Genotype–phenotype correlation in inherited
severe insulin resistance
Nicola Longo1,2,*, Yuhuan Wang2, Shelley A. Smith2, Sharon D. Langley2, Linda A. DiMeglio3
and Daniel Giannella-Neto4
1Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT, USA,2Division of
Medical Genetics, Department of Pediatrics, Emory University, Atlanta, GA, USA,3Department of Pediatrics,
Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA and
4Laboratory for Cellular and Molecular Endocrinology, Division of Endocrinology, University of Sa ˜o Paulo School of
Medicine, Sa ˜o Paulo, Brazil
Received February 27, 2002; Revised and Accepted March 27, 2002
The insulin receptor is a ligand-activated tyrosine kinase. Mutations in the corresponding gene cause the rare
inherited insulin-resistant disorders leprechaunism and Rabson–Mendenhall syndrome. Patients with the
most severe syndrome, leprechaunism, have growth restriction, altered glucose homeostasis and early death
(usually before 1 year of age). Rabson–Mendenhall syndrome is less severe, with survival up to 5–15 years of
age. These disorders are transmitted as autosomal recessive traits. Here we report six new patients and
correlate mutations in the insulin receptor gene with survival. Patients with leprechaunism were homozygous
or compound heterozygous for mutations in the extracellular domain of the insulin receptor and their cells
had markedly impaired insulin binding (<10% of controls). Mutations in their insulin receptor gene inserted
premature stop codons (E124X, R372X, G650X, E665X and C682X), resulting in decreased levels of mature
mRNA, or affected the extracellular domain of the receptor (R86P, A92V, DN281, I898T and R899W). Three
patients with Rabson–Mendenhall syndrome had at least one missense mutation in the intracellular domain
of the insulin receptor (P970T, I1116T, R1131W and R1174W). Expression studies in CHO cells indicated that
the R86P, A92V, DN281, I898T, R899W and R1131W mutations markedly impaired insulin binding (<5% of
control), while the P970T, I1116T and R1174W mutant receptors retained significant insulin-binding activity.
These results indicate that mutations in the insulin receptor retaining residual insulin-binding correlate with
prolonged survival in our series of patients with extreme insulin resistance.
The human insulin receptor is a heterotetramer composed of
two extracellular a subunits that bind insulin and two b
subunits that span the plasma membrane and have an
intracellular tyrosine kinase domain (1,2). Insulin binding to
the a subunit of the receptor stimulates b-subunit autopho-
sphorylation and kinase activity. A single gene located on
chromosome 19 codes for both the a and b subunits of the
receptor (3). Mutations in this gene cause the insulin-resistant
syndromes leprechaunism, Rabson–Mendenhall syndrome and
type A insulin resistance (4,5). Leprechaunism (OMIM
246200), the most severe of these syndromes, is characterized
by intrauterinegrowth restriction, loss of glucose homeostasis,
hyperinsulinemia, and dysmorphic features, with prominent
eyes, thick lips, upturned nostrils, low-set posteriorly rotated
ears, thick skin with lack of subcutaneous fat, distended
abdomen, and enlarged genitaliain themaleand cystic ovaries
in the female (6–8). Cells from most patients with leprechau-
nism have absent insulin binding, although recent exceptions
were reported (9,10). Patients with the slightly less severe
Rabson–Mendenhall syndrome(OMIM 262190) havedifferent
dysmorphic features, with premature or dysplastic teeth and
gingival hyperplasia (11). In addition, they present pineal
hyperplasia. In both cases, children have initially postprandial
hyperglycemia and fasting hypoglycemia. The paradoxical
fasting hypoglycemia is caused by inappropriately elevated
insulin levels at the time of fasting, due to the excessive
productionof insulinby thepancreas of thesepatients, coupled
to the prolonged half-life of the hormone for the inability of
peripheral tissues to bind and remove circulating insulin (12).
Patients with Rabson–Mendenhall syndrome survive beyond
1 year of age and, with time, develop constant hyperglycemia
followed by diabetic ketoacidosis and death. This is caused by
*Towhomcorrespondenceshould beaddressed at: Divisionof Medical Genetics, Department of Pediatrics, University of Utah, 2C412 SOM, 50 North
Medical Drive, Salt Lake City, UT 84103, USA. Tel: þ 1801 587 9071; Fax: þ 1801 585 0956; Email: Nicola.Longo@hsc.utah.edu
# 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No. 121465–1475
by guest on June 12, 2013
a progressive decline of insulin levels, which become
insufficient to prevent glucose synthesis in the liver and
prevent release of fatty acid by adipocytes (12).
A number of different mutations in the insulin receptor gene
have been reported in patients with leprechaunism and
Rabson–Mendenhall syndrome (5). Although both syndromes
are inherited as autosomal recessive traits, a clear correlation
between genotype and phenotype has not yet been established
(5). Thedifficulty in establishing genotype–phenotypecorrela-
tion is due in part to the rarity of these syndromes and to the
patients. Inaddition, several of themutations reportedhavenot
been analyzed in vitro in the patient’s cells or in transfected
cells to determine their effect on insulin binding or signaling.
