Original Paper
HOR MON E RE SE ARCH I N PÆDIATRIC S
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
Received: February 11, 2014 Accepted: June 25, 2014 Published online: January 31, 2015
SHOX Gene Variants: Growth Hormone/ Insulin-Like Growth Factor-1 Status and Response to Growth Hormone Treatment Sofia Shapiro a Genna W. Klein a Michelle L. Klein a Elizabeth J. Wallach a Ye Fen b James H. Godbold b Robert Rapaport a a
Division of Pediatric Endocrinology and Diabetes and b Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, N.Y., USA
Abstract Context: Short stature homeobox-containing gene (SHOX) variants of unknown clinical significance occur frequently among children with short stature, yet their growth hormone (GH)/insulin-like growth factor-1 (IGF-1) status and response to GH have not been studied. Objective: To define GH and IGF-1 status in children with SHOX variants and assess their response to GH. Patients and Methods: This is a retrospective review of children with short stature. Children with SHOX variants were compared to those with no variants. Height standard deviation scores (SDS) and IGF-1 SDS at baseline and during GH treatment at 6, 12, and 24 months were analyzed. Growth velocity (GV), maximum GH dose, IGF-BP3, and changes in height SDS, IGF-1 SDS, and GV were compared. Results: Among 355 children, 83 (23%) had SHOX variants. Nineteen different SHOX variants were detected. There was no difference in age, height SDS, IGF-1 SDS, or IGFBP3 between children with SHOX variants and those with normal SHOX. Height SDS, IGF-1 SDS, IGF-BP3, GV, and GH dose were not different between patients with SHOX vari-
© 2015 S. Karger AG, Basel 1663–2818/15/0831–0026$39.50/0 E-Mail [email protected] www.karger.com/hrp
ants and those without. Conclusions: The GH and IGF-1 characteristics of children with short stature were not different between children with SHOX+ variants and children with no variants. Although these findings suggest that SHOX variants are polymorphisms, studies prospectively comparing individual SHOX variants are needed. © 2015 S. Karger AG, Basel
Introduction
The short stature homeobox-containing gene (SHOX) has been the focus of research in the multifactorial genetics of linear growth. Located within the pseudoautosomal region of the X and Y chromosomes, SHOX encodes a homeodomain transcriptional regulator that localizes to nuclei in fetal and prepubertal growth plate chondrocytes [1–5]. SHOX escapes X inactivation, requiring the expression of both alleles. Homozygous SHOX deficiency is known to cause Langer type mesomelic dysplasia [6, 7], and SHOX haploinsufficiency is responsible for the short stature seen in Turner syndrome and Léri-Weill dyschondrosteosis (LWD) [8–11]. Deletions in SHOX have been found in patients with idiopathic short stature (ISS) [12–14]. Growth hormone (GH) treatment is indicated in Sofia Shapiro, MD Pediatric Endocrinology Mount Kisco Medical Group 111 Bedford Road, Katonah, NY 10536 (USA) E-Mail sshapiro @ mkmg.com
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Key Words SHOX gene · Allelic variants · Insulin-like growth factor-1 · Growth hormone deficiency · Growth hormone treatment
Patients and Methods
SHOX variant negative (SHOX–) was defined as the result ‘no mutation detected’. All other results were defined as SHOX variant positive (SHOX+) and included single-nucleotide substitutions, partial gene deletions, and duplications. Mutations were checked with those published in the human SHOX allelic variant database [23]. Age, height standard deviation score (SDS), IGF-1 SDS, IGFBP3, and growth velocity (GV) were compared at baseline between SHOX+ and SHOX– patients. Among GH-treated children, GH dose (mg/kg/week), IGF-1 SDS, IGF-BP3, GV, and distance to the target height were compared at 6, 12, and 24 months of treatment. Patients were classified into 3 groups based on their response to GH stimulation testing with 2 agents, i.e. arginine and L-dopa, with the highest GH peak used to define the response as follows: A, classic GH deficiency (GHD; peak GH G c.676T>C (SHOXb) c.785T>C IVS1 – 84C>G 5′UTR–428C>A IVS5 – 3T>C 3′UTR+6A>G (SHOXb) IVS2 – 19C>T IVS1 – 96G>A
