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The genetics of familial glucocorticoid deficiency
Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 159–165
Contents lists available at ScienceDirect
Best Practice & Research Clinical Endocrinology & Metabolism journal homepage: www.elsevier.com/locate/beem
2
The genetics of familial glucocorticoid deficiency Adrian J.L. Clark, DSc, FRCP, Professor of Medicine *, Li F. Chan, MB, BDhir, MRCP, MRC, Clinical Training Fellow, Teng-Teng Chung, MBBS, MRCP, MRC, Clinical Training Fellow, Louise A. Metherell, PhD, Lecturer in Endocrine Genetics Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, London EC1M 6BQ, UK
Keywords: adrenal failure neonatal hypoglycaemia ACTH resistance melanocortin 2 receptor
Familial glucocorticoid deficiency is an autosomal recessive disorder resulting from defects in the action of adrenocorticotropic hormone (ACTH) to stimulate glucocorticoid synthesis in the adrenal. Production of mineralocorticoids by the adrenal is normal. Patients present in early life with low or undetectable cortisol and – because of the failure of the negative feedback loop to the pituitary and hypothalamus – grossly elevated ACTH levels. About half of all cases result from mutations in the ACTH receptor (melanocortin 2 receptor) or from mutations in the melanocortin 2 receptor accessory protein (MRAP), but other genetic causes of this potentially lethal disorder remain to be discovered. Ó 2008 Elsevier Ltd. All rights reserved.
Familial glucocorticoid deficiency (FGD) – also sometimes known as isolated glucocorticoid deficiency or hereditary unresponsiveness to adrenocorticotropic hormone (ACTH) – is a rare, potentially lethal, autosomal recessive disorder that usually presents in the neonatal period or in early childhood. As long as a diagnosis of adrenal failure is considered, treatment is relatively straightforward and the long-term prognosis is good. Perhaps one of the most valuable aspects of FGD is in what it teaches us about the actions of ACTH on the adrenal. In this article we will review both the established and some of the emerging aspects of the clinical diagnosis, and then consider the developing understanding of the role of the two genes so far identified to cause FGD.
* Corresponding author. Tel.: þ44 2078826202; Fax: þ44 278826197. E-mail address: [email protected] (A.J.L. Clark). 1521-690X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.beem.2008.09.006
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A.J.L. Clark et al. / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 159–165
The clinical diagnosis The majority of patients with FGD have episodes of hypoglycaemia in the neonatal period. These will often respond quickly to improved or more frequent feeding regimens, and in many cases a clinical diagnosis will not be made at this time. Those that are diagnosed as neonates will often have more persistent or severe hypoglycaemia, which prompts further investigation, or in a few cases excessive skin pigmentation will be recognized at this early stage. Patients who are not identified at this time will usually re-present over the next few years with a recurrence of hypoglycaemic symptoms, with skin pigmentation, or with a poor response to an infective episode. The oldest patient at presentation in our experience was aged 7.6 years. In a small number of patients hypoglycaemia can be sufficiently severe and persistent that it leads to serious long-term neurological damage or death.1–6 The diagnosis is readily confirmed with a low plasma cortisol measurement in the presence of an elevated ACTH level and normal aldosterone and plasma renin measurements. Electrolytes are normal. With this biochemistry and the absence of other symptoms a diagnosis of FGD is fairly secure. The triple-A syndrome (OMIM #231550) may present in a similar manner, although other symptoms – including alacrima, achalasia or various neurological defects – will usually be suggestive of this alternative diagnosis.6 It is very unusual for congenital adrenal hyperplasia or adrenoleucodystrophy to present with isolated glucocorticoid deficiency, although measurement of 17a-hydroxyprogesterone and very-long-chain fatty acids (respectively) should exclude these diagnoses. Addison’s disease will usually be associated with loss of mineralocorticoid production with consequent electrolyte imbalance. It is not unusual to see slightly elevated plasma renin levels at the time of diagnosis of FGD, and it is possible that this persuades clinicians to favour a diagnosis of combined glucocorticoid and mineralocorticoid deficiency as might occur in Addison’s disease. This may reflect a degree of volume depletion in a sick or shocked child at this time, and if there is doubt about this aspect, confirmation of renin and aldosterone levels at a later stage after stabilization of the patient may be valuable. There are anecdotes of FGD patients being treated for several years with fludrocortisone replacement before they themselves begin to omit it, with no ensuing disturbance of salt and water metabolism. This is unlikely to be a major problem, however, and in a recent study of 40 patients with a clinical diagnosis of Addison’s disease without autoantibodies or an autoimmune family history, none was found to have mutations in genes associated with FGD (Dias et al, unpublished). A clinical feature sometimes observed in FGD patients is tall stature.7,8 The underlying mechanisms are not clear and do not seem to be related to over-activity of the growth hormone–insulin-like growth factor I (IGF-I) axis. There is a suggestion