Sequencing analysis of insulin receptor defects and detection of two novel mutations in INSR gene.

Mutations in the insulin receptor gene cause the inherited insulin resistant syndromes Leprechaunism and Rabson-Mendenhall syndrome. These recessive conditions are characterized by intrauterine and post-natal growth restrictions, dysmorphic features, altered glucose homeostasis, and early demise. The insulin receptor gene (INSR) maps to the short arm of chromosome 19 and is composed of 22 exons. Here we optimize the conditions for sequencing this gene and report novel mutations in patients with severe insulin resistance.
METHODS: PCR amplification of the 22 coding exons of the INSR gene was performed using M13-tailed primers. Bidirectional DNA sequencing was performed with BigDye Terminator chemistry and M13 primers and the product was analyzed on the ABI 3100 genetic analyzer. Data analysis was performed using Mutation Surveyor software comparing the sequence to a reference INSR sequence (Genbank NC_000019).
RESULTS: We sequenced four patients with Leprechaunism or Rabson-Mendenhall syndromes as well as seven samples from normal individuals and confirmed previously identified mutations in the affected patients. Three of the four mutations identified in this group caused premature insertion of a stop codon. In addition, the INSR gene was sequenced in 14 clinical samples from patients with suspected insulin resistance and one novel mutation was found in an infant with a suspected diagnosis of Leprechaunism. DISCUSSION: Leprechaunism and Rabson-Mendenhall syndrome are very rare and difficult to diagnose. Diagnosis is currently based mostly on clinical criteria. Clinical availability of DNA sequencing can provide an objective way of confirming or excluding the diagnosis.

Introduction

The insulin receptor is a membrane protein composed of two extracellular α subunits that bind insulin and two β subunits which span the plasma membrane and have an intracellular tyrosine kinase domain [1], [2]. Insulin binding to the α-subunits causes a conformational change that results in the activation of the kinase activity of the β-subunits with subsequent autophosphorylation and activation of kinase activity toward intracellular substrates [1], [2]. A single gene codes for both subunits. The resulting preprotein is post-translationally cleaved into mature alpha and beta subunits that assemble together as a heterotetramer to generate the mature insulin receptor [1], [2], [3]. The INSR gene maps to the short arm of chromosome 19 and is composed of 22 exons. Alternative splicing of the 36 base pair exon 11 results in two isoforms which differ in sequence at the C-terminal end of the insulin-binding alpha-subunit [3].

Mutations in INSR cause the insulin-resistant syndromes Leprechaunism, also known as Donohue syndrome [4], Rabson–Mendenhall syndrome and type A insulin resistance [5], [6]. Leprechaunism, (OMIM 246200), the most severe of the insulin resistant syndromes, is characterized by intrauterine growth restriction (IUGR), 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 genitalia in the male and cystic ovaries in the female [7], [8], [9]. Cells from most patients with Leprechaunism have markedly reduced insulin binding, although exceptions were reported [10], [11].

The slightly less severe Rabson–Mendenhall syndrome (OMIM 262190) was first described in three siblings with dental and skin abnormalities, abdominal distension, phallic enlargement, early dentition, coarse senile-looking facies, striking hirsutism, intellectual disability, prognathism, thick fingernails and acanthosis nigricans. Insulin-resistant diabetes mellitus, ketoacidosis, intercurrent infections, pineal hyperplasia and ovarian tumor [12]. Children have initial postprandial hyperglycemia and fasting hypoglycemia, caused by inappropriately elevated insulin levels at the time of fasting [6], [13]. Patients with Rabson–Mendenhall syndrome can survive beyond 1 year of age and, with time, develop constant hyperglycemia followed by diabetic ketoacidosis and death. This is accompanied by a progressive decline of insulin levels, which become insufficient to prevent liver glucose synthesis and release of fatty acids by adipocytes [13].

Mutations in the insulin receptor can cause disease with a dominant pattern of inheritance as well. For example, a mutation (p.Gly996Val) in a conserved Gly-X-Gly-X-X-Gly motif impairs tyrosine kinase activity of the insulin receptor and is associated with insulin-resistant diabetes mellitus and acanthosis nigricans, suggesting a dominant-negative pathogenesis [14], [15], [16]. A different mutation (p.Arg1174Gln) with unknown functional effects in INSR is implicated in familial hyperinsulinemic hypoglycemia type 5 in a few patients (HHF5) [17].

Leprechaunism and Rabson–Mendenhall syndrome are inherited as autosomal recessive traits. There is some correlation between genotype and phenotype, with mutations that markedly impair insulin binding resulting in the most severe phenotypes, while the presence of at least one mutation leaving residual insulin binding activity is associated with longer survival [6], [18]. Definitive genotype–phenotype correlation for INSR defects is difficult to establish primarily due to the rarity of these syndromes [6], a paucity of functional studies to determine the effect of mutations on insulin binding or signaling, and difficulty in establishing a precise molecular diagnosis due to the lack of clinically validated INSR gene sequencing [6], [19].

