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Reduced Longevity in Untreated Patients with Isolated Growth Hormone Deficiency

Departments of Paediatrics, Paediatric Endocrinology, and Diabetology (A.B., S.S., A.J., R.H., P.S.M., P.E.M.) and Molecular Genetics (S.G.), University Children’s Hospital, Inselspital, CH-3010 Bern, Switzerland

Address all correspondence and requests for reprints to: Prof. Dr. Primus E. Mullis, University Children’s Hospital, Inselspital, CH 3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch.

	Abstract

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Increased longevity of hypopituitary dwarf mice and GH- resistant knockout mice appears to be in contrast with observations made in clinical practice. In humans, on one hand hypopituitarism and GH deficiency (GHD) are believed to constitute risk factors for cardiovascular disease and, therefore, early death. But on the other hand, patients with a PROP-1 gene mutation, presenting with a combined pituitary-derived hormonal deficiency, can survive to a very advanced age, apparently longer than normal individuals in the same population. The aim of this study was to analyze the impact of untreated GHD on life span. Hereditary dwarfism was recognized in 11 subjects. Genetic analysis revealed an underlying 6.7-kb spanning deletion of genomic DNA encompassing the GH-1 gene causing isolated GHD. These patients (five males and six females) were never treated for their hormonal deficiency and thus provide a unique opportunity to compare their life span and cause of death directly with their unaffected brothers and sisters (11 males and 14 females) as well as with the normal population (100 males and females). Although the cause of death did not vary between the two groups, median life span in the GH-deficient group was significantly shorter than that of unaffected brothers and sisters [males, 56 vs. 75 yr (P < 0.0001); females, 46 vs. 80 yr (P < 0.0001)]. Therefore, with the wealth of information regarding the beneficial effects of GH replacement and the dramatic findings of this study, GH treatment in adult patients suffering from either childhood- or adult-onset GHD is crucially important.

	Introduction

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INCREASED LONGEVITY OF hypopituitary dwarf mice and GH-resistant knockout mice appears to be in contrast with observations made in clinical practice (1, 2). In humans, hypopituitarism and GH deficiency are believed to constitute risk factors for cardiovascular disease and early death (3, 4, 5, 6). Most intriguing in this matter, however, is the fact that patients with a PROP-1 gene mutation, presenting with the absence of four adenohypophyseal cell types (somato-, lacto-, thyro-, and gonadotropes), as has been reported in the "little people of Krk," can survive to a very advanced age, apparently longer than normal individuals in the same population (7). These findings may challenge not only the importance and effectiveness of GH as an antiaging drug but also the notion that GH deficiency either isolated or in combination with a lack of any pituitary-derived hormone may shorten life span (3, 4, 5, 6). All the reports so far have focused on patients with hypopituitarism, and no data are available on life span in patients suffering from isolated GHD (IGHD) only. Therefore, the aim of our study was to analyze the impact of untreated IGHD (type IA) on life span.

	Subjects and Methods

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Subjects

Hereditary dwarfism was recognized in 11 related inhabitants of two isolated valleys in Switzerland in the late 19th and early 20th centuries (Fig. 1). As shown in Fig. 2, these affected patients belonged to two pedigrees and, importantly, they were never treated for their growth disorder (Fig. 3). To obtain more information on the underlying genetic defect and its consequences on life span as well as on the cause of death, we traced their direct descendents in Switzerland as well as in neighboring countries such as Austria and principality of Liechtenstein. The study was accepted and approved by the local Ethical Committees as well as from the individual governments. All the subjects involved gave verbal, witnessed, and written informed consent. Furthermore, to compare the life span of the affected as well as unaffected family members (Fig. 2), 100 males and females were randomly selected of the normal population living in this valley at that time (middle of the 20th century).

View larger version (29K): [in this window] [in a new window]   FIG. 1. In this illustration the remote area, where the large pedigree is originated from, is depicted. Just before World War I, a road was built to join this valley of Samnaun with Switzerland. Before that time, there was only a steep trail over the mountains, which connected Samnaun with Switzerland.

View larger version (15K): [in this window] [in a new window]   FIG. 2. The two pedigrees are presented. Full squares (males) and circles (females) indicate affected subjects (6.7-kb deletion of the GH gene cluster). A, Dotted lines present the possible hereditary way of the founder gene deletion (full circle), which is partly a speculation because files relating to these subjects were destroyed by a fire in the early 18th century. As all the affected family members were already dead at the time of analysis, we traced and studied the descendents of the following generation (not shown) looking for heterozygosities. B, The second pedigree is shown.

View larger version (162K): [in this window] [in a new window]   FIG. 3. A photograph of two affected family members sitting on the oven is shown.

As in these subjects clinically either a GH receptor, or GH-1 gene defect was suggested we analyzed the descendents (n = 78) of several families looking for heterozygosities within this two possible genes causing this hereditary form of dwarfism (8, 9). The GH receptor gene (10, 11, 12, 13) as well as the GH gene cluster was analyzed as previously reported (9, 14, 15, 16).

Preparation of DNA from leukocytes

DNA was isolated from peripheral leukocytes as previously described (9, 16). The concentration of each sample was determined by measuring the optical density of the purified DNA at 260 and 280 nm.

Screening for GH-1 gene defect

Amplification of DNA and restriction endonuclease analysis of PCR products. Unequal recombination between two highly homologous regions of 1250-bp 5' flanking and 3' flanking to the human GH-1 (hGH) gene are the main cause for 6.7-kb deletions of genomic DNA containing the hGH-1 gene (17). DNA amplification of these highly homologous regions was performed using the PCR, as described by Vnencak-Jones et al. (14). The primers used were 5'-GGA TCC AGC CTC AAA GAG CTT AC-3'; 3'-AGG TAA CGA GTT CCG AGA CCC TTA AG-5'. After amplification, 25 µl of each reaction mixture were digested with the restriction endonuclease SmaI. The digested PCR products were visualized after electrophoresis on a 0.8% agarose gel.

