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The RM syndrome belongs to the group of genetic syndromes of extreme insulin resistance(1). Patients with RM syndrome have the clinical and biologic signs characteristic of extreme insulin resistance, such as acanthosis nigricans, virilization, impaired glucose tolerance, and/or diabetes mellitus associated with marked hyperinsulinemia. In addition, they present with typical coarse facial features, marked growth retardation, advanced dentition, and abdominal protuberance. It seems now well established that molecular alterations of the IR gene are involved in the pathogenesis of the RM syndrome.

The IR gene, which has been allocated on chromosome 19p13.2-p13.3(2, 3), comprises 22 exons distributed over 130 kb of DNA(4). This gene encodes a transmembrane glycoprotein of α2β2 structure. Insulin binding to the extracellular α-subunit results in activation of the intracellular tyrosine kinase of the β-subunit, thus initiating events that lead to the final metabolic and mitogenic effects of insulin(5).

Mutations in the two alleles of the IR gene have been reported in five patients with RM syndrome who were either homozygous(6, 7) or compound heterozygous(8–10). In two other patients, only one mutant IR allele has been characterized so far(11, 12).

Mutations in the two alleles of this gene have been also reported in patients with leprechaunism and type A insulin resistance(11, 13). However, the morbidity and mortality of the three affections markedly differ. Because the genotype at the IR locus(nature and localization of the mutations) does not fully account for the phenotype, it is hypothetized that additional factors, genetic and/or environmental, can modulate the clinical characteristics of these syndromes.

In the present study, we describe a patient with RM syndrome who had a primary cellular resistance to insulin due to a homozygous mutation in the IRα-subunit. Interestingly, glucose homestasis varied markedly during the day in this patient.

METHODS

Subject. Patient RM-3 was a Caucasian boy of Algerian origin, born after 8 mo of gestation. He was the only child of a father deceased from unknown cause and a mother with no specific past or medical history. His paternal grandfather had been diabetic and was treated with insulin. Information on the family tree was unavailable. Little was known about the first years' medical records of patient RM-3. He had been hospitalized at the ages of 1, 2, and 3 y for unexplained dysmorphism, growth failure, and developmental retardation (he sat up at 10 mo and walked after he was 3 y old).

At the age of 7 y, he presented with polyuria, polydipsia, massive glycosuria, blood glucose levels greater than 17 mmol/L, and ketonuria. He was again admitted to the hospital and treated with insulin. Because of failure to control glycemic levels, doses were increased rapidly up to 8 U/kg/d. Postprandial glucose levels were up to 30 mmol/L, minimal values were 6.6 mmol/L around 0600 h, and glycosuria was 150-200 g/d with persistent ketonemia and ketonuria (about 0.4 g/L). At 7 y and 3 mo of age, he was diagnosed as presenting a RM syndrome: height was 85 cm (-7 SD), weight was 13.5 kg (-3.5 SD), and head circumference was 49 cm (-3.5 SD). The bone age was of 18 mo. Physical examination revealed dysmorphic features such as facial hypoplasia, dental dysplasia with advanced dental maturation estimated at 12 y, and abnormal enamel structure (Fig. 1). The skin was thickened and dry. Acanthosis nigricans was found in the cervical, axillary, periumbilical, and inguinal regions. Hair and nails were normal. There was no lipoatrophy. Moderate hirsutism was noted on the back and the face. The abdomen was hypotonic and distended with marked hepatomegaly and perception of bilateral lumbar masses. The testes were prepubertal but the penis was enlarged (6 cm). All skeletal radiographies, cardiac echography, and cerebral tomodensitometry were normal. Abdominal echography and tomodensitometry confirmed hepatomegaly with normal structure and bilateral nephromegaly. The liver histology was normal. Intestinal malabsorption was excluded due to normal bone density and xylose test and absence of steatorrhea. Eye fundoscopy and renal function were normal. Anti-islet cell antibodies were positive but at low levels. Anti-IR antibodies were absent. Hormonal investigations revealed normal levels of thyroid hormones, cortisol, ACTH, dehydroepiandrosterone sulfate, testosterone, aldosterone, prolactin, LH, FSH, urinary steroids, catecholamines, and melatonine. The patient died at the age of 14 y. The study was conducted with the approval of an institutional review board.

Figure 1
figure 1

Patient RM-3 at the age of 7 y and 3 mo.