that excessive growth is curtailed after patients are started on glucocorticoid replacement7, possibly suggesting that either glucocorticoid deficiency itself or excessively high ACTH levels may have a causative role. A number of melanocortin receptors are expressed in bone and the cartilaginous growth plate, and a reasonable hypothesis is that ACTH at high concentrations activates these receptors and stimulates growth.9 Tall stature is not observed in a probable variant form of FGD found in the Irish Traveller population. This is a relatively inbred group with a high prevalence of several genetic disorders, including FGD. There is clear documentation of the development of adrenal failure after several years of normal adrenal function in some of these children as well as the possible association with a typical facial phenotype.10 Genetic causes of FGD ACTH receptor – FGD type 1 FGD is an autosomal recessive disorder and consequently is most often encountered in consanguineous families. Some of the earliest clinical reports of patients with FGD speculated that it may result from a defect in the membrane receptor for ACTH3,4, although one study suggested the existence of a defect downstream from the receptor.11 The cloning of the putative ACTH receptor – thereafter named the melanocortin 2 receptor (MC2R) in 199212 – paved the way for investigating the possibility of mutations in MC2R in FGD. FGD patients with homozygous13 or compound heterozygous defects14 were soon identified. Over the following years many mutations of the MC2R were identified. It is of
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interest that the majority of these were missense mutations, and nonsense mutations were usually compounded with a missense mutation on the other allele. The presence of a homozygous nonsense mutation is uncommon. This raises the possibility that homozygous nonsense mutations either lead to reduced survival in utero, or are associated with a different phenotype. Although the finding of homozygous mutations in affected individuals is highly suggestive of a causative role in the disease, proof that this is the case requires some form of functional assessment. This has been particularly hard to achieve in the case of the MC2R. Transfection of MC2R DNA into a wide range of mammalian cell lines failed to produce a functional receptor that would support an ACTH-dependent cAMP signal. This was the case despite the use of highefficiency transfection techniques and evidence that the transfected DNA was being expressed and translated into protein.15,16 Some success was achieved by Naville et al using the melanoma-derived M3 cell line17, although this line had the disadvantage of expressing endogenous melanocortin 1 receptors (MC1R) which also respond to ACTH. However, it was the use of certain adrenal-derived cell lines which lacked endogenous MC2R expression, but which retained the machinery for generating a cAMP signal, that was particularly helpful. These cell lines – the Y6 and OS3 lines – were sister clones to the widely used ACTH-responsive Y1 cell line, differing only in the absence of the endogenous MC2R for reasons that remain unclear.18 Unfortunately these cell lines transfect inefficiently, and for most purposes it is necessary to make stable cell lines that have incorporated the transfected MC2R into their genome. Using this model Elias et al were able to characterize a number of FGD-associated mutant MC2Rs, showing them to be associated with either loss of ligand binding or loss of signal-generating ability in comparison to the wild-type receptor, and thereby confirming the causative nature of these mutations.19 An interesting report is that of the first promoter variant in the MC2R causing FGD. Tsiotra et al described a patient with a nonsense mutation in one allele (a frameshift following glycine 217) and a T-to-C substitution in the 2 position of the MC2R promoter. Functional analysis of this substitution suggested that the promoter activity was reduced by 15%, which – assuming that the nonsense allele has zero activity – indicates that reduction of MC2R expression to 35% normal leads to disease.20 In contrast, earlier investigation of heterozygous carriers of missense mutations suggested that they retained entirely adequate MC2R activity and had no glucocorticoid deficiency.21 However, this promoter variant has been found in w10% of MC2R alleles in the population22 and has been proposed to influence pituitary–adrenal axis activity in normal adults and children.22,23
Mouse model Until recently no animal model of FGD existed. In 2007 Chida et al reported the characterization of a mouse Mc2r knockout.24 In most respects this animal exactly mimicked human FGD in having elevated ACTH and undetectable corticosterone. There was a high rate of neonatal lethality as a result of hypoglycaemia, and examination of adrenal histology was similar to that in humans, showing an atrophic zona fasciculata with a relatively preserved zona glomerulosa. Interestingly these mice do not show excessive longitudinal growth and in this sense do not parallel patients with MC2R mutations. The major difference between knockout mice and MC2R-deficient patients was that mice were deficient in aldosterone and their adrenals showed low expression levels of aldosterone synthase. Mineralocorticoid deficiency was also seen in POMC knockout animals, supporting the view that in rodents, ACTH plays a more significant role in zona glomerulosa development and function than is the case in the human.25,26 The basis for this distinction between the mouse and human is not clear. A recent report has suggested in two FGD patients with a homozygous nonsense mutation of the MC2R there is a degree of mineralocorticoid deficiency as indicated by a raised renin level.27 As mentioned, totally inactivating mutations such as these are very uncommon in FGD, but it is interesting to note that in our own studies 4/6 patients with homozygous nonsense or frameshift mutations had elevated plasma renin, and 1/6 had aldosterone deficiency even when stabilized on glucocorticoid replacement (Chan et al, unpublished). Despite the clear causative link between MC2R mutations and FGD that these studies provide, it remains clear that only a fraction of FGD – around 25% in our experience – is associated with biallelic MC2R mutation. Those cases linked to the MC2R are known as FGD type 1. Two studies have
