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Abnormal vascular and neural retinal morphology in congenital lifetime isolated growth hormone deficiency
Growth Hormone & IGF Research 30–31 (2016) 11–15
Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
Abnormal vascular and neural retinal morphology in congenital lifetime isolated growth hormone deficiency Virginia M. Pereira-Gurgel a, Augusto C.N. Faro b, Roberto Salvatori c,⁎, Thiago A. Chagas b, José F. Carvalho-Junior b, Carla R.P. Oliveira a, Ursula M.M. Costa a, Gustavo B. Melo d, Ann Hellström e, Manuel H. Aguiar-Oliveira a a
Division of Endocrinology, Federal University of Sergipe, Aracaju, SE 49060-100, Brazil Division of Ophthalmology Federal University of Sergipe, Aracaju, SE 49060-100, Brazil Division of Endocrinology, Diabetes and Metabolism, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA d Hospital de Olhos de Sergipe, Aracaju, SE, Brazil e Sahlgrenska Academy, The Queen Silvia Children's Hospital, Göteborg, Sweden b c
a r t i c l e
i n f o
Article history: Received 12 June 2016 Received in revised form 10 July 2016 Accepted 26 July 2016 Available online 27 July 2016 Keywords: Isolated GH deficiency GH IGF-I Retina
a b s t r a c t Objective: Experimental models demonstrate an important role of GH in retinal development. However, the interactions between GH and the neuro-vascularization of the human retina are still not clear. A model of untreated congenital isolated GH deficiency (IGHD) may clarify the actions of GH on the retina. The purpose of this work was to assess the retinal neuro-vascularization in untreated congenital IGHD (cIGHD). Design: In a cross sectional study, we performed an endocrine and ophthalmological assessment of 25 adult cIGHD subjects, homozygous for a null mutation (c.57 + 1G N A) in the GHRH receptor gene and 28 matched controls. Intraocular pressure measurement, retinography (to assess the number of retinal vascular branching points and the optic disc and cup size), and optical coherence tomography (to assess the thickness of macula) were performed. Results: cIGHD subjects presented a more significant reduction of vascular branching points in comparison to controls (91% vs. 53% [p = 0.049]). The percentage of moderate reduction was higher in cIGHD than in controls (p = 0.01). The percentage of individuals with increased optic disc was higher in cIGHD subjects in comparison to controls (92.9% vs. 57.1%). The same occurred for cup size (92.9% vs. 66.7%), p b 0.0001 in both cases. There was no difference in macula thickness. Conclusions: Most cIGHD individuals present moderate reduction of vascular branching points, increase of optic disc and cup size, but have similar thickness of the macula. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction The visual system is fundamental for the neuro-motor development, environmental adaptation and survival capacity. Visual acuity depends on a well-developed eye that is capable to form the image on the retina and process it in the central nervous system. Body size is heavily influenced by the effect of circulating GH and its main effector IGF-I on bone and cartilage tissues. It has been proposed that retinal development may reflect autocrine or paracrine ocular production of GH, IGFI, IGF type II (IGF-II) and other peptides like fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) [1–5]. Accordingly, in mice, rats and chickens
⁎ Corresponding author at: Division of Endocrinology, Johns Hopkins University School of Medicine, 1830 East Monument street suite # 333, Baltimore, MD 21287, United States. E-mail address: [email protected] (R. Salvatori).
http://dx.doi.org/10.1016/j.ghir.2016.07.001 1096-6374/© 2016 Elsevier Ltd. All rights reserved.
