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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 10 3592-3596
Copyright © 1998 by The Endocrine Society

Original Studies

Exclusion of the Adrenocorticotropin (ACTH) Receptor (MC2R) Locus in Some Families with ACTH Resistance but No Mutations of the MC2R Coding Sequence (Familial Glucocorticoid Deficiency Type 2)

Danielle Naville, Angela Weber, Emmanuelle Genin1, Philippe Durand, Adrian J. L. Clark and Martine Bégeot

Unité INSERM/INRA U418 (D.N., P.D., M.B.), Hopital Debrousse, 69322 Lyon Cedex 05, France; Children’s Hospital (A.W.), Technical University, 01307 Dresden, Germany; Unité INSERM U155 (E.G.), Château de Longchamp, Bois de Boulogne, 75016 Paris Cedex, France; and Department of Chemical Endocrinology (A.J.L.C.), St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom

Address all correspondence and requests for reprints to: Danielle NAVILLE, INSERM-INRA U 418, Hôpital Debrousse, 29 Rue Soeur Bouvier, 69322 LYON Cedex 05, France. E-mail: naville{at}lyon151.inserm.fr


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several mutations in the coding exon of the ACTH receptor (MC2R) gene have been reported in cases of familial glucocorticoid deficiency or FGD. However, many patients with a similar syndrome do not present any mutation in the coding region of this gene. This is the case in 11 families we have investigated. Patients in these families present the typical clinical features of FGD, but no mutation was found in the coding exon of the ACTH receptor.

To determine whether mutations on MC2R gene, but outside the coding region, may be involved in FGD in these families, we have performed a linkage analysis. Using three markers flanking MC2R gene on chromosome 18, we were able to exclude linkage in a region of 12 centimorgans around the gene. This result clearly indicates that FGD is genetically heterogeneous. Defects in gene(s) different from MC2R gene are implicated in this syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FAMILIAL isolated glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder first described in 1959 (1), which is characterized by severe glucocorticoid deficiency, with a failure of adrenal responsiveness to ACTH without mineralocorticoid deficiency (because the renin-angiotensin system is not affected). After the cloning of the MC2R gene (2), some cases of FGD have been reported to be the consequence of mutations located in the coding exon of the gene (3, 4, 5, 6, 7). These could be classified as FGD type 1. However, many patients do not have mutations within the coding exon of the MC2R gene, and they could be classified as FGD type 2 (4, 7, 8). In a previous report, Weber et al. (8) have shown that linkage of FGD type 2 to the MC2R locus was excluded in three out of four families. However, these data do not exclude the possibility for other families that mutations outside the coding region of this gene (e.g. in the promoter) could cause the syndrome. In the absence of linkage of the syndrome to the MC2R locus, this would confirm and extend the notion that another locus could be implicated in the syndrome of FGD type 2.

To investigate these hypotheses, 11 families from different countries were selected on a clinical basis and on the basis that they lack mutations in the coding region of the MC2R gene. Linkage analysis was performed on these families with three polymorphic dinucleotide repeat markers flanking the MC2R gene, which is localized to human chromosome 18 p 11.2 (9, 10).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
FGD families

The diagnosis of FGD was established on the basis of typical clinical features and laboratory findings of isolated glucocorticoid deficiency. The pedigrees of the 11 families are presented in Fig. 1Go. There were 6 families with 2 or 3 affected children, giving a total number of 18 affected individuals with 22 normal siblings. Among these families, 4 were consanguineous, with a marriage between first cousins.



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  		Figure 1. A, Pedigree and haplotypes of families 1–6. {blacksquare} •, affected; {square} {circ}, not affected. The underlined number in family 5 may suggest a recombination event for the second sibling. B, Pedigree and haplotypes of families 7–11. {blacksquare} •, affected; {square} {circ}, not affected.

 
DNA preparation and search for ACTH receptor (ACTH-R) mutations

Genomic DNA was prepared from blood leukocytes collected from affected patients and from unaffected siblings and relatives. PCR amplification, cloning, and sequencing of the whole coding exon of the ACTH-R have been previously described in detail (4, 7, 8).

Genotyping

Three markers from the Genethon panel (11) were used (D18S1104, D18S1107, and D18S1116) (Fig. 2Go).