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A.J.L. Clark et al. / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 159–165 ACTH C
N
N
3
ACTH
N
C
C
SIGNAL
2 C
C
N
N
N
MRAP
Plasma Membrane
N
Endoplasmic Reticulum N C
N C
C
C
MC2R
1
Fig. 1. Schematic diagram of the possible actions of melanocortin 2 receptor accessory protein (MRAP) in supporting melanocortin 2 receptor (MC2R) function. (1) MRAP exists as an anti-parallel homodimer, and may have a chaperone-like function in assisting the correct folding of the MC2R in the endoplasmic reticulum. (2) MRAP may have an ‘escort’ function in assisting the trafficking of the correctly folded MC2R to the plasma membrane. (3) Finally, MRAP may form a trimeric structure with the MC2R at the cell surface and may be required for adrenocorticotropic hormone (ACTH) interaction and binding, or for generation of an intracellular signal.
investigated the possibility that the non-MC2R cases are not linked to the receptor, and both have shown that there is another genetic cause of FGD.28,29 MRAP – FGD type 2 Genetic studies using the technique of homozygosity mapping, in which a large number of informative genetic polymorphisms spread throughout the genome are studied, revealed a potential locus for FGD on chromosome 21. Analysis of all the genes in the critical interval at this locus revealed that a single gene was expressed only in the adrenal, but not in brain or liver. Sequence analysis of this gene in the index family showed a significant splice site mutation that was highly likely to lead to a defective protein. Sequencing of this gene in additional FGD families showed similar mutations in several other cases.30 The gene in question encoded a small protein of essentially unknown function. It had originally been identified by Xu et al as a protein that was induced when mouse 3T3-L1 cells were differentiated into adipocytes; it had been called the ‘fat-associated low-molecular-weight protein’ or Falp.31 We were able to show that this protein was physically associated with the MC2R as demonstrated by co-immunoprecipitation when both were transfected into heterologous cells. On confocal imaging it co-localized with MC2R at both the endoplasmic reticulum and at the plasma membrane. Importantly, in heterologous cells, when the Mc2r was co-transfected with the gene encoding Falp, a significant ACTH responsiveness was achieved.30 In comparison, Mc2r expression alone was unresponsive to ACTH. The mouse Y1 cell expresses this gene and has a functional endogenous ACTH response, which is lost when the gene is knocked down by RNA interference.32 Thus we believe that this gene – which we re-named ‘melanocortin 2 receptor accessory protein’ (MRAP) – is an essential cofactor for the MC2R. It is difficult to see how this advance could have been made without the study of FGD patients. It is still not entirely clear how MRAP works. Possible modes of action are summarized in Fig. 1 and are as follows:
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(a) it functions as a chaperone – a factor that assists the correct folding of the comparatively small and highly hydrophobic MC2R at the level of the endoplasmic reticulum (Fig. 1, point 1); (b) it functions as an escort protein, essentially escorting the MC2R to its destination at the plasma membrane (Fig. 1, point 2); (c) it acts as a co-receptor enabling the MC2R to bind to or signal in response to ACTH (Fig. 1, point 3). Existing data lend some support to each type of activity, and this is the subject of ongoing research. It seems highly probable that MRAP exhibits each of these functions to some extent. Perhaps one of the most interesting novel observations about MRAP is that it preferentially exists as an anti-parallel homodimer. Theoretical prediction tools suggest that the orientation of this small single transmembrane domain protein is with its N-terminus lying inside the cell. In elegant studies combining immunodetection and glycosylation analysis, Sebag & Hinkle demonstrated that it existed in a relatively stable dimer with each molecule in the dimer in opposing orientations.33 This is a unique structure in eukaryotes. Independently, Cooray et al had confirmed the dimer structure with a combination of co-immunoprecipitation and mass spectroscopic analysis of potential dimeric bands on gel electrophoresis.32 MRAP mutations do not account for a large proportion of FGD patients. In our studies about 15% of all FGD patients have biallelic MRAP mutations, and this is now known as FGD type 2. Others have also identified MRAP mutations34, and the possibility that an obese phenotype may characterize FGD patients with MRAP mutations has been raised.35 At this time it is very difficult to be clear about this, but there are certainly MRAP patients who are not overweight.
