J. Endocrinol. Invest. 35: 937-944, 2012 DOI: 10.3275/8647
REVIEW ARTICLE
Growth hormone variants: A potential avenue for a better diagnostic characterization of growth hormone deficiency in children A.E. Rigamonti1, M. Bozzola2, G. Banfi3, C. Meazza2, E.E. Müller1, and S.G. Cella1 1Department
of Medical Biotechnologies and Translational Medicine, University of Milan, Milan; 2Department of Internal Medicine and Therapeutics, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia; 3Department of Health Technologies, University of Milan, and Orthopedic Institute Galeazzi, Milan, Italy ABSTRACT. Human GH (hGH) is a heterogeneous protein hormone consisting of several isoforms. This heterogeneity is the consequence of multiple hGH genes, mRNA splicing, post-translational modifications, and peripheral metabolism, and it represents one important reason for the disparity among GH assay results from different laboratories. However, other factors are involved: a) interference from endogenous GH binding proteins; b) different specificities of antiGH (monoclonal and polyclonal) antibodies; c) different ma-
trix effects among the calibrators; d) the use of different calibrators. The measurement of GH levels in response to provocative testing is an essential part of the diagnosis of GH deficiency. For this purpose, an accurate, reproducible and universally valid GH measurement would be highly desirable, but, despite a huge number of efforts in clinical biochemistry, this goal remains elusive. (J. Endocrinol. Invest. 35: 937-944, 2012) ©2012, Editrice Kurtis
DIAGNOSIS OF GH DEFICIENCY: A “BIOCHEMICAL” PROBLEM
art in GH assays, discussing some of the reasons for variability of results and the negative implications in the diagnostics of GHD in pediatric endocrinology, and suggesting possible solutions for minimizing assay discrepancies. The same problems do apply to the diagnosis of adult GHD and the monitoring of acromegaly, two endocrinopathies that will not be considered in the present review.
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K e c i Y r L t N i O d E E S U , 2 AL 1 N O S © 20 R PE The measurement of circulating levels of GH in response to provocative testing is an essential part of the diagnosis of GH deficiency (GHD) in children having short stature. An arbitrary cut-off limit of 10 ng/ml in childhood is commonly used to classify patients as GH-deficient (1, 2). The measurement of serum GH by various immunoassays discloses, however, controversial findings: a difference of more than double in circulating GH levels has been reported among laboratories (3). The assay discrepancies, which represent a relevant clinical problem, frequently underestimated by endocrinologists, are caused by several factors: a) the heterogeneity of circulating GH; b) interference from endogenous GH-binding proteins (GHBP); c) different specificities of the anti-GH (monoclonal and polyclonal) antibodies for the different GH isoforms; d) different matrix effect among the calibrators; e) the use of different calibrators (4-6). An accurate, reproducible, and universally valid GH measurement would be highly desirable (7), but, despite a huge number of efforts in clinical biochemistry, this goal remains elusive. Unfortunately, the variability of results in GH measurement among immunoassays has increased rather than decreased over the past two decades (8). Aim of this article will be that of reviewing the molecular basis of the GH heterogeneity and the current state-of-the-
PHYSIOLOGY OF GH SYSTEM GH heterogeneity Human GH (hGH) is a heterogeneous protein that consists of several molecular isoforms. Heterogeneity arises at the level of the GH gene, mRNA splicing, post-translational processing, and peripheral GH metabolism. The existence of multiple isoforms might underlie a complexity in GH bioactivity and explain the difficulties for accurate measurement of GH in body fluids, and for assay standardization (9). The hGH gene cluster contains 5 genes: 3 placental lactogens (PL) (PL-A, PL-B, and PL-L or PL-V) and 2 GH genes [GH normal (GH-N or GH-1) and GH variant (GHV or GH-2)], which evolved from a common ancestral precursor by consecutive recombination events. The cluster, which spans 66 kb, is located on chromosome 17 (q22q24). The individual genes are transcriptionally orientated in the same direction; 5 exons and 4 introns compose each gene. The order of the genes from 5’ to 3’ is GH-N, PL-L, PL-A, GH-V, and PL-B (Fig. 1). The products of the GH gene cluster are GH-N, PL [PL-A, PL-B, and PL-L, also known as chorionic somatomammotropin (CS)], and GH-V (also known as placental GH). The PL genes and GH-V are expressed exclusively in the placenta, whereas GH-N in the pituitary (10). The PL-L gene in the cluster codes for a PL-like-protein that is expressed at low levels and may not be translated efficiently. The function – or non-function – of this
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Key-words: Analytical methods, biochemical diagnosis, GH deficiency, GH isoforms, growth hormone. Correspondence: A.E. Rigamonti, MD, PhD, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32 – 20129 Milan, Italy. E-mail:
[email protected] Accepted September 5, 2012. First published online October 1, 2012.
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22 kDa, whose primary structure is 85% identical to the related product of the GH-N gene. The mRNA for PL-A and PL-B are among the most abundant mRNA in the placenta, comprising approximately 3.5% of the total mRNA. PL has very low affinity for the GH receptor (GHR) and, therefore, at physiological levels, does not seem to act as an agonist/antagonist of this receptor. In clinical biochemistry there are immunoassays capable of measuring specifically GH and PL (15). The present review of GH isoforms will not further discuss PL.
3’ GH-N
PL-L
PL-A Pituitary
GH-V PL-B Placenta
Fig. 1 - The human GH gene cluster. The orientation and tissuespecific expression of the 5 genes comprising the cluster are shown from 5’ to 3’. Please, note that the locus is not drawn to scale. Modified from (15). See text for further details. GH-N: GH normal; GH-V: GH variant; PL: placental lactogen.
The GHBP Blood contains two GHBP, one with high affinity for GH and the other with low affinity (9). In humans, the high-affinity GHBP derives from a specific post-translational proteolytic cleavage of the extracellular portion of the cell membrane-embedded GH receptor (GHR). The high-affinity GHBP binds approximately 50% of circulating GH under physiological conditions. Hence, there is significant unoccupied GHBP in the serum, explaining the strong interference of this protein in GH immunoassay (see below). GHBP binds 22-kDa GH with a dissociation constant in the nanomolar range. While GHBP binds GH-V with an affinity equal to that of 22-kDa GH, the binding with 20k-Da GH is of considerably lower affinity. About 25% of 20-kDa GH is bound to this GHBP under physiological conditions. The low-affinity GHBP corresponds to α2-macroglobulin (16). A low percentage of circulating 22-kDa GH (
