Growth Hormone & IGF Research 24 (2014) 180–186
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Autosomal recessive form of isolated growth hormone deficiency is more frequent than the autosomal dominant form in a Brazilian cohort☆ Andria C.V. Lido a,b,1, Marcela M. França a,1, Fernanda A. Correa a, Aline P. Otto a, Luciani R. Carvalho a, Elisangela P.S. Quedas b, Mirian Y. Nishi a, Berenice B. Mendonca a, Ivo J.P. Arnhold a, Alexander A.L. Jorge b,⁎ a Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular LIM/42 do Hospital das Clinicas, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Sao Paulo, 05403-900 Sao Paulo, Brazil b Unidade de Endocrinologia Genetica, Laboratorio de Endocrinologia Celular e Molecular LIM/25, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, 01246-903 Sao Paulo, Brazil
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Article history: Received 9 May 2014 Received in revised form 21 July 2014 Accepted 22 July 2014 Available online 30 July 2014 Keywords: Short stature Isolated GH deficiency (IGHD) Mutation in Growth hormone 1 gene (GH1) Growth hormone (GH) secretion status Brazilian children
a b s t r a c t Background: In most studies, the autosomal dominant (type II) form of isolated growth hormone deficiency (IGHD) has been more frequent than the autosomal recessive (type I) form. Our aim was to assess defects in the GH1 in short Brazilian children with different GH secretion status. Subjects and methods: We selected 135 children with postnatal short stature and classified according to the highest GH peak at stimulation tests in: severe IGHD (peak GH ≤ 3.3 μg/L, n = 38, all with normal pituitary magnetic resonance imaging); GH peak between 3.3 and 10 μg/L (n = 76); and GH peak N10 μg/L (n = 21). The entire coding region of GH1 was sequenced and complete GH1 deletions were assessed by Multiplex Ligation Dependent Probe Amplification and restriction enzyme digestion. Results: Patients with severe IGHD had a higher frequency of consanguinity, were shorter, had lower levels of IGF-1 and IGFBP-3, and despite treatment with lower GH doses had a greater growth response than patients with GH peak ≥3.3 μg/L. Mutations were found only in patients with severe IGHD (GH peak b 3.3 μg/L). Eight patients had autosomal recessive IGHD: Seven patients were homozygous for GH1 deletions and one patient was compound heterozygous for a GH1 deletion and the novel c.171+5GNC point mutation in intron 2, predicted to abolish the donor splice site. Only one patient, who was heterozygous for the c.291+1GN T mutation located at the universal donor splice site of intron 3 and predicts exon 3 skipping, had an autosomal dominant form. Conclusion: Analysis of GH1 in a cohort of Brazilian patients revealed that the autosomal recessive form of IGHD was more common than the dominant one, and both were found only in severe IGHD. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Circulating growth hormone (GH) is secreted by the anterior pituitary gland and is one of the most important hormonal regulators of post-natal linear growth. It is encoded by the Growth Hormone 1 gene (GH1; OMIM *139250). GH1 is located on chromosome 17q22–q24 and comprises five exons within a 66 kb cluster region, where four other highly homologous genes are positioned: CSH1, CSH2, CSHP1 and GH2. Several GH1 defects
☆ Acknowledgments and grants: This work was supported by Grants 2013/03236-5 (to A.A.L.J.), 05/04726-0 and 07/56490-5 (to M.M.F.) from the Sao Paulo Research Foundation (FAPESP) and Grants 307922/2013-8 (to I.J.P.A.), 301339/2008-9 (to B.B.M.), and 304678/ 2012-0 (to A.A.L.J.) from the National Council for Scientific and Technological Development (CNPq). ⁎ Corresponding author at: Faculdade de Medicina da USP (LIM-25), Av. Dr. Arnaldo, 455 5º Andar Sala 5340, CEP 01246-903 Sao Paulo, SP, Brazil. Tel./fax: +55 11 3061 7252. E-mail addresses: [email protected], [email protected] (A.A.L. Jorge). 1 ACVL and MMF contributed equally to this article.
http://dx.doi.org/10.1016/j.ghir.2014.07.001 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
have been described in patients with short stature with different growth hormone secretory status and variable height deficit [1]. Complete deletions of GH1 are responsible for a severe autosomal recessive form of isolated growth hormone deficiency (IGHD type IA, OMIM 262400). Affected patients have a complete lack of GH secretion, classical GHD phenotype and during human GH (hGH) therapy develop growth-attenuating anti-GH antibodies, which often interfere with the action of exogenous GH [2]. The deletions described are heterogeneous and are caused by homologous recombination within the GH-cluster regions. The most frequently found GH1 deletion extends over 6.7 kb, followed by 7.6 kb, 7.0 kb, 4.5 kb and other infrequent deletions [2]. Other molecular defects, such as frameshift mutations and homozygous nonsense mutations can also cause IGHD type IA [3]. This form of IGHD is rare, being more frequent in families with a history of consanguinity or in specific geographic groups [2,3]. Usually missense, nonsense, frameshift and splice site mutations in GH1 are responsible for the recessive (type IB, OMIM 612781), whereas specific missense, splice site or splice enhancer mutations are responsible for the dominant (type II, OMIM 173100) forms of IGHD [3]. IGHD type II
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is caused mainly by point mutations located in the first six base pairs of the 5′ region of intron 3 of GH1 [4]. These mutations result in exon 3 skipping in the mutated allele, leading to the expression of the 17.5 kDa GH isoform, which exerts a dominant negative effect on the bioactive GH isoforms [4]. Patients with IGHD type II usually present great variability in the severity of GH deficiency and in phenotypic expression [5–8], even among members of the same family [5]. GH peak in general is low but detectable, can vary on different occasions and can be above 5 μg/L [5–8]. This variability in phenotype and secretory response of GH may increase the difficulty in establishing the diagnosis of IGHD. Furthermore, specific missense mutations in GH1 can result in a biologically inactive mutant protein [9–11], which affect GH action and cause insensitivity to endogenous GH (Kowarski syndrome, OMIM 262650), mainly characterized by growth retardation, normal or slightly increased GH secretion, low IGF-I levels and catch-up growth on rhGHreplacement therapy [12]. The frequency and the type of GH1 alteration identified in different studies depend on the ethnic background and the population selection criteria (frequency of consanguinity, severity of short stature and growth hormone secretory status) [13–15]. Generally, the autosomal dominant form is the most frequent genetic form of IGHD reported in the literature [1,3]. However, previous molecular-genetic studies in our Brazilian cohort of patients with IGHD identified only patients homozygous for GHRHR mutations or GH1 deletions [16,17]. In Brazil, the diagnosis of GHD is usually established using lower cutoff values of GH peak in stimulation tests than in several other countries (3.3 or 5 μg/L vs. 7 or 10 μg/L). It is possible that the high frequency of IGHD Type I and absence of Type II observed in our patients could be related to the adopted criteria to define a patient as IGHD or to an intrinsic characteristic of our population. Thus, in the present study, we investigated the frequency of defects in GH1 in Brazilian children with different GH secretion status and evaluated the first year growth response to recombinant GH (rhGH) therapy according to this status. 