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Clinical study of glucose metabolism. Insulin resistance was not evaluated using the hyperinsulinemic euglycemic clamp technique, because there were no reference values for a 7-y-old and 13.5-kg child. Instead we evaluated the circadian insulin needs using a blood glucose-controlled insulin infusion system (Biostator, Life Science Instruments, Miles Laboratories Inc., Elkhart, IN). On the 1st d, two indwelling catheters were introduced into forearm veins, one for the double lumen sampling catheter, and the other one for insulin infusion. The Biostator algorithms were set to maintain blood glucose at 5 mmol/L. During the 48 h of automated i.v. insulin infusion, the patient was given a balanced diet including breakfast, lunch, mid-afternoon snack, and dinner with his usual caloric intake. Insulin and C-peptide plasma concentrations were measured by RIA as previously described(14). Plasma glucagon was measured using the Hypolab kit.

In vivo GH, IGF-I, and IGFBP serum levels. GH was measured by RIA after several stimulation tests: ornithine hydrochloride (30-min i.v. infusion of 15 g/m2), DOPA (ingestion of 125 mg of L-DOPA), and GH-releasing hormone (i.v. injection of 2 μg/kg). Serum IGF-I was measured by RIA after acidic gel filtration(15) in basal and GH-stimulated states (three intramuscular injections of 2 mg of GH). IGFBP-2 and IGFBP-3 were analyzed by Western ligand and immunoblotting(16, 17). IGFBP-1 was measured by RIA(17).

Cell culture. Fibroblast cultures were established from forearm skin biopsies. Cells were grown in monolayers in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Eragny, France) and 10% FCS, as previously described(18). In each experiment, patient fibroblasts were studied in parallel with fibroblasts from four controls. For the assays described below, cells were deprived of serum for 16 h.

Insulin binding. 125I-Labeled insulin binding to erythrocytes was performed according to Gambhir et al.(19). Insulin binding to adherent fibroblasts was performed as previosuly described(18). Incubations were carried out in a total volume of 0.4 mL for 4 h at 15 °C in the presence of 125I-labeled insulin (0.8 ng,(3-[125I]iodotyrosylA14)insulin, Amersham, Les Ulis, France). Nonspecific binding was determined in the presence of an excess of unlabeled insulin (10 μg).

Metabolic effects of insulin. Hexose transport in the absence or presence of increasing concentrations of insulin was evaluated with 2-deoxy-D-[U-14C]glucose, a nonmetabolizable glucose analog, as previously described(18). Glycogen synthesis was estimated from the level of D-[U-14C]glucose incorporated into cellular glycogen, as previously described(18). The effect of insulin on amino acid transport was measured with aminoisobutyric acid, a nonmetabolizable analog of alanine, as previously described(18).

DGGE analysis. IR exons 2-22 and the surrounding intronic sequences were amplified by the PCR technique using genomic DNA as template, and analyzed by DGGE, as previously described(20). Briefly, amplified fragments were subjected to electrophoresis at 160 V in 6% polyacrylamide gels containing a linearly increasing denaturant gradient (100% denaturant = 7 M urea and 40% formamide). Gels were stained with ethidium bromide and examined under UV. The sequence of PCR products showing an altered migration pattern on denaturing gels was determined by direct sequencing.

Statistical analysis. Results are the means ± SEM for at least three independently performed experiments. Differences between control and patient values were evaluated by t test.

RESULTS

Clinical study of glucose metabolism. All the following investigations were conducted between the age of 7 y and 7 y and 5 mo. The precent of glycosylated Hb (HbA1c) was 9.8% (normal value at 4.2± 0.6%). Figure 2 shows the variations in blood glucose levels and insulin-infusion rates during the first 24 h of automated i.v. insulin infusion. Similar results were obtained during the next 24 h of insulin infusion. During the day, blood glucose was raised from near normal values before breakfast to levels between 18 and 28 mmol/L after meals. It never returned below 15 mmol/L before meals despite the infusion of very large doses of i.v. insulin, between 10 and 40 U/h (12.5-50 mU/kg·min) for a total of 290 and 350 U/d for the 2 d of infusion, respectively. In contrast, during both nights, as no more food was given, blood glucose returned progressively to normal with only 1.5 and 0.75 U/h of insulin (1.8 and 0.9 mU/kg·min) during the last 6 h of each night, respectively. Marked ketonuria persisted during the day under massive insulin therapy. It declined during the night but remained slightly positive in the postabsorptive state. The levels of glucagon measured during i.v. insulin infusion were increased to 0.50 ng/mL (adult control values between 0.05 and 0.25 ng/mL) and 1.91 ng/mL in basal and postprandial states, respectively. Under insulin therapy, endogenous insulin secretion was totally inhibited (C-peptide < 0.1 nmol/L). Basal insulin and C-peptide levels were elevated to 911 pmol/L(normal values between 35 and 100 pmol/L) and 3.5 nmol/L (normal values between 0.2 and 0.8 nmol/L), respectively, when treatment with insulin was stopped.