Herein we develop a clinically validated sequencing method to discover mutations in the INSR gene. Bidirectional sequencing with BigDye terminator and M13 primers was used to examine mutations in the coding regions and exon–intron boundaries of the INSR gene. A combination of the biochemical and DNA tests can provide accurate diagnosis for the insulin receptor deficiency.

Materials and methods

Patients/samples

DNA from 11 unrelated individuals (7 controls and 4 patients with Leprechaunism) was used to determine performance characteristics of this INSR full gene sequencing assay. Of these four patients with Leprechaunism, three of them, referred to here as 452, NY1, and 5880, had previously been described [6], [7], [23] Fibroblasts from each of these patients were received and DNA was extracted by MagNA Pure. The fourth patient with Leprechaunism, SLC, was not previously described but fit the clinical criteria. The diagnosis of Leprechaunism for all four patients was established from clinical presentation (failure to thrive, growth retardation, markedly elevated insulin levels, hirsutism, and acanthosis nigricans) and markedly reduced insulin binding to patients' fibroblasts. The samples were de-identified following an Institutional Review Board (IRB)-approved protocol. Fourteen additional samples referred to the ARUP Sequencing Laboratory by the patients' clinicians for INSR mutation detection were sequenced and analyzed.

DNA sequencing of the INSR gene

DNA was extracted from leukocytes in blood using MagNAPure Compact instrument (Roche Applied Science, Indianapolis, IN). Nucleic acid sequencing for the INSR gene coding region was performed by standard dideoxy termination. PCR primers were developed for the 22 exons of the INSR isoform containing exon 11 (NM_002082). Eighteen sets of PCR primers used in this validation were previously published [20], however, in the current study four sets of primers were re-designed to optimize PCR and sequencing results (see Table 1). We added another internal primer set to exon three interior to the homopolymer region to obtain cleaner sequence. In addition, exons 18 and 19 were consolidated into one amplicon. polymerase chain reaction of the 22 coding exons of the INSR gene was performed using M13-tailed primers Premix D (Epicentre, Madison, WI), and Platinum Taq (Invitrogen, Carlsbad, CA) using PCR conditions shown in Table 2 below. Unused PCR primers and unincorporated nucleotides were inactivated by incubation with ExoSAP (USB Corporation, Cleveland, OH). Bidirectional DNA sequencing was performed with BigDye Terminator chemistry (ABI, Foster City, CA) and M13 primers (IDT, Coralville, IA) and the product was analyzed on the ABI 3730. Data analysis was performed using Mutation Surveyor software (SoftGenetics, State College, PA) and GenBank reference sequence NG_008852.1.

Table 1

Sequence of INSR primers used in the current study. Lower case letters represent the M13 tail sequence.

Primer name	Sequence
IR E1F	tgtaaaacgacggccagtCGCGCTCTGATCCGAGGAGA
IR E1R	caggaaacagctatgaccAGGGTTCTCAGTCCACAAGC
IR E2F #2	tgtaaaacgacggccagtTCTTGCTTTCTGTTCATTTTC
IR E2R #2	caggaaacagctatgaccACGAGACACTGCTTAGAACC
IR E3F #2	tgtaaaacgacggccagtCAGACAGGAATTGGACAAA
IR E3F Int	tgtaaaacgacggccagtGACCATCTGTAAGTCACACG
IR E3R	caggaaacagctatgaccAGCAGAGACCTCACTCATAGCCAA
IR E4F	tgtaaaacgacggccagtGCCTGAGATGTCTGAAGGAC
IR E4R	caggaaacagctatgaccGCCACTGAACGACCATCCTA
IR E5F	tgtaaaacgacggccagtCTCACCATGGAGAATCATGA
IR E5R	caggaaacagctatgaccCTAATACACGAACTTCCTAG
IR E6F #2	tgtaaaacgacggccagtCACACCATCTTGGAGTTGTA
IR E6R	caggaaacagctatgaccTGTAATGCACTTGAATCATGCTG
IR E7F #2	tgtaaaacgacggccagtTTGGTCTGAAACTACACTGAAA
IR E7R	caggaaacagctatgaccAAACGTAGCAAGCACAGAGC
IR E8F	tgtaaaacgacggccagtCGGTCTTGTAAGGGTAACTG
IR E8R #2	caggaaacagctatgaccGCCAATAACCATATCAAGGA
IR E9F	tgtaaaacgacggccagtGCACACTGTTTCTCATGATG
IR E9R	caggaaacagctatgaccAGAGGTGAAGCAAAGTGCAT
IR E10F	tgtaaaacgacggccagtTGTTCAGCCGCAGAGACTTG
IR E10R	caggaaacagctatgaccCGGTCCCTAAGTAATGACCT
IR E11F	tgtaaaacgacggccagtGTGGTCTGTCTAATGAAGTT
IR E11R	caggaaacagctatgaccGAATTGGTGAAGCATCTGCT
IR E12F	tgtaaaacgacggccagtTGATGGTGATGGTGTCATCATA
IR E12R	caggaaacagctatgaccTGTCCTTGGTCAGCCTTGATGT
IR E13F #2	tgtaaaacgacggccagtCAATCTTGTGGGATGAGTTT
IR E13R	caggaaacagctatgaccTACTAATAGCACAGTACCTG
IR E14F	tgtaaaacgacggccagtTGGACACTCCCAGATGTGCA
IR E14R	caggaaacagctatgaccACCATGCTCAGTGCTAAGCA
IR E15F	tgtaaaacgacggccagtGTGAACTTTGTTGGAAACACATTG
IR E15R	caggaaacagctatgaccCCTATACCTATATCAAGGCATG
IR E16F	tgtaaaacgacggccagtTCTGCTGGTAAGGGCTGCCA
IR E16R	caggaaacagctatgaccCTCACTCAATGGTGAAGGCA
IR E17F	tgtaaaacgacggccagtCCAAGGATGCTGTGTAGATAAG
IR E17R	caggaaacagctatgaccTCAGGAAAGCCAGCCCATGTC
IR E18–19F	tgtaaaacgacggccagtGGAGAACCCTGGTGAGTC
IR E18–19R	caggaaacagctatgaccTCCTTCTGAAATCAAACCTG
IR E20F	tgtaaaacgacggccagtAGGTTAAGAGCGTGTGAACCT
IR E20R	caggaaacagctatgaccGAATTCAAGCCCAGCGTCCAT
IR E21F	tgtaaaacgacggccagtTGTTACTACTATCAACTGTC
IR E21R	caggaaacagctatgaccACCTGTAACATACAGCATGC
IR E22F	tgtaaaacgacggccagtACTCACCCAGGACGTGTCCTTCT
IR E22R	caggaaacagctatgaccACCAGAGGAAAGCGAAAATG