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  		Figure 2. Multipoint linkage analysis of FGD type 2 with the markers D18S1107, D18S1104, and D18S1116.

 
PCR amplification was carried out in a total vol of 50 µL, containing 50–60 ng genomic DNA, 125 µM deoxynucleotide triphosphates, 1 U Taq DNA polymerase, and primers 1 µM (final concentrations). PCR conditions consisted of 35 cycles, with 40 sec at 94 C, 30 sec at 55 C, and a brief passage at 72 C. The PCR products were run on 6% denaturing polyacrylamide gels in 1x TBE for 3 h. The gels were then transferred overnight to nylon membranes, and hybridization was performed overnight using peroxidase-labeled poly-AC at 42 C. Autoradiography was performed using ECL (Amersham Life Science, Amersham, les Ulis, France) for genotyping.

Linkage analysis

Haplotypes for the 3 markers were constructed in the 11 families (see Fig. 1Go). Two-point lod scores were computed for each marker using the MLINK program of the 5.1 version of the LINKAGE program (12). Multipoint lod scores were computed using the GENEHUNTER program (13). Linkage analyses were performed assuming a fully penetrant recessive model and a frequency of 10-5 for a mutation being responsible for the FGD syndrome. Marker allele frequencies were estimated from the data using the unrelated individuals who were typed in each family.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Case ascertainment (Table 1Go)

Families 1 and 3 have already been described in a previous report (8).


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  		Table 1. Clinical and biochemical findings in seven representative probands from independent families with FGD without ACTH receptor mutations

 
Clinical findings. For all patients of the 11 families, pregnancy was normal; and they were born full term. Most of the patients presented with recurrent hypoglycemic episodes early in life, which are frequent in this syndrome because of the severe glucocorticoid deficiency illustrated in 8 representative probands of unrelated families (Table 1Go). Hyperpigmentation of the skin was observed in all studied patients, caused by the high plasma ACTH levels. Tests for achalasia and alacrima were negative for all patients (which excludes Allgrove’s syndrome) (14).

Laboratory findings. The basal plasma cortisol level was undetectable in more than 70% of the studied patients or very low in the other cases. In contrast, greatly elevated plasma ACTH levels were always detected. Moreover, an impaired response to short- or long-term Synacthen administration was observed for all studied patients, which is the most consistent feature in this syndrome. However, the level of plasma renin activity was in the normal range, as well as the plasma aldosterone level, except in cases 2 and 4 (where the aldosterone level was slightly decreased).

Analysis of the coding exon of the ACTH-R gene in the 11 selected families

No abnormality in the coding exon of this receptor was detected in any of the affected patients from the above selected families, except for family 1, where one patient and several unaffected members carried a substitution of proline 27 by arginine in a heterozygous form, as reported previously (8).

Linkage analysis

Haplotypes for the 3 markers were constructed in the 11 families (Fig. 1Go). Two-point lod scores were computed for markers D18S1104, D18S1107, and D18S1116 (data not shown). For each marker, linkage can be excluded up to a recombination fraction ({theta}) of 0.05 on the whole sample of families. For marker D18S1104, linkage may even be excluded for {theta} = 0.08. Because this marker is known to be located at 4 cM from the ACTH-R locus (Fig. 2Go), this also excludes the region of the ACTH-R gene. Lod scores were not very dependent on the marker allele frequencies used in the analysis and were not noticeably changed by increasing the frequency of the mutation to 10-4.

A multipoint analysis was performed with the 3 markers. Lod scores were computed every 0.1 cM in the 9-cM interval between D18S1104 and D18S1116, using GENEHUNTER. The MC2R locus has been mapped, relative to the 3 other markers. Results for the whole sample of families are shown in Fig. 2Go. Lod scores are smaller than -6.0 in the entire region; and thus, linkage may be excluded in a 12-cM region around the MC2R gene. Linkage was clearly excluded in 2 single pedigrees (family 1 and family 7). Four other pedigrees showed negative lod scores in the whole region. Positive multipoint lod scores were obtained in 5 pedigrees (families 4, 5, and 9–11). Multipoint lod scores obtained for each of the 3 markers, as well as the presumed position of the MC2R gene, are shown in Table 2Go for the 11 families.