Herewe report several new mutations in the insulin receptor
gene of patients with leprechaunism and Rabson–Mendenhall
syndrome. Themutant receptors wereanalyzed in thepatients’
cells and expressed in mammalian cells to define the residual
insulin-binding and signaling properties of the mutant insulin
receptor. In our patient population, mutations markedly
impairing insulinbinding resultedinthemostseverephenotype
with early demise, while mutations leaving residual insulin-
binding activity were associated with longer survival.
Insulin binding to cultured cells
Table 1 reports insulin binding to fibroblasts obtained from
patients with leprechaunism and patient ATL-2 with Rabson–
Mendenhall syndrome. Insulinbinding wasreducedtolessthan
10% of controls in fibroblasts derived from all patients with
leprechaunism, while cells from patient ATL-2 with Rabson–
Mendenhall syndromeretained about 18% of normal binding.
Table 2 reports insulin binding to lymphoblasts obtained
from patients with Rabson–Mendenhall syndrome and patient
MtSinai withleprechaunism. CellsfrompatientswithRabson–
Mendenhall syndrome retained significant residual insulin
binding, which was 18–27% of the normal average. By
contrast, insulin binding to lymphoblasts of patient Mt Sinai
with leprechaunism was negligible.
Mutations in the insulin receptor gene
Wesequenced each oneof the22 exons of theinsulinreceptor
gene using PCR and primers in the flanking introns. PCR
products were sequenced directly without subcloning and the
mutations identified confirmed, when possible, by restriction
analysis. Mutations are summarized in Table 3.
Patient FL-1 with leprechaunism was a compound heter-
ozygote for a single-base-pair change (356C >T) converting
the codon for Ala92 to that for Val (A92V) and a 2774T >C
transversion converting the codon for Ile898 to that for Thr
(I898T). TheA92V mutationremovedaCfoI sitefromexon2,
while the I898T mutation added a novel HinfI site in exon 14.
Both mutations were confirmed by restriction analysis.
Patient GE was a compound heterozygote for two single-
nucleotide insertions in exon 10. The paternal mutation
inserted an extra G in position 2187 of the insulin receptor
cDNA, converting the codon for Thr657 into that for Asp and
causing aframeshiftof thedownstreamsequence. Thisresulted
in the premature insertion of a stop codon in position 665
(665X). The maternal mutation inserted an A in position 2263
of theinsulinreceptorcDNA, converting thecodonforCys682
to a stop codon (C682X). The paternal insertion (2187insG)
created a novel BsmFI site in exon 10 of the insulin receptor
gene, whilethematernal insertion(2263insA) abolishedoneof
the two MwoI sites in exon 10 of the insulin receptor gene.
Both mutations were confirmed by restriction analysis in the
patient and both parents.
Patient NY-1 was homozygous for a single-base-pair change
in exon 2 of the insulin receptor (451G >T), converting the
codonforGlu124(GAG) toastopcodon(TAG) (E124X). This
mutation abolished a BanII restriction site in exon 2 of the
insulin receptor gene.
Patient VA with leprechaunism was a compound heter-
ozygote for a four base-pair deletion in exon 9 removing bp
2136–2139 of the insulin receptor cDNA. The 4bp deletion
caused a frameshift that inserted a premature stop codon at
codon 650 of the insulin receptor cDNA (650X). This
deletion created a novel PvuII restriction site. The second
mutation was a C-to-T transition in exon 14 converting the
codon for Arg899 to that for Trp (R899W). This mutation
abolished one of two MspI cut sites in exon 14 of the insulin
Table 1. Insulin binding to fibroblasts of patients with inherited severe insulin resistance
Survival age Specific binding of insulina
(fmol/mg cell protein)
Fibroblastswereculturedandevaluatedforequilibriumbinding of [125I]insulin(1ng/ml) for2hat20?C. Non-specific binding, measuredinthepresenceof 5mg/ml
of cold ligand, was subtracted from each point. Data are means?SE of triplicates.
aNormal range (n¼18) 1.01–2.20; average 1.52?0.15.
bP<0.01 versus normal range, using analysis of variance.
1466Human Molecular Genetics, 2002, Vol. 11, No. 12
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Mendenhall syndrome. The paternal mutation was a C-to-A
transversion in exon 16 that changed the codon for Pro970 to
that for Thr(P970T). This mutation neither created nor
destroyedarestrictionenzymesite. This mutationwashowever
confirmed by reading in both directions in independent PCR
productsfromthepatientandhisfather. Thematernal mutation
was a C-to-T transition in exon 19 that changed the codon for
Arg1131 to that for Trp (R1131W). This mutation abolished a
SfaNI siteandwas previously describedinanother patient with
Rabson–Mendenhall syndrome (11,12).
Patient CG-1 with Rabson–Mendenhall syndrome was
heterozygous for a C-to-T transition in exon 20 converting
the codon for Arg1174 to that for Trp (R1174W). We were
unable to identify the second mutation in this patient.
All other exons of the insulin receptor genewere sequenced,
and no other variations fromthe published sequenceor known
polymorphisms were detected. Southern blot analysis on DNA
frompatientsNY-1 andCG-1 failedtoidentify abnormal bands
Reduced insulin receptor mRNA in fibroblasts of patients
with leprechaunism with mutations creating premature
Mutations introducing premature stop codons in the insulin
receptor decrease insulin receptor mRNA levels (6,13). To test
the effect of the new mutations identified, we evaluated the
levels of mature insulin receptor mRNA by RNase protection
assay (Fig. 1), northern blot analysis (Fig. 2) or RT–PCR
(Fig. 3) in fibroblasts of patients with leprechaunism.
