Genetic Steroid Disorders || Animal Models of Adrenal Genetic Disorders
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peutic approaches.. Thereby, SF1 marks the steroidogenic precursors, which later become adrenal cortex and somatic cells in Genetic Steroid Disorders. http://dx.doi.org/10.1016/B978-0-12-416006-4.00026-0 Copyright 2014 Elsevier Inc. All rights reserved.323
MOUSE MODELS FOR CONGENITAL ADRENAL HYPOPLASIA
Cells within the coelomic epithelium give rise to the adrenogonadal primordium, which resembles the primi-tive organs for the adrenals and gonads. Proper adrenal development is dependent upon a cascade of molecular events that includes sequential gene expression, pres-ence of signaling molecules, and paracrine and endo-crine factors . Accordingly, defects in either of these mechanisms can result in adrenal agenesis or aplasia . In fact, a number of mouse models have been described in which absence of the adrenal gland has been part of
the gonads. Mice deficient in Sf1 die soon after birth as a result of primary adrenal insufficiency owing to complete absence of adrenal glands in addition to loss of gonads, pituitary gonadotropes, and ventrome-dial hypothalamus. The essential role of Sf1 for adrenal growth has also been underscored by studies in haplo-insufficient animals, which display a defect in specific adrenal growth paradigms  as well as in transgenic models where increase in Sf1 dosage has been shown to induce adrenal tumorigenesis . Interestingly, high expression levels of SF1 can also be regarded as a prognostic marker in human adrenal carcinogenesis . Taken together, these studies have placed SF1 as INTRODUCTION
Mouse models exhibit a wide range of possibilities for the investigation of adrenocortical function and growth. Incidental discovery of adrenal tumors or disorders in steroidogenesis in genetically modified animals can provide clues on pathways involved in adrenal func-tion that would not have been predicted on the basis of structural analysis or in vitro exploration. Mouse mod-els can also be used to verify functional significance of a given candidate gene for adrenal growth and steroido-genesis in vivo through targeted genetic modification. Furthermore, incidence of adrenal disorders in inbred mouse strains can serve as the starting point for genetic approaches to identify the underlying genetic cause. In contrast, differences in phenotypic alterations in humans and mice following the same genetic cause can provide important insights into genetic or environmental modi-fiers that impact disease severity. In this chapter we will provide an overview on adrenocortical disease models relevant for either mechanistic studies or future thera-
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13Animal Models of Adr
Felix BeEndocrine Research Unit, Medizinische Klinik und Poliklinitheir phenotype. These alterations mostly include tran-scription factors that are involved in the developmental program of the urogenital ridge as the common precur-sor of the adrenal cortex, the gonads, and the kidney, or, more specifically, that of the adrenogonadal primordium or the adrenal gland itself. Examples include Wilms tumor gene 1 (Wt1) , Pbx1 [4,5], steroidogenic factor 1 (Sf1) , and Cited2 . However, in many instances (including WT1, PBX1 and CITED2) an adrenal phe-notype in affected human patients has been absent or has not been reported. Conversely, in the case of DAX1 affected patients typically suffer from adrenal insuffi-ciency because of cytomegalic adrenal hypoplasia [8,9], while adrenal function is normal in mice with targeted deletions of Dax1 .
Despite these differences there are clear examples for the convergence of genotype/phenotype correla-tion among patients and the according mouse models (Table 13A.1). One of the best studied models includes that of mice with targeted deletions of Sf1 . In the mouse embryos, the adrenogonadal primordium starts to express SF1 at approximately 9.0 days post coitum
P T E R
Aenal Genetic Disordersuschleink IV, Klinikum der Universitt Mnchen, Munich, Germany
13A. ANIMAL MODELS OF ADRENAL GENETIC DISORDERS324
autosomal recessive disorder resulting from resistance to the action of adrenocorticotropic hormone (ACTH) on
clinical phenotype of FGD type 1 patients but similar to that of mice deficient of POMC , Mc2r knockout mice the adrenal cortex . Affected individuals present with signs of cortisol deficit such as severe hypoglycemia or severe infection in infancy or childhood. In contrast to other forms of primary adrenal insufficiency, mineralo-corticoid secretion is typically not compromised.
Mutations of the ACTH receptor (melanocortin 2 receptor, MC2R) are responsible for 25% of FGD cases (FGD type 1) , while mutations of the MC2R acces-sory protein MRAP, which plays a role in the traffick-ing of MC2R from the endoplasmic reticulum to the cell surface, account for around 15% of FGD cases (FGD type 2) [17,18]. Only recently, mutations in NNT (encoding nicotinamide nucleotide transhydrogenase) have been added to the list of genes causing FGD .
