| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
University-Children’s Hospital and Growth Research Center, 72076 Tübingen, Germany; University-Children’s Hospital, 04317 Leipzig, Germany; University-Children’s Hospital, 18055 Rostock, Germany; and Department of Pediatrics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
Address all correspondence and requests for reprints to: Gerhard Binder, M.D., Department of Pediatric Endocrinology, University-Children’s Hospital, Hoppe-Seyler-Str. 1, Tübingen 72076, Germany.
Abstract
Autosomal dominantly inherited isolated GH deficiency is caused by mutations of GH-1 that alter the normal structure of GH. We studied 16 familial cases and 1 sporadic case with isolated GH deficiency type II from 1 Dutch and 4 German families by direct sequencing of PCR-amplified genomic DNA and ectopic transcript analysis of lymphocyte mRNA. In addition, the clinical data of the affected individuals were analyzed. Two previously reported mutations and 1 novel splice site mutation in intron III of GH-1 (+1G to C and +1G to A; new, +2T to C) were detected that cause exon 3 skipping. We also discovered a novel G6191to T missense mutation in exon 4 of GH-1 that changes valine 110, which is highly conserved in mammalian and several nonmammalian GH, to phenylalanine. Splicing of the primary RNA transcript was not affected by this mutation, which is very likely to alter the normal GH structure at the protein level.
The onset of growth failure was earlier, and the degree was more severe in affected children with GH-1 splice site mutations compared with those in children with the GH-1 missense mutation. In addition, the severity of the phenotype was variable, even within the same family. The age at diagnosis was between 0.8–9.6 yr (median, 5.1 yr); height at diagnosis was between -2.5 and -8.1 SD score (median, -4.0). Most of the children were lean at diagnosis, with a body mass index ranging from -1.7 to +3.3 SD score (median, -0.5). The 5 affected adults had final heights between -1.8 and -4.5 SD score (median, -3.6), centripetal obesity, and muscular hypotrophy. Before therapy, IGF-I and IGF-binding protein-3 serum levels of all affected children were severely diminished (<<5th percentiles for age). The maximum GH peak in a total of 25 stimulation tests was between 0.1–5.0 µg/liter (median, 0.9), indicating severe GH deficiency. The height of the adenohypophysis studied by magnetic resonance imaging was normal in 2 affected children and mildly decreased in 2 others. Substitution with GH resulted in good catch-up growth in all treated children.
Children with severe GH and IGF-I deficiencies, but normal size of the adenohypophysis should be examined for GH-1 splice site and missense mutations. The observed discrepancy between the very homogeneous hormone data proving severe GH and IGF-I deficiencies and the high variability of growth failure even within the same family suggests that the onset and predominance of GH-dependent growth during infancy are individually different and modified by as yet unknown factors.
THE MAJORITY OF cases with isolated GH deficiency (IGHD) are idiopathic (1). Monogenetic recessive inheritance of IGHD was shown to be caused by complete deletions of the GH-1 (IGHD IA) (2) and, more recently, by nonsense mutations of the GHRH receptor gene (3). Until recently, dominant transmission (IGHD II) was exclusively found in the presence of GH-1 splice site mutations, which cause skipping of exon 3 (4, 5). This in-frame deletion results in the loss of 40 amino acids and a presumably misfolded del32–71GH. The prevalence of such mutations in families with IGHD II is high, up to 100% (6). The mechanism of the dominant negative effect of the mutant protein is only partly understood (7). In vitro studies suggested cell-specific mechanisms in neuroendocrine cells that included insufficient storage and secretion of the wild-type GH in the presence of the del32–71GH (8, 9). Seven different splice site mutations in intron 3 of GH-1 have been reported (4, 5, 10, 11, 12, 13, 14). In addition, two GH-1 missense mutation (P89L and R183H) were recently implicated in IGHD II (15, 16).
In this study we present molecular and clinical data on 16 affected individuals from 4 families and 1 sporadic case with IGHD II that provide new insights in IGHD.
Subjects
The pedigrees of the five unrelated families (no. 1–5) are
shown in Figs. 1
, 2, and 3.
Families 4 and 5 have been reported previously (5, 17).