Increasing amounts of normal RNA generated increasing
signals for both actin and the insulin receptor by RNase
protection assay (Fig. 1A). However, whentheinsulinreceptor
signal was normalized for the actin signal, no significant
variations were observed over a 4-fold concentration range
(Fig. 1C), confirming the linearity of the assay. When RNA
frompatients’ cells was analyzed, thelevels of insulinreceptor
mRNA in cells frompatient GE was reduced to less than 10%
of normal, as in cells frompatient Mt Sinai, previously shown
tohavereducedinsulinreceptor mRNA levels(6). By northern
blot analysis, the insulin receptor mRNA produces multiple
bands, ranging in size from 5 to 10kb (6,13,14). Control
human fibroblasts had two prominent bands of 7.5 and 9.5kb
(Fig. 2). Although bands of size similar to those seen in
controls were visible in cells from patient NY-1, the levels of
insulin receptor mRNA were reduced to15% or less of those
measured in control cells in fibroblasts from patient NY-1 and
patient GE (Fig. 2).
In the case of patient VA (Fig. 3), expression of the allele
containing the deletion and the premature insertion of a stop
codon (del 2136–2139, Fig. 3A) was compared against
expressionof theother allelecontaining themissensemutation
(R899W; Fig. 3B). The PCR-amplified insulin receptor cDNA
from normal controls had two bands of 262 and 325bp
(Fig. 3C) after digestion with MspI. By contrast, insulin
receptor cDNA from patient VA was missing one MspI site
abolished by the R899W mutation, and had bands of 325 and
286bp. The lack of the 262bp band in the insulin receptor
cDNA of patient VA indicated that the allele containing the
Table 3. Mutations in the insulin receptor gene in patients with leprechaunismand Rabson–Mendenhall syndrome
Patient DiagnosisMutation Restriction enzymeMutation Restriction enzyme
2989C >A, P970T
3601C >T, R1174W
356C >T, A92V
þ BsmFI, exon 10
7BanII, exon 2
þ PvuII, exon 9
—, exon 16
7RsaI, exon 20
2770C >T, R899W
3472C >T, R1131W
2774T >C, I898T
7BanII exon 2
7MspI exon 14
7SfaNI exon 19
þ HinfI exon 14
The 22 exons of the insulin receptor were amplified using flanking primers and sequenced (6). Mutations identified were confirmed by sequencing in both
directions and on additional PCR products. When available, parents were also sequenced. The table reports the changes observed from the published sequence.
Nucleotides intheinsulinreceptor cDNA arenumbered fromtheinitiating methionine. Historically, theamino acid positionis numberedfromthehistidine, after
removal of the27-amino-acidsignal peptide(1,2). Whenindicated, restrictionsitesadded( þ ) orremoved(7) by themutationintherelativeexonareindicated.
Table 2. Insulin binding to lymphoblasts of patients with inherited severe insulin resistance
Specific binding of insulina
(fmol/mg cell protein)
Equilibrium binding of [125I]Insulin (1ng/ml) to lymphoblasts was measured for 2h at 16?C as in fibroblasts, except that the assay medium contained EDTA.
Non-specific binding, measured in the presence of 5mg/ml of cold ligand, was subtracted from each point. Data are means?SE of triplicates.
aNormal range (n¼8) 3.15–8.92; average 5.45?0.7.
bP<0.01 versus normal range.
Human Molecular Genetics, 2002, Vol. 11, No. 121467
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deletion of nucleotides 2136–2139 produced undetectable
levels of stable mRNA.
abnormal splicing, we checked for the presence of abnormal
transcripts by PCR using cDNA primers located in the exons
flanking thenonsensemutations identified. After RT–PCR and
40 cycles of amplification, bands of the predicted size were
identified in control RNA, but not in RNA from patients GE
and NY-1 (not shown).
Functional expression of mutations in the insulin receptors
in Chinese hamster ovary (CHO) cells
To confirm their causative role, the mutations identified in the
insulin receptor gene of patients with insulin resistance were
recreated in the insulin receptor cDNA and stably expressed in
CHO cells. Insulin binding to cells expressing normal and
mutant receptors is reportedinTable4. Insulinbinding tocells
expressing the R86P, DN281, I898T, R899W and R1131W
Figure1. RNaseprotectionassay. Total RNA obtainedfromcontrol fibroblasts andfibroblasts frompatients Mt Sinai andGE withleprechaunismwas hybridized
withlabeledantisenseRNA specific fortheinsulinreceptor(425nucleotides) andactin(127nucleotides). Afterhybridization, thereactionwassubjectedtoRNase
H digestion. Double-stranded RNA was then separated by PAGE and visualized by autoradiography. Counts per minute(CPM) in each band were counted by an
InstantImager and are reported in the bar graphs on the right.
Figure2. ReducedinsulinreceptormRNA levelsinfibroblastsof patientsGE andNY-1 withleprechaunism. Poly(A)þRNA (1–5mg) fromnormal (lanes1and2)
andinsulin-receptordefective(lanes3and4) humanfibroblastswasanalyzedby northernblotanalysisusing theinsulinreceptorandactincDNAsasprobes. CPM
corresponding to actin and insulin receptor bands were counted in an InstantImager and are reported in the graph on the right.