Table 2

PCR conditions used in this study.

Temp (°C)	Rate (Δ°/cycle)	Time	Cycles
95		5 m
94		30 s
62	−0.5	45 s	10
72		1 m
94		30 s
57		45 s	25
72		5 m

Results

INSR mutation update

The INSR gene product contains 120 kilobases and is composed of 22 exons. There are three transcription initiation sites located at 276, 282 and 283 base pairs upstream of the translation initiation site. The alpha subunit is encoded by exons one through 11 (and part of exon 12) whereas the beta subunit is encoded by exons 12–22 [1], [2]. The insulin receptor is synthesized as a single protein that is post-translationally cleaved at a four amino acid site (p.759_762, RKRR, encoded by exon 12) to generate the mature alpha and beta subunit.

The INSR gene product contains a leader sequence of 27 amino acids. Cleavage of these amino acids results in the mature active protein. As a result of this cleavage, the nomenclature of reported variations differs depending on the author, time of publication, and source of their reference DNA sequence. For this reason, we reviewed the published literature for all known INSR variations to determine the consistent amino acid position using the current nomenclature, in both the immature and the mature protein. Absolute nucleotide positions were kept consistent with the beginning of the cDNA regardless of protein cleavage (recommendations of the Human Genome Variation Society, http://www.hgvs.org/rec.html). In addition, the INSR gene has two isoforms that differ only by the 12 amino acids encoded by the alternatively spliced exon 11. These isoforms have slightly different reported biological activity and different abundance in different tissues with the isoform containing exon 11 being predominant in the liver; the other in leukocytes; with similar expression levels in most other tissues such as skeletal muscle, placenta and adipose tissue [21].

To date, there are 132 reports of disease causing mutations in the INSR gene in the literature (Table 4). The majority of the mutations (64%, or 85 of 132) are missense mutations, 13% (17 of 132) are nonsense mutation, 4.7% are splice site mutations, 8.3% are deletions (11/132), 2.3% are insertions (3/132), 1.5% are insertions and deletions (indel, 2/132), 5.3% are gross deletions or complex gene rearrangements (7/132) (Fig. 1). Most of the mutations located in the first 11 exons result in Leprechaunism while the mutations in the beta subunit are found more frequently in patients with Rabson–Mendenhall syndrome.

Table 4

Compilation of reported INSR mutations.

