Edition 70 - October 2018 / Bibliographic Reviews

Bibliographic Reviews – Ed. 70

Marco A. Rivarola y Alicia Belgorosky

Servicio de Endocrinología, Hospital de Pediatria Garrahan, Buenos Aires, Argentina

We have selected the following publications for this issue of Endocrinología Pediátrica On line.

346

J Clin Endocrinol Metab 103: 2079–2082, 2018
45,X/46,XY Mosaicism Presenting With Isolated Unilateral Cryptorchidism and a Normal Blood Karyotype. Grazia Morandi,1,2* Manuela Cerbone,1,2* Elisa B. Lamback,1,2 Eleni Rapti,1,2, and Mehul T. Dattani1,2 , 1Department of Endocrinology, Great Ormond Street Hospital for Children, London WC1N 3JH, United Kingdom; and 2Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Program, University College, London Great Ormond Street Institute of Child Health, London WC1N 1EH, United Kingdom

Abstract
CONTEXT: 45,X/46,XY mosaicism is a disorder of sex development leading to abnormal gonadal development and to unpredictable genital phenotype, growth, and pubertal development. CASE DESCRIPTION: A 2-year-old male presented with a right impalpable testis. Blood karyotype was 46,XY. A laparoscopy performed for right orchidopexy revealed a right streak gonad with mullerian structures, which was removed, whereas on the left side, a normal descended testis was present. The karyotype of the removed gonad was 45,X/46,XY. The child grew along the second centile, within the midparental height (MPH) range, until the time of puberty, when linear growth worsened due to a lack of a pubertal growth spurt, and growth hormone (GH) therapy was initiated. He developed spontaneous puberty (13 years of age) and showed normal pubertal progression. However, from the age of 15 years, he had low normal testosterone, raised follicle-stimulating hormone, and reduction of inhibin B, possibly suggestive of declining testicular function. His final height was 22.24 standard deviation score (SDS) (22.4 SDS at GH start; MPH 21.6 SDS). CONCLUSIONS: On conclusions, this case describes a mild male phenotype associated with 45,X/46,XY mosaicism, characterized by unilateral cryptorchidism, spontaneous onset of puberty, and normal blood karyotype. The case illustrates the difficulties inherent in making a diagnosis of 45,X/46,XY mosaicism when there is no genital ambiguity and makes the point that growth and testicular impairment may occur, mostly manifesting during adolescence. An early diagnosis is crucial to initiate careful monitoring for growth and pubertal disorders, increased tumor risk, and fertility issues commonly seen in these children.

Comments.
45,X/46,XY mosaicism is a rare genetic finding, leading to complex management issues with respect to sex of rearing and to unpredictability of the genital and gonadal phenotype of the affected children. In this particular individual, blood karyotype was 46,XY, compatible with the normal masculinization of external genitalia which took place during his fetal life. Moreover, he showed one normally descended testis but a non-palpable testis on the other side. At surgery, at 2 years of age, one streak gonad and a normally looking testis were observed in the scrotum. Interestingly, a 46,XY/45,X karyotype was found in the streak gonad. This tissue mosaicism could be present in other organs, such as the brain, liver, heart, etc., with unpredictable future health consequences for this boy. So far, he has had a normal male spontaneous puberty, but although the preserved testis maintains sufficient Leydig cell function to induce puberty, the current hormonal profile suggests declining testicular function, raising the possibility for more impairment in the future, and concerns for future fertility.
45,X/46,XY patients exhibit an increased tumor risk, not only when abdominal undifferentiated gonads are present but also in dysgenetic or “apparently normal” intrascrotal testes. In dysgenetic gonads, the formation of in situ gonadoblastoma or of an invasive germ cell tumor occurs in 15% to 52% of cases. Although in the past gonadectomy was recommended, a more conservative approach is now preferred to allow endogenous hormone production and therefore spontaneous puberty and to delay irreversible surgery until adulthood.
Growth seems to be consistently impaired in these patients. Variable outcomes have been reported on GH treatment, with a recent review concluding that although there may be some short-term to midterm improvement in growth, long-term data on adult height are disappointing. However, supra-physiological GH doses and earlier commencement might result in better outcomes, as could have possibly been the case in this patient.