In 2007 mice with targeted deletions of the Mc2r were described which resemble most of the features of patients with FGD type 1 . Interestingly, while most knockout animals die from lung failure, depending on the back-ground strain, some Mc2r-deficient animals survive until adulthood even without glucocorticoid substitution .
were found to secrete aldosterone at reduced levels.These findings are indicative of a more significant role
of ACTH in zona glomerulosa development and func-tion in rodents than is the case in the human. However, there are examples of FGD patients with a homozygous nonsense mutation of the MC2R with a slight degree of mineralocorticoid deficiency as indicated by a raised renin level . Therefore, it is possible that the pheno-type in Mc2r null animals represents a disease severity that is not necessarily found in patients with some resid-ual MC2R activity.
While no mouse model dedicated to FGD type 2 has yet been introduced in the literature, animals with NNT mutations have been described in the context of NNTs newly established role in adrenal steroidogenesis . NNT represents a highly conserved gene encoding an integral protein of the inner mitochondrial membrane involved in NADPH production. Upon identification of NNT mutations in families affected by FGD, the adre-nal phenotype of mice carrying a natural occurring NNT the crucial determinant of adrenocortical growth and differentiation.
MOUSE MODELS FOR FAMILIAL GLUCOCORTICOID DEFICIENCY (ACTH
Familial glucocorticoid deficiency (FGD) [Online Mendelian Inheritance in Man (OMIM) #202200] is an
TABLE 13A.1 Examples of Single Gene Defects in Inherited FormsHyperplasia
Congenital adrenal hypoplasia 300200300473
Familial glucocorticoid deficiency Type 1 Type 2 Type 3 Triple A syndrome
Congenital adrenal hyperplasia 21-Hydroxylase deficiency 11-Hydroxylase deficiency 3-Hydroxysteroid dehydrogenase deficiency 17-Hydroxylase deficiency Lipoid adrenal hyperplasia
X-linked adrenoleucodystrophy 300371
Autoimmune polyglandular syndrome type 1 240300
* No reported adrenal phenotype;# no functional relevant adrenal phenotype.(Adapted from ref. )In addition to its steroidogenic-inducing properties, ACTH has been implicated to be required for proper adrenal development and growth . Therefore, it is not surprising that Mc2r knockout mice are affected with significant adrenocortical hypoplasia in comparison to wild-type controls . Ultrastructural investigation of cells from the zona fasciculata in Mc2r knockout mice revealed a diminished number of lipid droplets as well as structural changes in mitochondrial appearance com-pared with that of wild-type animals. In contrast to the
of Primary Adrenal Insufficiency and/or Congenital Adrenal
Gene Mouse model Reference
NR0B1 (DAX1)NR5A1 (SF1)
ABCD1 Knockout  *
AIRE Knockout  #
MouSE MoDElS FoR ConGEnI
mutation was characterized . Thereby, mutant mice were found to have lower basal and stimulated levels of corticosterone than their wild-type counterparts. While adrenal cortices of affected animals displayed a slightly disorganized zona fasciculata with higher levels of apoptosis, no observable differences in the levels of the steroidogenic enzymes CYP11A1 and CYP11B1 were evident. Overall, these findings suggest that defects in oxidative stress response can result in impaired adreno-cortical function.
In line with this pathophysiological mechanism, adrenal insufficiency in the context of triple A syndrome (also known as Allgrove syndrome; OMIM #231550) has been associated with an impaired oxidative stress response. The triple A syndrome is a rare autosomal recessive disorder characterized by the clinical triad of achalasia of the cardia, alacrima, and ACTH-resistant adrenal insufficiency. The predicted product of AAAS, ALADIN (for alacrimaachalasiaadrenal insufficiency neurologic disorder), belongs to the WD-repeat fam-ily of regulatory proteins . Defects in this nuclear pore protein result in impaired nuclear import of DNA repair and antioxidant proteins, thereby rendering the cells more susceptible to oxidative stress . Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome . Therefore, the wide range of disease severity, the obvious lack of a genotype/phenotype relationship in human patients, and the absence of a drastic phenotype in Aaas knock-out animals suggest that additional factors, such as environmental influences or modifier genes, contribute to the disease course to a greater extent than previously anticipated.