– – G21G – L290L – – – – – R168G X226R V262A – – – – – –
– – synonymous 3′UTR synonymous intron intron 3′UTR 3′UTR intron missense read-through missense intron 5′UTR intron 3′UTR intron intron
6 3 1 3 5 46 2 3 1 2 1 3 1 1 1 1 2 1 4
– noa yesb no no yesb no no no yes no no no no yes yes no no no
yes yes
yes
176
I
II
III
IV
Novel
Color version available online
Table 5. SHOX variants detected and their relative frequencies in this study
292
V
VIa 225
VIb
33 children with GHD and 6 out of 44 children with partial GHD did not go on GH therapy. Reasons included, but were not limited to, the family’s decision not to pursue treatment, an adequate GV despite a low GH response, diagnosis of a concomitant medical condition, a switch in pediatric endocrinology providers, and loss to follow-up. Still, it is clinically evident that most GHD and partial GHD children were indeed treated with GH compared to ISS children who were not GHD and only 53 of 124 received GH. SHOX variants were common; 83 (23%) of the children in our study had at least one variant. SHOX haploinsufficiency due to mutations has been reported in 2–15% of patients with ISS [5, 11, 19–22]. The higher fre32
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
quency in our study may be explained by ascertainment bias or by a true increased frequency of SHOX variants. Comparison of mutation prevalences in prior studies is difficult due to inconsistent definitions of ISS, the inclusion of patients with skeletal abnormalities, different mutational analyses (single-strand conformational polymorphism vs. direct sequencing), differences in the regions of the SHOX gene analyzed, and population diversity. Among 83 SHOX+ children, 18 different SHOX variants were detected, 3 of which were novel. Most of the SHOX variants found were single-amino acid substitutions, consistent with Niesler et al. [23], authors of the SHOX allelic variant database. The database currently lists 1,169 mutations, 94% of which are single-nucleotide Shapiro /Klein /Klein /Wallach /Fen / Godbold /Rapaport
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Adapted from Ross et al. [28]. the database.
a Subsequently reclassified as a normal result. b Considered a polymorphism in
Table 6. SHOX variant IVS2 + 17G>T comparison
SHOX– (n = 272) SHOX+ (n = 42) p value Group A: GHD SHOX– (n = 25) IVS2 + 17G>T (n = 5) Group B: partial GHD SHOX– (n = 31) IVS2 + 17G>T (n = 5) Group C: ISS SHOX– (n = 91) IVS2 + 17G>T (n = 19)
Age, years
Height SDS
IGF-1 SDS
10.56 ± 3.54 9.14 ± 2.66 0.0034
–2.05 ± 0.76 –1.95 ± 0.60 0.3607
–1.39 (–5.29 to 5.60) –1.90 (0.78) 0.0124
10.80 ± 2.97 9.30 ± 2.71
–2.06 ± 0.56 –2.20 ± 0.24
–2.26 (–3.43 to 0.58) –2.13 (–2.38 to –1.22)
9.01 ± 2.34 10.62 ± 0.77
–2.20 ± 0.60 –2.27 ± 0.90
–2.26 (–3.76 to 0.60) –1.32 (–2.41 to –0.79)
10.26 ± 3.30 8.18 ± 2.53a
–2.22 ± 0.69 –1.79 ± 0.56b
–1.63 (–5.29 to 2.54) –2.05 (–3.55 to 1.19)
substitutions. Many of these point mutations are diseasecausing variants located within coding regions of the gene – detailed information on the location and frequency of the documented mutations is provided, and deleterious mutations linked to Turner syndrome, LWD, and ISS are specified. However, for most of the mutations listed, the functional consequences are unknown. Only 22 variants are classified as polymorphisms, leaving many of the variants classified as being of unknown clinical significance. As the variants detected in the children of our study did not translate into clinically significant differences in GH/IGF-1 status and the response to GH, it could be surmised that most of these variants are neutral polymorphisms. However, it has yet to be elucidated that the GH/IGF-1 status can be affected by mutations of the SHOX gene. The variants in this study are a heterogeneous group, and the relatively small number of children within each variant did not allow for individual variant comparisons, with the exception of C.277+17G>T of intron 2, a known polymorphism [23]. Further analyses comparing specific variants are needed to determine which mutations are ‘clinically neutral’ and which truly confer changes in gene expression and/or protein activity, in turn leading to clinical manifestations. Also, the spectrum of phenotype expressivity may be quite diverse for these variants. Recent studies have found that genetic elements outside of the coding regions of the SHOX gene may influence SHOX expression. Sabherwal et al. [26] found a highly conserved region downstream of the SHOX gene,