347

J Clin Res Pediatr Endocrinol. 2017 Jun 1;9(2):95-100. doi: 10.4274/jcrpe.3908.
Hypogonadotropic Hypogonadism due to Novel FGFR1 Mutations. Akkus G, Kotan LD, Durmaz E, Mengen E, Turan İ, Ulubay A, Gürbüz F, Yüksel B, Tetiker T, Topaloğlu AK. Çukurova University Faculty of Medicine, Division of Pediatric Endocrinology, Adana, Turkey, E-mail: ktopaloglu@cu.edu.tr.

Abstract
OBJECTIVE: The underlying genetic etiology of hypogonadotropic hypogonadism (HH) is heterogeneous. Fibroblast growth factor signaling is pivotal in the ontogeny of gonadotropin-releasing hormone neurons. Loss-of-function mutations in FGFR1 gene cause variable HH phenotypes encompassing pubertal delay to idiopathic HH (IHH) or Kallmann syndrome (KS). As FGFR1 mutations are common, recognizing mutations and associated phenotypes may enhance clinical management. METHODS: Using a candidate gene approach, we screened 52 IHH/KS patients. RESULTS: We identified three novel (IVS3-1G>C, p.W2X, and p.R209C) FGFR1 gene mutations. Despite predictive null protein function, patients from the novel mutation families had normosmic IHH with variable reproductive and non-reproductive phenotype. CONCLUSION: These findings further emphasize the great variability of FGFR1 mutation phenotypes in IHH/KS.

Comments.
To date, at least 17 genes have been associated with KS and these include KAL1, FGFR1, PROK2, PROKR2, FGF8, HS6ST1, CHD7, WDR11, SEMA3A, FGF17, IL17RD, DUSP6, SPRY4, FLRT3, NELF, FEZF1, and CCDC141.
KS is most commonly caused by mutations in anosmin 1 encoded by KAL1. FGFR1 encodes a tyrosine kinase receptor that mediates fibroblast growth factor signaling. Presence of various congenital anomalies which are not associated with the reproductive system such as defects in kidney and tooth differentiation, ear and palate morphogenesis. Development of interhemispheric or cortico-spinal axonal tracts, encountered in a proportion of KS patients, due to FGFR1 mutations indicates that the fibroblast growth factor signaling plays important roles in many other developmental processes. The precise roles played by KS genes in these processes are not known. FGFR1 has been shown to be a key factor for angiogenesis, embryogenic development, and wound healing.
They screened 52 IHH/KS patients (36 male, 16 female) in their cohort. The majority of patients (n=38) were normosmic IHH (nIHH) and the remaining 12 were cases of anosmic or hyposmic IHH (KS). Diagnosis of IHH/KS was based on delayed or absent spontaneous puberty by age 13 in girls (Tanner breast stage 1) and by age 14 in boys (testicular volume

Family 1
The proband (II-4) was a 17-year-old female patient who was referred for lack of breast development and primary amenorrhea. Chromosome analysis showed a 46,XX karyotype. Her pelvic ultrasonography and cranial magnetic resonance imaging (MRI) results were normal. One of her sisters (II-2), a 22-year-old female who had complaints of absent breast enlargement and primary amenorrhea was also diagnosed as a case of IHH. Her sense of smell was normal. Her karyotype was 46,XX. Her serum plasma estradiol, LH, and FSH levels were prepubertal. Their parents were healthy cousins. One of the proband’s half-sisters (II-1), a 16-year-old female whose chief complaints were delayed puberty and primary amenorrhea, was also diagnosed to have IHH.
The affected three sisters and their unaffected mother from family 1 were found to have the heterozygous IVS3-1G>C (g.G38886C) novel mutation. This splicing mutation in intron 3 is predicted to cause skipping of exon 4, eventually resulting in a totally different protein product

Family 2
The proband (II-1) was an 18-year-old female patient who was also referred for lack of breast enlargement and primary amenorrea. Her sense of smell was normal. GnRH stimulation test elicited peak levels of FSH and LH as 2.8 and 2.1 mIU/ mL, respectively. One of her siblings (II-2), a 15-year-old boy, was also referred for delayed puberty. His testicular volumes were 2 mL bilaterally. Axillary and pubic hair were at stage 1. He reported a normal sense of smell. His reproductive hormone levels were prepubertal. Her affected siblings had the p.W2X (c.G6A) mutation in the heterozygous state.