2. Patients and methods 2.1. Subjects This study was approved by the local Ethics Committee and the patients and/or guardians gave their written informed consent before initiating the genetic studies. The studied cohort consisted of selected patients referred for clinical and/or genetic evaluation at the Developmental Endocrinology Unit of the Hospital das Clinicas of the University of Sao Paulo School of Medicine, Brazil. One hundred thirty-five short children met the following criteria: 1) normal birth weight and length for gestational age; 2) height SDS ≤− 2 [19] and/or height velocity SDS ≤− 1, observed over a period of six months; 3) normal body proportions [20]; 4) had at least one GH stimulation test available; 5) absence of a known cause of short stature, except IGHD and 6) absence of other pituitary hormone deficiencies [16]. Patients were classified according to the highest GH peak obtained in stimulation tests. Patients were diagnosed as GHD based on clinical and auxological parameters and failure to respond to two GH stimulation tests (GH peak ≤ 3.3 μg/L) [18]. Additionally, patients with the typical clinical phenotype of severe GHD (such as severe short stature, hypoglycemia, protruding forehead, saddle nose, central obesity and high-pitched voice) and failure to respond to one GH stimulation test were also diagnosed as GHD. Magnetic resonance imaging (MRI) of the pituitary region was performed in all patients with a GH peak ≤3.3 μg/L and only patients with eutopic posterior lobe and an intact stalk were selected for this study [16]. 2.2. Clinical evaluation Evaluations were performed at the same period of the day and included measurements of height (measured with a stadiometer) and weight (measured with a digital scale). Body mass index (BMI) was
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calculated as weight (Kg) divided by height (m) squared. Height and body mass index (BMI) were expressed as SDS [19]. Target height was calculated [(father's height + mother's height + 13 cm for boys or -13 cm for girls) / 2] and expressed as SDS. Left hand and wrist X-rays for bone age determination were assessed by the method of Greulich and Pyle [21]. Sixty-two patients were treated with rhGH administered subcutaneously (sc) at a dose range of 30–50 μg/kg/day, which was adjusted according to changes in weight at each visit. All children were evaluated at baseline and every 3–4 months during rhGH treatment. First year growth velocity was determined after an observation period of at least 11 months. 2.2.1. Hormone assays GH was measured by immunofluorometric assay (IFMA) (AutoDELFIA, Wallac, Turku, Finland) method with monoclonal antibodies. The GH secretion status was evaluated by provocative tests, usually with Clonidine and/or insulin-induced hypoglycemia, following standard protocols. IGF-I was measured by RIA after ethanol extraction (Diagnostic Systems Laboratories, Webster, TX) or by chemiluminescence assays (CLIA) (Immulite; Diagnostic Products Corp., Los Angeles, CA). IGFBP-3 was measured by IRMA (Diagnostic Systems Laboratories) or CLIA (Immulite). IGF-I and IGFBP-3 levels were expressed as SDS for age and sex according to reference values provided by the respective assay kits. The absence of deficiencies of the other pituitary hormones was established by the normal basal values of total T4 and/or free T4, cortisol, prolactin, TSH, gonadotropins, and testosterone (postpubertal boys) or estradiol (postpubertal girls) as well as the TSH, prolactin, and cortisol responses to combined insulin-TRH infusion [16,22]. 2.2.2. Molecular studies Genomic DNA was extracted from peripheral-blood leucocytes of all patients using standard techniques. The entire coding region and all introns of the GH1 (GenBank accession number NC_000017.10 for genomic and NM_000515.3 for mRNA sequences) were amplified by polymerase chain reaction (primer sequences and amplification protocols are available upon request). Amplification products were bidirectionally sequenced with the dideoxy chain-termination method using a dye terminator kit and analyzed in an ABI Prism 3130 automated sequencer (Applied Biosystems, Foster City, CA). Large GH1 deletions were assessed by multiplex ligation-dependent probe amplification (MLPA) and by restriction endonuclease analysis using SmaI as previously described [23,24]. MLPA analysis of GH1 was carried out using the commercial Salsa Kit P216 Growth Hormone Deficiency (MCR Holland, Amsterdam, The Netherlands). This kit contains 4 GH1 probes locate at intron 1, exon 3, exon 4 and exon 5. Data generated by MLPA were intra-normalized against every single probe and assay. Patients' and parents' sample normalized peak area was then divided by the average normalized peak area from control samples. Dosage quotient areas outside the range 0.70–1.3 were considered abnormal. In addition, visual comparison of peak profiles was performed. 2.2.3. In silico prediction of mutation effects To identify the potential effects of sequence variants identified in GH1 on splicing and on protein function or structure, the wild type and variant sequences were submitted to NetGene2 Server (http://www.cbs.dtu.dk/services/NetGene 2) [25] and Mutation Taster (http://www.mutationtaster.org) [26]. 2.2.4. Statistical analysis Results were expressed as mean ± SD. Differences between groups were tested by t-test or Kruskal–Wallis and chi-square or Fisher exact test, as appropriate. A p value b 0.05 was considered statistically significant. All statistical calculations were carried out using SigmaStat version 3.5 (Systat Software Inc., Chicago, IL).
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3. Results
Table 2 Phenotype of patients treated with rhGH regarding their GH secretory status.