Figure 2
figure 2

Variations in blood glucose levels and insulin infusion rates during the first 24 h of automated i.v. insulin infusion. Glycemia(black lines) and insulin needs (shaded areas) were evaluated in a control subject (upper panel) and in patient RM-3 (lower panel) with a blood glucose-controlled insulin infusion system (Biostator).

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In vivo GH, IGF-I, and IGFBP levels. The response of GH to ornithine stimulation was absent in the first test (maximal value at 3μg/L). and normal in the second (peak at 15 μg/L). GH levels were increased after GH-releasing hormone stimulation (up to 31 μg/L) but not after L-DOPA ingestion (maximal value at 5.2 μg/L).

IGF-I was undetectable in the basal state as well as after GH stimulation. IGFBP-2 expression was increased in serum from patient RM-3(Fig. 3A), whereas IGFBP-3 was undetectable (Fig. 3B). The IGFBP-1 level was normal (54 ng/mL; normal value for the age at 68 ± 8 ng/mL).

Figure 3
figure 3

Ligand and immunoblots of serum IGFBPs. (A) Ligand blot analysis. Serum samples from a control and patient RM-3 were submitted to 11% SDS-PAGE without reducing agent. The binding proteins were electroblotted onto nitrocellulose and, after incubation with125 I-labeled IGF-I, identified by autoradiography. (B) Immunoblot analysis. Serum samples from a control and patient RM-3 were submitted to 11% SDS-PAGE without reducing agent. The binding proteins were transferred onto nitrocellulose and immunoblotted with IGFBP-3 antiserum.

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Insulin binding. Insulin binding to the patient's erythrocytes was studied during insulin therapy, and 1 mo after cessation of the treatment. It was markedly decreased in both cases (2.6 and 2.1% of insulin bound/4.5× 109 cells, respectively; normal values between 8 and 10%). The apparent IR affinity (ED50) was normal (2 ng/mL; normal values between 1 and 6 ng/mL). Scatchard analysis of the data revealed a marked decrease in high affinity binding sites (data not shown).

Insulin binding on cultured fibroblasts was also evaluated. The maximal binding of a tracer amount of 125I-labeled insulin to patient cells was significantly decreased (11.6 ± 1.2 pg of insulin bound/mg of protein) as compared with control fibroblasts (18.9 ± 2.0 pg of insulin bound/mg of protein; p < 0.02).

Insulin action. Basal and insulin-stimulated 2-deoxyglucose transport were comparatively studied in control and patient cells (Fig. 4). In control cells, insulin (0-10-6 mol/L) gradually increased glucose transport. In patient cells, neither basal nor maximally activated hexose transport were significantly different from that obtained in control cells. However, at 10-9 and 10-8 mol/L insulin, activation of glucose transport was significantly decreased in patient fibroblasts. Similar observations were made when glycogen synthesis and amino acid uptake were examined in patient fibroblasts (data not shown).

Figure 4
figure 4

Insulin stimulation of 2-deoxyglucose transport in control (open symbols) and patient (full symbols) fibroblasts. Statistically significant differences between control and patient cells are indicated:*p < 0.05. Basal levels of transport were 7386 ± 1027 and 5634 ± 1335 dpm/mg of protein in control and patient cells, respectively.

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Thus, patient cells were resistant to the stimulation of several metabolic processes by physiologic concentrations of insulin, such concentrations acting specifically through the IR. At high concentrations, which involve insulin binding to the IGF-I receptors, patient cells responded normally, suggesting that IGF-I signaling was preserved in these cells.