A. Missense/nonsense mutations
Location	Mutation type	Nucleotide change	Amino acid change (HGVS nomenclature)	Amino acid change (legacy, mature protein)	Phenotype	Reference
Exon 1	Nonsense	c.90C > A	p.Tyr30Term	Tyr3Term	Rabson–Mendenhall syndrome	[30]
Exon 2	Missense	c.121C > T	p.Arg41Trp	Arg14Trp	Rabson–Mendenhall syndrome	[31]
Exon 2	Missense	c. 126C > A	p.Asn42Lys	Asn15Lys	Leprechaunism	[32]
Exon 2	Missense	c.164T > C	p.Val55Ala	Val28Ala	Leprechaunism	[33]
Exon 2	Missense	c.172G > A	p.Gly58Arg	Gly31Arg	Leprechaunism	[34]
Exon 2	Missense	c.257A > G	p.Asp86Gly	Asp59Gly	Insulin resistance	[35]
Exon 2	Missense	c.266T > C	p.Leu89Pro	Leu62Pro	Insulin resistance	[35]
Exon 2	Missense	c.338G > C	p.Arg113Pro	Arg86Pro	Leprechaunism	[8]
Exon 2	Nonsense	c.337C > T	p.Arg113Term	Arg86Term	Insulin resistance	[36]
Exon 2	Missense	c.356C > T	p.Ala119Val	Ala92Val	Leprechaunism	[6]
Exon 2	Missense	c.359T > A	p.Leu120Gln	Leu93Gln	Insulin resistance	[37]
Exon 2	Missense	c.425G > T	p.Gly142Val	Gly115Val	Leprechaunism	This report
Exon 2	Missense	c.433C > T	p.Arg145Cys	Arg118Cys	Insulin resistance A	[38]
Exon 2	Missense	c.438C > G	p.Ile146Met	Ile119Met	Insulin resistance	[39]
Exon 2	Nonsense	c.442A > T	p.Lys148Term	Lys121Term	Leprechaunism	[40]
Exon 2	Nonsense	c.451G > T	p.Glu151Term	Glu124Term	Leprechaunism	[6]
Exon 2	Nonsense	c.479G > A	p.Trp160Term	Trp133Term	Insulin resistance	[32]
Exon 2	Missense	c.499G > T	p.Val167Leu	Val140Leu	Insulin resistance A	[41]
Exon 2	Missense	c.511T > A	p.Tyr171Asn	Tyr144Asn	Diabetes, NIDDM	[42]
Exon 2	Missense	c.515T > G	p.Ile172Ser	Ile145Ser	Diabetes, NIDDM	[42]
Exon 2	Missense	c.557G > T	p.Cys186Phe	Cys159Phe	Rabson–Mendenhall syndrome	[19]
Exon 2	Missense	c.586T > A	p.Cys196Ser	Cys169Ser	Diabetes, NIDDM	[42]
Exon 2	Missense	c.628T > A	p.Trp210Arg	Trp183Arg	Diabetes, NIDDM	[42]
Exon 3	Missense	c.659C > T	p.Pro220Leu	Pro193Leu	Leprechaunism	[43]
Exon 3	Missense	c.679G > A	p.Gly227Ser	Gly200Ser	Diabetes, NIDDM	[42]
Exon 3	Missense	c.694G > A	p.Gly232Ser	Gly205Ser	Diabetes, NIDDM	[42]
Exon 3	Missense	c.707A > G	p.His236Arg	His209Arg	Leprechaunism	[32]
Exon 3	Missense	c.712G > A	p.Glu238Lys	Glu211Lys	Rabson–Mendenhall syndrome	[30]
Exon 3	Missense	c.766C > T	p.Arg256Cys	Arg229Cys	Rabson–Mendenhall syndrome	[19]
Exon 3	Missense	c.779T > C	p.Leu260Pro	Leu233Pro	Insulin resistance	[44]
Exon 3	Missense	c.835C > T	p.Arg279Cys	Arg252Cys	Insulin resistance	[45]
Exon 3	Missense	c.836G > A	p.Arg279His	Arg252His	Insulin resistance	[37]
Exon 3	Missense	c.839G > A	p.Cys280Tyr	Cys253Tyr	Insulin resistance A	[46]
Exon 3	Nonsense	c.895C > T	p.Gln299Term	Gln272Term	Leprechaunism	[47]
Exon 3	Missense	c.902G > A	p.Cys301Tyr	Cys274Tyr	Leprechaunism	[48]
Exon 3	Missense	c.932G > A	p.Cys311Tyr	Cys284Tyr	Rabson–Mendenhall syndrome	[49]
Exon 4	Missense	c.1049C > T	p.Ser350Leu	Ser323Leu	Insulin resistance	[50]
Exon 4	Nonsense	c.1072C > T	p.Arg358Term	Arg331Term	Insulin resistance	[51]
Exon 4	Nonsense	c.1114C > T	p.Arg372Term	Arg345Term	Insulin resistance A	[52]
Exon 5	Missense	c.1156G > A	p.Gly386Ser	Gly359Ser	Rabson–Mendenhall syndrome	[53]
Exon 5	Missense	c.1177G > A	p.Gly393Arg	Gly366Arg	Leprechaunism	[54]
Exon 5	Nonsense	c.1195C > T	p.Arg399Term	Arg372Term	Insulin resistance	[55]
Exon 5	Missense	c.1225T > G	p.Phe409Val	Phe382Val	Insulin resistance	[56]
Exon 5	Nonsense	c.1246C > T	p.Arg416Term	Arg389Term	Leprechaunism	[57]
Exon 6	Missense	c.1316G > C	p.Trp439Ser	Trp412Ser	Leprechaunism	[58]
Exon 6	Missense	c.1372A > G	p.Asn458Asp	Asn431Asp	Insulin resistance	[37]
Exon 6	Missense	c.1459A > G	p.Lys487Glu	Lys460Glu	Leprechaunism	[59]
Exon 6	Missense	c.1466A > G	p.Asn489Ser	Asn462Ser	Insulin resistance	[32]
Exon 8	Missense	c.1627A > T	p.Thr543Ser	Thr516Ser	Diabetes, NIDDM	[42]
Exon 8	Missense	c.1650G > A	p.Ala550Ala	Ala523Ala	Association with reduced diastolic blood pressure	[60]
Exon 9	Missense	c.1975T > C	p.Trp659Arg	Trp632Arg	Leprechaunism	[61]
Exon 10	Nonsense	c.2095C > T	p.Gln699Term	Gln672Term	Leprechaunism	[59]
Exon 10	Missense	c.2201A > C	p.Asp734Ala	Asp707Ala	Leprechaunism	[62]