1468 Human Molecular Genetics, 2002, Vol. 11, No. 12
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mutations increased only minimally abovethe levels measured
inuntransfectedCHO cellsandtolessthan10% of theincrease
observedincellsexpressing thenormal insulinreceptorcDNA.
By contrast, cells expressing the P970T, I1116T and R1174W
mutations increased their insulin binding significantly: up to
63% of the level measured in cells expressing the normal
insulinreceptor. Therefore, themajor mechanismby which the
R86P, DN281, I898T, R899W and R1131W impair insulin
action is by impairing insulin binding.
To evaluate whether the P970T, I1116T and R1174W
mutations impaired insulin signaling, a dose–response curve
for insulin stimulation of glucose transport was obtained.
Glucose transport was half-maximally stimulated by insulin at
10.2?2.7pM in cells expressing the normal insulin receptor
cDNA, as compared with 320?15pM in untransfected CHO
cells. Cells expressing the P970T, I1116T and R1174W
mutations had dose–response curves for insulin stimulation
of glucose transport comparable to those measured in
untransfected CHO cells (half-maximal stimulation of glucose
transport was observed at 290–450pM of insulin), indicating
that the transfected receptors were not functional in signaling
glucose transport (not shown).
Of the patients shown in Tables 1 and 2, those with a clinical
diagnosis of leprechaunismdied before3 years of age, with an
average survival of 10.2?9.5 months. Patients clinically
diagnosed with Rabson–Mendenhall syndrome survived at
least 9?1.4 years. The mutations identified in patients with
leprechaunism all markedly impaired insulin binding to less
than 5% of normal both in the patients’ cells (Tables 1 and 2)
and when expressed in CHO cells (Table 4). By contrast, at
least one of the mutations identified in patients with longer
Figure 3. Comparison of expression of insulin receptor alleles in fibroblasts from patient VA with leprechaunism. (A) PvuII digestion of exon 9 of the insulin
receptor gene. Thedeletionof bp 2136–2139 created anovel PvuII restriction sitein exon 9 of patient VA and resulted in theappearanceof a novel 182bp band
in addition to thenormal 285bp band. (B) MspI digestionof PCR-amplified exon 14 of theinsulin receptor gene. TheR899W mutationabolished a MspI sitein
exon 14 of the insulin receptor gene and resulted in the appearance of a 145bp band upon enzymatic digestion. Patient VA was heterozygous for the R899W
mutationandhisDNA hadbotha122 anda145bpband. (C) cDNA analysis. TheinsulinreceptorcDNA was amplifiedbetweenbp2482 and3174 using specific
primers. TheDNA fragmentobtainedwas digestedwithMspI. DNA containing theR899W mutationgenerateda286bpband, insteadof thenormal 262bpband.
Patient VA had only the abnormal 286bp band, indicating that the other allele was not expressed.
Table 4. Insulin binding to CHO cells expressing normal and mutant insulin
Cells Specific insulin binding
(fmol/mg cell protein)
Parental CHO cells
Mutations in the insulin receptor gene were introduced by site-directed mut-
agenesis in the insulin receptor cDNA and stably transfected into CHO cells.
Insulin(25pM) binding was measured for 3h at 20?C. Dataaremeans?SE of
triplicates. Percentage of wild type is calculated after subtracting insulin
binding by untransfected cells.
aP<0.01 versus parental CHO cells.
Human Molecular Genetics, 2002, Vol. 11, No. 12 1469
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survival left residual insulin binding in the patients’ cells
(Tables 1 and 2) and when expressed in CHO cells (Table 4).
In this paper, we characterize several patients with inherited,
severe insulin resistant syndromes and different survival,
ranging from a few weeks to several years (Table 1). Two
major phenotypes are recognized in these patients: leprechau-
nism and Rabson–Mendenhall syndrome. Both disorders are
characterized by peculiar dysmorphic features and usually
different outcomes, with patients with Rabson–Mendenhall
syndrome surviving longer than patients with leprechaunism.
Both syndromes are inherited as autosomal recessive traits.
Although the clinical severity of patients with these severe
syndromes varies among different families, the phenotype
varies only slightly among multipleaffectedsiblingswithinthe
samefamily, suggesting that genotypeis themajor predictor of
phenotype (14–18). This differs frommilder insulin-resistance
syndromes, such as type A insulin resistance, in which other
genes in addition to the insulin receptor, or environmental
factors affect the severity of the phenotype (5).
Cells from all our patients with extreme insulin resistance
had defectiveinsulinbinding (Tables 1 and2). This defect was
complete in cells from patients with leprechaunism and
incomplete (with 18–27% residual binding) in fibroblasts or
lymphoblasts from patients with Rabson–Mendenhall
Sequence analysis and expression studies in CHO cells
confirmed that the mutations in the insulin receptor gene
identified in these patients affected insulin binding (Tables 3
and 4). Patients whose cells failed to bind insulin were
homozygous or compound heterozygous for mutations abol-
ishing insulin binding, either for the premature insertion of a
stop codon or for a structural alteration in the insulin receptor
preventing insulin binding.