GH and GH receptor gene expression was documented in developing neural retina [3]. More than a half century has passed since the first description of regression of neovascularization in diabetic retinopathy after pituitary infarction and resultant GH deficiency, which led to the use of hypophysectomy as a therapy for proliferative diabetic retinopathy (GHD) [6]. However, the interaction between GH/IGFs and vascularization of the human retina is still not completely understood [5]. More recently, reduced retinal vascularization was shown in GHD children [7] and in GH insensitivity syndrome (Laron syndrome) [8], suggesting that the GH-IGF-I axis is critical for normal vascularization of the human retina. Nevertheless, in both studies there was some overlap between patients and controls, suggesting that other factors may affect the pattern of vascularization. The consequences of GHD or Laron syndrome in human retina may not be identical. While both models cause very low serum IGF-I levels, in the former there is often some GH secretion, albeit low, whereas the action of GH is completely impaired in Laron
12
V.M. Pereira-Gurgel et al. / Growth Hormone & IGF Research 30–31 (2016) 11–15
syndrome. Furthermore, congenital GHD can be isolated or associated to other pituitary deficits, and caused by several genetics or embryological insults, with direct consequences to the visual system [9,10]. Therefore, it is important to assess if individuals with inherited (therefore not caused by intrauterine insults) isolated GHD (IGHD) exhibit abnormalities in retinal neuro-vascularization. However, IGHD is a rare disease, occurring in 1:3480 to 1:10,000 live births [11], and most cases are treated with GH replacement during childhood. We have described in rural Northeastern Brazil a cohort of congenital IGHD (cIGHD) individuals due to a homozygous mutation (c.57 + 1 G N A) in the GHRHR gene (GHRHR) [12]. Despite severe short stature with adult height ranging from 1.07 to 1.36 m in pooled genders [13], these individuals cope well with daily challenges, do not exhibit neurodevelopmental problems, and have normal life expectancy [14]. Therefore, we hypothesized that these individuals present satisfactory retinal health, contributing to their normal survival. The objective of this study is to assess retinal neuro-vascularization in these untreated IGD individuals. 2. Subjects and methods
the branching point reduction in the initial categories: no reduction, mild reduction, moderate reduction and severe reduction. As no cIGHD had no reduction, and no control had mild reduction, we pooled no reduction and mild reduction in the final classification as minimal reduction. The same examiner evaluated the optic disc size (decreased, normal and increased) and cup size (normal or increased) in 14 cIGHD and 28 controls (one control had only the optic disc assessed). 2.5. OCT Twenty-five cIGHD subjects and 22 controls were analyzed and the average of each right and left macular area were calculated before comparison. Three patients had only one eye examined: two in cIGHD group (one due to toxoplasmosis and another because retinal detachment) and one in control group (severe cataract). We measured the thickness of the fovea and eight more macular areas: inner temporal, inner superior, inner nasal, inner inferior, outer temporal, outer superior, outer nasal, and outer inferior.
2.1. Subjects
2.6. Statistical analysis
In a cross sectional study, adult treatment-naïve cIGHD subjects, and age and sex-matched controls were recruited by advertising in the local Dwarfs Association, and by word of mouth among inhabitants of Itabaianinha County. Inclusion criterion for cIGHD was genotype-proven homozygosis for the c.57 + 1 G N A GHRHR mutation, whereas for controls was proven homozigosity for the wild-type allele. Genotyping method was described before [12]. Exclusion criteria for both groups were previous GH replacement, the presence of other genetic or acquired diseases that could alter the eye fundus appearance, hypertension requiring more than four drugs, diabetic retinopathy and glaucoma. Twenty-five cIGHD and 28 controls volunteered. The Federal University of Sergipe Institutional Review Board approved this study, and all subjects gave written informed consent.
Continuous variables were expressed as mean and standard deviation. Categorical variables were expressed in absolute number and percentage. The Student's t-test was used to compare continuous variables. Fisher's exact test was used to compare the categorical variables. Statistical analysis was performed using the statistical software IBM®SPSS® Version 20. Probability values b0.05 on a two tailed test were considered statistically significant.
2.2. Endocrine assessment Before the eye protocol, all the subjects fulfilled a clinical questionnaire and collected blood after an overnight fast for total cholesterol, triglycerides, glucose and IGF-I. Total cholesterol, triglycerides, and glucose were measured by standard techniques. IGF-I was measured by a solid-phase, enzyme-labeled chemiluminescent immunometric assay (IMMULITE 2000, Siemens Healthcare Diagnostics Products Ltd., Malvern, PA, USA), with a with intra- and inter-assay variabilities of 4.2 and 5.1%. 2.3. Study protocol All individuals underwent a complete eye examination, including applanation tonometry for intraocular pressure (Kowa Applanation Tonometer HA-2, Kowa, Japan), retinography (fundus photography) (Visucam 500, Carl Zeiss Meditec AG, Jena, Germany) and optical coherence tomography (OCT), (Stratus 3000, Carl Zeiss Meditec Inc. Dublin, CA, USA), under drug induced mydriasis. All fundus photographs were digitally recorded, sent to Göteborg, Sweden and analyzed by a unique examiner with clinical long-term experience (A. H.) blinded to the GH status. 2.4. Retinography Branching point reduction was assessed in all the visualized area in only well-focused photographs where all vessels were clearly visualized (11 cIGHD individuals and 17 controls). Both eyes were examined and there was no discrepancy between the two eyes of each patient regarding vascular branching points. The examiner classified in a blind fashion
3. Results Table 1 shows the clinical, biochemical and intraocular pressure in the cIGHD and controls subjects. Only the height, weight and IGF-I levels exhibited severe reduction in cIGHD group in comparison to control subjects. Table 2 shows the vascular branching point reduction in cIGHD subjects and controls in absolute number (n) and percentage (%) in the initial categories. Fig. 1 shows the final classification of the rate of vascular branching point reduction. Fisher's exact test revealed that cIGHD subjects presented more reduction of vascular branching points in comparison to controls (p = 0.049). The percentage of moderate reduction in cIGHD was higher than in control (91% vs. 53%, p = 0.01). Figs. 2 and 3 show that the rates of individuals with increased optic disc and cup size were increased in cIGHD in comparison to controls (p b 0.0001 in both cases). The percentage of individuals with increased optic and cup was significantly higher in cIGHD than in controls (p = 0.005, and p = 0.028, respectively). Fig. 4 shows the vascular branching point reduction and the optical disc and cup increase in an cIGHD individual and in a control. Table 3 shows that there was no difference in the thickness of the macula between the groups (fovea and eight more areas). Table 1 Clinical, biochemical and intraocular pressure (IOP) in the IGHD and control subjects.