Exon 12	Missense	c.2286G > T	p.Arg762Ser	Arg735Ser	Insulin resistance	[63]
Exon 12	Nonsense	c.2437C > T	p.Arg813Term	Arg786Term	Leprechaunism	[64]
Exon 12	Missense	c.2453A > C	p.Tyr818Cys	Tyr791Cys	Leprechaunism	[65]
Exon 13	Missense	c.2572A > G	p.Thr858Ala	Thr831Ala	Diabetes, NIDDM	[66]
Exon 13	Missense	c.2621C > T	p.Pro874Leu	Pro847Leu	Leprechaunism/Rabson–Mendenhall syndrome	[31]
Exon 13	Nonsense	c.2668C > T	p.Arg890Term	Arg863Term	Leprechaunism	[65]
Exon 13	Missense	c.2669G > C	p.Arg890Pro	Arg863Pro	Diabetes, NIDDM	[42]
Exon 13	Nonsense	c.2673T > A	p.Tyr891Term	Tyr864Term	Insulin resistance A	[46]
Exon 14	Missense	c.2717C > G	p.Ala906Gly	Ala879Gly	Diabetes, NIDDM	[42]
Exon 14	Nonsense	c.2770C > T	p.Arg924Term	Arg897Term	Leprechaunism	[23]
Exon 14	Missense	c.2774T > C	p.Ile925Thr	Ile898Thr	Leprechaunism	[6]
Exon 14	Missense	c.2776C > T	p.Arg926Trp	Arg899Trp	Leprechaunism	[6]
Exon 14	Missense	c.2810C > T	p.Thr937Met	Thr910Met	Leprechaunism	[67]
Exon 16	Missense	c.2971C > A	p.Leu991Ile	Leu964Ile	Leprechaunism	This report
Exon 16	Missense	c.2989C > A	p.Pro997Thr	Pro970Thr	Rabson–Mendenhall syndrome	[6]
Exon 17	Missense	c.3034G > A	p.Val1012Met	Val985Met	Diabetes, NIDDM	[68]
Exon 17	Missense	c.3059G > A	p.Arg1020Gln	Arg993Gln	Insulin resistance	[69]
Exon 17	Missense	c.3067A > T	p.Ile1023Phe	Ile996Phe	Insulin resistance	[70]
Exon 17	Nonsense	c.3079C > T	p.Arg1027Term	Arg1000Term	Insulin resistance	[32]
Exon 17	Missense	c.3104G > T	p.Gly1035Val	Gly1008Val	Diabetes, NIDDM	[14]
Exon 17	Missense	c.3143G > A	p.Gly1048Asp	Gly1021Asp	Insulin resistance	[71]
Exon 17	Missense	c.3160G > A	p.Val1054Met	Val1027Met	Leprechaunism	[61]
Exon 17	Missense	c.3164C > T	p.Ala1055Val	Ala1028Val	Insulin resistance A	[41]
Exon 17	Missense	c.3224C > A	p.Ala1075Asp	Ala1048Asp	Insulin resistance	[72]
Exon 17	Missense	c.3255C > T	p.His1085His	His1058His	Association with polycystic ovary syndrome in lean women	[29]
Exon 17	Missense	c.3257T > A	p.Val1086Glu	Val1059Glu	Diabetes, NIDDM	[42]
Exon 18	Missense	c.3283A > G	p.Lys1095Glu	Lys1068Glu	Diabetes, NIDDM	[68]
Exon 18	Missense	c.3220G > C	p.Glu1074Gln	Glu1047Gln	Rabson–Mendenhall syndrome	[31]
Exon 18	Missense	c.3356G > A	p.Arg1119Gln	Arg1092Gln	Leprechaunism	[11]
Exon 18	Missense	c.3355C > T	p.Arg1119Trp	Arg1092Trp	Leprechaunism	[49]
Exon 19	Missense	c.3428T > C	p.Ile1143Thr	Ile1116Thr	Rabson–Mendenhall syndrome	[13]
Exon 19	Missense	c.3436G > C	p.Gly1146Arg	Gly1119Arg	Insulin resistance	[71]
Exon 19	Missense	c.3439A > T	p.Met1147Leu	Met1120Leu	Insulin resistance A	[73]
Exon 19	Nonsense	c.3447C > A	p.Tyr1149Term	Tyr1122Term	Insulin resistance	[37]
Exon 19	Missense	c.3470A > G	p.His1157Arg	His1130Arg	Insulin resistance	[74]
Exon 19	Missense	c.3471T > A	p.His1157Gln	His1130Gln	Diabetes, NIDDM	[42]
Exon 19	Missense	c.3473G > A	p.Arg1158Gln	Arg1131Gln	Insulin resistance	[75]
Exon 19	Missense	c.3472C > T	p.Arg1158Trp	Arg1131Trp	Rabson–Mendenhall syndrome	[13]
Exon 19	Missense	c.3481G > A	p.Ala1161Thr	Ala1134Thr	Insulin resistance	[76]
Exon 19	Missense	c.3485C > A	p.Ala1162Glu	Ala1135Glu	Insulin resistance	[77]
Exon 20	Missense	c.3540G > A	p.Met1180Ile	Met1153Ile	Insulin resistance	[78]
Exon 20	Missense	c.3572G > A	p.Arg1191Gln	Arg1164Gln	Diabetes, NIDDM	[79]
Exon 20	Missense	c.3602G > A	p.Arg1201Gln	Arg1174Gln	Insulin resistance	[80]
Exon 20	Missense	c.3601C > T	p.Arg1201Trp	Arg1174Trp	Leprechaunism	[81]
Exon 20	Missense	c.3614C > T	p.Pro1205Leu	Pro1178Leu	Insulin resistance	[82]
Exon 20	Missense	c.3618G > C	p.Glu1206Asp	Glu1179Asp	Insulin resistance	[83]
Exon 20	Missense	c.3616G > A	p.Glu1206Lys	Glu1179Lys	Leprechaunism	[49]
Exon 20	Missense	c.3659G > T	p.Trp1220Leu	Trp1193Leu	Insulin resistance	[83]
Exon 21	Missense	c.3680G > C	p.Trp1227Ser	Trp1200Ser	Insulin resistance	[76]
Exon 21	Nonsense	c.3769C > T	p.Gln1257Term	Gln1230Term	Insulin resistance A	[73]
Exon 22	Missense	c.4082A > G	p.Tyr1361Cys	Tyr1334Cys	Diabetes, NIDDM	[66]
Exon 22	Missense	c.4133G > A	p.Arg1378Gln	Arg1351Gln	Insulin resistance	[50]