Structure of the IR gene. We searched for point mutations in the IR exons 2-22 and splice site junctions by means of DGGE. PCR-amplified exon 3 from patient RM-3 showed an abnormal pattern of migration on a denaturing gel when compared with DNA from a control subject (Fig. 5A). After direct sequencing of PCR fragments, a G-to-A substitution was identified at nucleotide 1070 (Fig. 5B). Therefore, patient RM-3 was homozygous for a missense mutation converting Cys284 to Tyr. This mutation has not been previously reported in patients with inherited extreme insulin resistance and was not present in 30 controls, indicating that it is not a common polymorphism in the normal population. DGGE analysis of the remaining IR exons and flanking introns evidenced neither homozygous nor heterozygous nucleotide variation. In addition, Southern blot analysis did not reveal any major rearrangement in the IR gene from patient RM-3 (data not shown).

Figure 5
figure 5

Detection of a point mutation in exon 3 of the IR gene.(A) DGGE analysis of IR exon 3. Exon 3 from a control subject and patient RM-3 was amplified by PCR and analyzed on a 40-80% denaturing gel for 4 h at 160 V. The migration shift observed with DNA fragment from patient RM-3 reveals the presence of a homozygous nucleotide variation. (B) Sequence analysis of IR exon 3. PCR-amplified exon 3 from a control subject and patient RM-3 was directly sequenced. A homozygous G-to-A substitution was identified at nucleotide 1070 in DNA from patient RM-3 which replaced Cys284 by Tyr.

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DISCUSSION

A new homozygous mutation substituting Tyr284 for Cys was identified in the IR α-subunit from patient RM-3. Because mutant IR alleles are very rare in the general population, homozygosity suggests that patient RM-3 was the issue of a consanguineous marriage. The absence of heterozygous polymorphisms in the IR gene from this patient supports the hypothesis. The Cys-to-Tyr substitution at position 284 is likely a pathogenic mutation, because it is the unique amino acid change detected in the IR sequence and because it is not present in normal subjects. In addition, Cys284 is a highly conserved residue among the family of tyrosine kinase receptors and resides in a domain playing a role in insulin binding and IR biosynthesis.

Indeed, Cys284 is one of the 26 cysteine residues located in the cysteine-rich domain of the α-subunit (residues 155-312). Some of these cysteines are thought to participate in the maintenance of the structure of the insulin-binding site by establishing disulfide bonds betweenα-subunits(5). However, as discussed by Roach et al.(10), the cysteine-rich domain does not seem to contain direct insulin/IR contact sites. The characterization of the naturally occurring point mutations Pro193 → Leu(7, 21), His209 → Arg(22), and Leu233 → Pro(23) also suggests that this domain is important for IR processing and transport to the cell surface.

The Tyr284 mutation identified in the IR from patient RM-3 resulted in a reduction of maximal insulin binding on erythrocytes and cultured fibroblasts. Total levels of IR mRNA evaluated by reverse transcription-PCR as previously described(24) were comparable in control and patient fibroblasts (data not shown). Scatchard analysis of the binding data suggested that the number of cell-surface IRs was decreased in patient erythrocytes. By analogy with the effect of other mutations in the cysteine-rich domain, we speculate that the Tyr284 mutation may inhibit posttranslational processing and intracellular transport of the IR to the cell surface. Consistent with defective insulin binding, patient fibroblasts were resistant to physiologic concentrations of insulin for the stimulation of different metabolic effects.

Concordantly, most IR mutations identified in patients with RM map in theα-subunit: Asn15 → Lys(8), Pro193 → Leu(7), Ser323 → Leu(6, 10), insertion of 4 amino acids at codon 262(9), and mutation in the splice acceptor site of intron 4(11). Nonsense mutations leading to a premature chain termination have been also reported(8–10). The functional significance of some of these structural alterations has been evaluated and consists in defective insulin binding as a result of altered IR biosynthesis, IR processing, and/or insulin-binding affinity.

In addition to the RM syndrome, leprechaunism and some cases of type A insulin resistance are associated with a molecular defect in both alleles of the IR gene. However, these three affections markedly differ in morbidity and mortality. In particular, patients with leprechaunism generally do not survive beyond the 1st y of life whereas those with the RM syndrome live several years, and those with type A insulin resistance reach adulthood. The nature and localization of the structural alterations in the IR may account for these clinical differences although other factors, genetic and/or environmental, have undoubtedly to be considered.