Restriction endonuclease digestion and Southern blotting analysis

Samples of DNA (5 µg) were digested to completion with the restriction enzymes BamHI and HindIII. After electrophoresis in 0.5–1.2% (wt/vol) agarose gels, the DNA fragments were transferred onto nylon membranes (Hybond-N, Amersham, International, Buckinghamshire, UK), hybridized to the hGH probe as previously described (9, 16). The filters were then washed at 65 C and autoradiographed at -70 C using intensifying screens (9, 16).

Statistics

All data are expressed as mean values (median, range). Data were compared using Student’s unpaired two-tailed t test (normal population groups). To compare the life span among the affected and nonaffected family members deriving from five subfamilies (Fig. 2), in which the assumption of independence is not given, a linear mixed-effects model using S-PLUS 6.0 software (Insight Corp., Seattle, WA) was applied which takes the intrafamilial dependency into account (17). P values less than 0.05 were considered statistically significant.

	Results

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Gene defect: 6.7-kb deletion of the GH-1 gene

GH receptor gene analysis revealed no abnormalities. As shown in Fig. 4, the DNA analysis of the GH gene cluster revealed an identical heterozygous pattern for a 6.7-kb deletion of genomic DNA encompassing the GH-1 gene throughout all the families tested. This supported the fact that the hereditary dwarfism was caused by the most commonly reported homozygous 6.7-kb deletion within the GH gene cluster resulting in the GH-1 gene deletion and, therefore, in IGHD (9). These data were confirmed by the finding of a homozygous pattern in the analysis of a blood spot of a patient (Fig. 4).

View larger version (53K): [in this window] [in a new window]   FIG. 4. Staining and autoradiograph patterns of DNA fragments. Open circle, Homozygote normal female; hatched circle, heterozygote for the 6.7-kb GH-1 gene deletion; closed circle, homozygote for the 6.7-kb GH-1 gene deletion. A, Ethidium bromide staining patterns of DNA fragments obtained by PCR amplification after digestion with SmaI and agarose gel electrophoresis (14 ). B, Autoradiograph patterns obtained after digestion with HindIII and hybridization to the hGH cDNA probe (9 ).

These data of a 6.7-kb gene deletion within the GH gene cluster were confirmed by Southern blot analysis. Inasmuch, as both hGH-1 gene and hCS-L gene reside in a 25-kb HindIII-derived fragment, the size of the majority of the deletion (6.7 kb) can be determined after HindIII digestion of the DNA. The data obtained by Southern blotting were identical to the PCR data (9, 16).

Life span

As the original patients (five males and six females) were never treated for their hormonal deficiency, this genetic isolate provides a unique opportunity to compare their life span and cause of death directly with their unaffected brothers and sisters (11 males and 14 females) (Fig. 2). After a normal life for that time in the Swiss Alps, they died in the middle of the 20th century. The cause of death was analyzed both by interviewing still living children of families with affected members (n = 21; mean age, 82 yr; median, 84 yr; range, 74–96 yr) and going through the files of the church as well as of the community where all these data are kept. Although these data are not detailed in strict medical terms, we can conclude that there was no difference in cause of death between the affected and unaffected brothers and sisters. The main causes were heart problems and infectious diseases. Focusing on life span, we found a highly significant difference—the longevity of the affected, GH-1 gene deleted and, therefore, IGHD patients was significantly reduced. The data are summarized in Table 1. Furthermore, the normal females (n = 100) at that time (unaffected females of normal stature belonging to the same population living in this valley at the same time, beginning of 20th century) had a significantly longer life span than the unaffected males (n = 100) (P < 0.05; 75.3 yr vs. 70.2 yr). This finding holds also true in the unaffected brothers and sisters where the same significantly different longer life span (P < 0.05) between females and males (74.2 yr vs. 70.9 yr) could be found as it was in the normal population. Interesting is the finding that the affected males had an increased life span (mean, 57.4 yr; median, 56 yr) when compared with the affected females (mean, 47.4 yr; median, 46 yr), which is in contrast to the findings in subjects without GHD. This difference, however, was not statistically different when correctly a linear mixed-effects model was applied taking dependency of life span among siblings and/or cousins into account.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Further, one has to assume that theoretically 50% of the unaffected family members presenting with normal stature were heterozygous for the same gene defect and that this defect may not have a significant impact on life span. In addition, as the cause of death did not differ among the two groups, one might suggest that the lack of GH might have a major impact on aging at least in patients suffering from a life-long absence of GH. As nowadays these patients are diagnosed as children and treated with recombinant human GH during childhood, we do not know for the time being what will happen in terms of life span in all these patients not being treated thereafter in adulthood. Given the fact that a lack of GH in adulthood has an impact on muscle and fat mass, on bone mineral content, and perhaps also on cognitive function, in addition to its negative effect on well being, the continuation of the GH replacement, especially in patients with childhood-onset GHD, is of high importance. This statement is further underlined by the wealth of information regarding the beneficial effects of GH replacement in GHD adults and by the dramatic findings of this study.

	Acknowledgments

 
We are most grateful to Dr. Liz Buergi for reviewing this article. In addition, we thank Dr. Roland A. Ammann for his support for the statistical analysis of our data.

	Footnotes

 
This work was supported by Swiss National Science Foundation Grant SNF 3200-064623.01 (to P.E.M.).

Abbreviations: GHD, GH deficiency; hGH, human GH; IGHD, isolated GHD.

Received December 10, 2002.

Accepted May 5, 2003.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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