responsible for a kindred with normal SHOX yet an LWD phenotype [26], and deletions downstream of an intact SHOX gene were thought to cause growth failure in both ISS and LWD patients [22, 27]. These data signify the evolving role of genetic testing in diagnosing the causes of growth failure as new genetic regions unrelated to the coding sequences of the SHOX gene are found to have important clinical manifestations. To assist in characterizing a specific SHOX mutation as functional or nonfunctional, researchers have looked at the prevalence of SHOX mutations in normal-stature children as well as the parents of short children who carry the same mutation [12]. Finding a mutation in a person of normal height renders the mutation a nonfunctional polymorphism. However, due to the variable expressivity of SHOX mutations, as evidenced by SHOX deletions manifesting across a wide spectrum from LWD to ISS, or growth failure of unknown etiology, larger population studies are needed to verify the classification of a specific variant as a polymorphism. Limitations of this study include its retrospective design and the relatively small number of patients with each variant. The response to GH was evaluated up to 24 months of treatment, which is a relatively short duration. Not all of our children with short stature had a height SD below –2.0, as the mid-parental height as well as the height SD were used to define short stature; this may have added heterogeneity to our study cohort. Increased sitting height and sitting height-to-total height ratios were noted to occur with a higher frequency in children with
SHOX Variants: GH/IGF-1 Status and GH Treatment
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
33
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Group A: peak GH
HOR MON E RE SE ARCH I N PÆDIATRIC S
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
Received: February 11, 2014 Accepted: June 25, 2014 Published online: January 31, 2015
SHOX Gene Variants: Growth Hormone/ Insulin-Like Growth Factor-1 Status and Response to Growth Hormone Treatment Sofia Shapiro a Genna W. Klein a Michelle L. Klein a Elizabeth J. Wallach a Ye Fen b James H. Godbold b Robert Rapaport a a
Division of Pediatric Endocrinology and Diabetes and b Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, N.Y., USA
Abstract Context: Short stature homeobox-containing gene (SHOX) variants of unknown clinical significance occur frequently among children with short stature, yet their growth hormone (GH)/insulin-like growth factor-1 (IGF-1) status and response to GH have not been studied. Objective: To define GH and IGF-1 status in children with SHOX variants and assess their response to GH. Patients and Methods: This is a retrospective review of children with short stature. Children with SHOX variants were compared to those with no variants. Height standard deviation scores (SDS) and IGF-1 SDS at baseline and during GH treatment at 6, 12, and 24 months were analyzed. Growth velocity (GV), maximum GH dose, IGF-BP3, and changes in height SDS, IGF-1 SDS, and GV were compared. Results: Among 355 children, 83 (23%) had SHOX variants. Nineteen different SHOX variants were detected. There was no difference in age, height SDS, IGF-1 SDS, or IGFBP3 between children with SHOX variants and those with normal SHOX. Height SDS, IGF-1 SDS, IGF-BP3, GV, and GH dose were not different between patients with SHOX vari-
© 2015 S. Karger AG, Basel 1663–2818/15/0831–0026$39.50/0 E-Mail [email protected] www.karger.com/hrp
ants and those without. Conclusions: The GH and IGF-1 characteristics of children with short stature were not different between children with SHOX+ variants and children with no variants. Although these findings suggest that SHOX variants are polymorphisms, studies prospectively comparing individual SHOX variants are needed. © 2015 S. Karger AG, Basel
Introduction
