Everlayny F. Costalonga

Novel Heterozygous Nonsense GLI2 Mutations in Patients with Hypopituitarism and Ectopic Posterior Pituitary Lobe without Holoprosencephaly

CONTEXT GLI2 is a transcription factor downstream in Sonic Hedgehog signaling, acting early in ventral forebrain and pituitary development. GLI2 mutations were reported in patients with holoprosencephaly (HPE) and pituitary abnormalities. OBJECTIVE The aim was to report three novel frameshift/nonsense GLI2 mutations and the phenotypic variability in the three families. SETTING The study was conducted at a university hospital. PATIENTS AND METHODS The GLI2 coding region of patients with isolated GH deficiency (IGHD) or combined pituitary hormone deficiency was amplified by PCR using intronic primers and sequenced. RESULTS Three novel heterozygous GLI2 mutations were identified: c.2362_2368del p.L788fsX794 (family 1), c.2081_2084del p.L694fsX722 (family 2), and c.1138 G>T p.E380X (family 3). All predict a truncated protein with loss of the C-terminal activator domain. The index case of family 1 had polydactyly, hypoglycemia, and seizures, and GH, TSH, prolactin, ACTH, LH, and FSH deficiencies. Her mother and seven relatives harboring the same mutation had polydactyly, including two uncles with IGHD and one cousin with GH, TSH, LH, and FSH deficiencies. In family 2, a boy had cryptorchidism, cleft lip and palate, and GH deficiency. In family 3, a girl had hypoglycemia, seizures, excessive thirst and polyuria, and GH, ACTH, TSH, and antidiuretic hormone deficiencies. Magnetic resonance imaging of four patients with GLI2 mutations and hypopituitarism showed a hypoplastic anterior pituitary and an ectopic posterior pituitary lobe without HPE. CONCLUSION We describe three novel heterozygous frameshift or nonsense GLI2 mutations, predicting truncated proteins lacking the activator domain, associated with IGHD or combined pituitary hormone deficiency and ectopic posterior pituitary lobe without HPE. These phenotypes support partial penetrance, variable polydactyly, midline facial defects, and pituitary hormone deficiencies, including diabetes insipidus, conferred by heterozygous frameshift or nonsense GLI2 mutations.

The −202 A Allele of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Promoter Polymorphism Is Associated with Higher IGFBP-3 Serum Levels and Better Growth Response to Growth Hormone Treatment in Patients with Severe Growth Hormone Deficiency

CONTEXT Genetic factors that influence the response to recombinant human GH (rhGH) therapy remain mostly unknown. To date, only the GH receptor gene has been investigated. OBJECTIVE The aim of the study was to assess the influence of a polymorphism in the IGF-binding protein-3 (IGFBP-3) promoter region (-202 A/C) on circulating IGFBP-3 levels and growth response to rhGH therapy in children with GH deficiency (GHD). DESIGN AND PATIENTS -202 A/C IGFBP3 genotyping (rs2854744) was correlated with data of 71 children with severe GHD who remained prepubertal during the first year of rhGH treatment. MAIN OUTCOME MEASURES We measured IGFBP-3 levels and first year growth velocity (GV) during rhGH treatment. RESULTS Clinical and laboratory data at the start of treatment were indistinguishable among patients with different -202 A/C IGFBP3 genotypes. Despite similar rhGH doses, patients homozygous for the A allele presented higher IGFBP-3 sd score levels and higher mean GV in the first year of rhGH treatment than patients with AC or CC genotypes (first year GV, AA = 13.0 +/- 2.1 cm/yr, AC = 11.4 +/- 2.5 cm/yr, and CC = 10.8 +/- 1.9 cm/yr; P = 0.016). Multiple linear regression analyses demonstrated that the influence of -202 A/C IGFBP3 genotype on IGFBP-3 levels and GV during the first year of rhGH treatment was independent of other variables. CONCLUSION The -202 A allele of IGFBP3 promoter region is associated with increased IGFBP-3 levels and GV during rhGH treatment in prepubertal GHD children.

Exon 3‐deleted genotype of growth hormone receptor (GHRd3) positively influences IGF‐1 increase at generation test in children with idiopathic short stature

Context   A GHR‐exon 3 polymorphism has been reported to influence the growth response to hGH therapy in short stature children. None of these studies provided data on IGF‐1 generation test.

The sitting height/height ratio for age in healthy and short individuals and its potential role in selecting short children for SHOX analysis.

