Type A Insulin Resistance Syndrome- Novel insulin receptor gene mutation and familiar phenotypic variability

Type A Insulin Resistance Syndrome is due to heterozygous mutations in the insulin receptor (INSR) gene or its signaling pathway. We present a premenarcheal 14 year-old girl with normal BMI, severe hirsutism, acanthosis nigricans, clitoral hypertrophy, deep voice, enlarged polycystic ovaries, severe hyperinsulinemia and biochemical hyperandrogenism. We identifi ed a novel heterozygous missense variant in the tyrosine kinase domain of INSR (p.Leu1150Pro) and an heterozygous missense variant in SH2B adapter protein 1 involved in the insulin pathway (p.Ala663Val). Interestingly, the patients’ mother and brother had the same INSR mutation although of a milder phenotype, reason why their IR went undiagnosed. The novel heterozygous p.Leu1150Pro mutation in the INSR gene appears to be the cause of the type A insulin resistance syndrome; the SH2B1 mutation, likely to synergistically affect the insulin pathway, may contribute to explain the more severe presentation of the phenotype in the patient and the phenotypic variability of the syndrome within this family. Case Report Type A Insulin Resistance SyndromeNovel insulin receptor gene mutation and familiar phenotypic variability Analía V Freire1*, Paula Scaglia1,2, Mirta G Gryngarten1, Mariana Gutiérrez1, Andrea J Arcari1, Laura Suarez1, María Gabriela Ballerini1, Laura Valinotto2,3, Mónica I Natale2,4, Kenny Y Del Toro Camargo5, Ignacio Bergadá1, Rodolfo A Rey1,2 and María Gabriela Ropelato1,2 1Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET-FEI-División de Endocrinología, Hospital de Niños “Ricardo Gutiérrez” 2Laboratorio de Medicina Traslacional-Hospital de Niños Ricardo Gutiérrez. 3Consejo Nacional de Investigaciones Científi cas y Técnicas (CONICET) 4Facultad de Medicina, Universidad de Buenos Aires 5Unidad Médica Villa Country, Barranquilla, Colombia. Received: 30 March, 2019 Accepted: 12 June, 2019 Published: 13 June, 2019 *Corresponding author: Analía Verónica Freire, M.D. Ph.D., Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE). CONICET-FEI-División de Endocrinología, Hospital de Niños “Ricardo Gutiérrez”, Gallo 1330, C1425EFD, Buenos Aires. Argentina, Tel: +5411-49635931, ext: 231; Fax: +5411-49635930; E-mail:


Introduction
Insulin resistance (IR) generally occurs in obese individuals with a favorable genetic background; IR is rare in lean individuals. Most cases of this syndrome are due to mutations in the insulin receptor gene (INSR) or its signaling pathway [1,2]. Patients with Type A IR syndrome present severe IR, hyperandrogenism and acanthosis nigricans in the absence of obesity or lipoatrophy. Molecular studies help characterize this mostly monogenic condition. A 14 year-old caucasian girl consulted for hirsutism, acanthosis nigricans and absence of menarche despite thelarche start at age 9. She was born from non-consanguineous parents at term and with normal weight. Developmental milestones and her past medical history were normal; the patient´s health had been good and she had always had good appetite. No family history of diabetes was reported.

Patient report
Two years before admission the patient began to complain of progressive whole body and facial hirsutism associated to seborrhea and a deepening of her voice. Her height was 158.5 cm (50 th centile), weight 42.5 kg (10 th centile) and BMI was 14.3 kg/m 2 (-1.3 SD). At physical examination she showed normal muscular habitus, severe hirsutism (Ferriman-Gallwey score=32) and acanthosis nigricans on the neck, axillae and knees (Figure 1 (Table 1A). OGTT showed fasting glucose level of 73 mg/dL and 136 mg/dL at 120 min, with severe hyperinsulinemia (47.7 and 600 μUI/mL, respectively) (Table 1B).
After these molecular fi ndings, the family was evaluated. The mother (39 y-o) presented normal weight (BMI 23 kg/m 2 ), her menarche had been at age 16, she had never been hirsute and had had no problems in conceiving. A mild acanthosis nigricans on the armpits was found at the physical exam. The father (38 y-p) was overweight (BMI 27.9 kg/m 2 ) without any relevant fi ndings. The prepubertal brother (11.8 y-o) was overweight (BMI 25.5kg/m 2 ) and presented acanthosis nigricans on the neck and hands.
The mother and brother underwent similar baseline laboratory evaluations and OGTT; the father could not be evaluated due to social reasons. Hyperandrogenia (testosterone: 87 ng/dl and androstenodione: 441 ng/dL) and severe hyperinsulinemia were found in the mother, without changes in the glucose responses to the OGTT. Severe hyperinsulinism was found for the brother at OGTT (Table 1B) although with normal glucose responses. His androgenic profi le was normal.
With the diagnosis of Type A IR syndrome, the patient was started on Metformin 1500 mg/day. Her menarche occurred 3 months later. She was lost to follow up due to social reasons.