Other genetic loci Our genotyping analysis clearly shows that in many patients FGD does not segregate with MRAP or the MC2R, implying that other genes may cause the disease. Prior to the work that identified MRAP, a microsatellite-based whole genome analysis had suggested that two FGD families had FGD that localized to the long arm of chromosome 8.29 Since that time it has not been possible to link many other families to this locus, and although a number of candidate genes in this region have been studied, none has been shown to carry mutations. There also remain many families who clearly do not map to this region of the genome; hence further FGD genes remain to be identified.
Practice points primary adrenal failure in a child with a normal rennin–angiotensin–aldosterone axis is highly suggestive of a diagnosis of familial glucocorticoid deficiency persistent hypoglycaemia in a neonate should raise the suspicion of glucocorticoid deficiency establishing a genetic diagnosis of familial glucocorticoid deficiency is important both in providing reassurance that mineralocorticoid replacement is unnecessary and for genetic prediction and counselling over-replacement of glucocorticoids in an attempt to treat an elevated ACTH carries significant risk of increased morbidity, including growth retardation
Research agenda identify new genes for familial glucocorticoid deficiency develop an understanding of how MRAP works in enabling MC2R expression
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References 1. Shepard TH, Landing BH & Mason DG. Familial Addison’s disease. A.M.A. Journal of Diseases of Children 1959; 97: 154–162. 2. Stempfel RS & Engel FL. A congenital, familial syndrome of adrenocortical insufficiency without hypoaldosteronism. The Journal of Pediatrics 1960; 57: 443–451. 3. Migeon CJ, Kenny FM, Kowarski A et al. The syndrome of congenital adrenocortical unresponsiveness to ACTH. Report of six cases. Pediatric Research 1968; 2: 501–513. 4. Franks RC & Nance WE. Hereditary adrenocortical unresponsiveness to ACTH. Pediatrics 1970; 45: 43–48. 5. Moshang T, Rosenfield RL, Bongiovanni AM et al. Familial glucocorticoid insufficiency. The Journal of Pediatrics 1973; 82: 821–826. 6. Clark AJ & Weber A. Adrenocorticotropin insensitivity syndromes. Endocrine Reviews 1998; 19: 828–843. 7. Elias LL, Huebner A, Metherell LA et al. Tall stature in familial glucocorticoid deficiency. Clinical Endocrinology 2000; 53: 423–430. 8. Imamine H, Mizuno H, Sugiyama Y et al. Possible relationship between elevated plasma ACTH and tall stature in familial glucocorticoid deficiency. The Tohoku Journal of Experimental Medicine 2005; 205: 123–131. 9. Evans JF, Shen CL, Pollack S et al. Adrenocorticotropin evokes transient elevations in intracellular free calcium ([Ca2þ]i) and increases basal [Ca2þ]i in resting chondrocytes through a phospholipase C-dependent mechanism. Endocrinology 2005; 146: 3123–3132. 10. O’Riordan SM, Lynch SA, Hindmarsh PC et al. A novel variant of familial glucocorticoid deficiency prevalent among the Irish traveler population. The Journal of Clinical Endocrinology and Metabolism 2008; 93: 2896–2899. 11. Yamaoka T, Kudo T, Takuwa Y et al. Hereditary adrenocortical unresponsiveness to adrenocorticotropin with a postreceptor defect. The Journal of Clinical Endocrinology and Metabolism 1992; 75: 270–274. *12. Mountjoy KG, Robbins LS, Mortrud MT et al. The cloning of a family of genes that encode the melanocortin receptors. Science 1992; 257: 1248–1251. *13. Clark AJ, McLoughlin L & Grossman A. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet 1993; 341: 461–462. *14. Tsigos C, Arai K, Hung W et al. Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene. The Journal of Clinical Investigation 1993; 92: 2458–2461. 15. Weber A, Kapas S, Hinson JP et al. Functional characterization of the cloned human ACTH receptor: impaired responsiveness of a mutant receptor in familial glucocorticoid deficiency. Biochemical and Biophysical Research Communications 1993; 197: 172–178. *16. Noon LA, Franklin JM, King PJ et al. Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor. The Journal of Endocrinology 2002; 174: 17–25. *17. Naville D, Barjhoux L, Jaillard C et al. Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency. The Journal of Clinical Endocrinology and Metabolism 1996; 81: 1442–1448. 18. Schimmer BP, Kwan WK, Tsao J et al. Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor. Journal of Cellular Physiology 1995; 163: 164–171. *19. Elias LL, Huebner A, Pullinger GD et al. Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: poor correlation of phenotype and genotype. The Journal of Clinical Endocrinology and Metabolism 1999; 84: 2766–2770. 