Age (years) Sex (M) Smoking Height (m) Weight (kg) Systolic BP (mm Hg) Diastolic BP (mm Hg) IGF-I (ng/ml) Glucose (mg/dl) Cholesterol (mg/dl) Tryglicerides (mg/dl) IOP (mm Hg)
IGHD
Control
p
50.1 (15.9) 13 1 1.2 (0.18) 39.3 (8.7) 122.1 (18.9) 78.8 (9.3) 1.9 (0) 105.5 (22.7) 227.0 (65.1) 142.0 (95.5) 15.2 (3.1)
51.1 (14.0) 15 2 1.6 (0.1) 71.8 (14.4) 124.4 (15.0) 80.4 (6.5) 132 (55) 105.9 (70.5) 210.7 (28.2) 135.3 (51.3) 14.6 (1.9)
0.799 0.764 1 b0.0001 b0.0001 0.622 0.486 b0.0001 0.975 0.257 0.762 0.510
V.M. Pereira-Gurgel et al. / Growth Hormone & IGF Research 30–31 (2016) 11–15
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Table 2 Branching point reduction in IGHD individuals and controls in absolute number (n) and percentage (%).
IGHD Control
0
1
2
Total
0/0 8 (47%)
1 (9.1%) 0 (0%)
10 (90.9%) 9 (53%)
11 17
Branch reduction was thus code: 0 = no reduction; 1 = mild reduction; 2 = moderate reduction. Fisher's exact test revealed that IGHD subjects present more reduction of vascular branching points in comparison to controls (p = 0.0491). The percentage of moderate reduction in IGHD was higher than in control group (p = 0.01).
4. Discussion This work describes three main ocular findings in adults with lifetime, untreated, inherited cIGHD. First, a moderate reduction of vascular branching points; second, a mild increase in the optic disc and cup size; and third, the normal thickness of the macula. As groups were matched for demographic, clinical and biochemical variables and intraocular pressure, the obtained retinal findings may reflect the true impacts of cIGHD in the human retina. Only one paper has previously reported the retinal vascularization status in 39 children (3.6–18.7 years old) with congenital GHD (six with multiple pituitary deficiencies), most of them without genetic testing and already on GH replacement [7]. Another study looked at 11 individuals with Laron syndrome (10–49 years) [8]. In both studies, a reduction of vascular branching points was found, apparently more marked in Laron syndrome. Since IGF-I deficiency is common to both conditions, this factor has been hypothesized to be critical for the normal vascularization of the human retina. Our cIGHD data seem to agree with those findings, although the magnitude of reduction was moderate. This finding suggests possible compensatory mechanism(s) in cIGHD due to a GHRHR mutation. As the retinal vascularization normally occurs during fetal development [15], it can be influenced more by IGF-II, thought to be more relevant for fetal somatic and ocular development [1] than IGF-I. Interestingly, the Itabaianinha cIGHD individuals exhibit an up-regulation of serum IGF-II [16]. This could influence retinal vessels development, partially counteracting the consequences of GHD and IGF-I reduction. The more marked reduction of vascular branching points in Laron syndrome can reflect a lack of a possible direct angiogenic GH effect on retinal vessels. Together, our and previous findings show a reduction of vascular branching points in cIGHD, with a slow progression to advanced stages of vascular branching points reduction. The second finding of this work is the simultaneous increase of the optic disc and cup size, probably with little effect on the rim area (the area between the cup and the border of the optic disc), indicating preservation of enough retinal ganglion cell number (RGC). RGC survival is influenced by ocular GH, as both GH and GH receptors were
