Older Individuals Heterozygous for a Growth Hormone-Releasing Hormone Receptor Gene Mutation Are Shorter Than Normal Subjects

ORIGINAL ARTICLE

Older individuals heterozygous for a growth hormone-releasing hormone receptor gene mutation are shorter than normal subjects

Manuel H Aguiar-Oliveira1, Marco A Cardoso-Filho1, Rossana MC Pereira1, Carla RP Oliveira1, Anita HO Souza1, Elenilde G Santos1, Viviane C Campos1, Eugênia HO Valença1, Francielle T de Oliveira1, Luiz A Oliveira-Neto1, Miburge B Gois-Junior1, Alecia A Oliveira-Santos1 and Roberto Salvatori2

Growth hormone (GH)-releasing hormone (GHRH) is the most important stimulus for GH secretion by the pituitary gland. Subjects homozygous for GHRH receptor (GHRHR) gene (GHRHR) inactivating mutations have severe GH deﬁciency, resulting in severe short stature if not treated. We previously reported that young adults heterozygous for the c.57+1G4A null GHRHR mutation (MUT/N) have reduced weight and body mass index (BMI) but normal stature. Here we have studied whether older MUT/N have an additional phenotype. In a cross-sectional study, we measured height, weight and blood pressure, and calculated BMI in two groups (young, 20–40 years of age) and old (60–80 years) of individuals heterozygous for the same GHRHR mutation, and compared with a large number of individuals of normal genotype residing in the same geographical area. Standard deviation score (SDS) of weight was lower, and BMI had a trend toward reduction in young heterozygous compared with young normals, without signiﬁcant difference in stature. Conversely, SDS of height was lower in older heterozygous individuals than in controls, corresponding to a reduction of 4.2 cm. These data show a reduced stature in older subjects heterozygous for the c.57 +1G4A GHRHR mutation, indicating different effects of heterozygosis through lifespan. Journal of Human Genetics (2015) 60, 335–338; doi:10.1038/jhg.2015.25; published online 12 March 2015

INTRODUCTION The c.57+1G4A GHRHR mutation, found in Itabaianinha county, The activity of the growth hormone (GH)/insulin-like growth factor-I in the northeastern Brazilian state of Sergipe, is by far the one with the axis is a major determinant of infantile and childhood growth, and of highest number of homozygous (MUT/MUT) affected individuals adult stature. GH secretion is low in early childhood, peaks in late (~100 over seven generations).8 This mutation–originating from the adolescence, achieves a plateau at the beginning of adult life and same founder9–has spread over the years in this community owing to declines 14% per decade thereafter.1 After adult stature is reached, age- minimal population mobility and the high prevalence of consangui- related height reduction occurs between 60 and 80 years of age, at neous marriages.8 The untreated adults present severe short stature, nearly 2 cm per decade,2 due to flattening of vertebral disks and with height standard deviation scores (SDS) ranging from –9.6 to –5.1. bodies, and the accentuation of the sagittal curvature of the spinal Their phenotype includes increased fat mass percentage and marked column.3 A better understanding of the causes of aging-associated reduction in muscle mass (MM).10 Despite this, they have normal height reduction is very important as it is linked to frailty, a public volumetric bone mineral density both below11 and above 60 years of health problem. age.12 Many genomic regions have been linked to variations in stature, but Although the consequences of bi-allelic GHRHR mutations are few of these regions have been confirmed.4,5 GH-releasing hormone obvious, the phenotype of heterozygous carriers (MUT/N) may be (GHRH) is necessary for the secretion of GH by the pituitary gland. revealed only in childhood and senescence, when the activity of the Indeed, homozygous GHRH receptor (GHRHR) gene (GHRHR) GH/insulin-like growth factor-I axis is sub-maximal. In agreement inactivating mutations are an important genetic cause of autosomal with this argument, MUT/N individuals of the Pakistani cohort of recessive congenital isolated GH deficiency (IGHD), resulting–when Sindh (a smaller kindred with 18 affected individuals carrying a not treated by GH replacement–in severe short stature.6 In one study, different mutation in the same gene) showed short stature only in haplotype GHRHR variations have been shown to be an important children and adolescents.13 Unfortunately, height of elderly individuals genetic factor for height variation in normal population.7 was not studied in that paper. Similarly, short stature has been

1Department of Medicine, Federal University of Sergipe Aracaju, Sergipe, Brazil and 2Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, The Johns Hopkins University School of Medicine (R.S), Baltimore, MD, USA Correspondence: Dr R Salvatori, Division of Endocrinology, Diabetes and Metabolism, Johns Hopkins University School of Medicine, 1830 East Monument street suite #333, Baltimore, MD 21287, USA. E-mail: [email protected] Received 12 January 2015; revised 1 February 2015; accepted 13 February 2015; published online 12 March 2015 Stature in GHRHR mutation heterozygosity MH Aguiar-Oliveira et al 336

Table 1 Height SDS and absolute value of height, weight, BMI, absolute values of SBP, DBP, mean (s.d.) and frequencies of categories of smoking, alcohol use and diabetes by age and genotype