20. Tsiotra PC, Koukourava A, Kaltezioti V et al. Compound heterozygosity of a frameshift mutation in the coding region and a single base substitution in the promoter of the ACTH receptor gene in a family with isolated glucocorticoid deficiency. Journal of Pediatric Endocrinology & Metabolism 2006; 19: 1157–1166. 21. Weber A, Toppari J, Harvey RD et al. Adrenocorticotropin receptor gene mutations in familial glucocorticoid deficiency: relationships with clinical features in four families. The Journal of Clinical Endocrinology and Metabolism 1995; 80: 65–71. 22. Slawik M, Reisch N, Zwermann O et al. Characterization of an adrenocorticotropin (ACTH) receptor promoter polymorphism leading to decreased adrenal responsiveness to ACTH. The Journal of Clinical Endocrinology and Metabolism 2004; 89: 3131–3137. 23. Lappalainen S, Utriainen P, Kuulasmaa T et al. ACTH receptor promoter polymorphism associates with severity of premature adrenarche and modulates hypothalamo-pituitary-adrenal axis in children. Pediatric Research 2008; 63: 410–414. *24. Chida D, Nakagawa S, Nagai S et al. Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis. Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 18205–18210. 25. Karpac J, Ostwald D, Bui S et al. Development, maintenance, and function of the adrenal gland in early postnatal proopiomelanocortin-null mutant mice. Endocrinology 2005; 146: 2555–2562. 26. Coll AP, Fassnacht M, Klammer S et al. Peripheral administration of the N-terminal pro-opiomelanocortin fragment 1-28 to Pomc-/- mice reduces food intake and weight but does not affect adrenal growth or corticosterone production. The Journal of Endocrinology 2006; 190: 515–525. 27. Lin L, Hindmarsh PC, Metherell LA et al. Severe loss-of-function mutations in the adrenocorticotropin receptor (ACTHR, MC2R) can be found in patients diagnosed with salt-losing adrenal hypoplasia. Clinical Endocrinology 2007; 66: 205–210. *28. Weber A & Clark AJ. Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency. Human Molecular Genetics 1994; 3: 585–588. 29. Genin E, Huebner A, Jaillard C et al. Linkage of one gene for familial glucocorticoid deficiency type 2 (FGD2) to chromosome 8q and further evidence of heterogeneity. Human Genetics 2002; 111: 428–434. *30. Metherell LA, Chapple JP, Cooray S et al. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nature Genetics 2005; 37: 166–170. 31. Xu A, Choi KL, Wang Y et al. Identification of novel putative membrane proteins selectively expressed during adipose conversion of 3T3-L1 cells. Biochemical and Biophysical Research Communications 2002; 293: 1161–1167.
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32. Cooray SN, Almiro Do Vale I, Leung KY et al. The melanocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse Y1 cell line. Endocrinology 2008; 149: 1935–1941. *33. Sebag JA & Hinkle PM. Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers. Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 20244–20249. 34. Modan-Moses D, Ben Zeev B, Hoffmann C et al. Unusual presentation of familial glucocorticoid deficiency (FGD) with a novel MRAP mutation. The Journal of Clinical Endocrinology and Metabolism 2006; 91: 3713–3717. 35. Rumie´ H, Metherell LA, Clark AJ et al. Clinical and biological phenotype of a patient with familial glucocorticoid deficiency type 2 caused by a mutation of melanocortin 2 receptor accessory protein. European Journal of Endocrinology 2007; 157: 539–542.
Contents lists available at ScienceDirect
Best Practice & Research Clinical Endocrinology & Metabolism journal homepage: www.elsevier.com/locate/beem
2
The genetics of familial glucocorticoid deficiency Adrian J.L. Clark, DSc, FRCP, Professor of Medicine *, Li F. Chan, MB, BDhir, MRCP, MRC, Clinical Training Fellow, Teng-Teng Chung, MBBS, MRCP, MRC, Clinical Training Fellow, Louise A. Metherell, PhD, Lecturer in Endocrine Genetics Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, London EC1M 6BQ, UK
Keywords: adrenal failure neonatal hypoglycaemia ACTH resistance melanocortin 2 receptor
Familial glucocorticoid deficiency is an autosomal recessive disorder resulting from defects in the action of adrenocorticotropic hormone (ACTH) to stimulate glucocorticoid synthesis in the adrenal. Production of mineralocorticoids by the adrenal is normal. Patients present in early life with low or undetectable cortisol and – because of the failure of the negative feedback loop to the pituitary and hypothalamus – grossly elevated ACTH levels. About half of all cases result from mutations in the ACTH receptor (melanocortin 2 receptor) or from mutations in the melanocortin 2 receptor accessory protein (MRAP), but other genetic causes of this potentially lethal disorder remain to be discovered. Ó 2008 Elsevier Ltd. All rights reserved.