demonstrated in these cells [17]. Conversely, pituitary GH and circulating IGF-I are not critical to birth weight, as cIGHD babies due to GHRHR mutations are born of normal size [12]. It was suggested that GH and GH receptor activity in the early embryonic retina and the effect on RGC development might explain the decreased peripapillary nerve fiber thickness in some children with congenital GHD [17], which was not assessed in our study. An increase in optic disc is generally considered a marker or abnormal neurodevelopment. Normal [17], reduced, or increased [18] optic disc size were previously described in GHD, possibly reflecting the variety of causes of GHD. While optic nerve hypoplasia is often linked to septo-optic dysplasia [9], it was suggested that an increased optic disc could predict the possibility of GHD in a child with severe short stature [18]. Our findings seem to corroborate this hypothesis. Therefore, an increased optic disc size, similar to voice [19] and cephalometric findings [20] can facilitate the clinical diagnosis of cIGHD. Furthermore, our findings suggest that the presence of an increased optical disc in patients on GH replacement therapy, but without signs of increased intracranial pressure, radial pericapillary bleeding or blurring of retinal vessels in the disc edges, does not indicate pseudotumor cerebri, and does not need treatment suspension or costly investigations as suggested in a small number of patients [18]. Neuroophthalmologists and endocrinologists should incorporate this information into their practices. The third finding of this work is the normal thickness of the macula, assessed by OCT. This technique provides detailed information of cross sectional retinal anatomy and quantification of retinal morphology [21,22]. There is no previous published data about OCT in congenital IGD. However, OCT data in Laron syndrome also show normal thickness of the all macula regions and normal retinal architecture [23]. Together with Laron syndrome findings, our data suggests that postnatal circulating IGF-I is not critical for the macula architecture, at least as assessed by OCT.
Fig. 1. Vascular branching point reduction rate in IGHD and controls.
Fig. 3. Rate of cup size in IGHD and controls.
Fig. 2. Rate of optic disc size in IGHD and controls.
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V.M. Pereira-Gurgel et al. / Growth Hormone & IGF Research 30–31 (2016) 11–15
Fig. 4. Photo of one IGHD subject due to GHRH receptor deficiency and one control.
Our work has some limitations. First, the non-uniformity in the number of individuals used in all evaluations for retinal vascularization, due to the fact that half of the fundus images were not suitable for scoring. Another limitation was the use, for logistical reasons, of a semiquantitative system to assess the number of vascular branching points and optic disc size and excavation. However, this problem has been alleviated by using a simple and robust categorization system of these variables, performed by an ophthalmologist with solid clinical longterm experience in retinal assessment of GH deficiency and insensitivity syndromes [7,8].
Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Acknowledgments The authors thank the Associação do Crescimento Físico e Humano de Itabaianinha and Hospital Ocular in Aracaju, Sergipe, for assistance. References
5. Conclusions Adults with congenital, lifetime, untreated and isolated IGHD present a moderate reduction of vascular branching points, an increase of optic disc and cup size, with normal thickness of the macula. These data feature a distinct retinal phenotype in congenital IGHD due to an inactivating GHRHR mutation.
Declaration of interest R.S. serves in the advisory board of Novo Nordisk, Pfizer and Ionis. Prevention of retinopathy of prematurity by administering IGF-I are covered by patent owned by or licensed to Premacure AB, Uppsala, Sweden. AH owns shares in a company with financial interest in Premacure AB. AH work as consultants for Shire pharmaceuticals.
Table 3 Macula OCT map thickness (microns) in IGHD and control subjects. Data are expressed as mean (standard deviation).