Age 20–40 years Age 60–80 years

N/N MUT/N N/N MUT/N N = 461 N = 39 P-value N = 319 N = 24 P-value

SDS height − 1.2 (1) − 1.4 (1.2) 0.300 − 2.4 (1) − 2.8 (1.1) 0.040 Height (cm) 163.1 (9) 160.8 (9) 0.160 156.0 (9) 151.8 (8.0) 0.020 Weight (Kg) 65.3 (13) 62.5 (20) 0.047 62.8 (12) 59.8 (13) 0.250 SDS weight − 0.1 (1.4) − 0.5 (1.8) 0.030 − 0.5 (1.6) − 0.8 (1.9) 0.520 BMI (Kg m − 2) 24.4 (4.4) 24.2 (6.5) 0.192 25.8 (4.5) 26.2 (6.1) 0.379 SDS BMI 0.33 (1.4) 0.08 (1.4) 0.253 0.7 (1.4) 0.71 (1.4) 0.435 SBP (mm Hg) 123 (18) 122. (17) 0.750 144 (22) 147 (23) 0.650 DBP (mm Hg) 80 (13) 79 (10) 0.490 85 (14) 86 (12) 0.460 Non-smoker 363 (84) 17 (85) 0.170 230 (74) 12 (75) 0.870 Smoker 66 (15) 2 (10) 68 (22) 4 (25) Former Smoker 3 (1) 1 (5) 11 (4) 0 (0) Unanswered 29 19 10 8 No alcohol 188 (44) 13 (65) 0.150 233 (75) 11 (73) 0.150 Mild 136 (32) 2 (10) 55 (18) 1 (7) Moderate 61 (14) 2 (10) 12 (4) 2 (13) High 40 (9) 3 (15) 5 (2) 1 (7) Formeruser 5(1) 0(0) 5(2) 0(0) Unanswered 31 19 9 9 Not diabetic 441 (99) 34 (100) 0.990 262 (85) 23 (96) 0.230 Diabetic 5 (1) 0 (0) 48 (15) 1 (4) Unanswered 15 5 9 0

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure; SDS, standard deviation score.

reported in heterozygous carriers of mutations in the GH1 gene, Height, weight and BMI were expressed in absolute values and SDS, encoding for human GH.14 calculated by subtracting the mean value of the respective gender of the ‘British In a previous study of the Itabaianinha kindred, we have shown that 1990 Growth Reference Data’ and dividing this value by the SD of the adult MUT/N subjects have reduced weight, body mass index (BMI) respective gender, by using the site: www.phsim.man.ac.uk/SDSCalculator/ SDSCalculator.aspx and MM when compared with homozygous normal (N/N) subjects, Blood pressure was the mean value of three measurements after 10 min in fi 15 but their adult stature is not signi cantly reduced. However, only seated position. Data are expressed in mean (SD). Student’s t-test was used to ⩾ 10% of those individuals were 60 years of age. We hypothesized that compare the variables with normal distribution, and Mann–Whitney’s U-test, the height reduction may manifest itself during senescence. The main for those with not normal distribution (weight in young, BMI and SDS BMI in objective of this work was to assess the stature of older MUT/N both young and old). Mann–Whitney’s U-test was also used to compare the age individuals from this kindred. Secondary aims were to evaluate the of the N/N and MUT/N groups. Fisher test was used to compare possible fl consequences of heterozygosity on weight, BMI and blood pressure. factors in uencing the phenotype in each group, namely alcoholism, smoking and self-reported diagnosis of diabetes. Institutional Review Boards of both the Johns Hopkins University and the SUBJECTS AND METHODS Federal University of Sergipe approved the protocol, and all subjects provided Advertising by broadcast and loudspeakers in front of the voting station, we written informed consent. invited normal statured Itabaianinha county residents to volunteer for this study on the day of the October 2008 municipal elections (voting in Brazil is RESULTS compulsory, causing very high voting rates: 86.8% in the 2008 elections). There was no difference in the prevalence of the MUT/N genotype in – We enrolled 843 normal-appearing volunteers, 500 young (20 40 years) (255 the two groups: young 39/500 (7.8%) and older 24/343 (7.0%). – females, 51%) and 343 old (60 80 years) (164 females, 48%). A cross-sectional Although there was a statistical difference in age between young N/N study was carried out to compare height, body weight, BMI and blood pressure. and MUT/N (29.6 (6.2) vs 32.3 (5.8) years, P = 0.009) there was no Owing to the high number of enrollees, we could not collect any additional age difference between N/N and MUT/N in the old groups (68.3 (6.9) demographic or racial information on the volunteers. The population of P = fi ‘ vs 68.0 (7.72) 0.84. Itabaianinha County includes 38 910 inhabitants, ~ 62% classi ed as mixed – race’, 31% white and 7% black (http://cidades.ibge.gov.br/painel/painel.php? We compared the 39 young (20 40 years) MUT/N with 461 young – codmun = 280300). All subjects were genotyped from buccal swabs for the c.57 N/N subjects, and 24 old (60 80 years) MUT/N with 319 old N/N +1G4A GHRHR mutation as previously described.16 Height (cm) and body subjects. Results are shown in Table 1. SDS height was lower in older − − P = weight (kg) were measured with a portable stadiometer and a portable scale. MUT/N than N/N individuals ( 2.79 (1.1) and 2.37 (1.0), 0.04) BMI was calculated using the formula: weight/height kg m − 2. BMI was corresponding to a height reduction of 4.16 cm (P = 0.02). There was expressed in absolute values and SDS, calculated using the British 1990 Growth no significant difference in SDS height in young MUT/N (−1.4 (1.2) Reference Data, by using the site http://www.phsim.man.ac.uk/SDSCalculator/ and − 1.2 (1) vs N/N), corresponding to 2.35 cm. Weight, both in SDSCalculator.aspx. absolute value and SDS, was lower in younger (P = 0.047 and 0.03,