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  		Table 2. Multipoint lod scores obtained for each of the markers and for the presumed MC2R locus considering all the information on the three markers. Results are reported family-by-family, with the total in the last column

 
Linkage heterogeneity was tested on the multipoint lod scores, using the admixture test of Smith (15). Results are reported in Table 3Go. The test was not significant [but close to significance (see H2 vs. H1: P = 0.06 and P = 0.03 when using the correction proposed by Ott (16) to account for the one-sided nature of the test]. However, even when accounting for heterogeneity, there was no significant evidence of linkage with the region, because the maximum lod score is only 0.76. The only conclusion we can draw from this test is that linkage may not be excluded in all families.


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  		Table 3. Results of the test of linkage heterogeneity Chi-squares are given for the different tests performed: heterogeneity given linkage (H2 vs. H1), linkage given no heterogeneity (H1 vs. H0), and linkage given heterogeneity (H2 vs. H0)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FGD is characterized by a severe glucocorticoid deficiency, which may also be a common feature in the triple-A syndrome, although the latter is more complex with inter- and intrafamily variability (14). In contrast, patients with FGD share a common phenotype. The MC2R gene was an obvious candidate, because all patients showed resistance to ACTH administration. Recently, the gene responsible for triple-A syndrome was mapped to chromosome 12q13, thereby excluding the MC2R gene (17, 18, 19). Despite the common features of FGD, at least 2 subtypes can be distinguished. Defects in the coding exon of the MC2R gene have been reported in some cases of FGD (3, 4, 5, 6, 7), and this syndrome has been referred to as FGD type 1. The term FGD type 2 has been used to describe patients with this syndrome who do not carry defects in the coding exon of this gene. It is important to know whether these patients with FGD type 2 could harbor mutations in the same gene but outside the coding sequence (e.g. in the promoter) or whether another gene or genes at different loci could be responsible for the same phenotype. In a previous report, linkage of FGD type 2 to the MC2R gene was excluded in 3 out of 4 families (8). In the present study, 11 FGD type 2 families were selected on clinical and biochemical findings and by the absence of defects in the coding exon of the MC2R gene. The haplotypes were constituted after genotyping using the 3 chosen markers, and two-point and multipoint analyses were performed in a 12-cM region around the MC2R gene.

Examination of the 3 locus haplotypes in the 11 families (Fig. 1Go) shows that in families 1, 3, 6, and 7, some affected and nonaffected siblings share their 2 haplotypes (providing strong evidence against linkage). None of the inbred affected individuals in families 2, 7, and 8 are homozygous. This makes it unlikely that the disease locus is in this region, and this explains the negative lod scores obtained with these families.

In family 9, the inbred affected individual is homozygous and carries a haplotype different from that of his nonaffected brother. Linkage can thus not be excluded in this family; and indeed, multipoint lod scores are greater than 1.0 all over the region. In families 4, 5, and 11, the two affected sibs carry the same haplotypes and linkage of the disease with this region also cannot be excluded. In family 10, the two nonaffected individuals have different haplotypes from those of their affected brother, so that linkage also cannot be excluded.

In conclusion, in our series of families, we have excluded linkage of FGD type 2 to a 12-cM region around the MC2R locus in 6 of 11 families. However, linkage cannot be clearly excluded in the other families. Families for whom the MC2R gene has been excluded will be submitted to a whole genome scanning approach.


   Acknowledgments
 
This project would not have been possible without the helpful cooperation of the families and the physicians. This work has been performed with Genethon (1 Rue de l’Internationale, BP 60 91002 Evry Cedex, France), and we would like to thank Sylvie Marchand for her helpful technical assistance. We also thank Joëlle Bois and Marie-Ange Di Carlo for their secretarial assistance.


   Footnotes
 
1 Present address: Department of Integrative Biology, 3060 Valley Life Science Building, University of California, Berkeley, California 94720. Back

Received March 30, 1998.

Revised June 12, 1998.