Twopatients (GE andNY-1) werehomozygous orcompound
heterozygotes for null insulinreceptoralleles. All thenonsense
mutations identified in this study (E124X, 650X, 665X and
682X) and in one of our previous patients (R372X) (6) were
associated with greatly reduced insulin receptor mRNA levels
(Figs 1–3). Other mutations in the insulin receptor gene
resulting inprematurestop codons also reduceinsulin receptor
mRNA levels (13,16). However, this is not always the case.
Among patients homozygous for nonsense mutations in the
insulin receptor gene, cells from patient Qatar-1 (855X) had
normal mRNA levels (19). A second patient (Richmond)
homozygous for the R786X nonsense mutation had absent
PCR amplification of insulin receptor cDNA (20), while RNA
was not evaluatedinthethirdpatient (Cam-1) homozygous for
the K121X nonsense mutation (21). Thus, reduced mRNA
the insulin receptor gene. Figure 4 reports nonsense mutations
in the insulin receptor whose effect on mRNA levels was
determined either by measurement of mRNA levels or absence
of the relevant cDNA. Some nonsense mutations were the
result of single nucleotide changes, and others the result of
frameshifts or small deletions or insertions (Table 5). Natural
mutations reported in the insulin receptor gene introduce 10
UGA (opal), 5 UAG (amber), and 1 UAA (ochra) premature
termination codons. There was no clear association between
specific types of stop codons and effect on mRNA levels, even
though the single ochra codon, similar to the natural
termination codon of the insulin receptor gene, was associated
withnormal mRNA levels. ReducedinsulinreceptormRNA or
Figure 4. Effect of nonsense mutations on insulin receptor mRNA levels. The
figure is a schematic of the insulin receptor gene located on chromosome
19p13, with boxes representing individual exons. The location of nonsense
mutations within each exon is indicated. Mutations whose effect on insulin
receptormRNA levelswasdefinedeitherby northernblotanalysisorby cDNA
studies arereportedontherightwhenthey reducematuremRNA levels andon
the left of the gene if they do not reduce mRNA levels.
1470 Human Molecular Genetics, 2002, Vol. 11, No. 12
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cDNA was associated with E124X (this paper), W133X (22),
R372X (6), 650X (this paper), 665X (this paper), C682X (this
paper), R786X (20), 801X (23), R897X (13), R1000X (22) and
1118X (23) mutations. By contrast the presence of normal
levels of insulin receptor mRNA or cDNA was reported with
the 34X (24), R86X (25), Q672X (26) and 855X (19)
mutations. Importantly, multiple nonsense mutations within
exons 2 (R86X, E124X and W133X) and 10 (665X, Q672X
andC682X) hadadiscordant effect onmRNA levels, withtwo
mutations (R86X and Q672X) not reducing transcript levels
The reduction of mRNA levels with premature termination
codons occurs through nonsense-mediated RNA decay (27,28)
and is not expected to result in protein formation. Termination
codons located more than 55 nucleotides upstream of the 30-
most exon–exon junction usually mediate nonsense-mediated
mRNA decay (27,28). The recognition of abnormally termi-
nated mRNA is probably mediated by ribonucleoproteins that,
at time of splicing, bind close to the exon–exon junction and
flag to the mRNA surveillance machinery messages prema-
turely terminated (29). The additional nonsense mutations
identified in this study, when added to those already known in
the insulin receptor gene, indicate that the mechanism seems
morecomplex andthatthereareotherfactors, inadditiontothe
location of the premature stop codon, that are important
determinants of RNA stability. Thestudy of theeffect onRNA
stability of theseandothernatural nonsensemutationscanshed
light on the nature of these factors.
The missense mutations identified in our patients with
leprechaunism and shorter survival affected the extracellular
portion of the insulin receptor and abolished or markedly
reducedinsulinbinding (Fig. 5). TheA92V mutationidentified
Table 5. Effect of nonsense mutations in the insulin receptor gene on mRNA
Patient Mutation Nonsense codonmRNA levelsRef.
337C >T R86X
442A >T K121X
1195C >T R372X
2095C >T Q672X
2437C >T R786X
3079C >T R1000X
Nucleotides affected by the mutations are numbered from the starting
methionine. Historically, the amino acid position is numbered from the
histidine after removal of the 27-amino-acid signal peptide (1,2).
Figure5. Mutations intheinsulinreceptorgeneinpatientswithleprechaunismandRabson–Mendenhall syndrome. Theinsulinreceptorgeneis represented, with
each one of the 22 exons shown. Missense mutations are reported on the left-hand side and nonsense mutations on the right-hand side.
Human Molecular Genetics, 2002, Vol. 11, No. 12 1471
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in patient FL-1 is located close to the R86P mutation and one
of the major insulin binding sites of the receptor (7,30). The
DN281 mutation, found in two unrelated patients with
leprechaunism (8,31), was associated with absent insulin
binding to the patient’s cells in one study (8), but near-normal
insulinbinding inanotherstudy (31). Insulin-binding studiesin
fibroblasts can be difficult to interpret given the low levels of
expression of the insulin receptor gene. Expression studies
were not conducted in either case, and our data in transfected
CHO cells indicate that the DN281-mutant insulin receptor
does not bind insulin (Table 3). The I898T and R899W
mutations are located in the extracellular portion of the b
subunit of the insulin receptor and close to the T910M
mutation identified inanother patient with leprechaunism(23).