Fig. 1

Summary of types of mutations found in the INSR gene.

Sequencing INSR

Four patients with known mutations were verified by the above sequencing protocol. The first patient, NY1, with clinically-confirmed Leprechaunism [6], [22], had a homozygous G to T variation at nucleotide 451 converting Glu 151 to a premature stop codon (c.451G > T,p.Glu151term). The second patient, 452, was a female infant with symptoms including repeated transient hypoglycemic episodes, prominent female genitalia, marked hirsutism, breast hyperplasia, loose and pachydermatous skin, decreased adipose tissue, acanthosis nigricans, and abdominal distention [7]. Sequencing results showed a heterozygous C to T nucleotide change at position 1195 coding for a premature stop codon at amino acid position 399 (c.1195C > T, p.Arg399term). A second mutation could not be detected by the assay as in the initial publication [7]. The third patient, 5880, had physical features of Leprechaunism and his lymphoblasts had a 90% decrease in the number of insulin receptors. This patient had a heterozygous C to T nucleotide change at position 2734 resulting in a change of arginine 924 to a premature stop codon (c.2770C > T, p.Arg924term) [23]. A second mutation could not be found even in this patient as in the original manuscript [24].

The forth patient, SLC, died before one year of age and had physical features of Leprechaunism. Insulin binding was reduced to about 4% of normal in fibroblasts from this patient. A novel G to T missense mutation was identified at nucleotide position 425 resulting in a change of glycine 142 to a valine (c.425G > T, p.Gly142Val). Computational prediction with the program Polyphen 2 (Harvard) predicts that a glycine to valine amino acid change at this position is “possibly damaging” with a score of 0.814 while SIFT (J Craig Venter Institute) predicts that the substitution is “damaging”. A second mutation could not be identified in this patient either.