Familial glucocorticoid deficiency (FGD) – also sometimes known as isolated glucocorticoid deficiency or hereditary unresponsiveness to adrenocorticotropic hormone (ACTH) – is a rare, potentially lethal, autosomal recessive disorder that usually presents in the neonatal period or in early childhood. As long as a diagnosis of adrenal failure is considered, treatment is relatively straightforward and the long-term prognosis is good. Perhaps one of the most valuable aspects of FGD is in what it teaches us about the actions of ACTH on the adrenal. In this article we will review both the established and some of the emerging aspects of the clinical diagnosis, and then consider the developing understanding of the role of the two genes so far identified to cause FGD.
* Corresponding author. Tel.: þ44 2078826202; Fax: þ44 278826197. E-mail address: [email protected] (A.J.L. Clark). 1521-690X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.beem.2008.09.006
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The clinical diagnosis The majority of patients with FGD have episodes of hypoglycaemia in the neonatal period. These will often respond quickly to improved or more frequent feeding regimens, and in many cases a clinical diagnosis will not be made at this time. Those that are diagnosed as neonates will often have more persistent or severe hypoglycaemia, which prompts further investigation, or in a few cases excessive skin pigmentation will be recognized at this early stage. Patients who are not identified at this time will usually re-present over the next few years with a recurrence of hypoglycaemic symptoms, with skin pigmentation, or with a poor response to an infective episode. The oldest patient at presentation in our experience was aged 7.6 years. In a small number of patients hypoglycaemia can be sufficiently severe and persistent that it leads to serious long-term neurological damage or death.1–6 The diagnosis is readily confirmed with a low plasma cortisol measurement in the presence of an elevated ACTH level and normal aldosterone and plasma renin measurements. Electrolytes are normal. With this biochemistry and the absence of other symptoms a diagnosis of FGD is fairly secure. The triple-A syndrome (OMIM #231550) may present in a similar manner, although other symptoms – including alacrima, achalasia or various neurological defects – will usually be suggestive of this alternative diagnosis.6 It is very unusual for congenital adrenal hyperplasia or adrenoleucodystrophy to present with isolated glucocorticoid deficiency, although measurement of 17a-hydroxyprogesterone and very-long-chain fatty acids (respectively) should exclude these diagnoses. Addison’s disease will usually be associated with loss of mineralocorticoid production with consequent electrolyte imbalance. It is not unusual to see slightly elevated plasma renin levels at the time of diagnosis of FGD, and it is possible that this persuades clinicians to favour a diagnosis of combined glucocorticoid and mineralocorticoid deficiency as might occur in Addison’s disease. This may reflect a degree of volume depletion in a sick or shocked child at this time, and if there is doubt about this aspect, confirmation of renin and aldosterone levels at a later stage after stabilization of the patient may be valuable. There are anecdotes of FGD patients being treated for several years with fludrocortisone replacement before they themselves begin to omit it, with no ensuing disturbance of salt and water metabolism. This is unlikely to be a major problem, however, and in a recent study of 40 patients with a clinical diagnosis of Addison’s disease without autoantibodies or an autoimmune family history, none was found to have mutations in genes associated with FGD (Dias et al, unpublished). A clinical feature sometimes observed in FGD patients is tall stature.7,8 The underlying mechanisms are not clear and do not seem to be related to over-activity of the growth hormone–insulin-like growth factor I (IGF-I) axis. There is a suggestion that excessive growth is curtailed after patients are started on glucocorticoid replacement7, possibly suggesting that either glucocorticoid deficiency itself or excessively high ACTH levels may have a causative role. A number of melanocortin receptors are expressed in bone and the cartilaginous growth plate, and a reasonable hypothesis is that ACTH at high concentrations activates these receptors and stimulates growth.9 Tall stature is not observed in a probable variant form of FGD found in the Irish Traveller population. This is a relatively inbred group with a high prevalence of several genetic disorders, including FGD. There is clear documentation of the development of adrenal failure after several years of normal adrenal function in some of these children as well as the possible association with a typical facial phenotype.10 Genetic causes of FGD ACTH receptor – FGD type 1 FGD is an autosomal recessive disorder and consequently is most often encountered in consanguineous families. Some of the earliest clinical reports of patients with FGD speculated that it may result from a defect in the membrane receptor for ACTH3,4, although one study suggested the existence of a defect downstream from the receptor.11 The cloning of the putative ACTH receptor – thereafter named the melanocortin 2 receptor (MC2R) in 199212 – paved the way for investigating the possibility of mutations in MC2R in FGD. FGD patients with homozygous13 or compound heterozygous defects14 were soon identified. Over the following years many mutations of the MC2R were identified. It is of