All affected children were prepubertal at diagnosis and had normal free
T4, TSH, PRL, and cortisol levels. Basal serum
levels of IGF-I and IGF-binding protein-3 (IGFBP3) were determined in
all but 1 patient. Two independent GH stimulation tests were performed
in 7 patients, the insulin test only was performed in 3 patients, and
no test was performed because of young age (<1.0 yr) in 2 patients, of
whom 1 had a pathologically low GH level during spontaneous
hypoglycemia. Of the 12 affected children, 11 were treated with
recombinant human GH sc (median dose, 0.17 mg/kg·wk). Magnetic
resonance imaging with narrow scanning of the pituitary region and
gadolinium injection was performed in 4 patients and 1 affected parent
from 3 families. Blood samples for genetic analysis were taken after
obtaining informed consent from parents and patients.
|
|
|
Hormone measurements
Serum GH levels were measured in different clinical centers by several assays (RIA, ELISA, and enzyme immunoassay) that had the same cut-off level of 10 µg GH/liter for the normal response to stimulation. IGF-I and IGFBP-3 concentrations in families 1, 2, 3, and 5 were determined using the same assays by Blum et al. (18).
DNA and RNA extraction
Genomic DNA was extracted from 5 ml frozen EDTA blood using an extraction kit (Genomix Blood Scale-Up, Talent, Triest, Italy) that was based on chloroform extraction after initial blood lysis. Total RNA from peripheral lymphocytes was extracted according to the method described by Chomczynski and Sacchi (19).
Oligonucleotide primers
For the RT reaction, the primer used corresponded to nucleotides 6578–6600 (GH3.2) of the reported GH-1 sequence (20). The PCR was performed using GH3.2 and the upstream primer 5503–5525 (GH5.1). The upstream primer of the nested PCR corresponded to nucleotides 5555–5577 (GH5.2), and the downstream primer corresponded to nucleotides 6547–6568 (GH3.4). For restriction digest analysis with MvnII, NlaIII, and DdeI, the nested PCR was performed with the upstream primer GH5.7 (5816–5835) and the downstream primer GH3.7 (6121–6140). The primers used for sequencing were GS5.8 (5629–5648) and GS3.8 (6495–6515).
RT-PCR of RNA
RNA (5 µg) was reversibly transcribed in PCR buffer, and the total cDNA was amplified by nested PCR as previously described (5). The PCR product (10 µl of the reaction volume) were electrophoresed on 8% PAGE.
PCR of genomic DNA
Genomic DNA was amplified by nested PCR. The first PCR was performed with 0.2 µg gDNA, 100 pmol of each primer GH5.1 and GH3.1, 2.5 U Taq DNA polymerase (QIAGEN, Hamburg, Germany), and 0.2 mmol/liter of each deoxy-NTP in QIAGEN PCR buffer with a final volume of 50 µl. The reaction mixture was cycled 30 times (95 C for 60 sec, 65 C for 60 sec, and 72 C for 90 sec). An aliquot of this reaction (0.2 µl) was amplified in the nested PCR using the upstream primer GH5.2 and the downstream primer GH3.4 or, alternatively (for restriction analysis), the sense primer GH5.7 and the antisense primer GH3.7.
Restriction digest
The 325-bp fragment of GH-1 (5816–6140) containing the complete intron 3 was digested with 20 U MvnII, 20 U NlaIII, or 10 U DdeI (Roche Molecular Biochemicals, Mannheim, Germany) in a volume of 30 µl containing 10 µl PCR product for 3 h. The 1014-bp fragment of GH-1 (5555–6568) containing the genomic sequence from exon 2 to exon 5 was digested with 4 U MaeII (Roche Molecular Biochemicals) under the same conditions. The fragments were visualized by ethidium bromide staining after electrophoresis on an 8% polyacrylamide gel.
Direct sequencing
Both strands of the PCR products were directly sequenced with the Thermo Sequenase cycle sequencing kit containing 7-deaza-deoxy-GTP. The reaction was performed according to the manufacturer’s recommendations (Amersham Pharmacia Biotech, Freiburg, Germany). The sequencing primers were 5'-labeled with IR-800 fluorescent dye. The products were run under denaturing conditions on a Li-COR DNA automatic sequencer 4200.
Results
Direct sequencing of the PCR-amplified genomic DNA resulted
in the detection of a heterozygous point mutation of the first base of
the donor splice site of intron 3 in the affected individuals of
families 1, 4, and 5. In detail, we found a G to A transition in family
1 and the G to C transversion reported previously by us in families 4
and 5 (5, 17). A new splice site mutation with a T to C
transition of the second base of the intron 3 donor splice site was
detected in family 2. Restriction fragment analysis of a 327-bp DNA
fragment (5816–6140) containing the complete intron 3 with
NlaII (family 1) and MvnI (family 2; Fig. 1
), and
with DdeI (families 4 and 5) (5, 17) detected
the mutation in all affected, but in no unaffected, individuals of the
four families. Ectopic transcript analysis of lymphocyte mRNA was
performed in the proband of family 5 and revealed the presence of a
shortened mRNA lacking exon 3 (Fig. 4, lane 1) (5).
|
).