3.1. Patients' characteristics
Maximum GH peak
The one hundred thirty-five children were divided into three groups according to values of GH peak response in stimulation tests: GH peak b3.3 μg/L (or severe IGHD group, n = 38), between 3.3 and 10 μg/L (n = 76), and N10 μg/L (n = 21). The clinical and laboratory features of all patients selected for GH1 study regarding the growth hormone secretory status are summarized in Table 1. The patients with IGHD showed slightly higher prevalence of females, whereas in the other GH secretion status groups there was a clear male preponderance. This IGHD group also had higher frequency of consanguinity and presence of anterior pituitary hypoplasia at MRI (48.5% vs 12.5%), was shorter and had lower levels of IGF-1 and IGFBP-3 than patients with GH peak ≥3.3 μg/L. The clinical and laboratory features of sixty-two patients who were treated with rhGH are shown in Table 2. Patients with IGHD were treated with lower rhGH dose, had higher growth velocity and greater changes in height SDS during the first year of rhGH therapy than patients with GH peak ≥3.3 μg/L. Patients with GH peak between 3.3 and 10 μg/L or N10 μg/L had similar growth response to rhGH therapy. 3.2. Molecular results We identified mutations in GH1 only in patients classified as severe IGHD (GH peak b 3.3 μg/L). The results of the molecular study obtained with the MLPA technique demonstrated in seven patients a complete absence of peaks corresponding to intron 1, exon 3 and exon 4 of GH1 probes, indicating homozygous deletions of this gene (Fig. 1C). A modest peak related to GH1 exon 5-probe was observed in all these patients, suggesting a nonspecific hybridization. All GH1 deletions were confirmed by SmaI restriction endonuclease that showed in 6 patients a 6.7 kb deletion and in one patient a restriction pattern compatible with a 7.6 kb deletion, all in homozygous state (Fig. 1). Consanguinity between parents was reported in 3 of 7 patients homozygous for GH1 deletions. Families with the 6.7 kB deletion are unrelated to one another. Five of these patients stopped responding to treatment with rhGH due to the development of neutralizing antibodies, whereas two patients had a successful long term growth response to rhGH therapy. The MLPA analysis in all obligatory heterozygous parents of patients with GH1 deletions, revealed a 50% reduction of peak height of intron 1, exon 3 and exon 4 of GH1 probes in comparison with controls, which is compatible with hemizygous genotype in this locus. This MLPA profile was also observed in one
Table 1 Phenotype of patients selected for GH1 study regarding their GH secretory status. Maximum GH peak
Male:female Consanguinity (%) Target height SDS Family history of short stature — n (%) Data on first evaluation Chronological age (y) Height SDS BMI SDS Prepubertal:pubertal ratio GH peak at stimulation test (μg/L) IGF-1 SDS IGFBP-3 SDS
b3.3 μg/L
3.3–10 μg/L
N10 μg/L
17:21a 12 (32%)b −1.1 ± 1.0 9 (24%)
52:24 8 (11%) −1.3 ± 0.8 25 (33%)
16:5 1 (0.5%) −1.2 ± 0.7 7 (33%)
9.5 ± 5.2 −4.5 ± 1.8c −0.4 ± 1.4 31 : 7 1.0 ± 0.9 −2.4 ± 1.5c −2.8 ± 2.1c
9.8 ± 4.2 −3.0 ± 0.7 −1.1 ± 1.6 46 : 30 6.5 ± 2.2 −1.6 ± 1.3 −0.8 ± 1.0
10.9 ± 3.1 −2.9 ± 0.6 −1.0 ± 1.3 16 : 5 22.4 ± 8.7 −1.1 ± 1.0 −0.6 ± 1.0
a Children with GH peak b3.3 μg/L (GHD) vs. children with GH peak ≥3.3 μg/L; p b 0.019. b Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.005. c Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.001.
Male:female Target height SDS Baseline Chronological age (y) Bone age delay (y) Height SDS Prepubertal:pubertal stage 1st yr of rhGH treatment Dose (μg/kg/d) Growth velocity (cm/y) Changes in height SDS
b3.3 μg/La
3.3–10 μg/L
N10 μg/L
16:9 −1.2 ± 0.9
17:12 −1.3 ± 0.8
7:1 −1.4 ± 0.5
9.5 ± 4.7 3.5 ± 2.1b −4.2 ± 1.4c 13:6
9.5 ± 3.1 2.4 ± 1.5 −3.2 ± 0.7 21:8
11.8 ± 1.9 2.5 ± 1.5 −3.4 ± 0.4 6:1
35 ± 9d 10.8 ± 4.9b 1.1 ± 1.0e
49 ± 6 8.8 ± 2.3 0.6 ± 0.4
43 ± 9 7.1 ± 1.1 0.4 ± 0.2
a In the treated group, we excluded patients who were treated with human-derived pituitary GH or developed growth-attenuating anti-GH antibodies. b Children with GH peak b3.3 μg/L (GHD) vs. children with GH peak ≥3.3 μg/L; p = 0.018. c Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.002. d Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.001. e Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p = 0.009.
additional patient and SmaI restriction endonuclease confirming a 6.7 kb GH1 heterozygous deletion. MLPA was unable to distinguish patients with GH1 deletion of 6.7 kb or 7.6 kb; however MLPA was able to identify heterozygous 7.6 kb deletion, which produces a SmaI digestion pattern similar to that of control samples (Fig. 1A and B). Additionally, we also found two heterozygous intronic mutations in GH1 in patients with IGHD: In the patient who is heterozygous for the 6.7 kB GH1 deletion, we identified a heterozygous nucleotide change at the fifth nucleotide of intron 2 (c.171+5GNC). This allelic variation was not found in either our controls (140 alleles) or in a public database (1000 Genomes, http://browser.1000genomes.org). Segregation analysis showed that the c.171+5GN C mutation was inherited from the mother, whereas the 6.7 kb GH1 deletion was inherited from the father, both parents with normal height (Fig. 2). In silico analysis predicted that the mutant c.171 +5GNC allele would result in the abolition of the splice donor site. In another patient we identified only a heterozygous nucleotide substitution in the first nucleotide of intron 3 (c.291+ 1GN T). This allelic variation was previously associated with IGHD type II [27]. Segregation analysis was not possible in this family but the variant was absent in 140 alleles from our control population. In silico analysis predicted that the mutant c.291 + 1G N T disrupts the 5′ splice site consensus sequence and causes complete exon 3 skipping and preferential expression of the 17.5 kD GH isoform. In total, we identified pathogenic GH1 defects in 9 of 38 patients with IGHD (23%), eight with the autosomal recessive and one with the autosomal dominant inheritance form of IGHD. All our patients with Type IA IGHD had typical features observed in patients with severe congenital GHD. Anterior pituitary hypoplasia was observed in 71% of these patients. Additionally, patients with Type IA IGHD were diagnosed at younger age (3.1 ± 2.0 vs. 9.5 ± 4.2 years, p = 0.001), with a more severe short stature (height SDS of −5.7 ± 1.4 vs. −4.0 ± 1.1, p = 0.003) and had lower GH peak (0.3 ± 0.5 vs. 1.2 ± 0.8 μg/L, p = 0.018) than other IGHD patients. 4. Discussion Defects in GH1 are responsible for autosomal recessive (Type I) and autosomal dominant (Type II) IGHD, as well as occasionally being responsible for the generation of a biologically inactive GH molecule. In previous studies that investigated GH1 in large cohorts of patients with IGHD, mutations responsible for autosomal dominant IGHD were the most frequently observed in Caucasians [13,14]. In contrast,