The T910M mutation was found to affect insulin binding by
impairing receptor processing (23), and it is likely that thetwo
novel mutations identified in this study reduce insulin binding
via a similar mechanism.
In our patients with Rabson–Mendenhall syndrome and
prolonged survival, at least one mutation was located in the
intracellular portion of the receptor bsubunit. The R1131W
mutation, identified in two unrelated patients with Rabson–
Mendenhall syndrome (ATL-2 and CIN), abolished insulin
binding when expressed in CHO cells despite its intracellular
location. Three other mutations in intracellular residues
(P970T, I1116T and R1174W) reduced insulin binding to
about 50% of normal. These data are compatiblewith insulin-
binding dataincellsfromthesepatientsshowing areductionof
insulin binding to about 25% of normal (Tables 1 and 2)
(11,12). Therefore, even intracellular mutations in the insulin
receptor can reduce insulin signaling by impairing, at least in
part, insulin binding. The R1174W mutation was previously
identified in a patient with leprechaunism(32), and was found
not to impair insulin binding in transiently transfected CHO
cells when binding was normalized to receptor number.
However, the R1174W-mutant receptors were degraded more
rapidly than normal receptors (32,33). This might result in
reduced steady-state levels of insulin receptors on the plasma
membrane, resulting inthedecreaseininsulinbinding reported
here in the patient’s cells and in transfected CHO cells.
The P970T, I1116T and R1174W substitutions prevented
insulin stimulation of glucose transport, indicating their
Themechanismby which this occurs has not yet beendefined.
These substitutions affect conserved domains of the insulin
the receptor or its interaction with intracellular substrates.
Specifically, the P970T mutation affects the consensus
sequence (969-NPEY-972) for the binding of insulin receptor
substrate 1 (IRS-1) (34,35), and is the first natural mutation
reported in such an important area of the insulin receptor. The
I1116T substitution affects an a-helical region to which the
catalytic loop of the insulin receptor kinase is attached (36). A
hydrophobic amino acid is present in this position inanumber
of tyrosine kinases, including the epidermal growth factor,
platelet-derived growth factor and fibroblast growth factor
receptors(EGFR, PDGFR andFGFR), andthetyrosinekinases
c-Src, c-Abl and cAPK (36). The R1174W substitution affects
theactivationloop of thetyrosinekinasedomainof theinsulin
receptor (36). A positively charged amino acid in this position
is conserved among several tyrosine kinases as well (36).
Previous studies of this latter mutation haveconfirmed that the
R1174W substitution abolishes insulin-stimulated receptor
autophosphorylation and kinase activity (32).
Taken together, our data indicate that, within the limits of
our patient population, the most severe phenotype with early
demise was observed when both mutations completely
abolished insulin binding and subsequent insulin action. By
contrast, when one mutation retained residual insulin-binding
activity, the survival was longer. While the majority of
patients reported to date seem to fit within this classification,
there are notable exceptions. There are patients with
leprechaunism in whom the mutations affect the intracellular
portion of the insulin receptor (9,10). However, in one case,
the mutations identified were not expressed in CHO cells (9),
and an effect on insulin binding cannot be excluded in view
of our results with the R1131W mutation. In the second case,
the patient was older than 1 year of age at the time of the
study and an affected brother died at 7 years of age with the
same syndrome (10), despite a phenotype of leprechaunism.
The prolonged survival of patients with leprechaunism within
this family indicates that residual insulin binding (and
probably action) correlates with survival, rather than the
specific phenotype of leprechaunism or Rabson–Mendenhall
syndrome. Therefore, the two diseases should be considered
as a continuous of a spectrum, in which the specific mutation
and the degree of impairment of insulin action predicts
survival, rather than the type of syndrome.
MATERIALS AND METHODS
Several new patients are reported in this manuscript. A brief
description of each new patient will follow. Details on patients
ATL-1 (7,14), ATL-2 (11,12), Mt Sinai (6) and NZ (8) can be
found in previous papers.
Patient GE with leprechaunism was a Caucasian female, the
first child of healthy unrelated parents of European descent.
Pregnancy was complicated by oligohydramnios and the
patient was born at 40 weeks of gestation via Caesarean
section. The proband weighed 1960g (small for gestational
age), her length was 44cm and the head circumference was
34.5cm. At birth, the infant was noted to have dysmorphic
features, such as a small face with prominent eyes and thick
lips, large, pointed ears, depressed nasal bridge, wide nostrils,
absence of subcutaneous fat, wrinkled and loose skin,
hirsutism, acanthosis nigricans, disproportionately large geni-
taliawithbilateral inguinal hernia, andaprotuberant abdomen.
A systolic ejection murmur was also noted. This prompted
further diagnostic evaluation, which revealed hypertrophic
obstructivecardiomyopathy. Unstablebloodsugars withsevere
hypoglycemia (down to 21mg%) and elevated insulin levels
(up to 800mU/ml) were noted shortly after birth. Abdominal
ultrasound revealed bilateral polycystic ovaries, with liver,
spleen, kidneys, bladder and uterus of normal size and
laparotomy and surgical removal of the ovaries shortly after 7
1472Human Molecular Genetics, 2002, Vol. 11, No. 12
by guest on June 12, 2013
weeks of age. The peritoneal cavity was almost completely
occupied by large cystic ovaries, which were removed.