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interest that the majority of these were missense mutations, and nonsense mutations were usually compounded with a missense mutation on the other allele. The presence of a homozygous nonsense mutation is uncommon. This raises the possibility that homozygous nonsense mutations either lead to reduced survival in utero, or are associated with a different phenotype. Although the finding of homozygous mutations in affected individuals is highly suggestive of a causative role in the disease, proof that this is the case requires some form of functional assessment. This has been particularly hard to achieve in the case of the MC2R. Transfection of MC2R DNA into a wide range of mammalian cell lines failed to produce a functional receptor that would support an ACTH-dependent cAMP signal. This was the case despite the use of highefficiency transfection techniques and evidence that the transfected DNA was being expressed and translated into protein.15,16 Some success was achieved by Naville et al using the melanoma-derived M3 cell line17, although this line had the disadvantage of expressing endogenous melanocortin 1 receptors (MC1R) which also respond to ACTH. However, it was the use of certain adrenal-derived cell lines which lacked endogenous MC2R expression, but which retained the machinery for generating a cAMP signal, that was particularly helpful. These cell lines – the Y6 and OS3 lines – were sister clones to the widely used ACTH-responsive Y1 cell line, differing only in the absence of the endogenous MC2R for reasons that remain unclear.18 Unfortunately these cell lines transfect inefficiently, and for most purposes it is necessary to make stable cell lines that have incorporated the transfected MC2R into their genome. Using this model Elias et al were able to characterize a number of FGD-associated mutant MC2Rs, showing them to be associated with either loss of ligand binding or loss of signal-generating ability in comparison to the wild-type receptor, and thereby confirming the causative nature of these mutations.19 An interesting report is that of the first promoter variant in the MC2R causing FGD. Tsiotra et al described a patient with a nonsense mutation in one allele (a frameshift following glycine 217) and a T-to-C substitution in the 2 position of the MC2R promoter. Functional analysis of this substitution suggested that the promoter activity was reduced by 15%, which – assuming that the nonsense allele has zero activity – indicates that reduction of MC2R expression to 35% normal leads to disease.20 In contrast, earlier investigation of heterozygous carriers of missense mutations suggested that they retained entirely adequate MC2R activity and had no glucocorticoid deficiency.21 However, this promoter variant has been found in w10% of MC2R alleles in the population22 and has been proposed to influence pituitary–adrenal axis activity in normal adults and children.22,23
Mouse model Until recently no animal model of FGD existed. In 2007 Chida et al reported the characterization of a mouse Mc2r knockout.24 In most respects this animal exactly mimicked human FGD in having elevated ACTH and undetectable corticosterone. There was a high rate of neonatal lethality as a result of hypoglycaemia, and examination of adrenal histology was similar to that in humans, showing an atrophic zona fasciculata with a relatively preserved zona glomerulosa. Interestingly these mice do not show excessive longitudinal growth and in this sense do not parallel patients with MC2R mutations. The major difference between knockout mice and MC2R-deficient patients was that mice were deficient in aldosterone and their adrenals showed low expression levels of aldosterone synthase. Mineralocorticoid deficiency was also seen in POMC knockout animals, supporting the view that in rodents, ACTH plays a more significant role in zona glomerulosa development and function than is the case in the human.25,26 The basis for this distinction between the mouse and human is not clear. A recent report has suggested in two FGD patients with a homozygous nonsense mutation of the MC2R there is a degree of mineralocorticoid deficiency as indicated by a raised renin level.27 As mentioned, totally inactivating mutations such as these are very uncommon in FGD, but it is interesting to note that in our own studies 4/6 patients with homozygous nonsense or frameshift mutations had elevated plasma renin, and 1/6 had aldosterone deficiency even when stabilized on glucocorticoid replacement (Chan et al, unpublished). Despite the clear causative link between MC2R mutations and FGD that these studies provide, it remains clear that only a fraction of FGD – around 25% in our experience – is associated with biallelic MC2R mutation. Those cases linked to the MC2R are known as FGD type 1. Two studies have
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2 C
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Fig. 1. Schematic diagram of the possible actions of melanocortin 2 receptor accessory protein (MRAP) in supporting melanocortin 2 receptor (MC2R) function. (1) MRAP exists as an anti-parallel homodimer, and may have a chaperone-like function in assisting the correct folding of the MC2R in the endoplasmic reticulum. (2) MRAP may have an ‘escort’ function in assisting the trafficking of the correctly folded MC2R to the plasma membrane. (3) Finally, MRAP may form a trimeric structure with the MC2R at the cell surface and may be required for adrenocorticotropic hormone (ACTH) interaction and binding, or for generation of an intracellular signal.