Because the G6191 to T mutation was inside an
exonic motif that was homologous to a sequence reported to act as an
exonic splicing enhancer in the human IgM gene (21), we
performed ectopic transcript analysis of peripheral lymphocyte RNA. The
main GH mRNA fragment found was identical to controls, excluding a
defect of primary RNA splicing (Fig. 4, lane 2). The missense mutation
results in an amino acid exchange from valine to phenylalanine at
position 110 of the mutant GH (V110F GH). The alignment of GH sequences
revealed that Val110 is highly conserved in
mammalian and several nonmammalian GH (Fig. 5).
|
. The SD scores were taken
from the report by Prader et al. (22). For
better comparison of the genotype-phenotype relation, the clinical data
from the children with GH-1 splice site mutations (families
1, 2, 4, and 5) and from the children affected by the GH-1
missense mutation (family 3) are shown separately. The age at diagnosis
and the degree of short stature were variable, even in members of the
same family. This variability could not be correlated to the IGF-I and
IGFBP-3 serum levels or to the GH peaks reached in stimulation tests
(Table 1). Overall, children with GH-1 splice site mutations
showed a tendency to an earlier onset and more severe degree of growth
failure than children with the GH-1 missense mutation (Table 1).
|
). The stimulated
GH levels were very low in all tested probands regardless of the
stimulus. A GH level above 3 µg/liter was reached in 3 of 25
stimulation tests; the highest level was 5.0 µg/liter (normal cut-off
for the used assays, 10 µg/liter; Table 1). The median bone age delay
was 1.6 yr (range, 0.5–3.6). Only 2 children (5.1 and 7.1 yr old at
diagnosis) were overweight with body mass index SD scores
of 2.2 and 3.3. Therapy with 0.17 mg/kg recombinant human GH sc weekly
has been initiated in 10 children for more than 2 yr. The median height
velocity during the first 2 yr of therapy increased to 10.0 cm/yr
(range, 7.0–12.5), which was equivalent to a median amplification of
the growth velocity by 2.1-fold (range, 1.7–3.6). Magnetic
resonance imaging of the hypophysis showed normal morphology and
normal adenohypophyseal height according to Argyropoulou et
al. (23) in 2 affected children from families 2 and 5
(-0.5 and -0.6 SD scores) and a normal
hypophysis with mildly reduced height in 2 children from families 1 and
2 (-2.5 and -2.6 SD scores) and in one adult
(family 1). The 5 affected adults with GH-1 splice site
mutations (4 females) had short stature with a height
SD score of -3.6 (-2.7 to -4.2), centripetal
obesity, and muscular hypotrophy, but normal fertility. The IGF-I serum
levels were severely reduced. The mother with V110F GH had short
normal stature (-1.8 SD score), but also
centripetal obesity and muscular hypotrophy. Her 7 pregnancies were
reportedly uneventful. Discussion
All affected individuals of the five families whose pedigrees suggested an autosomal dominant transmission of IGHD carried a deleterious point mutation of GH-1, suggesting a very high prevalence of these mutations in IGHD II. One novel splice site mutation found in one family affected the highly conserved second base of the intron 3 donor splice site (+2T to C). The predicted effect of this mutation, as shown for mutations of the first base of intron 3, is the skipping of exon 3 during splicing (4, 5). The resultant del32–71 GH protein exerts a dominant negative effect on the wild-type GH that is cell specific and not observed in nonsecretory cell types such as Epstein-Barr virus-transformed lymphocytes (7) or COS cells (8, 9). Heterodimer formation between wild-type GH and del32–71 GH that lacks one cysteine at position 53 was the first hypothetical proposal for the dominant negative effect (4). This theory has recently been questioned by the finding that the recombinant double mutant del32–71,C165A GH that lacks the unpaired cysteine at position 165 had the same effect in vitro (9). Neither del32–71 GH nor wild-type GH accumulates in neuroendocrine cell lines, indicating a decrease in intracellular stability (9) whose molecular basis is still unknown (24). In this context it is of importance that missense mutations of GH-1 also result in a mutant GH with a dominant negative effect.