Molecular study
Genomic DNA was isolated from peripheral blood by CTAB method [3]. The patient´s clinical exome was studied by next generation sequencing (NGS) using the TruSight One assay in a NextSeq500 system Illumina. Resulting variants were prioritized, considering mutation impact, population frequency, and a list of candidate genes according to the pathway suspected to be altered (list of genes available upon request). The variants identifi ed were confi rmed in the patient and her relatives by PCR amplifi cation and Sanger sequencing to evaluate its segregation (primers available upon request).
In silico bioinformatic analysis: Missense variants on candidate genes identifi ed by NGS sequencing were classifi ed based on their potential impact on protein function or structure using fi ve in silico bioinformatics tools (Polyphen-2, Mutation Taster, SIFT, MutPred, and CADD prediction). Sequence variants were classifi ed according to ACMG guidelines [4].
A novel heterozygous missense variant in exon 19 of INSR gene (NM_000208.3: c.3449T>C, p.Leu1150Pro) was identifi ed in the proband. This variant is in the tyrosine kinase domain of the insulin receptor. Sanger sequencing confi rmed the fi ndings in the patient and revealed that her mother and her brother were heterozygous for the same variant.
The variant found in the INSR gene is classifi ed as likely pathogenic according to ACMG guidelines. This mutation has not been reported in publicly available 1000G, ExAC, EVS and NCBI dbSNP databases and was predicted to be pathogenic by fi ve in silico bioinformatics tools. By the multiple alignment analysis of the insulin receptor sequences in several species, we found that Leu1150 was conserved http://www.ibi.vu.nl/ programs/pralinewww/(Supplementary Figure 1). Introduction of proline in position 1150 is predicted to have helixbreaking effects according to PSIPRED [5]. Furthermore, in silico molecular 3D modeling of -Subunit of IR showed that   or tolerated (SIFT) to potential high impact (CADD score=2.5).

Discussion
We describe a premenarcheal patient that presented with severe hyperandrogenism, acanthosis nigricans and severe IR. Although the criteria of severe IR in OGTT is not formally established, a peak insulin higher than 260 μUI/mL in individuals with BMI < 30 kg/m is the value considered in practice [1,2]. The patient was diagnosed as a classical phenotype of the Type A IR syndrome (OMIM # 610549*).  [7]. According to the results of the largest cohort study of this rare condition, hyperinsulinemia correlates with the degree of hyperandrogenism, ovarian enlargement [6] and the degree of acanthosis nigricans [8]. The pathogenesis of this skin disorder is not completely clear; it has been proposed that high levels of insulin activate IGF1R to increase the mitotic activity of keratinocytes causing darkening and thickening in folds such as neck, armpit, and extension areas such as knees and fi ngers knuckles [8].
Our patient´s laboratory results support the absence of fatty liver and show a normal lipid profi le. This is an element of differential diagnosis with lipodystrophy where the patients showed severe dyslipidemia [9]. Interestingly, the patient showed increased serum levels of adiponectin. This fi nding is usually described in patients with severe IR due to receptor defects in contrast to prevalent forms of IR associated with obesity where adiponectin levels are reduced. Hyperinsulinemia is able to suppress the adiponectin gene expression in adipose tissue if the elements of the cellular insulin signal network are intact [10].
Although no functional data were reported for the variant identifi ed in this family, we assume that it is likely to be causal of the severe IR for several reasons. The Leu1150 residue affects the tyrosine kinase activity and functional studies have shown that mutations in tyrosine kinase domain impair insulin action [11,12], this very rare variant is absent from the controls that has been proposed in other mutations affecting the tyrosine kinase activity of the insulin receptor [13]. Mutants and wild type heterodimers of insulin receptor associated randomly might lead to a 75% reduction in insulin receptor tyrosine kinase activity [8].
Sexual dimorphism is associated with type A IR syndrome, which is more often diagnosed in females during infancy or adolescence than in males, since females show more health problems associated with this condition (especially hyperandrogenism) while males only present acanthosis nigricans, symptomatic postprandial hypoglycemia and commonly go undiagnosed, or simply diagnosed with "type 2 diabetes" in mid-life [1]. In the case of this family, if the proband had not been diagnosed, the patient's brother would probably have gone undiagnosed.
Our patient presented more severe phenotype than her mother, who had had no clinical manifestations of hyperandrogenism during adolescence and nowadays only shows a mild acanthosis nigricans. We assume that the phenotypical differences could be explained by the coexistence of two variants in different genes of the insulin pathway.  In our patient, the additional variant may translate into a more severe phenotype, compared to her mother who only had the insulin gene variant defect but did not present hirsutism during adolescence.

Conclusion
We identifi ed a novel heterozygous mutation (p.Leu1150Pro) in the INSR gene using an NGS approach in an adolescent with Type A IR syndrome. The fi nding of other variant of the SH2B1 gene, likely to synergistically affect the insulin pathway, may contribute to explain the more severe presentation of the phenotype in the patient and the phenotypic variability of the syndrome within this family.