Family 3
The proband (II-1) is a 14-year-old male patient who was referred for micropenis and absence of erections. He had a decreased sense of smell. Pubic and axillary hair were at stage 3. Testicular volumes were 2 mL bilaterally. He had no midline anomalies. His height and weight were 54 kg (25-50th percentile) and 160 cm (25-50th percentile), respectively. His basal testosterone and gonadotropin levels were prepubertal. His karyotype was 46,XY. His cranial MRI was normal. A paternal uncle (I-2) of his also reportedly suffers from absent puberty and anosmia. This family is ethnically Arabic.
Three cases have been reported so far of discordance between blood (46,XY) and gonadal (45,X/46,XY) karyotype. Two patients presented with ambiguous genitalia at the age of 1 year and with a left undescended testis and penoscrotal hypospadias at the age of 16 years, respectively. The third case was a neonate with ambiguous genitalia in whom chromosome Y–derived sequences were detected in the dysgenetic gonad and skin fibroblasts. Sanger sequence analysis of the entire coding regions of FGFR1 (HGNC:3688 NM_001174063, NP_001167534) revealed three novel mutations. Patient showed biochemical features of possible declining testicular function. It is well known that most 45,X/46,XY patients born with ambiguous genitalia are infertile. Even in patients with mild phenotypes in childhood, spermatogenesis may be impaired in adulthood

348

J Clin Endocrinol Metab. 2018 Apr 1;103(4):1273-1276. doi: 10.1210/jc.2017-02636.
Discordance in the Dependence on Kisspeptin Signaling in Mini Puberty vs Adolescent Puberty: Human Genetic Evidence. Shahab M1, Lippincott M1, Chan YM2, Davies A1, Merino PM3, Plummer L1, Mericq V3, Seminara S1.1 Harvard Reproductive Sciences Center and Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts. 2 Division of Endocrinology, Department of Medicine, Boston Children’s Hospital, Boston, Massachusetts. 3 Institute of Maternal and Child Research, University of Chile, Santiago, Chile.

Abstract
Context: Hypothalamic kisspeptin signaling plays a critical role in the initiation and maintenance of reproductive function. Biallelic mutations in the coding sequence of KISS1R (GPR54) have been identified in patients with idiopathic hypogonadotropic hypogonadism, but it is unknown whether biallelic variants can also be associated with related reproductive disorders. Case Description:
A missense homozygous variant (c.890G>T p.R297L) in KISS1R was identified in a child who presented with microphallus and bilateral cryptorchidism. This variant has been reported to reduce, but not abolish, postreceptor signaling in vitro. Biochemical evaluation during the neonatal period revealed low testosterone levels. By 11 years and 8 months, the boy began demonstrating increases in testicular volume. By 17 years and 3 months, his testicular volume was 20 mL; his penile length was 7.3 cm; and he had adult levels of circulating gonadotropins and testosterone. Conclusion: This case report associates biallelic loss-of-function mutations in KISS1R with normal timing of adolescent puberty. Because these coding sequence variants occurred in a patient with microphallus and cryptorchidism, they demonstrate different levels of dependence of the hypothalamic-pituitary-gonadal cascade on kisspeptin signaling at distinct times in the reproductive life span. The suppression of the hypothalamic-pituitary-gonadal cascade during early life but not adolescence suggests that the mini puberty of infancy depends more on kisspeptin-induced, gonadotropin-releasing hormone-induced luteinizing hormone secretion than does adolescent puberty.