Some clinical and metabolic features are more often observed in patients with RM than in patients with other inherited syndromes of extreme insulin resistance. At first, uncontrolled diabetes often occurs in childhood with glycemic levels between 9 and 28 mmol/L, marked glycosuria and resistance to exogenous insulin(6,9,10,25,26) (this study). In contrast, patients with leprechaunism do not display overt diabetes mellitus but rather exhibit only mildly impaired glucose tolerance(1). However, the short life expectancy of leprechaun patients precludes comparative studies with patients with RM.

Second, unexpected elevated levels of ketonemia and ketonuria have been reported in several patients with RM(1, 9, 26) (this study), whereas FFA and glycerol levels were normal or low(27). This suggests that increased ketone bodies result from increased liver ketogenesis, possibly in response to excessive glucagon levels, as this was surprisingly found in patient RM-3.

Third, patients with RM present with considerable and permanent growth restriction. Consistent with this observation, blood IGF-I levels are markedly reduced in these patients, although GH levels are normal or moderately decreased(12, 26) (this study). This points out an alteration in GH signaling at the liver level. Such an alteration has been directly assessed by the failure of rhGH to stimulate growth and to increase levels of circulating IGF-I in a patient with RM(12). One may suggest that the altered GH effect is secondary to the markedly abnormal metabolic status caused by defective IRs. Cellular insulin resistance would produce a state of nutritional and insulin deprivation(28). In addition, insulin resistance and reduced amounts of IGF-I could explain the increased levels of IGFBP-2 and the decreased levels of IGFBP-3 seen in patients with RM(12, 28, 29). They could also account for the increased levels of glucagon, because insulin and IGF-I are known to inhibit the glucagon secretion(30).

It has been shown that treatment with rhIGF-I fails to restore growth velocity in a patient with RM(12). The lack of rhIGF-I effect may be explained by an increased clearance of rhIGF-I as a result of low levels of IGFBP-3. In addition, defective rhIGF-I action may also be related to IGF-I resistance in target cells, cellular IGF-I signaling being possibly inhibited by mutant IRs. However, the latter hypothesis is not supported by other in vitro and in vivo studies. IGF-I binding(12, 28) and probably IGF-I signaling(9) (this study) are normal in cultured cells from patients with RM. Administration of rhIGF-I in a patient with RM is able to decrease FFA production(27). Otherwise, IGF-I receptor signaling appears to be preserved in some cell types because patients with RM present with acanthosis nigricans, advanced dentition, and thickened nails, all lesions presumed to result from activation of IGF-I receptors by excessive concentrations of insulin on keratinocytes and dermal fibroblasts(31) and on some other cell types from mesenchymal origin(1). Interestingly, IGF-I signaling pathways have been often reported to be defective in patients suffering from leprechaunism(see Desbois-Mouthon et al.(32), and references cited herein). Thus, in patients with RM, the presence of IGF-I receptors responsive to high concentrations of insulin could account for the lesser clinical severity of the syndrome compared with leprechaunism.

Patient RM-3 displayed circadian variations of glucose homeostasis despite marked primary insulin resistance due to the homozygous Tyr284 mutation. In vivo insulin requirements were enormous during daytime and minimal at the end of the night. This is in good agreement with the observations we made in another type of extreme insulin resistance(14). Indeed, we showed that the fasting plasma glucose level does not depend primarily on the severity of peripheral insulin resistance, which is principally responsible for the postprandial glucose intolerance. Fasting hyperglycemia rather depends on increased hepatic glucose production.

Marked hyperglycemia after eating suggests that hepatic glucose output was not suppressed, and also that insulin did not stimulate glucose uptake into muscle. Renal excretion of glucose could contribute toward the fall in blood glucose concentration during the latter part of the day, but there could be also a mass action effect in promoting glucose uptake and storage in the liver and muscles.

In conclusion, we describe a patient with RM syndrome and primary in vitro insulin resistance consecutive to a structural alteration in the IRα-subunit. Nevertheless, our study reveals that primary insulin resistance accounts for only a part of the in vivo alterations of glucose homeostasis. Other abnormalities, likely secondary to insulin resistance such as lack of IGF-I, increased glucagon levels, and marked variations in metabolite concentrations, undoubtedly play an important role in the major disturbance of glucose homeostasis observed during the day. Although it is not feasible for the moment to undertake correction of the IR molecular defects involved in the pathogenesis of the RM syndrome, therapeutic control of the metabolic and/or hormonal consequences of severe insulin resistance should help to improve the clinical features of this syndrome.