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publications (~ 6.1% [13,14,30]), which included patients classified as IGHD using higher GH peaks at stimulation tests. The only patient in our cohort diagnosed with IGHD type II had severe GH deficiency with very low levels of IGF-1 and GH peak of 0.2 μg/L. Two other patients previously described with the same GH1 mutation (c.291+ 1G NT) had a similar phenotype [27]. Different from IGHD Type I patients, where the peak GH is typically b1 μg/L, patients with IGHD type II can have variable GH responses at stimulation tests, occasionally reaching a GH peak N 3.3 μg/L [13,27]. However, the absence of autosomal dominant mutation in GH1 in patients with GH peak between 3.3 and 10 μg/L in the present study suggests that IGHD type II is infrequent in our population. This finding stands in opposition to previous studies, which reported a preponderance of Type II IGHD in Caucasian patients. The Brazilian population consists of a broad ethnic heterogeneity, mainly formed by European, African and native Amerindian descendants [31]. This characteristic of Brazilian population may explain our results. Interestingly, in our cohort, patients with GH peak between 3.3 and 10 μg/L and N10 μg/L had similar basal characteristics and growth response to rhGH therapy, whereas patients classified as severe IGHD (GH peak b 3.3 μg/L) had a more severe short stature and a better
previous studies in Brazilian patients with IGHD identified only complete GH1 deletions or mutations in GHRHR, both inherited in autosomal recessive fashion [16,17]. This divergence may represent a real difference in frequency among different populations or alternatively can be caused by different criteria used to define the patient as IGHD. The cutoff value of peak GH (b3.3 μg/L) used to diagnose IGHD in our group is one of the most stringent adopted [18]. In the present study, we studied the GH1 in a group of 135 patients with different GH secretion status, including a group of patients with severe IGHD (GH peak b 3.3 μg/L) with normal pituitary-hypothalamic imaging and a group of patients with short stature with GH peak between 3.3 and 10 μg/L. Mutations in GH1 were identified only in patients with GH peak b3.3 μg/L, being eight Type I and one Type II IGHD. Complementary studies also identified an additional five patients from this cohort with homozygous (n = 3) or compound heterozygous (n = 2) mutations in GHRHR [28,29]. This elevated frequency of IGHD type I (13:38, 34%) can be explained by a high frequency of inbreeding in our cohort (32%), the prevalence of certain mutations in our population [28] and by a referral bias, as more severe cases of IGHD are more likely to be referred. Conversely, the frequency of IGHD type II (1:38, 2.6%) in our cohort was lower than the frequency observed in other
A GH1
1900 bp
1919 bp
7.6 kb 6.7 kb PCR Products 711
1474 bp
19 18 bp
7.6 kb deletion
1900 bp
446 bp
Normal Control
Parent heterozygous for 7.6kb deletion
Parent heterozygous for 7.6kb deletion
Patient homozygous for 7.6kb deletion
Parent heterozygous for 6.7kb deletion Patient heterozygous for 6.7kb deletion
Patient homozygous for 6.7kb deletion
Restriction site for SmaI
Patient homozygous for 6.7kb deletion
Parent heterozygous for 6.7kb deletion
Patient homozygous for 6.7kb deletion
PCR product without SmaI
1kb DNA leader
B
762
1919 bp
448 bp
6.7 kb deletion
Primers
negative
e
1900 bp
Normal
1900/1919 bp 1474 bp 762 bp 711 bp 446/448 bp
Fig. 1. (A): Schematic representation of the restriction enzyme digestion of PCR products by SmaI in the presence of the normal allele and alleles containing a deletion of 6.7 kb or 7.6 kb GH1. (B): Agarose gel electrophoresis of PCR products digested with SmaI restriction enzymes. Lane 1: negative control; Lane 2: 1-kb DNA marker; Lanes 3–12: samples with homozygous or heterozygous deletions of GH1; and Lane 13: control sample. (C): Electropherogram of MLPA runs obtained for a normal control individual and for patient heterozygous or homozygous for GH1 deletion. Each peak corresponds to amplification of one probe.
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C Probe 1 Probe 2 Probe 3 Probe 4 (intron1) (exon 3) (exon 4) (exon 5)
Control
Heterozygous
Homozygous
Fig. 1 (continued).
growth response to GH therapy, despite using lower rhGH doses than the other patients with GH peak ≥ 3.3 μg/L. Previous studies had shown that the GH peak response to stimulation tests is inversely related to the response to rhGH treatment, being more relevant in patients with GH peak b 5.0 μg/L [32]. Of note, only one previous case report evaluated the GH1 deletion by MLPA technique [33]. The MLPA correctly identified the homozygous and heterozygous deletions in the index case and his parents, respectively, but no information on the type of GH1 deletion was provided. In the current study we evaluated patients homozygous for 6.7 kb and 7.6 kb GH1 deletion as well as their carrier parents. Our results validated the use of MLPA to identify homozygous and heterozygous GH1 deletions. However, it is noteworthy that the interpretation should be based on the results of probes for intron 1, exon 3 and exon 4 since the probe for exon 5 may result in nonspecific
hybridization. The PCR amplification of GH1-exons in patients homozygous for GH1 gene deletion usually resulted in the absence of PCR products. However, we occasionally observed a faint band, which corresponded to non-specific amplification of homologous regions of CSH2 (data not shown). For this reason, we recommend a specific test for the GH1 gene deletion. Additionally, in the present study we describe a novel GH1 mutation located at the 5′-splice donor site of intron 2 (c.171 + 5G NC). This mutation was identified in compound heterozygous state with a 6.7 kb GH1 deletion, which is compatible with an autosomal recessive model of inheritance. The parents heterozygous for either mutation had a height within normal limits. In summary, the autosomal recessive form of IGHD was more common in this cohort of Brazilian patients than the autosomal dominant form, indicating that IGHD type II is infrequent in Brazil.
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A
B
G/C
G/del
Height SDS -0.3
Height SDS -1.5
G/C
G/del
C/del
Height SDS -0.1
Height SDS -0.7
Height SDS - 5.1
Exon2
185
Intron 2
Father
Patient
Mother
Fig. 2. (A): Pedigree of the patient with compound heterozygous GH1 mutation. Patient with isolated growth hormone deficiency is indicated by solid symbols. The height SDS and corresponding GH1 genotype for each family member are indicated: G or C at the fifth nucleotide of intron 2 (c.171+5GNC) and “del” for the presence of allele containing a 6.7 kb GH1 deletion. (B): Representative genomic DNA sequence electropherogram of GH1 exon 2–intron 2 in the patient and his parents.