Comments
Loss-of-function mutations in KISS1R have been described in idiopathic hypogonadotropic hypogonadism (IHH), a disease characterized by deficient release of gonadotropin releasing hormone (GnRH). Despite the importance of kisspeptin signaling in modulating GnRH secretion, coding sequence mutations in KISS1R are relatively rare causes of IHH compared with mutations in other genes associated with the disease. To determine the breadth of reproductive phenotypes associated with KISS1R variants, authors examined the coding sequence of KISS1R in >1600 patients with a spectrum of reproductive phenotypes.
Subjects (N = 1337 probands) with IHH were referred to the Reproductive Endocrine Unit at Massachusetts General Hospital for participation in genetic studies.
Of the 1337 individuals, nine probands (eight male, one female) were identified with biallelic rare variants in the coding sequence of KISS1R (eight homozygous, one compound heterozygous). Eight of the nine probands had IHH; however, one proband, the subject of this case report, did not have IHH. He is described as it follows:
Of Chilean descent, he was born at term (body weight (3.02 kg, with microphallus and bilateral cryptorchidism He had a normal karyotype, and normal olfactory bulbs by brain imaging. At 2 months, the boy had low testosterone (13 ng/dL), with LH level of 2.9 mIU/mL and FSH level of 1.9 mIU/mL. The subject was subsequently treated with testosterone enanthenate (75 mg/mo for 3 months); his penile length increased to 1.7 cm. He underwent right orchidopexy at 2.5 y and left orchidopexy at 4 y of age. He received additional testosterone treatment for microphallus during the rest of prepuberty, and finally developed a spontaneous, but only partial puberty. The subject later underwent screening of 14 genes associated with IHH, which revealed a rare homozygous missense variant R297L in KISS1R. This variant has a frequency of one in 4320. A R297L mutant construct was shown to lead to a modest but significant reduction in kisspeptin-dependent Ca++ mobilization in vitro. R297L has a maximal activity of 85% of the wild type. Both unaffected parents carried the same mutation in a heterozygous form.
Loss-of-function variants in KISS1R were initially identified in patients with hypogonadotropic hypogonadism. Two case reports have also described KISS1R variants with an abnormal mini puberty. Taken together, the literature appears to support a role for kisspeptin as a powerful stimulus of GnRH-induced LH secretion across reproductive life.
The current index subject challenges the notion that mini puberty and adolescent puberty are equally dependent on kisspeptin signaling. Subject 233 had an abnormal mini puberty, as evidenced by low testosterone levels; however, he underwent normal timing of adolescent puberty. The discordance between the mini puberty and adolescent puberty demonstrates that the functioning of GnRH neurons in mini puberty may be more sensitive to the integrity of kisspeptin signaling compared with the functioning of GnRH neurons during adolescent puberty.
This report demonstrates diversity in the phenotypic expression of biallelic KISS1R mutations and demonstrates that such mutations can be associated with absent mini puberty but with normal onset of adolescent puberty. Furthermore, this report suggests that although pubertal activation of the hypothalamic pituitary-gonadal cascade requires kisspeptin signaling, even more robust kisspeptin drive is necessary for activation of the cascade in infancy.

 

349

Horm Res Paediatr. 2018;89(1):13-21. doi: 10.1159/000481777.
Recurrent Copy Number Variants Associated with Syndromic Short Stature of Unknown Cause. Homma TK1,2, Krepischi ACV3, Furuya TK4, Honjo RS5, Malaquias AC6, Bertola DR5, Costa SS3, Canton AP1, Roela RA4, Freire BL2, Kim CA5, Rosenberg C3, Jorge AAL1,2. 1 Unidade de Endocrinologia Genetica, Laboratorio de Endocrinologia Celular e Molecular LIM25, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), Sao Paulo, Brazil. 2 Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular LIM42, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), Sao Paulo, Brazil. 3 Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo (IB-USP), Sao Paulo, Brazil. 4 Laboratorio de Oncologia Experimental LIM24, Departamento de Radiologia e Oncologia, Centro de Investigação Translacional em Oncologia do Instituto do Cancer do Estado de Sao Paulo (CTO/ICESP), Faculdade de Medicina da Universidade de São Paulo (FMUSP), Sao Paulo, Brazil. 5 Unidade de Genetica do Instituto da Criança, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), Sao Paulo, Brazil. 6 Unidade de Endocrinologia Pediatrica, Departamento de Pediatria, Irmandade da Santa Casa de Misericórdia de São Paulo, Faculdade de Ciências Médicas da Santa Casa de São Paulo, Sao Paulo, Brazil.

Abstract
BACKGROUND/AIMS: Genetic imbalances are responsible for many cases of short stature of unknown etiology. This study aims to identify recurrent pathogenic copy number variants (CNVs) in patients with syndromic short stature of unknown cause. METHODS: We selected 229 children with short stature and dysmorphic features, developmental delay, and/or intellectual disability, but without a recognized syndrome. All patients were evaluated by chromosomal microarray (array-based comparative genomic  hybridization/single nucleotide polymorphism array). Additionally, we searched databases and previous studies to recover recurrent pathogenic CNVs associated with short stature. RESULTS: We identified 32 pathogenic/probably pathogenic CNVs in 229 patients. By reviewing the literature, we selected 4 previous studies which evaluated CNVs in cohorts of patients with short stature. Taken together, there were 671 patients with short stature of unknown cause evaluated by chromosomal microarray. Pathogenic/probably pathogenic CNVs were identified in 87 patients (13%). Seven recurrent CNVs, 22q11.21, 15q26, 1p36.33, Xp22.33, 17p13.3, 1q21.1, 2q24.2, were observed. They are responsible for about 40% of all pathogenic/probably pathogenic genomic imbalances found in short stature patients of unknown cause. CONCLUSION: CNVs seem to play a significant role in patients with short stature. Chromosomal microarray should be used as a diagnostic tool for evaluation of growth disorders, especially for syndromic short stature of unknown cause.