Here, we report for the first time the V110F mutation of
GH-1. Its genetic basis is a C to T transition in a CpG
dinucleotide that is a general hotspot for mutations in vertebrate
genomes (25). This mutation changes a valine that is
completely conserved in mammalians and in some amphibians and birds to
phenylalanine. Valine is located next to the N-terminal beginning of
the third 
-helix and is integrated in the closely packed core of the
four--helix bundle of GH (26). The more bulky
phenylalanine at this position is very likely to introduce steric
hindrance. Two other GH-1 missense mutations that
cosegregate with IGHD II were recently reported: P89L and R183H
(15, 16). Similar to V110F, both mutations change highly
conserved amino acids. Proline 89 forms a kink in the second -helix
of GH that leucine is not able to form (24). Arginine 183
is next to the disulfide bond of cysteine 182, whose formation may be
disturbed by the more bulky histidine (24). However, the
effects of the two missense mutations in cultured neuroendocrine cells
have yet not been shown.
In the past screening for GH-1 defects was performed if severe growth failure with a height below -4.5 SD score at diagnosis was present (6). Severe short stature according to this definition was only present in one third of our affected individuals at diagnosis, indicating that growth failure in IGHD II is less severe than would be expected. The children with the splice site mutations were younger and shorter at diagnosis than their counterparts with the missense mutation. Moderate growth failure was also reported for the family with the P89L mutation (16). In addition, we observed in three of our families a pronounced intrafamilial variability between siblings in both mutation groups with height differences exceeding 3.0 SD score. This variability in growth did not correlate with the severity of GH or IGF-I deficiency, because the hormone levels were very low in all tested subjects, including those affected individuals with the highest growth velocity and only minor growth failure. There is no substantial evidence for a course of slow progression of GH deficiency in IGHD II as was described for the dominantly inherited vasopressin deficiency (24). Factors other than systemic GH and IGF-I levels must be responsible for the differences in growth failure. These unknown factors obviously modulate the start and predominance of GH-dependent growth. Some of these unknown factors may also be involved in the high variability of growth failure reported in Laron syndrome (27).
Data on the systematic examination of the pituitary anatomy in monogenetic disorders are scarce (28). Approximately 50% of the hormone-producing cells of the anterior pituitary are somatotrope cells (29). Recent magnetic resonance imaging observations in children with idiopathic GHD suggested a positive relationship between the volume of the adenohypophysis and the secretory GH capacity (30). This is not the case in IGHD II; the four children examined by magnetic resonance imaging showed a normal adenohypophysis in two cases and mild hypoplasia in the two others. Normal size of the anterior hypophysis was also reported in two children affected with IGHD IA, suggesting that the presence of GH is not a prerequisite for normal size of the adenohypophysis (31).
The combination of normal or almost normal height of the adenohypophysis and the presence of severe GH and IGF-I deficiencies is highly suggestive of the presence of a causative GH-1 mutation. In such cases the molecular diagnosis establishes the basis for genetic counseling and the recommendation of a life-long substitution with GH. The high variability in growth failure in the presence of severe GH and IGF-I deficiencies, even within the same family, suggests that the onset and predominance of GH-dependent growth during infancy are individually different and modified by as yet unknown factors.
Acknowledgments
We thank B. Schütt for fruitful discussions, P. Schwarze for language editing, and C. Urban for technical assistance.
Footnotes
Abbreviations: IGFBP, IGF-binding protein; IGHD, isolated GH deficiency.
Received December 14, 2000.
Accepted April 20, 2001.