The short stature homeobox-containing gene (SHOX) has been the focus of research in the multifactorial genetics of linear growth. Located within the pseudoautosomal region of the X and Y chromosomes, SHOX encodes a homeodomain transcriptional regulator that localizes to nuclei in fetal and prepubertal growth plate chondrocytes [1–5]. SHOX escapes X inactivation, requiring the expression of both alleles. Homozygous SHOX deficiency is known to cause Langer type mesomelic dysplasia [6, 7], and SHOX haploinsufficiency is responsible for the short stature seen in Turner syndrome and Léri-Weill dyschondrosteosis (LWD) [8–11]. Deletions in SHOX have been found in patients with idiopathic short stature (ISS) [12–14]. Growth hormone (GH) treatment is indicated in Sofia Shapiro, MD Pediatric Endocrinology Mount Kisco Medical Group 111 Bedford Road, Katonah, NY 10536 (USA) E-Mail sshapiro @ mkmg.com
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Key Words SHOX gene · Allelic variants · Insulin-like growth factor-1 · Growth hormone deficiency · Growth hormone treatment
Patients and Methods
SHOX variant negative (SHOX–) was defined as the result ‘no mutation detected’. All other results were defined as SHOX variant positive (SHOX+) and included single-nucleotide substitutions, partial gene deletions, and duplications. Mutations were checked with those published in the human SHOX allelic variant database [23]. Age, height standard deviation score (SDS), IGF-1 SDS, IGFBP3, and growth velocity (GV) were compared at baseline between SHOX+ and SHOX– patients. Among GH-treated children, GH dose (mg/kg/week), IGF-1 SDS, IGF-BP3, GV, and distance to the target height were compared at 6, 12, and 24 months of treatment. Patients were classified into 3 groups based on their response to GH stimulation testing with 2 agents, i.e. arginine and L-dopa, with the highest GH peak used to define the response as follows: A, classic GH deficiency (GHD; peak GH G c.676T>C (SHOXb) c.785T>C IVS1 – 84C>G 5′UTR–428C>A IVS5 – 3T>C 3′UTR+6A>G (SHOXb) IVS2 – 19C>T IVS1 – 96G>A
– – G21G – L290L – – – – – R168G X226R V262A – – – – – –
– – synonymous 3′UTR synonymous intron intron 3′UTR 3′UTR intron missense read-through missense intron 5′UTR intron 3′UTR intron intron
6 3 1 3 5 46 2 3 1 2 1 3 1 1 1 1 2 1 4
– noa yesb no no yesb no no no yes no no no no yes yes no no no
yes yes
yes
176
I
II
III
IV
Novel
Color version available online
Table 5. SHOX variants detected and their relative frequencies in this study
292
V
VIa 225
VIb
33 children with GHD and 6 out of 44 children with partial GHD did not go on GH therapy. Reasons included, but were not limited to, the family’s decision not to pursue treatment, an adequate GV despite a low GH response, diagnosis of a concomitant medical condition, a switch in pediatric endocrinology providers, and loss to follow-up. Still, it is clinically evident that most GHD and partial GHD children were indeed treated with GH compared to ISS children who were not GHD and only 53 of 124 received GH. SHOX variants were common; 83 (23%) of the children in our study had at least one variant. SHOX haploinsufficiency due to mutations has been reported in 2–15% of patients with ISS [5, 11, 19–22]. The higher fre32
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
quency in our study may be explained by ascertainment bias or by a true increased frequency of SHOX variants. Comparison of mutation prevalences in prior studies is difficult due to inconsistent definitions of ISS, the inclusion of patients with skeletal abnormalities, different mutational analyses (single-strand conformational polymorphism vs. direct sequencing), differences in the regions of the SHOX gene analyzed, and population diversity. Among 83 SHOX+ children, 18 different SHOX variants were detected, 3 of which were novel. Most of the SHOX variants found were single-amino acid substitutions, consistent with Niesler et al. [23], authors of the SHOX allelic variant database. The database currently lists 1,169 mutations, 94% of which are single-nucleotide Shapiro /Klein /Klein /Wallach /Fen / Godbold /Rapaport
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Adapted from Ross et al. [28]. the database.