Aims: To determine the presence of abnormal body proportion, assessed by sitting height/height ratio for age and sex (SH/H SDS) in healthy and short individuals, and to estimate its role in selecting short children for SHOX analysis. Methods: Height, sitting height and weight were evaluated in 1,771 healthy children, 128 children with idiopathic short stature (ISS), 58 individuals with SHOX defects (SHOX-D) and 193 females with Turner syndrome (TS). Results: The frequency of abnormal body proportion, defined as SH/H SDS >2, in ISS children was 16.4% (95% CI 10-22%), which was higher than in controls (1.4%, 95% CI 0.8-1.9%, p < 0.001). The SHOX gene was evaluated in all disproportionate ISS children and defects in this gene were observed in 19%. Among patients with SHOX-D, 88% of children (95% CI 75-100%) and 96% of adults had body disproportion. In contrast, SH/H SDS >2 were less common in children (48%, 95% CI 37-59%) and in adults (28%, 95% CI 20-36%) with TS. Conclusion: Abnormal body proportions were observed in almost all individuals with SHOX-D, 50% of females with TS and 16% of children considered ISS. Defects in SHOX gene were identified in 19% of ISS children with SH/H SDS >2, suggesting that SH/H SDS is a useful tool to select children for undergoing SHOX molecular studies.

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca2 + pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition.

Relatively high frequency of non‐synonymous GLI2 variants in patients with congenital hypopituitarism without holoprosencephaly

GLI2 is a downstream transcription factor in Sonic Hedgehog signalling, acting early in ventral forebrain and pituitary development. Heterozygous nonsense GLI2 mutations have been reported in patients with isolated or combined pituitary hormone deficiency (CPHD), with or without holoprosencephaly. The aim of this study was to screen for GLI2 mutations in a large cohort of patients with congenital GH deficiency.

Growth hormone pharmacogenetics: the interactive effect of a microsatellite in the IGF1 promoter region with the GHR-exon 3 and-202 A/C IGFBP3 variants on treatment outcomes of children with severe GH deficiency

Insulin-like growth factor type 1 (IGF1) is a mediator of growth hormone (GH) action, and therefore, IGF1 is a candidate gene for recombinant human GH (rhGH) pharmacogenetics. Lower serum IGF1 levels were found in adults homozygous for 19 cytosine–adenosine (CA) repeats in the IGF1 promoter. The aim of this study was to evaluate the influence of (CA)n IGF1 polymorphism, alone or in combination with GH receptor (GHR)-exon 3 and −202 A/C insulin-like growth factor binding protein-3 (IGFBP3) polymorphisms, on the growth response to rhGH therapy in GH-deficient (GHD) patients. Eighty-four severe GHD patients were genotyped for (CA)n IGF1, −202 A/C IGFBP3 and GHR-exon 3 polymorphisms. Multiple linear regressions were performed to estimate the effect of each genotype, after adjustment for other influential factors. We assessed the influence of genotypes on the first year growth velocity (1st y GV) (n=84) and adult height standard deviation score (SDS) adjusted for target-height SDS (AH-TH SDS) after rhGH therapy (n=37). Homozygosity for the IGF1 19CA repeat allele was negatively correlated with 1st y GV (P=0.03) and AH-TH SDS (P=0.002) in multiple linear regression analysis. In conjunction with clinical factors, IGF1 and IGFBP3 genotypes explain 29% of the 1st y GV variability, whereas IGF1 and GHR polymorphisms explain 59% of final height-target-height SDS variability. We conclude that homozygosity for IGF1 (CA)19 allele is associated with less favorable short- and long-term growth outcomes after rhGH treatment in patients with severe GHD. Furthermore, this polymorphism exhibits a non-additive interaction with −202 A/C IGFBP3 genotype on the 1st y GV and with GHR-exon 3 genotype on adult height.

Genetic Predictors of Long-Term Response to Growth Hormone (GH) Therapy in Children With GH Deficiency and Turner Syndrome: The Influence of a SOCS2 Polymorphism