Seven additional samples from normal, healthy individuals displayed no INSR variants. However all samples (as well as the clinical samples above) were found to have a benign polymorphism at nucleotide position 5 changing alanine 2 to glycine (c.5C > G, p.Ala2Gly).

An additional fourteen clinical samples (one sample from cultured amniocytes, four samples from pediatric patients and nine from adult patients) were referred to our lab for INSR sequencing. The clinical phenotype and laboratory results are summarized in Table 3. According to patient history received by ARUP with the amniotic sample, it was previously tested for deletions and duplications using a SNP array at another laboratory and was found to have a 63 kb deletion at 19p13.2 (7,143,507-7,206,857), including deletion of several exons of the INSR gene. Sequencing analysis detected no additional mutations. The 13 pediatric and adult patients presented with anomalies including, intra-uterine growth restriction (IUGR), failure to thrive (FTT), dysmorphic features, distended abdomen, and acanthosis nigricans. Although the major symptom was insulin resistance, the nine oldest patients tested were disproportionately female (7:2) with gynecological symptoms including menstrual irregularities and cystic ovaries. One 16 year old male patient had a history of IUGR, FTT, dysmorphic features, and poor response to exogenous insulin. Thirteen samples had no mutation detected by Sanger sequencing in the coding regions and exon/intron boundaries. An eleven week old boy with suspected Leprechaunism was homozygous for a variant of unknown clinical significance, c.2971C > A, p. Leu991Ile. For this patient, no positions of heterozygosity were observed in INSR, therefore we cannot rule out a partial or complete gene deletion. The infant was in intensive care and presented with IUGR, bilateral club feet, congenital hydrocephalus and dysmorphic features. Patient had only the right kidney and renal tubular acidosis. The patient had sporadic hypoglycemia and was noted to have glucose levels decreasing to 40 mg/dL range after 4–5 h of fasting, but given the age and size of the patient, this is of uncertain clinical significance. This patient also had elevated beta-hydroxybutyric acid of 14.1 mg/dL (reference range: 0.0–3.0) and a random insulin level of 1 μU/mL (reference range: 3–19 μU/mL). This variant (rs150114699) has been seen in the general population with a frequency of 0.4% in 1000 genomes and 0.6% in 6500 exomes in African Americans. The homozygous variant, c.2971C > A, p. Leu991Ile, has never been reported in the literature; sequence prediction programs give conflicting results about whether this substitution is likely to be deleterious (SIFT: deleterious; PolyPhen2: benign at score: 0.442). The next residue, Y992 is a conserved phosphorylation site in a highly conserved region (DGPLGPLyASSNPEY, http://www.phosphosite.org/siteAction.do?id=13426). The putative amino acid change at position L991 to the branched amino acid isoleucine may result in steric hindrance and decreased transporter activity. In light of the fact that the patient has only one kidney and is hypoglycemic, sequencing of HNF1B in this patient may be appropriate.

Table 3

Clinical information and laboratory results for patient samples sequenced in this study.

Patient	Age	Ethnicity	Gender	Clinical and other findings
1	Fetus	Asian	NA	Reported advanced maternal age. A SNP array detected a 63 kb deletion involving deletion of exons 3–11 of the INSR gene: 19p13.2(7,143,507-7,206,857)x1; GRCh37/hg 19 sequencing of the coding exons ruled out second mutation
2	11 weeks	African-American	M	Possible IUGR, dysmorphic features, distended abdomen, can fast for only 4–5 h after which the glucose levels drop to 40 s, insulin 1 μU/mL (refa 3–19), renal tubular acidosis type 4, only one kidney, bilateral club feet, congenital hydrocephalus not requiring shunt; c.2971C > A, p.Leu991Ile
3	1 yr	b	F	IUGR, low glucose fasting (35–67 mg/dL), seizures; previous testing found “regions of homozygosity” in INSR region by SNP array
4	5 yr	Multi-ethnicity	M	Delivered at 27 weeks and has complications of prematurity, holoprosencephaly, absence of corpus; loss of white matter on both occipital lobes, FTT, insulin 379.6 μU/mL (ref 3–17)
5	11 yr	c	F	Hypertriglyceridemia, low HDL cholesterol, high LDL cholesterol, nonalcoholic steatohepatitis, acanthosis nigricans, glucose fasting normal, insulin 70.5 μU/mL (ref 3–12)
6	15 yr	NA	M	Extreme insulin resistance type A
7	15 yr	African-American	F	Acanthosis nigricans, amenorrhea, insulin 21 μU/mL (ref 3–19)
8	16 yr	African-American	M	IUGR, FTT, dysmorphic features, lack of subcutaneous fat, poor response to exogenous insulin or hyperforin
9	21 yr	African-American	F	Acanthosis nigricans, cystic ovaries, insulin 65.8 μU/mL (ref 2.6–24.9), severe insulin resistance, cystic ovaries. Medications: Trajenta, metformin, Depo–Provera therapy
10	28 yr	Caucasian	F	Cystic ovaries, glucose fasting 89 mg/dL, hx of heavy irregular periods, mental health symptoms, cystic ovaries
11	29 yr	Asian Indian	F	Amenorrhea, cystic ovaries
12	30 yr	Caucasian	F	Cystic ovaries
13	50 yr	NA	F	Glucose fasting, 277 mg/dL (ref 70–99) unknown if fasting, insulin antibody 1.9 U/mL(< 0.4), triglyceride 1302 mg/dL (ref 40–149); cholesterol 231 mg/dL (ref 120–199)
14	66 yr	Caucasian	F	Aggression, hyper-androgenism, gingival hyperplasia, thick skin, amenorrhea, distended abdomen, reported high fasting glucose fasting, high postprandial glucose 1501 mg/dL (ref < 180 mg/dL)