Histologic examination was consistent with bilateral juvenile
granulosa cell tumor of the ovaries. A liver biopsy was also
performed during surgery for the cholestatic appearance of the
liver. Liver histology was consistent with cytomegalovirus
hepatitis. Two days after surgery at 55 days of age, the patient
died of refractory heart failure. Autopsy was not performed. A
detailed clinical report of this patient has been published (37).
Bothparents of theproband werephenotypically normal. An
oral glucose tolerance test performed in both parents was
normal, but elevated insulin levels were measured before and
during the test, consistent with mild, asymptomatic insulin
first child of healthy unrelated parents. Pregnancy and delivery
were uncomplicated. The patient was born at 37 weeks of
gestation via natural delivery. Birthweight was 1800g (small
for gestational age), length was 44.5cm and head circumfer-
ence was 30cm. For the intrauterine growth restriction, she
remained in the hospital for 10 days. Initial studies indicated
presence of antibodies against cytomegalovirus. At about 1
month of age, she was readmitted to the hospital for fever
(40?C) andirritability. Physical examinationwassignificantfor
low weight (2.9kg), decreased subcutaneous fat, generalized
hirsutism, gingival hyperplasia, breast enlargement, heart
murmur, distended abdomen, and prominence of clitoris and
labia majora. Echocardiogram documented left ventricular
hypertrophy and an atrial septal defect. Abdominal ultrasound
demonstrated bilateral cystic ovaries. Routine laboratory
23.4mM). The fever responded to antibiotics, but the blood
glucose remained elevated. Insulin levels returned elevated on
several occasions (>500mU/ml), with elevated C-peptide
(20mg/l; normal 0.8–4.0mg/l). Insulin therapy (up to 10U/kg/
day) wastriedwithouteffect. Bloodglucoserespondedtoadiet
low in carbohydrates, and she was discharged home. She
8 months of age, she received a trial of insulin-like growth
factor I (IGF-I) (38), with no improvement.
PatientNY-1 withleprechaunismwasaHispanic femaleborn
at 36 weeks gestation to an 18-year-old mother who had
receivednoprenatal care. Delivery wasvaginal, withApgarsof
8 at 1 minuteand 8 at 5 minutes. Theinfant was noted to have
severe intrauterine growth restriction and to have dysmorphic
hyperglycemia (glucose 307mg/dl) at 2 months of age.
Chromosomes were normal (46,XX). Insulin levels were 885,
1194 and 1290mU/ml on separate occasions, with a C-peptide
level of 16.5mg/l (normal 0.5–2mg/l). The child failed to
respond to exogenous insulin. A gastric tube was placed to
facilitatefeeding. Thechildexpiredat4monthsof ageduring a
mild infection. No information was given on the father of
Patient VA with leprechaunismwas aCaucasianmaleborn at
38 weeks gestation via Caesarean section for intrauterine
growth restriction. Birthweight was 1729g (small for gesta-
tional age), length was 41.9cm, and head circumference was
33cm. Apgars were 7 at 1 minute and 9 at 5 minutes. He was
small for gestational age. He was noted to have dysmorphic
features at birth consistent with leprechaunism. He also had
several respiratory problems, apnea and bradycardia, choles-
tasis with direct hyperbilirubinemia, and a restrictive cardio-
myopathy. There was abnormal glucose homeostasis with
fasting hypoglycemia and postprandial hyperglycemia. Insulin
levels were elevated (535, 1084 and 4394mU/ml). After 2
weight of 2000g. He was fed via gastric tube a diet rich in
complex carbohydrates every 3 hours to prevent hypo- and
hyperglycemia. He had a progressive worsening of his
respiratory status, and failed to thrive. At 3 months of age,
he presented fever (38.5?C) and acute deterioration of his
respiratory status. A blood count indicated leukocytosis with
bandemia. He had normal blood pH and blood gases (on
oxygen) with normal electrolytes. His average blood glucose
was 106?66mg/dl (n¼10) with one value of 20 and one of
280mg/dl. He died of heart failure shortly after admission.
Both parents are phenotypically normal.
Patient CIN with Rabson–Mendenhall syndrome was born
after a full-term pregnancy complicated by preterm labor and
oligohydramnios. He was the first child of a 21-year-old
mother. Thebirthweightwas2138g (small forgestational age).
Hewas diagnosedwithdiabetes at3 weeks of ageandinitiated
on insulin therapy. This was stopped after verifying thelack of
responseanddeterminationof highcirculating levelsof insulin
(above 2000mU/ml), indicative of insulinresistance. He has
beenmanagedwithasugar-freediet, glipzide, andglucophage,
with glucoselevels between200 and 400mg/dl. Other medical
problemsincluderepeatedepisodesof toxic synovitisof theleft
hip, and repeated ear infections requiring placement of ear
tubesandtonsillectomy. Hewasnotedtohaveearly eruptionof
permanent teeth at 3 years of age. Developmentally, he is
mildly delayed. He sat at 9 months and walked at 2 years of
age. Speech development, however, was normal. On examina-
tionat 5 years of agehis weight was 13.9kg (<5thpercentile,
appropriate for a 32-month-old boy), height was 95.4cm
(<5th percentile, appropriate for a 34-month-old boy), head
circumferencewas 47.5cm(<5thpercentile, appropriatefor a
14-month-old boy). He appeared very tiny, with coarse facial
features. The skin was thickened and coarse. Acanthosis
nigricans was present in the neck, axilla and groin. Ears had
thickenedhelices. Thenosewasupturnedwithabroadtip. The
mouth was wide with thick lips. The palate was narrow and
high-arched. Eight permanent teeth were present, and were
irregularly placed and crowded. Cardiac and lung exam were
normal. The liver edge was palpable 2cm below the costal
margin. Genitaliawerenormal for amale, with alargephallus.