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Autosomal recessive form of isolated growth hormone deficiency is more frequent than the autosomal dominant form in a Brazilian cohort☆ Andria C.V. Lido a,b,1, Marcela M. França a,1, Fernanda A. Correa a, Aline P. Otto a, Luciani R. Carvalho a, Elisangela P.S. Quedas b, Mirian Y. Nishi a, Berenice B. Mendonca a, Ivo J.P. Arnhold a, Alexander A.L. Jorge b,⁎ a Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular LIM/42 do Hospital das Clinicas, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Sao Paulo, 05403-900 Sao Paulo, Brazil b Unidade de Endocrinologia Genetica, Laboratorio de Endocrinologia Celular e Molecular LIM/25, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, 01246-903 Sao Paulo, Brazil
a r t i c l e
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Article history: Received 9 May 2014 Received in revised form 21 July 2014 Accepted 22 July 2014 Available online 30 July 2014 Keywords: Short stature Isolated GH deficiency (IGHD) Mutation in Growth hormone 1 gene (GH1) Growth hormone (GH) secretion status Brazilian children
a b s t r a c t Background: In most studies, the autosomal dominant (type II) form of isolated growth hormone deficiency (IGHD) has been more frequent than the autosomal recessive (type I) form. Our aim was to assess defects in the GH1 in short Brazilian children with different GH secretion status. Subjects and methods: We selected 135 children with postnatal short stature and classified according to the highest GH peak at stimulation tests in: severe IGHD (peak GH ≤ 3.3 μg/L, n = 38, all with normal pituitary magnetic resonance imaging); GH peak between 3.3 and 10 μg/L (n = 76); and GH peak N10 μg/L (n = 21). The entire coding region of GH1 was sequenced and complete GH1 deletions were assessed by Multiplex Ligation Dependent Probe Amplification and restriction enzyme digestion. Results: Patients with severe IGHD had a higher frequency of consanguinity, were shorter, had lower levels of IGF-1 and IGFBP-3, and despite treatment with lower GH doses had a greater growth response than patients with GH peak ≥3.3 μg/L. Mutations were found only in patients with severe IGHD (GH peak b 3.3 μg/L). Eight patients had autosomal recessive IGHD: Seven patients were homozygous for GH1 deletions and one patient was compound heterozygous for a GH1 deletion and the novel c.171+5GNC point mutation in intron 2, predicted to abolish the donor splice site. Only one patient, who was heterozygous for the c.291+1GN T mutation located at the universal donor splice site of intron 3 and predicts exon 3 skipping, had an autosomal dominant form. Conclusion: Analysis of GH1 in a cohort of Brazilian patients revealed that the autosomal recessive form of IGHD was more common than the dominant one, and both were found only in severe IGHD. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Circulating growth hormone (GH) is secreted by the anterior pituitary gland and is one of the most important hormonal regulators of post-natal linear growth. It is encoded by the Growth Hormone 1 gene (GH1; OMIM *139250). GH1 is located on chromosome 17q22–q24 and comprises five exons within a 66 kb cluster region, where four other highly homologous genes are positioned: CSH1, CSH2, CSHP1 and GH2. Several GH1 defects
☆ Acknowledgments and grants: This work was supported by Grants 2013/03236-5 (to A.A.L.J.), 05/04726-0 and 07/56490-5 (to M.M.F.) from the Sao Paulo Research Foundation (FAPESP) and Grants 307922/2013-8 (to I.J.P.A.), 301339/2008-9 (to B.B.M.), and 304678/ 2012-0 (to A.A.L.J.) from the National Council for Scientific and Technological Development (CNPq). ⁎ Corresponding author at: Faculdade de Medicina da USP (LIM-25), Av. Dr. Arnaldo, 455 5º Andar Sala 5340, CEP 01246-903 Sao Paulo, SP, Brazil. Tel./fax: +55 11 3061 7252. E-mail addresses: [email protected], [email protected] (A.A.L. Jorge). 1 ACVL and MMF contributed equally to this article.
http://dx.doi.org/10.1016/j.ghir.2014.07.001 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
have been described in patients with short stature with different growth hormone secretory status and variable height deficit [1]. Complete deletions of GH1 are responsible for a severe autosomal recessive form of isolated growth hormone deficiency (IGHD type IA, OMIM 262400). Affected patients have a complete lack of GH secretion, classical GHD phenotype and during human GH (hGH) therapy develop growth-attenuating anti-GH antibodies, which often interfere with the action of exogenous GH [2]. The deletions described are heterogeneous and are caused by homologous recombination within the GH-cluster regions. The most frequently found GH1 deletion extends over 6.7 kb, followed by 7.6 kb, 7.0 kb, 4.5 kb and other infrequent deletions [2]. Other molecular defects, such as frameshift mutations and homozygous nonsense mutations can also cause IGHD type IA [3]. This form of IGHD is rare, being more frequent in families with a history of consanguinity or in specific geographic groups [2,3]. Usually missense, nonsense, frameshift and splice site mutations in GH1 are responsible for the recessive (type IB, OMIM 612781), whereas specific missense, splice site or splice enhancer mutations are responsible for the dominant (type II, OMIM 173100) forms of IGHD [3]. IGHD type II
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is caused mainly by point mutations located in the first six base pairs of the 5′ region of intron 3 of GH1 [4]. These mutations result in exon 3 skipping in the mutated allele, leading to the expression of the 17.5 kDa GH isoform, which exerts a dominant negative effect on the bioactive GH isoforms [4]. Patients with IGHD type II usually present great variability in the severity of GH deficiency and in phenotypic expression [5–8], even among members of the same family [5]. GH peak in general is low but detectable, can vary on different occasions and can be above 5 μg/L [5–8]. This variability in phenotype and secretory response of GH may increase the difficulty in establishing the diagnosis of IGHD. Furthermore, specific missense mutations in GH1 can result in a biologically inactive mutant protein [9–11], which affect GH action and cause insensitivity to endogenous GH (Kowarski syndrome, OMIM 262650), mainly characterized by growth retardation, normal or slightly increased GH secretion, low IGF-I levels and catch-up growth on rhGHreplacement therapy [12]. The frequency and the type of GH1 alteration identified in different studies depend on the ethnic background and the population selection criteria (frequency of consanguinity, severity of short stature and growth hormone secretory status) [13–15]. Generally, the autosomal dominant form is the most frequent genetic form of IGHD reported in the literature [1,3]. However, previous molecular-genetic studies in our Brazilian cohort of patients with IGHD identified only patients homozygous for GHRHR mutations or GH1 deletions [16,17]. In Brazil, the diagnosis of GHD is usually established using lower cutoff values of GH peak in stimulation tests than in several other countries (3.3 or 5 μg/L vs. 7 or 10 μg/L). It is possible that the high frequency of IGHD Type I and absence of Type II observed in our patients could be related to the adopted criteria to define a patient as IGHD or to an intrinsic characteristic of our population. Thus, in the present study, we investigated the frequency of defects in GH1 in Brazilian children with different GH secretion status and evaluated the first year growth response to recombinant GH (rhGH) therapy according to this status. 