investigated the possibility that the non-MC2R cases are not linked to the receptor, and both have shown that there is another genetic cause of FGD.28,29 MRAP – FGD type 2 Genetic studies using the technique of homozygosity mapping, in which a large number of informative genetic polymorphisms spread throughout the genome are studied, revealed a potential locus for FGD on chromosome 21. Analysis of all the genes in the critical interval at this locus revealed that a single gene was expressed only in the adrenal, but not in brain or liver. Sequence analysis of this gene in the index family showed a significant splice site mutation that was highly likely to lead to a defective protein. Sequencing of this gene in additional FGD families showed similar mutations in several other cases.30 The gene in question encoded a small protein of essentially unknown function. It had originally been identified by Xu et al as a protein that was induced when mouse 3T3-L1 cells were differentiated into adipocytes; it had been called the ‘fat-associated low-molecular-weight protein’ or Falp.31 We were able to show that this protein was physically associated with the MC2R as demonstrated by co-immunoprecipitation when both were transfected into heterologous cells. On confocal imaging it co-localized with MC2R at both the endoplasmic reticulum and at the plasma membrane. Importantly, in heterologous cells, when the Mc2r was co-transfected with the gene encoding Falp, a significant ACTH responsiveness was achieved.30 In comparison, Mc2r expression alone was unresponsive to ACTH. The mouse Y1 cell expresses this gene and has a functional endogenous ACTH response, which is lost when the gene is knocked down by RNA interference.32 Thus we believe that this gene – which we re-named ‘melanocortin 2 receptor accessory protein’ (MRAP) – is an essential cofactor for the MC2R. It is difficult to see how this advance could have been made without the study of FGD patients. It is still not entirely clear how MRAP works. Possible modes of action are summarized in Fig. 1 and are as follows:
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(a) it functions as a chaperone – a factor that assists the correct folding of the comparatively small and highly hydrophobic MC2R at the level of the endoplasmic reticulum (Fig. 1, point 1); (b) it functions as an escort protein, essentially escorting the MC2R to its destination at the plasma membrane (Fig. 1, point 2); (c) it acts as a co-receptor enabling the MC2R to bind to or signal in response to ACTH (Fig. 1, point 3). Existing data lend some support to each type of activity, and this is the subject of ongoing research. It seems highly probable that MRAP exhibits each of these functions to some extent. Perhaps one of the most interesting novel observations about MRAP is that it preferentially exists as an anti-parallel homodimer. Theoretical prediction tools suggest that the orientation of this small single transmembrane domain protein is with its N-terminus lying inside the cell. In elegant studies combining immunodetection and glycosylation analysis, Sebag & Hinkle demonstrated that it existed in a relatively stable dimer with each molecule in the dimer in opposing orientations.33 This is a unique structure in eukaryotes. Independently, Cooray et al had confirmed the dimer structure with a combination of co-immunoprecipitation and mass spectroscopic analysis of potential dimeric bands on gel electrophoresis.32 MRAP mutations do not account for a large proportion of FGD patients. In our studies about 15% of all FGD patients have biallelic MRAP mutations, and this is now known as FGD type 2. Others have also identified MRAP mutations34, and the possibility that an obese phenotype may characterize FGD patients with MRAP mutations has been raised.35 At this time it is very difficult to be clear about this, but there are certainly MRAP patients who are not overweight.
Other genetic loci Our genotyping analysis clearly shows that in many patients FGD does not segregate with MRAP or the MC2R, implying that other genes may cause the disease. Prior to the work that identified MRAP, a microsatellite-based whole genome analysis had suggested that two FGD families had FGD that localized to the long arm of chromosome 8.29 Since that time it has not been possible to link many other families to this locus, and although a number of candidate genes in this region have been studied, none has been shown to carry mutations. There also remain many families who clearly do not map to this region of the genome; hence further FGD genes remain to be identified.
Practice points primary adrenal failure in a child with a normal rennin–angiotensin–aldosterone axis is highly suggestive of a diagnosis of familial glucocorticoid deficiency persistent hypoglycaemia in a neonate should raise the suspicion of glucocorticoid deficiency establishing a genetic diagnosis of familial glucocorticoid deficiency is important both in providing reassurance that mineralocorticoid replacement is unnecessary and for genetic prediction and counselling over-replacement of glucocorticoids in an attempt to treat an elevated ACTH carries significant risk of increased morbidity, including growth retardation
Research agenda identify new genes for familial glucocorticoid deficiency develop an understanding of how MRAP works in enabling MC2R expression
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