Fingers were short and broad, with mild flexion contractures.
Neurological examination was normal. The child developed
ketoacidosis at 8 years of age.
The family history was significant for absence of consangui-
nity and distant relatives with diabetes mellitus. Glucose
tolerance tests on the parents revealed normal tolerance but
moderate hyperinsulinemia, with fasting insulin levels of 78.1
and 43.8mU/ml in the mother and father, respectively.
Patient CG-1 is an8-year-oldHispanic femalewithRabson–
Mendenhall syndrome. Only limitedinformationisavailableon
this patient. On physical examination, she had growth
restriction, with weight and height about 3 SD below average,
and mild developmental delay. She has dysmorphic features,
Human Molecular Genetics, 2002, Vol. 11, No. 12 1473
by guest on June 12, 2013
including a high arched, V-shaped palatewith dysplastic teeth,
hirsutism, acanthosis nigricans, breast enlargement and clitor-
omegaly. A random insulin level was 500mU/ml, with a
simultaneous glucose level of 85mg/dL. The mother has a
normal glucose tolerance test. No information is available
about the father.
Sera, growth media and trypsin solutions were fromSigma (St
Louis, MO). Radiochemicals, including [125I]insulin, were
from Amersham. Products for molecular biology were from
Roche Molecular Biochemicals (Indianapolis, IN). Sigma was
the source of other chemicals.
Fibroblast cultures were established from skin biopsies of
patients and their parents for diagnostic purposes. Control
fibroblastcultures(GM 003348andGM 005756) wereobtained
fromtheCoriell InstituteforMedical Research(Camden, NJ) or
The research use of these cells was approved by the Emory
University Institutional ReviewBoard.Fibroblastsfrompatients
ATL-1, ATL-2, Mt Sinai and NZ were obtained as previously
described (6–8,11). Cells were grown in Dulbecco–Vogt (DV)
mediumcontaining15% fetal bovineserum.Lymphoblastswere
growninRPMI mediumcontaining 15% fetal bovineserum.
Insulin binding to lymphoblasts and cultured fibroblasts was
performed at 20?C in Earle’s balanced salt solution (EBSS)
buffered with tris(hydroxymethyl)aminomethane (26mM,
pH7.4), as previously described (14). Ligand binding was
normalized to cell proteins, corrected for non-specific binding
(measured in the presence of 5mg/ml of cold ligand), and
expressedasfemtomolesof ligandboundpermilligramsof cell
DNA and RNA analysis
GenomicDNA wasamplifiedby PCR usingprimersflankingthe
22 exons of the insulin receptor gene (6). Amplified DNA was
purifiedandsequencedaspreviously described(6). Sequencing
results were confirmed by enzymatic digestion using the
gross deletions was performed using EcoRI and BamHI as
receptor cDNA probes covering thewholecoding region(39).
Cellular RNA was extracted with guanidinium thiocyanate
and poly(A)þRNA was analyzed by northern blot analysis
using a3kbfragmentcorresponding tothe30endof theinsulin
receptor cDNA as a probe (6,14). After autoradiography, the
radioactivity in each band was measured by an InstantImager
(Packard). The blot was then stripped and hybridized to the
actin cDNA for normalization.
RNase protection assay was performed using the manufac-
turer’sprotocol (Ambion) ontotal RNA using antisense-labeled
RNA to actin and insulin receptor. Bands on the gel were
quantified using the InstantImager (Packard) as above.
Mutagenesis of the insulin receptor cDNA
The mammalian expression vector containing the insulin
receptor cDNA (40) was mutagenized by site-directed
mutagenesis using the Quik Change system (Stratagene, La
Jolla, CA), following themanufacturer’s instructions. Thefinal
clones weresequenced to confirmthepresenceof themutation
and the absence of PCR artifacts. The clones were transfected
into CHO cells using lipofectamine. Cells were selected for 2
weeks in 0.8mg/ml of G418 and then used for insulin binding
(performed as described above) and insulin stimulation of
glucose transport. Insulin stimulation of glucose transport in
transfected CHO cells was measured and analyzed as
previously described (30,40).
This work was supportedinpart by NIH Grant R29 DK 48742
and by Grant 95-192 from the Genentech Foundation for
GrowthandDevelopment. Wethank DrRosannaGatti, Gaslini
Hospital, Genoa, Italy, Dr Generoso Andria, University of
Naples, Italy, Dr David Bick, Genetics and IVF Institute,
Fairfax, VA, Dr Susan Skowers-Brooks, Institute for Basic
Research in Developmental Disabilities, Staten Island, NY, Dr
Elizabeth Schorry, Children’s Hospital Medical Center, Cin-
cinnati, OH, Dr Gary Freidenberg, Indiana University Medical
Center, Indianapolis, IN and Dr Aline da Mota Rocha, Brazil
for referring patients with insulin resistance.
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