et al., 2018] and therefore not affecting the expression of the other two copies of SHOX.

Secondly, have all other causes of the proband’s phenotype been excluded? As LWD is specific to SHOX, the exclusion of an additional SHOX variant in an LWD proband is the sole requirement. However, ISS has multiple aetiologies, so ideally such individuals should have genome-wide testing such as aCGH and whole genome/exome sequencing (WGS/WES) in order to exclude other possible causes of short stature. However, these techniques do not have 100% coverage, they are unlikely to detect methylation abnormalities or deep intronic variants, and WGS/WES has been reported to provide a molecular genetic diagnosis in only 30–50% of cases [Yang et al., 2013; Gilissen et al., 2014; Soden et al., 2014; Srivastava et al., 2014], so excluding all other causes of ISS is very difficult, even if additional testing is performed. An ideal example is patient P6 who has had aCGH and WGS testing and the only detected variant was the SHOX duplication. However, he also has epilepsy and behavioral and sleep difficulties, so if a genetic cause for these symptoms has not been found we cannot be sure that another cause of ISS has also been missed.

Although it is clear from Figure 1 that apparently similar duplications do not always produce a consistent phenotype, the duplication size, regulatory element content and the proximity of one or more breakpoint to the SHOX gene may all contribute to the phenotypic consequences of SHOX whole gene duplications. With a limited number of positive patients, it is difficult to draw significant conclusions from the breakpoint data. However, five of the six individuals with a duplication of just the SHOX gene had LWD (n = 2) or ISS (n = 3), so breakpoints close to SHOX appear more likely to produce a SHOX haploinsufficiency phenotype. Patients with normal stature or tall stature have the largest average duplication size (and consequently the highest number of regulatory elements). However, while these two groups have the same average number of regulatory elements, they have a different distribution: tall stature duplications have an excess of upstream regulatory elements while normal stature duplications have an excess of downstream regulatory elements. Similarly, although duplications identified in patients with ISS and club foot also have the same average number of regulatory elements there is a difference in distribution: ISS duplications contain approximately equal numbers of upstream and downstream regulatory elements, while club foot duplications predominantly contain upstream regulatory elements.

Another possible explanation for the phenotypic discrepancy between apparently similar duplications is the influence of modifier genes such as CYP26C1 [Montalbano et al., 2016]. The pathogenic X:780,550–828,092 deletion that removes the flanking ZED SHOX regulatory element was shown to be inherited from a phenotypically normal parent in 43% of cases in one cohort [Bunyan et al., 2013], so high levels of phenotypic variability have previously been observed even in individuals with a known pathogenic SHOX variant. In Table 1, only two of the nine parents with the duplication (where the parental heights are known) have the same phenotype designation (ISS) as the proband, with the other seven falling into the 4th–96th centile range.

Alternatively, the two in cis copies of SHOX in these individuals are effectively competing for the same regulatory elements which may result in inefficient expression of both copies, ultimately leading to SHOX underexpression rather than overexpression. Duplications could also change the 3D structure, preventing efficient transcription. In these scenarios, the term triplosensitivity is, therefore, inappropriate and misleading. Distinguishing between phenotypic variability, ascertainment bias, and the physical location of the duplicated material makes any conclusive genotype-phenotype correlation difficult.

Without significant intra-familial segregation and/or re-evaluation of the SHOX ClinGen “triplosensitivity” score, these duplications will inevitably be classified as variants of uncertain clinical significance. Further studies are required to establish the significance of SHOX whole gene duplications that do not include the entire regulatory region. However, there is currently sufficient evidence to suggest that these duplications should not be discounted without considering the extent of the duplication and the patient phenotype, and should be included on diagnostic laboratory reports. However, given the uncertain pathogenicity of these duplications, any reports should encourage the exclusion of all other causes of short stature where possible.

We are grateful to all patients and clinicians who were involved in our SHOX and aCGH cohorts, and to Emma-Jane Cassidy for the interpretation of aCGH data and advice on copy number variant interpretation guidelines.

The new probands presented in this study were consented for SHOX gene testing or aCGH analysis as part of their routine clinical care within the UK National Health Service. Written informed consent was obtained from a parent or legal guardian of any participants under 16 years old prior to sample collection. This retrospective review of patient data did not require ethical approval in accordance with local/national guidelines.

The authors have no conflicts of interest to declare.

The authors have no funding sources to declare.

David J. Bunyan: conceptualisation, validation, investigation, writing - original draft, writing - review and editing. James I. Hobbs: validation, investigation. Philippa J. Duncan-Flavell: validation, investigation. Rachel J. Howarth: validation, investigation. Sarah Beal: validation, investigation. Diana Baralle: investigation, resources. Nicholas Simon Thomas: conceptualisation, validation, writing - original draft, writing - review and editing, supervision.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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