Journal of Human Genetics Stature in GHRHR mutation heterozygosity MH Aguiar-Oliveira et al 337 respectively) but not in older MUT/N. No significant difference was where reduced bone mineral density and an increased risk of fractures found in blood pressure, in neither the prevalence of alcoholism, have been reported.24 It is possible that MUT/N subjects (with normal smoking and self-reported diagnosis of diabetes between MUT/N and adult height and bone dimensions) may have weaker musculoskeletal N/N in any age group. system, with relatively excessive loading forces, possibly resulting in flattening of vertebral disks and bodies, and therefore, in height loss.25 DISCUSSION Future studies of MUT/N subjects will address this hypothesis. IGHD caused by mutations in the GHRHR is typically described as a Contrarily to stature, blood pressure was similar in N/MUT and classical autosomal recessive disease (IGHD type IB).6 Although most N/N subjects (both young and elderly groups), with the expected autosomal recessive disease manifests a phenotype only when both increase in blood pressure with aging in both genotypes. The normal alleles are defective, there are many examples of partial phenotype blood pressure pattern, combined with normal beta cell function and caused by a single allele receptor defect (‘gene dosage effect’). increased insulin sensitivity, we had previously noted in younger Examples include mutations in genes encoding the calcium-sensing MUT/N subjects15 may contribute to the normal longevity of MUT/N 17 18 receptor, the natriuretic peptide receptor-B and the melanocortin 4 individuals.16 19 receptor. In addition, heterozygous mutations in the GH receptor Interestingly, we did not find in these older MUT/N subjects the gene have been shown in ~ 5% children with idiopathic short same reduction in body weight and BMI that we have previously 20 stature. The possibility of a milder phenotype in heterozygous found in younger individuals (the SDS BMI in younger MUT/N group GHRHR 15 carriers of a mutation has only been suggested recently. was lower but failed to reach statistical significance).15 We had found In that early work we had not detected an effect on adult stature. That that MUT/N subjects have a reduction of both fat mass and fat-free population was, however, composed mostly by young adults. mass, but the reduction is more marked for fat-free mass. It is possible fi Although here we have con rmed that young MUT/N individuals that the natural reduction in MM (contributor to fat-free mass) that have similar SDS stature to N/N subjects (160.8 and 163.1 cm, occurs with aging in normal control individuals20 may obliterate any respectively), we found that older heterozygous subjects are shorter difference between MUT/N and N/N older groups. than individuals homozygous for the same allele, residing in the same A limitation of our study is its cross-sectional nature. For obvious P = P = area (151.8 and 156.0 cm, 0.002 in absolute values and 0.004 in reasons, we could not follow the same individuals throughout life, and SDS for height, respectively). These data confirm the known aging 2 therefore we cannot completely exclude that the older MUT/N were related height reduction in both groups, and show that such already shorter at younger age owing to some environmental or reduction is more accentuated in the MUT/N subjects, providing genetic factor, although this seems improbable because of the fact that evidence that genetic factors may contribute to age-related loss of controls came from the same geographical reason. stature. As age-related height loss is associated with sarcopenia, In conclusion, the reduced height found in older MUT/N shows osteoporosis and frailty,21 this finding may be very relevant in terms that the phenotypic expression of heterozygosity for a GHRHR of public health. mutation could be seen more clearly in later stages of life. The This age-related height reduction is not caused by age difference mechanism remains to be elucidated. between the two groups, and it is unlikely because of racial or genetic background differences, as all study subjects were drawn from the CONFLICT OF INTEREST same municipality, and most Northeastern Brazilians share similar The authors declare no conflict of interest. racial background.22 Interestingly, the height reduction we observed in older MUT/N individual is of a degree similar to that found in two ACKNOWLEDGEMENTS independent Scandinavian cohorts with non-disease causing GHRHR We thank the Court of Electoral Justice of Sergipe estate and the Catholic – 7 haplotypes (1.2 3.8 cm). The subjects in that study were of a broad Roman and First Baptist churches in Itabaianinha for allowing us to perform – fi age range (15 87 years), and no age strati cation was provided. 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