MOUSE MODELS FOR CONGENITAL ADRENAL HYPERPLASIA
In contrast to humans, the adrenal steroid biosynthe-sis in rodents is characterized by a lack of 17-hydroxy-lase activity . Therefore, pregnenolone is catalyzed by 21-hydroxylase (Cyp21) to corticosterone as the major glucocorticoid, and corticosterone is further hydroxyl-ated to aldosterone, which is the main active mineralo-corticoid in the mouse . Owing to the lack of adrenal 17-hydroxylase activity within the mouse adrenals, ste-roid precursors do not, however, shunt into the andro-gen pathway and no clinical effects (such as virilization of the external genitalia) can be expected in animals that lack upstream steroidogenic enzyme activity. Despite these well-known differences for a number of steroido-genic defects specific mouse models for congenital adrenal hyperplasia (CAH) have been generated. Their phenotypic work-up has provided a number of insights TAl ADREnAl HyPERPlASIA 325
into genetics and/or functional characteristics of the specific diseases.
21-Hydroxylase Deficiency (21-OHD)
The gene coding for human CYP21 (CYP21A2) is located in the major histocompatibility complex (MHC) on chromosome 6p21.3 while a non-functional pseu-dogene (CYP21A1-P) is located in close proximity . While both genes show a 98% sequence similarity, CYP21A1-P carries several inactivating mutations. In humans, mutations causing 21-OHD are most frequently the result of complex recombination events between CYP21A2 and CYP21A1-P. Interestingly, the human and murine 21-hydroxylase gene locus shows a high level of homology: in both species within the class III region of the MHC two structurally homologous genes for 21-hydroxylase are located in the same region .
In 1987 a deleterious phenotype in mice that had undergone meiotic recombination in parts of the H-2 class III region was described, and recombination between homologous chromosomes of the H-2a and H-2wm7 haplotypes causing the deletion of complement compo-nent C4 and the 21-hydroxylase gene was assumed . Following extensive genetic analysis of the Cyp21 locus, it was demonstrated that the underlying molecular defect of 21-OHD in the congenic H-2aw18 mouse strain in fact consists of a complex gene rearrangement caused by unequal crossing over, which generates a hybrid gene consisting of a truncated active gene and pseudo-gene . Multiple missense mutations and a nonsense point mutation resulting in a premature stop codon were found to be introduced in the hybrid gene neighboring several pseudogene-specific point mutations.
As expected from a phenotype including adrenal insufficiency, newborn aw18 homozygous mice (H-2aw18) did not survive the early postnatal stage. Furthermore, affected animals presented with evident morphological changes in the adrenal glands, indicating adrenocortical hyperplasia . Therefore, in addition to various ste-roid substitution regimens to prolong survival, H-2aw18 animals were also utilized to study therapeutic strate-gies such as transgenic rescues through overexpression of Cyp21 . Using this approach, prolonged survival without further substitution could be achieved in vari-ous proportions of the transgenic animals (between 20% and 80% depending on the integration of the transgene). Finally, H-2aw18 animals were utilized as a model of adre-nal insufficiency to dissect the interrelationship between adrenal cortical and medullary function. Specifically, it could be demonstrated that catecholamine secretion is severely affected in Cyp21-deficient animals . Of note, similar functional alterations between the model organism and affected patients were appreciated only after implementation of dedicated clinical protocols .
13A. ANIMAL MODELS OF ADRENAL GENETIC DISORDERS32611-Hydroxylase DeficiencyPatients with CAH due to mutations of the
11-hydroxylase gene (CYP11B1), the final enzyme in the glucocorticoid biosynthetic pathway, are characterized by glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamopituitary adrenal activity, which are shunted into the adrenal androgen synthesis pathway, and excess min-eralocorticoid activity caused by the accumulation of deoxycorticosterone .
Specific CAH mouse model animals were created by targeted replacement of Cyp11b1 with a gene for a fluo-rescent protein. As expected, the urinary steroid profile of Cyp11b1 knockout mice was similar to that of patients carrying null mutations of the 11-hydroxylase gene with evidence of glucocorticoid depletion and miner-alocorticoid and progesterone excess . Through a 30-fold increase in deoxycorticosterone its weak miner-alocorticoid activity resulted in a significant hypokale-mia, decrease in plasma renin concentration, and lower levels of aldosterone in affected mice. Furthermore, adrenal hyperplasia was evident in homozygous knock-out animals. Thereby, this mouse model resembles many aspects of the clinical phenotype seen in patients with CAH and could aid in future mechanistic or therapeutic studies.