Fovea Inner temporal Inner superior Inner nasal Inner inferior Outer temporal Outer superior Outer nasal Outer inferior
IGHD
Control
p
197.1 (34.8) 253.0 (29.6) 266.1 (34.8) 267.7 (36.0) 269.3 (35.5) 219.2 (20.9) 233.4 (23.6) 255.1 (26.7) 235.2 (20.4)
197.3 (23.2) 252.9 (21.3) 268.3 (18.8) 268.0 (19.7) 260.7 (21.5) 220.8 (17.7) 235.9 (17.3) 253.5 (26.0) 232.9 (18.4)
0.983 0.990 0.786 0.968 0.760 0.777 0.672 0.832 0.683
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Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
Abnormal vascular and neural retinal morphology in congenital lifetime isolated growth hormone deficiency Virginia M. Pereira-Gurgel a, Augusto C.N. Faro b, Roberto Salvatori c,⁎, Thiago A. Chagas b, José F. Carvalho-Junior b, Carla R.P. Oliveira a, Ursula M.M. Costa a, Gustavo B. Melo d, Ann Hellström e, Manuel H. Aguiar-Oliveira a a
Division of Endocrinology, Federal University of Sergipe, Aracaju, SE 49060-100, Brazil Division of Ophthalmology Federal University of Sergipe, Aracaju, SE 49060-100, Brazil Division of Endocrinology, Diabetes and Metabolism, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA d Hospital de Olhos de Sergipe, Aracaju, SE, Brazil e Sahlgrenska Academy, The Queen Silvia Children's Hospital, Göteborg, Sweden b c
a r t i c l e
i n f o
Article history: Received 12 June 2016 Received in revised form 10 July 2016 Accepted 26 July 2016 Available online 27 July 2016 Keywords: Isolated GH deficiency GH IGF-I Retina
a b s t r a c t Objective: Experimental models demonstrate an important role of GH in retinal development. However, the interactions between GH and the neuro-vascularization of the human retina are still not clear. A model of untreated congenital isolated GH deficiency (IGHD) may clarify the actions of GH on the retina. The purpose of this work was to assess the retinal neuro-vascularization in untreated congenital IGHD (cIGHD). Design: In a cross sectional study, we performed an endocrine and ophthalmological assessment of 25 adult cIGHD subjects, homozygous for a null mutation (c.57 + 1G N A) in the GHRH receptor gene and 28 matched controls. Intraocular pressure measurement, retinography (to assess the number of retinal vascular branching points and the optic disc and cup size), and optical coherence tomography (to assess the thickness of macula) were performed. Results: cIGHD subjects presented a more significant reduction of vascular branching points in comparison to controls (91% vs. 53% [p = 0.049]). The percentage of moderate reduction was higher in cIGHD than in controls (p = 0.01). The percentage of individuals with increased optic disc was higher in cIGHD subjects in comparison to controls (92.9% vs. 57.1%). The same occurred for cup size (92.9% vs. 66.7%), p b 0.0001 in both cases. There was no difference in macula thickness. Conclusions: Most cIGHD individuals present moderate reduction of vascular branching points, increase of optic disc and cup size, but have similar thickness of the macula. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction The visual system is fundamental for the neuro-motor development, environmental adaptation and survival capacity. Visual acuity depends on a well-developed eye that is capable to form the image on the retina and process it in the central nervous system. Body size is heavily influenced by the effect of circulating GH and its main effector IGF-I on bone and cartilage tissues. It has been proposed that retinal development may reflect autocrine or paracrine ocular production of GH, IGFI, IGF type II (IGF-II) and other peptides like fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) [1–5]. Accordingly, in mice, rats and chickens
⁎ Corresponding author at: Division of Endocrinology, Johns Hopkins University School of Medicine, 1830 East Monument street suite # 333, Baltimore, MD 21287, United States. E-mail address: [email protected] (R. Salvatori).
http://dx.doi.org/10.1016/j.ghir.2016.07.001 1096-6374/© 2016 Elsevier Ltd. All rights reserved.