Within normal reference range.

Patient from Haiti, ethnicity unknown.

Patient from Puerto Rico.

Discussion

Mutations in INSR can cause the insulin-resistant syndromes Leprechaunism, Rabson–Mendenhall syndrome, and type A insulin resistance [5], [6]. Diagnosis is established on clinical examination as well as laboratory diagnostic tests with markedly elevated insulin levels being a constant feature. Functional studies (insulin binding to cultured fibroblasts) and DNA analysis can be used for definitive confirmation, keeping in mind that certain mutations do not decrease insulin binding and that DNA analysis is still not identifying all putative mutations. Although there is no straightforward genotype–phenotype correlation, mutations affecting the alpha subunit of the receptor are associated with a more severe phenotype than the mutations affecting the beta subunit [25].

Due to the lack of a central repository of INSR mutations, we compiled a list of the published mutations, using currently accepted standards (Table 4). Our literature search of INSR mutations identified 132 causative variations. The vast majority of these variations are missense and nonsense mutations (78%) (Fig. 1). Interestingly, different missense mutations in the same codon have been reported to produce different phenotypes (Table 4). This highlights the need to expand the currently available databases to allow better understanding of the genotype–phenotype correlation.

There are five reports of large deletions within the INSR gene including an entire gene deletion [26]. Gross deletions and gene rearrangements account for about 5% of the mutations [26]. Large deletions, as in one of our patients, can be detected by CGH/SNP arrays. For this reason, development of a commercial test to detect single exon and whole gene deletions may be attractive. No commercial deletion/duplication testing is currently available in the US; however, deletion and duplication testing is offered at laboratories in the United Kingdom and Germany. A multiplex ligation dependent probe amplification (MLPA) assay could be used to detect single exon deletions in the INSR gene.

DNA sequencing can identify novel sequence variants of unknown clinical significance. In our study, we detected a novel c.425G > T, p. Gly142Val affecting the insulin binding alpha subunit of the insulin receptor. The evolutionary conservation analysis by Polyphen and SIFT predicts that a glycine to valine amino acid change at this position is “possibly damaging” or “damaging” to the function of the protein. Cells from this patient (TGB) failed to bind insulin, supporting a damaging role of the identified mutation. A second mutation in this patient could not be detected indicating the limitations of the current test in detecting mutations in the deep intronic or promoter regions or deletions, duplications, and rearrangements of the gene. In fact, sequencing failed to identify the second mutation in three patients with markedly reduced insulin binding in which previous studies also failed to detect the second pathogenic change [6], [13].

An additional sample of a pediatric patient referred for possible Leprechaunism was an apparent homozygous for c.2971C > A, p. Leu991Ile. A review of clinical data indicated normal to low insulin levels, a finding inconsistent with severe insulin resistance and indicating that the amino acid change is of unknown significance. As no positions of heterozygosity were observed in INSR, we cannot rule out a partial or complete gene deletion. The effect of a deletion of one copy of INSR in conjunction with this variant is yet to be studied.

It should be noted that only one mutation was detected in the 14 samples sent for clinical testing. This may be related to the poor clinical selection of patients whose phenotypes were inconsistent with insulin resistance but were nevertheless referred for this INSR mutation detection assay. More specific selection of candidate patients may enhance the utility of the assay.

Association studies show a strong correlation between single nucleotide polymorphism (SNP) in the INSR gene and a predisposition to type 2 diabetes [27]. An alternative isoform of exon 8 in the INSR gene in the Han population confers increased risk for central obesity, hypertension, glucose intolerance, hyperinsulinemia and type 2 diabetes [28], whereas variation in exon 17 is associated with insulin resistance, hyperandrogenemism and polycystic ovarian syndrome (PCOS) [29].

In conclusion, we report the development of a sequencing assay to detect mutations within the coding region and intron/exon boundaries of the INSR gene. Further development of deletion/duplication analysis is needed to detect deletions, duplications and large gene rearrangement of the INSR gene. A compilation of all the mutations reported to date using current terminology (Table 4) is the first step toward development of a publicly available online mutation database for the INSR gene.