2. Patients and methods 2.1. Subjects This study was approved by the local Ethics Committee and the patients and/or guardians gave their written informed consent before initiating the genetic studies. The studied cohort consisted of selected patients referred for clinical and/or genetic evaluation at the Developmental Endocrinology Unit of the Hospital das Clinicas of the University of Sao Paulo School of Medicine, Brazil. One hundred thirty-five short children met the following criteria: 1) normal birth weight and length for gestational age; 2) height SDS ≤− 2 [19] and/or height velocity SDS ≤− 1, observed over a period of six months; 3) normal body proportions [20]; 4) had at least one GH stimulation test available; 5) absence of a known cause of short stature, except IGHD and 6) absence of other pituitary hormone deficiencies [16]. Patients were classified according to the highest GH peak obtained in stimulation tests. Patients were diagnosed as GHD based on clinical and auxological parameters and failure to respond to two GH stimulation tests (GH peak ≤ 3.3 μg/L) [18]. Additionally, patients with the typical clinical phenotype of severe GHD (such as severe short stature, hypoglycemia, protruding forehead, saddle nose, central obesity and high-pitched voice) and failure to respond to one GH stimulation test were also diagnosed as GHD. Magnetic resonance imaging (MRI) of the pituitary region was performed in all patients with a GH peak ≤3.3 μg/L and only patients with eutopic posterior lobe and an intact stalk were selected for this study [16]. 2.2. Clinical evaluation Evaluations were performed at the same period of the day and included measurements of height (measured with a stadiometer) and weight (measured with a digital scale). Body mass index (BMI) was
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calculated as weight (Kg) divided by height (m) squared. Height and body mass index (BMI) were expressed as SDS [19]. Target height was calculated [(father's height + mother's height + 13 cm for boys or -13 cm for girls) / 2] and expressed as SDS. Left hand and wrist X-rays for bone age determination were assessed by the method of Greulich and Pyle [21]. Sixty-two patients were treated with rhGH administered subcutaneously (sc) at a dose range of 30–50 μg/kg/day, which was adjusted according to changes in weight at each visit. All children were evaluated at baseline and every 3–4 months during rhGH treatment. First year growth velocity was determined after an observation period of at least 11 months. 2.2.1. Hormone assays GH was measured by immunofluorometric assay (IFMA) (AutoDELFIA, Wallac, Turku, Finland) method with monoclonal antibodies. The GH secretion status was evaluated by provocative tests, usually with Clonidine and/or insulin-induced hypoglycemia, following standard protocols. IGF-I was measured by RIA after ethanol extraction (Diagnostic Systems Laboratories, Webster, TX) or by chemiluminescence assays (CLIA) (Immulite; Diagnostic Products Corp., Los Angeles, CA). IGFBP-3 was measured by IRMA (Diagnostic Systems Laboratories) or CLIA (Immulite). IGF-I and IGFBP-3 levels were expressed as SDS for age and sex according to reference values provided by the respective assay kits. The absence of deficiencies of the other pituitary hormones was established by the normal basal values of total T4 and/or free T4, cortisol, prolactin, TSH, gonadotropins, and testosterone (postpubertal boys) or estradiol (postpubertal girls) as well as the TSH, prolactin, and cortisol responses to combined insulin-TRH infusion [16,22]. 2.2.2. Molecular studies Genomic DNA was extracted from peripheral-blood leucocytes of all patients using standard techniques. The entire coding region and all introns of the GH1 (GenBank accession number NC_000017.10 for genomic and NM_000515.3 for mRNA sequences) were amplified by polymerase chain reaction (primer sequences and amplification protocols are available upon request). Amplification products were bidirectionally sequenced with the dideoxy chain-termination method using a dye terminator kit and analyzed in an ABI Prism 3130 automated sequencer (Applied Biosystems, Foster City, CA). Large GH1 deletions were assessed by multiplex ligation-dependent probe amplification (MLPA) and by restriction endonuclease analysis using SmaI as previously described [23,24]. MLPA analysis of GH1 was carried out using the commercial Salsa Kit P216 Growth Hormone Deficiency (MCR Holland, Amsterdam, The Netherlands). This kit contains 4 GH1 probes locate at intron 1, exon 3, exon 4 and exon 5. Data generated by MLPA were intra-normalized against every single probe and assay. Patients' and parents' sample normalized peak area was then divided by the average normalized peak area from control samples. Dosage quotient areas outside the range 0.70–1.3 were considered abnormal. In addition, visual comparison of peak profiles was performed. 2.2.3. In silico prediction of mutation effects To identify the potential effects of sequence variants identified in GH1 on splicing and on protein function or structure, the wild type and variant sequences were submitted to NetGene2 Server (http://www.cbs.dtu.dk/services/NetGene 2) [25] and Mutation Taster (http://www.mutationtaster.org) [26]. 2.2.4. Statistical analysis Results were expressed as mean ± SD. Differences between groups were tested by t-test or Kruskal–Wallis and chi-square or Fisher exact test, as appropriate. A p value b 0.05 was considered statistically significant. All statistical calculations were carried out using SigmaStat version 3.5 (Systat Software Inc., Chicago, IL).
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3. Results
Table 2 Phenotype of patients treated with rhGH regarding their GH secretory status.