GH and GH receptor gene expression was documented in developing neural retina [3]. More than a half century has passed since the first description of regression of neovascularization in diabetic retinopathy after pituitary infarction and resultant GH deficiency, which led to the use of hypophysectomy as a therapy for proliferative diabetic retinopathy (GHD) [6]. However, the interaction between GH/IGFs and vascularization of the human retina is still not completely understood [5]. More recently, reduced retinal vascularization was shown in GHD children [7] and in GH insensitivity syndrome (Laron syndrome) [8], suggesting that the GH-IGF-I axis is critical for normal vascularization of the human retina. Nevertheless, in both studies there was some overlap between patients and controls, suggesting that other factors may affect the pattern of vascularization. The consequences of GHD or Laron syndrome in human retina may not be identical. While both models cause very low serum IGF-I levels, in the former there is often some GH secretion, albeit low, whereas the action of GH is completely impaired in Laron
12
V.M. Pereira-Gurgel et al. / Growth Hormone & IGF Research 30–31 (2016) 11–15
syndrome. Furthermore, congenital GHD can be isolated or associated to other pituitary deficits, and caused by several genetics or embryological insults, with direct consequences to the visual system [9,10]. Therefore, it is important to assess if individuals with inherited (therefore not caused by intrauterine insults) isolated GHD (IGHD) exhibit abnormalities in retinal neuro-vascularization. However, IGHD is a rare disease, occurring in 1:3480 to 1:10,000 live births [11], and most cases are treated with GH replacement during childhood. We have described in rural Northeastern Brazil a cohort of congenital IGHD (cIGHD) individuals due to a homozygous mutation (c.57 + 1 G N A) in the GHRHR gene (GHRHR) [12]. Despite severe short stature with adult height ranging from 1.07 to 1.36 m in pooled genders [13], these individuals cope well with daily challenges, do not exhibit neurodevelopmental problems, and have normal life expectancy [14]. Therefore, we hypothesized that these individuals present satisfactory retinal health, contributing to their normal survival. The objective of this study is to assess retinal neuro-vascularization in these untreated IGD individuals. 2. Subjects and methods
the branching point reduction in the initial categories: no reduction, mild reduction, moderate reduction and severe reduction. As no cIGHD had no reduction, and no control had mild reduction, we pooled no reduction and mild reduction in the final classification as minimal reduction. The same examiner evaluated the optic disc size (decreased, normal and increased) and cup size (normal or increased) in 14 cIGHD and 28 controls (one control had only the optic disc assessed). 2.5. OCT Twenty-five cIGHD subjects and 22 controls were analyzed and the average of each right and left macular area were calculated before comparison. Three patients had only one eye examined: two in cIGHD group (one due to toxoplasmosis and another because retinal detachment) and one in control group (severe cataract). We measured the thickness of the fovea and eight more macular areas: inner temporal, inner superior, inner nasal, inner inferior, outer temporal, outer superior, outer nasal, and outer inferior.
2.1. Subjects
2.6. Statistical analysis
In a cross sectional study, adult treatment-naïve cIGHD subjects, and age and sex-matched controls were recruited by advertising in the local Dwarfs Association, and by word of mouth among inhabitants of Itabaianinha County. Inclusion criterion for cIGHD was genotype-proven homozygosis for the c.57 + 1 G N A GHRHR mutation, whereas for controls was proven homozigosity for the wild-type allele. Genotyping method was described before [12]. Exclusion criteria for both groups were previous GH replacement, the presence of other genetic or acquired diseases that could alter the eye fundus appearance, hypertension requiring more than four drugs, diabetic retinopathy and glaucoma. Twenty-five cIGHD and 28 controls volunteered. The Federal University of Sergipe Institutional Review Board approved this study, and all subjects gave written informed consent.
Continuous variables were expressed as mean and standard deviation. Categorical variables were expressed in absolute number and percentage. The Student's t-test was used to compare continuous variables. Fisher's exact test was used to compare the categorical variables. Statistical analysis was performed using the statistical software IBM®SPSS® Version 20. Probability values b0.05 on a two tailed test were considered statistically significant.