Accepted June 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shepard TH, Landing BM, Mason DG. 1959 Familial Addison’s disease. Am J Dis Child. 97:154–162.[Abstract/Free Full Text]
  2. Mountjoy KG, Robbins LS, Mortrud MT, Cone RG. 1992 The cloning of a family of genes that encode the melanocortin receptors. Science. 257:1248–1251.[Abstract/Free Full Text]
  3. Clark AJL, McLoughlin L, Grossman A. 1993 Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet. 341:461–462.[CrossRef][Medline]
  4. Weber A, Toppari J, Harvey RD, et al. 1995 Adrenocorticotropin receptor gene mutations in familial glucocorticoid deficiency: relationships with clinical features in four families. J Clin Endocrinol Metab. 80:65–71.[Abstract]
  5. Tsigos C, Arai K, Hung W, Chrousos GP. 1993 Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene. J Clin Invest. 92:2458–2461.
  6. Tsigos C, Arai K, Latronico C, DiGeorge AM, Rapaport R, Chrousos GP. 1995 A novel mutation of the adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency but no ACTH-R abnormalities in two families with the triple A syndrome. J Clin Endocrinol Metab. 80:2186–2189.[Abstract]
  7. Naville D, Barjhoux L, Jaillard C, et al. 1996 Demonstration by transfection studies that mutations in the adrenocorticotropin receptor are one cause of the hereditary syndrome of glucocorticoid deficiency. J Clin Endocrinol Metab. 81:1442–1448.[Abstract]
  8. Weber A, Clark AJL. 1994 Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency. Hum Mol Genet. 3:585–588.[Abstract/Free Full Text]
  9. Gantz I, Toshiro T, Barcroft C, et al. 1993 Localization of the genes encoding the melanocortin-2 (adrenocorticotropic hormone) and melanocortin-3 receptors to chromosomes 18 p11.2 and 20 q13.2-q13.3 by fluorescence in situ hybridization. Genomics. 18:166–167.[CrossRef][Medline]
  10. Magenis RE, Smith L, Nadeau JH, Johnson KR, Mountjoy KG, Cone RD. 1994 Mapping of the ACTH, MSH and neural (MC3 and MC4) melanocortin receptors in the mouse and human. Mamm Genome. 5:503–508.[CrossRef][Medline]
  11. Dib C, Fauré S, Fizanes C, et al. 1996 A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature. 380:152–154.[CrossRef][Medline]
  12. Lathrop CM, Lalouel JM, Julien C, Ott J. 1984 Strategies for multilocus linkage analysis analysis in humans. Proc Natl Acad Sci USA. 81:3443–3446.[Abstract/Free Full Text]
  13. Kruglyak L, Daly MJ, Reeve-Daly MB, Lander ES. 1996 Parametric and nonparametric linkage analyses: a unified multipoint approach. Am J Hum Genet. 58:1347–1363.[Medline]
  14. Allgrove J, Clayden GS, Grant DB, Macaulaay JC. 1978 Familial glucocorticoid deficiency with achalasia of the cardia and deficient tear production. Lancet. 1:1284–1286.[Medline]
  15. Smith CAB. 1963 Testing for heterogeneity of recombination values in human genetics. Ann Hum Genet. 27:175–182.[Medline]
  16. Ott JM. 1985 A chi-square to distinguish allelic association from other causes of phenotypic association between two loci. Genet Epidemiol. 2:79–84.[CrossRef][Medline]
  17. Stratekis CA, Lin JP, Pras E, Rennert OM, Bourdony CJ, Chan WY. 1997 Allgrove (triple-A) syndrome in Puerto Rican kindreds maps to chromosome 12 (12q13). Proc Assoc Am Physicians. 109:478–482.[Medline]
  18. Weber A, Wienker TF, Jung M, et al. 1996 Linkage of the gene for the triple A syndrome to chromosome 12q13 near the type II keratin gene cluster. Hum Mol Genet. 5:2061–2066.[Abstract/Free Full Text]
  19. Wu SM, Stratekis CA, Bourdony CJ, Rennert OM, Chan WY. 1997 Molecular genetics of adrenocorticotropin (ACTH) resistance novel mutations of the ACTH receptor (ACTH-R) gene in a hereditary glucocorticoid resistance patient and refinement of the Allgrove syndrome locus (chrom. 12q13). American Society of Human Genetics, 47th Annual Meeting, Baltimore MD. Am J Hum Genet. 61:A351.



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