3.1. Patients' characteristics
Maximum GH peak
The one hundred thirty-five children were divided into three groups according to values of GH peak response in stimulation tests: GH peak b3.3 μg/L (or severe IGHD group, n = 38), between 3.3 and 10 μg/L (n = 76), and N10 μg/L (n = 21). The clinical and laboratory features of all patients selected for GH1 study regarding the growth hormone secretory status are summarized in Table 1. The patients with IGHD showed slightly higher prevalence of females, whereas in the other GH secretion status groups there was a clear male preponderance. This IGHD group also had higher frequency of consanguinity and presence of anterior pituitary hypoplasia at MRI (48.5% vs 12.5%), was shorter and had lower levels of IGF-1 and IGFBP-3 than patients with GH peak ≥3.3 μg/L. The clinical and laboratory features of sixty-two patients who were treated with rhGH are shown in Table 2. Patients with IGHD were treated with lower rhGH dose, had higher growth velocity and greater changes in height SDS during the first year of rhGH therapy than patients with GH peak ≥3.3 μg/L. Patients with GH peak between 3.3 and 10 μg/L or N10 μg/L had similar growth response to rhGH therapy. 3.2. Molecular results We identified mutations in GH1 only in patients classified as severe IGHD (GH peak b 3.3 μg/L). The results of the molecular study obtained with the MLPA technique demonstrated in seven patients a complete absence of peaks corresponding to intron 1, exon 3 and exon 4 of GH1 probes, indicating homozygous deletions of this gene (Fig. 1C). A modest peak related to GH1 exon 5-probe was observed in all these patients, suggesting a nonspecific hybridization. All GH1 deletions were confirmed by SmaI restriction endonuclease that showed in 6 patients a 6.7 kb deletion and in one patient a restriction pattern compatible with a 7.6 kb deletion, all in homozygous state (Fig. 1). Consanguinity between parents was reported in 3 of 7 patients homozygous for GH1 deletions. Families with the 6.7 kB deletion are unrelated to one another. Five of these patients stopped responding to treatment with rhGH due to the development of neutralizing antibodies, whereas two patients had a successful long term growth response to rhGH therapy. The MLPA analysis in all obligatory heterozygous parents of patients with GH1 deletions, revealed a 50% reduction of peak height of intron 1, exon 3 and exon 4 of GH1 probes in comparison with controls, which is compatible with hemizygous genotype in this locus. This MLPA profile was also observed in one
Table 1 Phenotype of patients selected for GH1 study regarding their GH secretory status. Maximum GH peak
Male:female Consanguinity (%) Target height SDS Family history of short stature — n (%) Data on first evaluation Chronological age (y) Height SDS BMI SDS Prepubertal:pubertal ratio GH peak at stimulation test (μg/L) IGF-1 SDS IGFBP-3 SDS
b3.3 μg/L
3.3–10 μg/L
N10 μg/L
17:21a 12 (32%)b −1.1 ± 1.0 9 (24%)
52:24 8 (11%) −1.3 ± 0.8 25 (33%)
16:5 1 (0.5%) −1.2 ± 0.7 7 (33%)
9.5 ± 5.2 −4.5 ± 1.8c −0.4 ± 1.4 31 : 7 1.0 ± 0.9 −2.4 ± 1.5c −2.8 ± 2.1c
9.8 ± 4.2 −3.0 ± 0.7 −1.1 ± 1.6 46 : 30 6.5 ± 2.2 −1.6 ± 1.3 −0.8 ± 1.0
10.9 ± 3.1 −2.9 ± 0.6 −1.0 ± 1.3 16 : 5 22.4 ± 8.7 −1.1 ± 1.0 −0.6 ± 1.0
a Children with GH peak b3.3 μg/L (GHD) vs. children with GH peak ≥3.3 μg/L; p b 0.019. b Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.005. c Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.001.
Male:female Target height SDS Baseline Chronological age (y) Bone age delay (y) Height SDS Prepubertal:pubertal stage 1st yr of rhGH treatment Dose (μg/kg/d) Growth velocity (cm/y) Changes in height SDS
b3.3 μg/La
3.3–10 μg/L
N10 μg/L
16:9 −1.2 ± 0.9
17:12 −1.3 ± 0.8
7:1 −1.4 ± 0.5
9.5 ± 4.7 3.5 ± 2.1b −4.2 ± 1.4c 13:6
9.5 ± 3.1 2.4 ± 1.5 −3.2 ± 0.7 21:8
11.8 ± 1.9 2.5 ± 1.5 −3.4 ± 0.4 6:1
35 ± 9d 10.8 ± 4.9b 1.1 ± 1.0e
49 ± 6 8.8 ± 2.3 0.6 ± 0.4
43 ± 9 7.1 ± 1.1 0.4 ± 0.2
a In the treated group, we excluded patients who were treated with human-derived pituitary GH or developed growth-attenuating anti-GH antibodies. b Children with GH peak b3.3 μg/L (GHD) vs. children with GH peak ≥3.3 μg/L; p = 0.018. c Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.002. d Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p b 0.001. e Children with GH peak b3.3 μg/L vs. children with GH peak ≥3.3 μg/L; p = 0.009.
additional patient and SmaI restriction endonuclease confirming a 6.7 kb GH1 heterozygous deletion. MLPA was unable to distinguish patients with GH1 deletion of 6.7 kb or 7.6 kb; however MLPA was able to identify heterozygous 7.6 kb deletion, which produces a SmaI digestion pattern similar to that of control samples (Fig. 1A and B). Additionally, we also found two heterozygous intronic mutations in GH1 in patients with IGHD: In the patient who is heterozygous for the 6.7 kB GH1 deletion, we identified a heterozygous nucleotide change at the fifth nucleotide of intron 2 (c.171+5GNC). This allelic variation was not found in either our controls (140 alleles) or in a public database (1000 Genomes, http://browser.1000genomes.org). Segregation analysis showed that the c.171+5GN C mutation was inherited from the mother, whereas the 6.7 kb GH1 deletion was inherited from the father, both parents with normal height (Fig. 2). In silico analysis predicted that the mutant c.171 +5GNC allele would result in the abolition of the splice donor site. In another patient we identified only a heterozygous nucleotide substitution in the first nucleotide of intron 3 (c.291+ 1GN T). This allelic variation was previously associated with IGHD type II [27]. Segregation analysis was not possible in this family but the variant was absent in 140 alleles from our control population. In silico analysis predicted that the mutant c.291 + 1G N T disrupts the 5′ splice site consensus sequence and causes complete exon 3 skipping and preferential expression of the 17.5 kD GH isoform. In total, we identified pathogenic GH1 defects in 9 of 38 patients with IGHD (23%), eight with the autosomal recessive and one with the autosomal dominant inheritance form of IGHD. All our patients with Type IA IGHD had typical features observed in patients with severe congenital GHD. Anterior pituitary hypoplasia was observed in 71% of these patients. Additionally, patients with Type IA IGHD were diagnosed at younger age (3.1 ± 2.0 vs. 9.5 ± 4.2 years, p = 0.001), with a more severe short stature (height SDS of −5.7 ± 1.4 vs. −4.0 ± 1.1, p = 0.003) and had lower GH peak (0.3 ± 0.5 vs. 1.2 ± 0.8 μg/L, p = 0.018) than other IGHD patients. 4. Discussion Defects in GH1 are responsible for autosomal recessive (Type I) and autosomal dominant (Type II) IGHD, as well as occasionally being responsible for the generation of a biologically inactive GH molecule. In previous studies that investigated GH1 in large cohorts of patients with IGHD, mutations responsible for autosomal dominant IGHD were the most frequently observed in Caucasians [13,14]. In contrast,