2.2. Endocrine assessment Before the eye protocol, all the subjects fulfilled a clinical questionnaire and collected blood after an overnight fast for total cholesterol, triglycerides, glucose and IGF-I. Total cholesterol, triglycerides, and glucose were measured by standard techniques. IGF-I was measured by a solid-phase, enzyme-labeled chemiluminescent immunometric assay (IMMULITE 2000, Siemens Healthcare Diagnostics Products Ltd., Malvern, PA, USA), with a with intra- and inter-assay variabilities of 4.2 and 5.1%. 2.3. Study protocol All individuals underwent a complete eye examination, including applanation tonometry for intraocular pressure (Kowa Applanation Tonometer HA-2, Kowa, Japan), retinography (fundus photography) (Visucam 500, Carl Zeiss Meditec AG, Jena, Germany) and optical coherence tomography (OCT), (Stratus 3000, Carl Zeiss Meditec Inc. Dublin, CA, USA), under drug induced mydriasis. All fundus photographs were digitally recorded, sent to Göteborg, Sweden and analyzed by a unique examiner with clinical long-term experience (A. H.) blinded to the GH status. 2.4. Retinography Branching point reduction was assessed in all the visualized area in only well-focused photographs where all vessels were clearly visualized (11 cIGHD individuals and 17 controls). Both eyes were examined and there was no discrepancy between the two eyes of each patient regarding vascular branching points. The examiner classified in a blind fashion
3. Results Table 1 shows the clinical, biochemical and intraocular pressure in the cIGHD and controls subjects. Only the height, weight and IGF-I levels exhibited severe reduction in cIGHD group in comparison to control subjects. Table 2 shows the vascular branching point reduction in cIGHD subjects and controls in absolute number (n) and percentage (%) in the initial categories. Fig. 1 shows the final classification of the rate of vascular branching point reduction. Fisher's exact test revealed that cIGHD subjects presented more reduction of vascular branching points in comparison to controls (p = 0.049). The percentage of moderate reduction in cIGHD was higher than in control (91% vs. 53%, p = 0.01). Figs. 2 and 3 show that the rates of individuals with increased optic disc and cup size were increased in cIGHD in comparison to controls (p b 0.0001 in both cases). The percentage of individuals with increased optic and cup was significantly higher in cIGHD than in controls (p = 0.005, and p = 0.028, respectively). Fig. 4 shows the vascular branching point reduction and the optical disc and cup increase in an cIGHD individual and in a control. Table 3 shows that there was no difference in the thickness of the macula between the groups (fovea and eight more areas). Table 1 Clinical, biochemical and intraocular pressure (IOP) in the IGHD and control subjects.
Age (years) Sex (M) Smoking Height (m) Weight (kg) Systolic BP (mm Hg) Diastolic BP (mm Hg) IGF-I (ng/ml) Glucose (mg/dl) Cholesterol (mg/dl) Tryglicerides (mg/dl) IOP (mm Hg)
IGHD
Control
p
50.1 (15.9) 13 1 1.2 (0.18) 39.3 (8.7) 122.1 (18.9) 78.8 (9.3) 1.9 (0) 105.5 (22.7) 227.0 (65.1) 142.0 (95.5) 15.2 (3.1)
51.1 (14.0) 15 2 1.6 (0.1) 71.8 (14.4) 124.4 (15.0) 80.4 (6.5) 132 (55) 105.9 (70.5) 210.7 (28.2) 135.3 (51.3) 14.6 (1.9)
0.799 0.764 1 b0.0001 b0.0001 0.622 0.486 b0.0001 0.975 0.257 0.762 0.510
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Table 2 Branching point reduction in IGHD individuals and controls in absolute number (n) and percentage (%).
IGHD Control
0
1
2
Total
0/0 8 (47%)
1 (9.1%) 0 (0%)
10 (90.9%) 9 (53%)
11 17
Branch reduction was thus code: 0 = no reduction; 1 = mild reduction; 2 = moderate reduction. Fisher's exact test revealed that IGHD subjects present more reduction of vascular branching points in comparison to controls (p = 0.0491). The percentage of moderate reduction in IGHD was higher than in control group (p = 0.01).
4. Discussion This work describes three main ocular findings in adults with lifetime, untreated, inherited cIGHD. First, a moderate reduction of vascular branching points; second, a mild increase in the optic disc and cup size; and third, the normal thickness of the macula. As groups were matched for demographic, clinical and biochemical variables and intraocular pressure, the obtained retinal findings may reflect the true impacts of cIGHD in the human retina. Only one paper has previously reported the retinal vascularization status in 39 children (3.6–18.7 years old) with congenital GHD (six with multiple pituitary deficiencies), most of them without genetic testing and already on GH replacement [7]. Another study looked at 11 individuals with Laron syndrome (10–49 years) [8]. In both studies, a reduction of vascular branching points was found, apparently more marked in Laron syndrome. Since IGF-I deficiency is common to both conditions, this factor has been hypothesized to be critical for the normal vascularization of the human retina. Our cIGHD data seem to agree with those findings, although the magnitude of reduction was moderate. This finding suggests possible compensatory mechanism(s) in cIGHD due to a GHRHR mutation. As the retinal vascularization normally occurs during fetal development [15], it can be influenced more by IGF-II, thought to be more relevant for fetal somatic and ocular development [1] than IGF-I. Interestingly, the Itabaianinha cIGHD individuals exhibit