Comments
The genetic evaluation of short stature is important not only for diagnosis, but also to provide additional infor­mation to the patients and their families regarding natu­ral history, prognosis, available treatment, and precise ge­netic counseling.
Analysis of 229 Patients with Short Stature of Unknown Cause:
Among 229 patients with short stature studied by chromosomal microarray analysis, we observed 77 rare CNVs in 73 patients (1–4 per patient). We classified 25 CNVs as pathogenic and 7 as probably pathogenic.
Analysis of Recurrent CNVs Associated with Risk for Short Stature Reviewing the literature, we found 6,029 studies which used chromosome microarray technologies. Of these 258 included patients with growth disorders or short stature. We performed a manual curation based on abstracts. They were analyzed according to our including and ex­cluding criteria previously cited. This strategy resulted in a final selection of 4 eligible studies to be included in the CNV recurrence analysis [8–11] (Table 2). These studies and the present one reported a total of 671 patients with short stature of unknown cause evaluated by chromo­some microarray technologies (SNPa or aCGH). Patho­genic/probably pathogenic CNVs were identified in 87 patients (13%; 95% confidence interval of 10.4–15.5%). We identified 7 recurrent CNVs in short stature.
In conclusion, chromosomal microarray made it pos­sible to identify the etiology of syndromic short stature condition in 13% of the cases in whom the clinical ap­proach was unable to establish a diagnosis due to rela­tively nonspecific features or a mild phenotype. More­over, we observed some recurrent CNVs associated with this condition, corresponding to 40.2% of the total patho­genic or probably pathogenic CNVs described in all stud­ies in which the short stature phenotype was the main emphasized phenotype. Among these recurrent CNVs, we identified deletions involving genes clearly associated with the short stature phenotype: IGF1R and SHOX [24, 29]. Additionally, novel candidate genes were suggested, and further studies should be performed to evaluate their role in growth disorders.

 

350

J Clin Endocrinol Metab. 2018 Aug 1;103(8):2794-2801. doi: 10.1210/jc.2018-01205.
GH Therapy in Childhood Cancer Survivors: A Systematic Review and Meta-Analysis. Tamhane S1,2, Sfeir JG1,2, Kittah NEN2, Jasim S1,3, Chemaitilly W4, Cohen LE5, Murad MH1.
1 Evidence-Based Practice Center, Mayo Clinic, Rochester, Minnesota. 2 Division of Endocrinology, Mayo Clinic, Rochester, Minnesota. 3 Division of Endocrinology, Metabolism and Lipid Research, Washington University in St. Louis, School of Medicine, St. Louis, Missouri. 4 Department of Pediatric Medicine, Division of Endocrinology, St. Jude Children’s Research Hospital, Memphis, Tennessee. 5 Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts.

Abstract
Background: GH deficiency (GHD) is common among childhood cancer survivors (CCSs) with history of tumors, surgery, and/or radiotherapy involving the hypothalamus-pituitary region. They aimed to evaluate the effects of GH therapy (GHT) in CCSs on adult height, risk of diabetes mellitus, abnormal lipids, metabolic syndrome, quality of life, secondary tumors, and disease recurrence.
Methods: They searched multiple databases for randomized and observational studies. Pairs of reviewers independently selected studies and collected data. Random effects meta-analysis was used to pool outcomes across the studies.
Results: They included 29 observational studies at moderate to high risk of bias. Sixteen studies compared CCSs on GHT with those not on GHT (512 patients, GH dose: 0.3 to 0.9 IU/kg/week). GHT was significantly associated with height gain [standard deviation score, 0.61; 95% CI, 0.08 to 1.13] and was not significantly associated with the occurrence of secondary tumors [odds ratio (OR), 1.10; 95% CI, 0.72 to 1.67] or tumor recurrence (OR, 0.57; 95% CI, 0.31 to 1.02). Thirteen studies compared CCSs on GHT with normal age- or sex-matched controls or controls with idiopathic GHD or short stature. GHT was associated with either improved or unchanged risk of diabetes, lipid profiles, and metabolic syndrome. GHT was associated with improvements in quality of life. Conclusion: CCSs treated with GHT gain height compared with the untreated controls. GHT may improve lipid profiles and quality of life and does not appear to increase the risk of diabetes or the development of secondary tumors, although close monitoring for such complications remains warranted due to uncertainty in the current evidence.