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publications (~ 6.1% [13,14,30]), which included patients classified as IGHD using higher GH peaks at stimulation tests. The only patient in our cohort diagnosed with IGHD type II had severe GH deficiency with very low levels of IGF-1 and GH peak of 0.2 μg/L. Two other patients previously described with the same GH1 mutation (c.291+ 1G NT) had a similar phenotype [27]. Different from IGHD Type I patients, where the peak GH is typically b1 μg/L, patients with IGHD type II can have variable GH responses at stimulation tests, occasionally reaching a GH peak N 3.3 μg/L [13,27]. However, the absence of autosomal dominant mutation in GH1 in patients with GH peak between 3.3 and 10 μg/L in the present study suggests that IGHD type II is infrequent in our population. This finding stands in opposition to previous studies, which reported a preponderance of Type II IGHD in Caucasian patients. The Brazilian population consists of a broad ethnic heterogeneity, mainly formed by European, African and native Amerindian descendants [31]. This characteristic of Brazilian population may explain our results. Interestingly, in our cohort, patients with GH peak between 3.3 and 10 μg/L and N10 μg/L had similar basal characteristics and growth response to rhGH therapy, whereas patients classified as severe IGHD (GH peak b 3.3 μg/L) had a more severe short stature and a better
previous studies in Brazilian patients with IGHD identified only complete GH1 deletions or mutations in GHRHR, both inherited in autosomal recessive fashion [16,17]. This divergence may represent a real difference in frequency among different populations or alternatively can be caused by different criteria used to define the patient as IGHD. The cutoff value of peak GH (b3.3 μg/L) used to diagnose IGHD in our group is one of the most stringent adopted [18]. In the present study, we studied the GH1 in a group of 135 patients with different GH secretion status, including a group of patients with severe IGHD (GH peak b 3.3 μg/L) with normal pituitary-hypothalamic imaging and a group of patients with short stature with GH peak between 3.3 and 10 μg/L. Mutations in GH1 were identified only in patients with GH peak b3.3 μg/L, being eight Type I and one Type II IGHD. Complementary studies also identified an additional five patients from this cohort with homozygous (n = 3) or compound heterozygous (n = 2) mutations in GHRHR [28,29]. This elevated frequency of IGHD type I (13:38, 34%) can be explained by a high frequency of inbreeding in our cohort (32%), the prevalence of certain mutations in our population [28] and by a referral bias, as more severe cases of IGHD are more likely to be referred. Conversely, the frequency of IGHD type II (1:38, 2.6%) in our cohort was lower than the frequency observed in other
A GH1
1900 bp
1919 bp
7.6 kb 6.7 kb PCR Products 711
1474 bp
19 18 bp
7.6 kb deletion
1900 bp
446 bp
Normal Control
Parent heterozygous for 7.6kb deletion
Parent heterozygous for 7.6kb deletion
Patient homozygous for 7.6kb deletion
Parent heterozygous for 6.7kb deletion Patient heterozygous for 6.7kb deletion
Patient homozygous for 6.7kb deletion
Restriction site for SmaI
Patient homozygous for 6.7kb deletion
Parent heterozygous for 6.7kb deletion
Patient homozygous for 6.7kb deletion
PCR product without SmaI
1kb DNA leader
B
762
1919 bp
448 bp
6.7 kb deletion
Primers
negative
e
1900 bp
Normal
1900/1919 bp 1474 bp 762 bp 711 bp 446/448 bp
Fig. 1. (A): Schematic representation of the restriction enzyme digestion of PCR products by SmaI in the presence of the normal allele and alleles containing a deletion of 6.7 kb or 7.6 kb GH1. (B): Agarose gel electrophoresis of PCR products digested with SmaI restriction enzymes. Lane 1: negative control; Lane 2: 1-kb DNA marker; Lanes 3–12: samples with homozygous or heterozygous deletions of GH1; and Lane 13: control sample. (C): Electropherogram of MLPA runs obtained for a normal control individual and for patient heterozygous or homozygous for GH1 deletion. Each peak corresponds to amplification of one probe.
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C Probe 1 Probe 2 Probe 3 Probe 4 (intron1) (exon 3) (exon 4) (exon 5)
Control
Heterozygous
Homozygous
Fig. 1 (continued).
growth response to GH therapy, despite using lower rhGH doses than the other patients with GH peak ≥ 3.3 μg/L. Previous studies had shown that the GH peak response to stimulation tests is inversely related to the response to rhGH treatment, being more relevant in patients with GH peak b 5.0 μg/L [32]. Of note, only one previous case report evaluated the GH1 deletion by MLPA technique [33]. The MLPA correctly identified the homozygous and heterozygous deletions in the index case and his parents, respectively, but no information on the type of GH1 deletion was provided. In the current study we evaluated patients homozygous for 6.7 kb and 7.6 kb GH1 deletion as well as their carrier parents. Our results validated the use of MLPA to identify homozygous and heterozygous GH1 deletions. However, it is noteworthy that the interpretation should be based on the results of probes for intron 1, exon 3 and exon 4 since the probe for exon 5 may result in nonspecific
hybridization. The PCR amplification of GH1-exons in patients homozygous for GH1 gene deletion usually resulted in the absence of PCR products. However, we occasionally observed a faint band, which corresponded to non-specific amplification of homologous regions of CSH2 (data not shown). For this reason, we recommend a specific test for the GH1 gene deletion. Additionally, in the present study we describe a novel GH1 mutation located at the 5′-splice donor site of intron 2 (c.171 + 5G NC). This mutation was identified in compound heterozygous state with a 6.7 kb GH1 deletion, which is compatible with an autosomal recessive model of inheritance. The parents heterozygous for either mutation had a height within normal limits. In summary, the autosomal recessive form of IGHD was more common in this cohort of Brazilian patients than the autosomal dominant form, indicating that IGHD type II is infrequent in Brazil.
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A
B
G/C
G/del
Height SDS -0.3
Height SDS -1.5
G/C
G/del
C/del
Height SDS -0.1
Height SDS -0.7
Height SDS - 5.1
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185
Intron 2
Father
Patient
Mother
Fig. 2. (A): Pedigree of the patient with compound heterozygous GH1 mutation. Patient with isolated growth hormone deficiency is indicated by solid symbols. The height SDS and corresponding GH1 genotype for each family member are indicated: G or C at the fifth nucleotide of intron 2 (c.171+5GNC) and “del” for the presence of allele containing a 6.7 kb GH1 deletion. (B): Representative genomic DNA sequence electropherogram of GH1 exon 2–intron 2 in the patient and his parents.
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