an up-regulation of serum IGF-II [16]. This could influence retinal vessels development, partially counteracting the consequences of GHD and IGF-I reduction. The more marked reduction of vascular branching points in Laron syndrome can reflect a lack of a possible direct angiogenic GH effect on retinal vessels. Together, our and previous findings show a reduction of vascular branching points in cIGHD, with a slow progression to advanced stages of vascular branching points reduction. The second finding of this work is the simultaneous increase of the optic disc and cup size, probably with little effect on the rim area (the area between the cup and the border of the optic disc), indicating preservation of enough retinal ganglion cell number (RGC). RGC survival is influenced by ocular GH, as both GH and GH receptors were
demonstrated in these cells [17]. Conversely, pituitary GH and circulating IGF-I are not critical to birth weight, as cIGHD babies due to GHRHR mutations are born of normal size [12]. It was suggested that GH and GH receptor activity in the early embryonic retina and the effect on RGC development might explain the decreased peripapillary nerve fiber thickness in some children with congenital GHD [17], which was not assessed in our study. An increase in optic disc is generally considered a marker or abnormal neurodevelopment. Normal [17], reduced, or increased [18] optic disc size were previously described in GHD, possibly reflecting the variety of causes of GHD. While optic nerve hypoplasia is often linked to septo-optic dysplasia [9], it was suggested that an increased optic disc could predict the possibility of GHD in a child with severe short stature [18]. Our findings seem to corroborate this hypothesis. Therefore, an increased optic disc size, similar to voice [19] and cephalometric findings [20] can facilitate the clinical diagnosis of cIGHD. Furthermore, our findings suggest that the presence of an increased optical disc in patients on GH replacement therapy, but without signs of increased intracranial pressure, radial pericapillary bleeding or blurring of retinal vessels in the disc edges, does not indicate pseudotumor cerebri, and does not need treatment suspension or costly investigations as suggested in a small number of patients [18]. Neuroophthalmologists and endocrinologists should incorporate this information into their practices. The third finding of this work is the normal thickness of the macula, assessed by OCT. This technique provides detailed information of cross sectional retinal anatomy and quantification of retinal morphology [21,22]. There is no previous published data about OCT in congenital IGD. However, OCT data in Laron syndrome also show normal thickness of the all macula regions and normal retinal architecture [23]. Together with Laron syndrome findings, our data suggests that postnatal circulating IGF-I is not critical for the macula architecture, at least as assessed by OCT.
Fig. 1. Vascular branching point reduction rate in IGHD and controls.
Fig. 3. Rate of cup size in IGHD and controls.
Fig. 2. Rate of optic disc size in IGHD and controls.
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Fig. 4. Photo of one IGHD subject due to GHRH receptor deficiency and one control.
Our work has some limitations. First, the non-uniformity in the number of individuals used in all evaluations for retinal vascularization, due to the fact that half of the fundus images were not suitable for scoring. Another limitation was the use, for logistical reasons, of a semiquantitative system to assess the number of vascular branching points and optic disc size and excavation. However, this problem has been alleviated by using a simple and robust categorization system of these variables, performed by an ophthalmologist with solid clinical longterm experience in retinal assessment of GH deficiency and insensitivity syndromes [7,8].
Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Acknowledgments The authors thank the Associação do Crescimento Físico e Humano de Itabaianinha and Hospital Ocular in Aracaju, Sergipe, for assistance. References
5. Conclusions Adults with congenital, lifetime, untreated and isolated IGHD present a moderate reduction of vascular branching points, an increase of optic disc and cup size, with normal thickness of the macula. These data feature a distinct retinal phenotype in congenital IGHD due to an inactivating GHRHR mutation.
Declaration of interest R.S. serves in the advisory board of Novo Nordisk, Pfizer and Ionis. Prevention of retinopathy of prematurity by administering IGF-I are covered by patent owned by or licensed to Premacure AB, Uppsala, Sweden. AH owns shares in a company with financial interest in Premacure AB. AH work as consultants for Shire pharmaceuticals.
Table 3 Macula OCT map thickness (microns) in IGHD and control subjects. Data are expressed as mean (standard deviation).
Fovea Inner temporal Inner superior Inner nasal Inner inferior Outer temporal Outer superior Outer nasal Outer inferior
IGHD
Control
p
197.1 (34.8) 253.0 (29.6) 266.1 (34.8) 267.7 (36.0) 269.3 (35.5) 219.2 (20.9) 233.4 (23.6) 255.1 (26.7) 235.2 (20.4)
197.3 (23.2) 252.9 (21.3) 268.3 (18.8) 268.0 (19.7) 260.7 (21.5) 220.8 (17.7) 235.9 (17.3) 253.5 (26.0) 232.9 (18.4)
0.983 0.990 0.786 0.968 0.760 0.777 0.672 0.832 0.683
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