Comments
The survival rates of children treated for cancer have improved significantly over the years. GH deficiency (GHD) is common among these children, especially among childhood cancer survivors (CCSs) with tumors/surgery in the hypothalamic-pituitary (HP) region, CCSs exposed to HP radiation or CCSs exposed to cranial (CIR), craniospinal (CSI), or total body irradiation.
Eligibility criteria We searched for cohort studies, case series, randomized clinical trials, and meta-analysis evaluating outcomes related to adult height, risk of diabetes mellitus, abnormal lipids, metabolic syndrome, QOL, secondary tumors, and disease recurrence in CCSs receiving GHT compared with those not receiving GHT.
Main findings GHT in CCSs is associated with a statistically significant gain in height and no apparent increases in the occurrence of secondary tumors or recurrence. There was either an improvement or no difference in the risk of diabetes, lipid fractions, metabolic syndrome, and QOL
However, the effect of GHT on height was heterogeneous, CCSs gain variable improvements in height based on other factors, such as spinal injury (radiotherapy, scoliosis). CCSs treated with GHT showed either lower height gain or comparable growth velocity. In patients with medulloblastoma, adult heights but not sitting heights were improved with GHT.
The strengths of this review relate to the comprehensive literature search, a priori–established protocol and the duplicate process of study selection and appraisal. However, the available studies were observational with inherent limitations. Most studies assessing GHT have a relatively short follow-up duration, which can affect the outcome assessment dealing with the risk of recurrence and the incidence of secondary neoplasms. In the study by Swerdlow (36), however, there was no indication of increased cancer risk with longer follow-up durations. Furthermore, the relationship between the dose and duration response of GHT and cancer risks has been clearly evaluated in only a few studies, which did not note increased cancer risks. The meta-analysis for outcomes related to height and recurrence risks has moderate to high heterogeneity.

351

J Biol Chem. 2015 Nov 13;290(46):27972-85.
Adrenocorticotropic Hormone (ACTH) Responses Require (in Addition to its Receptor, MC2) Actions of the Melanocortin-2 Receptor Accessory Protein (MRAP) on the Extracellular Surface of the Plasma Membrane. Malik S1, Dolan TM1, Maben ZJ1, Hinkle PM2. 1 From the Department of Pharmacology and Physiology, University of Rochester Medical Center Rochester, New York 14642. 2 From the Department of Pharmacology and Physiology, University of Rochester Medical Center Rochester, New York 14642.

Abstract
The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal.

Comments
Familial glucocorticoid deficiency (FGD) or hereditary unresponsiveness to ACTH is characterized by isolated glucocorticoid deficiency. It is an autosomal recessive disorder resulting from ACTH resistance, typically presenting between the neonatal period and late childhood with hyperpigmentation, hypoglycaemia and seizure. The first inactivating melanocortin 2 receptor (MC2R) mutations in FGD were described in 1993. Since then, multiple mutations have been identified throughout the receptor, the majority of which are homozygous or compound heterozygous missense mutations; FGD resulting from MC2R mutations accounts for 25% of all FGD and is now known as FGD type 1 (OMIM#202200). FGD type 2 (OMIM*609196) describes a group of patients with normal MC2R but with mutation in the melanocortin 2 receptor accessory protein (MRAP) which is required for MC2R trafficking and function and this accounts for 20% of FGD and is now known as FGD type 2.
The fusion protein approach has provided conclusive evidence that signaling by the MC2 receptor requires MRAP actions on the outside of the cell where it is poised to affect ACTH binding and receptor activation. Two regions of ACTH are particularly important for activating the MC2 receptor: the His-Phe-Arg-Trp pharmacophore (residues 6–9 of ACTH) and Lys-Lys-Arg-Arg-Pro (residues16–20). Although no MC receptor structure has been solved, analyses of evolutionary patterns, responses to ACTH analogs, and effects of receptor mutations have led to structural models in which the ACTH peptide interacts with amino acids at the extracellular face of multiple transmembrane helices. Liang et al. (Gen. Comp. Endocrinol. 181, 203–210) have suggested that binding is a two-step process in which the KKRRP segment of ACTH engages the receptor first and causes conformational changes that enable HFRW binding and G protein coupling. The MRAP partner in the Nout orientation could contribute at either step.


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