a Subsequently reclassified as a normal result. b Considered a polymorphism in
Table 6. SHOX variant IVS2 + 17G>T comparison
SHOX– (n = 272) SHOX+ (n = 42) p value Group A: GHD SHOX– (n = 25) IVS2 + 17G>T (n = 5) Group B: partial GHD SHOX– (n = 31) IVS2 + 17G>T (n = 5) Group C: ISS SHOX– (n = 91) IVS2 + 17G>T (n = 19)
Age, years
Height SDS
IGF-1 SDS
10.56 ± 3.54 9.14 ± 2.66 0.0034
–2.05 ± 0.76 –1.95 ± 0.60 0.3607
–1.39 (–5.29 to 5.60) –1.90 (0.78) 0.0124
10.80 ± 2.97 9.30 ± 2.71
–2.06 ± 0.56 –2.20 ± 0.24
–2.26 (–3.43 to 0.58) –2.13 (–2.38 to –1.22)
9.01 ± 2.34 10.62 ± 0.77
–2.20 ± 0.60 –2.27 ± 0.90
–2.26 (–3.76 to 0.60) –1.32 (–2.41 to –0.79)
10.26 ± 3.30 8.18 ± 2.53a
–2.22 ± 0.69 –1.79 ± 0.56b
–1.63 (–5.29 to 2.54) –2.05 (–3.55 to 1.19)
substitutions. Many of these point mutations are diseasecausing variants located within coding regions of the gene – detailed information on the location and frequency of the documented mutations is provided, and deleterious mutations linked to Turner syndrome, LWD, and ISS are specified. However, for most of the mutations listed, the functional consequences are unknown. Only 22 variants are classified as polymorphisms, leaving many of the variants classified as being of unknown clinical significance. As the variants detected in the children of our study did not translate into clinically significant differences in GH/IGF-1 status and the response to GH, it could be surmised that most of these variants are neutral polymorphisms. However, it has yet to be elucidated that the GH/IGF-1 status can be affected by mutations of the SHOX gene. The variants in this study are a heterogeneous group, and the relatively small number of children within each variant did not allow for individual variant comparisons, with the exception of C.277+17G>T of intron 2, a known polymorphism [23]. Further analyses comparing specific variants are needed to determine which mutations are ‘clinically neutral’ and which truly confer changes in gene expression and/or protein activity, in turn leading to clinical manifestations. Also, the spectrum of phenotype expressivity may be quite diverse for these variants. Recent studies have found that genetic elements outside of the coding regions of the SHOX gene may influence SHOX expression. Sabherwal et al. [26] found a highly conserved region downstream of the SHOX gene,
responsible for a kindred with normal SHOX yet an LWD phenotype [26], and deletions downstream of an intact SHOX gene were thought to cause growth failure in both ISS and LWD patients [22, 27]. These data signify the evolving role of genetic testing in diagnosing the causes of growth failure as new genetic regions unrelated to the coding sequences of the SHOX gene are found to have important clinical manifestations. To assist in characterizing a specific SHOX mutation as functional or nonfunctional, researchers have looked at the prevalence of SHOX mutations in normal-stature children as well as the parents of short children who carry the same mutation [12]. Finding a mutation in a person of normal height renders the mutation a nonfunctional polymorphism. However, due to the variable expressivity of SHOX mutations, as evidenced by SHOX deletions manifesting across a wide spectrum from LWD to ISS, or growth failure of unknown etiology, larger population studies are needed to verify the classification of a specific variant as a polymorphism. Limitations of this study include its retrospective design and the relatively small number of patients with each variant. The response to GH was evaluated up to 24 months of treatment, which is a relatively short duration. Not all of our children with short stature had a height SD below –2.0, as the mid-parental height as well as the height SD were used to define short stature; this may have added heterogeneity to our study cohort. Increased sitting height and sitting height-to-total height ratios were noted to occur with a higher frequency in children with
SHOX Variants: GH/IGF-1 Status and GH Treatment
Horm Res Paediatr 2015;83:26–35 DOI: 10.1159/000365507
33
Downloaded by: Selçuk Universitesi 193.255.248.150 - 2/10/2015 7:04:27 PM
Group A: peak GH