References
This article has been cited by other articles:
 |
|
 |
|
  K. S. Alatzoglou, J. P. Turton, D. Kelberman, P. E. Clayton, A. Mehta, C. Buchanan, S. Aylwin, E. C. Crowne, H. T. Christesen, N. T. Hertel, et al. Expanding the Spectrum of Mutations in GH1 and GHRHR: Genetic Screening in a Large Cohort of Patients with Congenital Isolated Growth Hormone Deficiency J. Clin. Endocrinol. Metab., September 1, 2009; 94(9): 3191 - 3199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dateki, K. Hizukuri, T. Tanaka, N. Katsumata, P. Katavetin, and T. Ogata An immunologically anomalous but considerably bioactive GH produced by a novel GH1 mutation (p.D116E) Eur. J. Endocrinol., August 1, 2009; 161(2): 301 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Murphy, J. Baptista, J. Holly, A. M. Umpleby, S. Ellard, L. W. Harries, J. Crolla, T. Cundy, and A. T. Hattersley Severe Intrauterine Growth Retardation and Atypical Diabetes Associated with a Translocation Breakpoint Disrupting Regulation of the Insulin-Like Growth Factor 2 Gene J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4373 - 4380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Petkovic, D. Lochmatter, J. Turton, P. E. Clayton, P. J. Trainer, M. T. Dattani, A. Eble, I. C. Robinson, C. E. Fluck, and P. E. Mullis Exon Splice Enhancer Mutation (GH-E32A) Causes Autosomal Dominant Growth Hormone Deficiency J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4427 - 4435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Hess, Y. Hujeirat, M. P. Wajnrajch, S. Allon-Shalev, Z. Zadik, I. Lavi, and Y. Tenenbaum-Rakover Variable Phenotypes in Familial Isolated Growth Hormone Deficiency Caused by a G6664A Mutation in the GH-1 Gene J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4387 - 4393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. I Iliev, N. E Wittekindt, M. B Ranke, and G. Binder In vitro analysis of hGH secretion in the presence of mutations of amino acids involved in zinc binding J. Mol. Endocrinol., August 1, 2007; 39(2): 163 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Salemi, S. Yousefi, D. Lochmatter, A. Eble, J. Deladoey, I. C. A. F. Robinson, H.-U. Simon, and P. E. Mullis Isolated Autosomal Dominant Growth Hormone Deficiency: Stimulating Mutant GH-1 Gene Expression Drives GH-1 Splice-Site Selection, Cell Proliferation, and Apoptosis Endocrinology, January 1, 2007; 148(1): 45 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P G Turton, C. R Buchanan, I. C A F Robinson, S. J B Aylwin, and M. T Dattani Evolution of gonadotropin deficiency in a patient with type II autosomal dominant GH deficiency Eur. J. Endocrinol., December 1, 2006; 155(6): 793 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Vivenza, L. Guazzarotti, M. Godi, D. Frasca, B. di Natale, P. Momigliano-Richiardi, G. Bona, and M. Giordano A Novel Deletion in the GH1 Gene Including the IVS3 Branch Site Responsible for Autosomal Dominant Isolated Growth Hormone Deficiency J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 980 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salemi, S. Yousefi, K. Baltensperger, I. C A F Robinson, A. Eble, D. Simon, P. Czernichow, G. Binder, E. Sonnet, and P. E Mullis Variability of isolated autosomal dominant GH deficiency (IGHD II): impact of the P89L GH mutation on clinical follow-up and GH secretion Eur. J. Endocrinol., December 1, 2005; 153(6): 791 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E. Mullis, I. C. A. F. Robinson, S. Salemi, A. Eble, A. Besson, J.-M. Vuissoz, J. Deladoey, D. Simon, P. Czernichow, and G. Binder Isolated Autosomal Dominant Growth Hormone Deficiency: An Evolving Pituitary Deficit? A Multicenter Follow-Up Study J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2089 - 2096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E Mullis Genetic control of growth Eur. J. Endocrinol., January 1, 2005; 152(1): 11 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. C. Ryther, A. S. Flynt, B. D. Harris, J. A. Phillips III, and J. G. Patton GH1 Splicing Is Regulated by Multiple Enhancers Whose Mutation Produces a Dominant-Negative GH Isoform That Can Be Degraded by Allele-Specific Small Interfering RNA (siRNA) Endocrinology, June 1, 2004; 145(6): 2988 - 2996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. E. Meyer, D. Korthaus, W. Jagla, E. Cornali, J. Grosse, H. Fuchs, M. Klingenspor, S. Roemheld, M. Tschop, G. Heldmaier, et al. A Novel Missense Mutation in the Mouse Growth Hormone Gene Causes Semidominant Dwarfism, Hyperghrelinemia, and Obesity Endocrinology, May 1, 2004; 145(5): 2531 - 2541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. V. Fofanova, O. V. Evgrafov, A. V. Polyakov, A. B. Poltaraus, V. A. Peterkova, and I. I. Dedov A Novel IVS2 -2A>T Splicing Mutation in the GH-1 Gene in Familial Isolated Growth Hormone Deficiency Type II in the Spectrum of Other Splicing Mutations in the Russian Population J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 820 - 826. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. McGuinness, C. Magoulas, A. K. Sesay, K. Mathers, D. Carmignac, J.-B. Manneville, H. Christian, J. A. Phillips III, and I. C. A. F. Robinson Autosomal Dominant Growth Hormone Deficiency Disrupts Secretory Vesicles in Vitro and in Vivo in Transgenic Mice Endocrinology, February 1, 2003; 144(2): 720 - 731. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