BACKGROUND There is great interindividual variability in the response to GH therapy. Ascertaining genetic factors can improve the accuracy of growth response predictions. Suppressor of cytokine signaling (SOCS)-2 is an intracellular negative regulator of GH receptor (GHR) signaling. OBJECTIVE The objective of the study was to assess the influence of a SOCS2 polymorphism (rs3782415) and its interactive effect with GHR exon 3 and -202 A/C IGFBP3 (rs2854744) polymorphisms on adult height of patients treated with recombinant human GH (rhGH). DESIGN AND PATIENTS Genotypes were correlated with adult height data of 65 Turner syndrome (TS) and 47 GH deficiency (GHD) patients treated with rhGH, by multiple linear regressions. Generalized multifactor dimensionality reduction was used to evaluate gene-gene interactions. RESULTS Baseline clinical data were indistinguishable among patients with different genotypes. Adult height SD scores of patients with at least one SOCS2 single-nucleotide polymorphism rs3782415-C were 0.7 higher than those homozygous for the T allele (P < .001). SOCS2 (P = .003), GHR-exon 3 (P= .016) and -202 A/C IGFBP3 (P = .013) polymorphisms, together with clinical factors accounted for 58% of the variability in adult height and 82% of the total height SD score gain. Patients harboring any two negative genotypes in these three different loci (homozygosity for SOCS2 T allele; the GHR exon 3 full-length allele and/or the -202C-IGFBP3 allele) were more likely to achieve an adult height at the lower quartile (odds ratio of 13.3; 95% confidence interval of 3.2-54.2, P = .0001). CONCLUSION The SOCS2 polymorphism (rs3782415) has an influence on the adult height of children with TS and GHD after long-term rhGH therapy. Polymorphisms located in GHR, IGFBP3, and SOCS2 loci have an influence on the growth outcomes of TS and GHD patients treated with rhGH. The use of these genetic markers could identify among rhGH-treated patients those who are genetically predisposed to have less favorable outcomes.

The Interactive Effect of GHR-Exon 3 and −202 A/C IGFBP3 Polymorphisms on rhGH Responsiveness and Treatment Outcomes in Patients with Turner Syndrome

CONTEXT There is great interindividual variability in the response to recombinant human (rh) GH therapy in patients with Turner syndrome (TS). Ascertaining genetic factors can improve the accuracy of growth response predictions. OBJECTIVE The objective of the study was to assess the individual and combined influence of GHR-exon 3 and -202 A/C IGFBP3 polymorphisms on the short- and long-term outcomes of rhGH therapy in patients with TS. DESIGN AND PATIENTS GHR-exon 3 and -202 A/C IGFBP3 genotyping (rs2854744) was correlated with height data of 112 patients with TS who remained prepubertal during the first year of rhGH therapy and 65 patients who reached adult height after 5 ± 2.5 yr of rhGH treatment. MAIN OUTCOME MEASURES First-year growth velocity and adult height were measured. RESULTS Patients carrying at least one GHR-d3 or -202 A-IGFBP3 allele presented higher mean first-year growth velocity and achieved taller adult heights than those homozygous for GHR-fl or -202 C-IGFBP3 alleles, respectively. The combined analysis of GHR-exon 3 and -202 A/C IGFBP3 genotypes showed a clear nonadditive epistatic influence on adult height of patients with TS treated with rhGH (GHR-exon 3 alone, R² = 0.27; -202 A/C IGFBP3, R² = 0.24; the combined genotypes, R² = 0.37 at multiple linear regression). Together with clinical factors, these genotypes accounted for 61% of the variability in adult height of patients with TS after rhGH therapy. CONCLUSION Homozygosity for the GHR-exon3 full-length allele and/or the -202C-IGFBP3 allele are associated with less favorable short- and long-term growth outcomes after rhGH treatment in patients with TS.

Evidence of underdiagnosis and markers of high blood pressure risk in children aged 6 to 13 years

OBJECTIVE To estimate the prevalence of high blood pressure (BP) in schoolchildren, as well as the reported frequency of previous measurements of BP in these children, and to identify high BP risk markers in the sample. METHODS This was a cross-sectional study involving 794 children aged 6 to 13 years, enrolled in public elementary schools. A questionnaire was given to parents/guardians, consisting of perinatal, socioeconomic data, and information on previous measurements of BP in these children. Anthropometric measurements included weight, height, waist, hip, and arm and neck circumference, in addition to the three BP measurements. Classification of BP levels was carried out according to current international recommendations, established in 2004. RESULTS The prevalence of high BP (hypertension or prehypertension) was 7%. Only 21.7% of children had previously undergone BP measurements. The odds ratio of high BP among children with and without overweight was 2.9 (95% CI=1.7 to 5.0, p<0.001). None of the anthropometric measurements was superior to the Z-score of BMI as a predictor of high BP. History of hypertension during pregnancy (p<0.001), prematurity (p=0.006), maternal hypertension (p=0.01), and paternal hypertension (p=0.008) were also correlated with the presence of high BP in children. CONCLUSIONS Overweight and family history constitute the main risk markers of high BP in children. The low frequency of BP measurement in children observed in this municipality contributes to the underdiagnosis of the disease, with irreversible consequences for these individuals.