ABSTRACT

Most cases of hereditary severe insulin-resistance syndrome (H-SIRS) are linked to mutations in the insulin receptor (INSR) gene. Patients with H-SIRS typically manifest symptoms of hyperinsulinemia, insulin resistance, and diabetes mellitus. Other symptoms include impaired glucose regulation, hyperandrogenism, and the presence of acanthosis nigricans (AN). In this report, we present two cases of H-SIRS in female children exhibiting various symptoms, including hyperinsulinemia, fasting hypoglycemia, postprandial hyperglycemia, overweight, fatty liver, hyperandrogenism, and varying degrees of AN. One patient also presented with mental retardation. Gene sequencing identified specific mutations in the INSR gene for both patients: c.2663A > G (p.Tyr888Cys) in Patient 1 and c.38_61del (p.Pro13_Ala20del) in Patient 2. These mutations both have the potential to disrupt the interaction between the insulin receptor, INSR, and insulin, leading to abnormal insulin signaling, insulin resistance, and various clinical manifestations.

Introduction

Insulin resistance is defined as a decrease in the sensitivity of target organs to the normal physiological effects of insulin. Its characteristics include impaired glucose uptake in muscle and adipose tissue, increased hepatic gluconeogenesis and glycogen breakdown, increased risk of obesity, impaired glucose tolerance, abnormal blood lipid levels, and endothelial dysfunction (1). Hereditary severe insulin-resistance syndrome (H-SIRS) is an extreme form of insulin resistance, accounting for approximately 0.1% to 0.5% of hospitalized patients with diabetes. H-SIRS caused by mutations in the insulin receptor (INSR) gene can be classified into Donahue syndrome (DS), Rabson-Mendenhall syndrome (RMS), and type A insulin resistance syndrome (A-IR), while type B insulin resistance is associated with the production of insulin autoantibodies (2, 3). The clinical phenotypes of H-SIRS include hyperinsulinemia, abnormalities in glucose homeostasis, dyslipidemia, and acanthosis nigricans (AN). It is also characterized by ovarian dysfunction and excessive androgen levels in women. Most DS or RMS patients have bi-allelic gene variations resulting in abnormalities in the INSRα subunit, resulting in more severe symptoms, such as intrauterine and postnatal growth retardation, reduced subcutaneous fat, hirsutism, and characteristic facial changes. A-IR patients have heterozygous variations in the intracellular tyrosine kinase domain of the β subunit (2, 3).

Patients with AN typically exhibit pigmentation and excessive keratinization in skin folds, resulting in darkening, roughness, or a velvety texture in localized areas. In certain instances, it may progress to nipple- or wart-like patches. Furthermore, the presence of AN correlates with insulin resistance, metabolic syndrome, and polycystic ovary syndrome (PCOS) in overweight and obese children (4). A single-center study conducted among teenagers in the UK reported that patients with AN exhibited significantly higher median fasting insulin levels, average fasting blood glucose levels, and median insulin resistance index scores in comparison to the control group (215 pmol/L vs. 126 pmol/L; 4.7 mmol/L vs. 4.5 mmol/L; 6.4 vs. 3.7). AN has been reported to have an 81% positive predictive value for insulin resistance, suggesting its utility as a marker for type 2 diabetes mellitus (T2DM) in teenagers (5). The present study contributes two cases of adolescent H-SIRS patients with AN resulting from two novel INSR mutations, thereby expanding the genotype and phenotype spectrum of this disease.

Patients and Research Methods

Case Introduction

Patient 1

The medical history of Patient 1 was as follows. She was a 12 years old and initially presented to The First Hospital of Lanzhou University because of thickening of the skin of the neck, armpits, groin, and popliteal fossa with hyperpigmentation which had been evident for eight years. She was a full-term delivery, but birth weight and length were unknown. There was no history of complications during pregnancy, but she had exhibited an introverted personality since childhood, she had suspected intellectual disability, and had not started menstruation. There was no history of similar diseases in the family.

On physical examination she was 159 cm (75^th-90^th percentile for same age and gender), weight was 65 kg (over 97^th percentile for same age and gender), body mass index (BMI) was 25.71 kg/m² (over 97^th percentile for same age and gender) (6). Examination showed skin thickening with hyperpigmentation in the neck, armpits, groin, and popliteal fossa, appearing velvety with no skin tags, normal subcutaneous fat, no excessive body hair (Ferriman-Gallwey Score: 4) (7) and no striae. She had no physical deformities, had Tanner stage 2 breast development, there was no lactation, and she had normal external female genitalia with no clitoromegaly.

Oral glucose tolerance (OGTT) and insulin stimulation tests revealed fasting hypoglycemia, postprandial hyperglycemia, and hyperinsulinemia (Table 1). In addition, her karyotype was 46,XX. She had normal liver function, kidney function, blood lipid, and no relevant autoantibodies (Table 2). However, there was elevated serum uric acid and elevated testosterone levels with normal follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, prolactin androstenediol, and dehydroepiandrosterone. Moreover, normal adrenocorticotropic hormone (ACTH), cortisol rhythm and 24-hours urinary free cortisol level were all normal. Abdominal ultrasonography suggested a fatty liver, and hand radiography for bone age assessment indicated an advanced bone age (between the ages of 15 and 17 years) greater than the actual age (12 years old).

Patient 2

This girl first presented to the First Hospital of Lanzhou University at the age of 10 years due to the discovery of thickening of the skin of the neck, armpits, groin, and popliteal fossa with hyperpigmentation which had been present for five years. She was also a full-term delivery with a birth weight of 2.8 kg. Of note, menstruation had occurred twice when she was nine years old, with light red color, no dysmenorrhea, no blood clots, and lasted about 3-4 days each time. At the time of presentation there was no menstruation. There was no history of similar diseases in the family.

At presentation her height was 155 cm (over 97^th percentile for same age and gender), and weight was 60 kg (over 97^th percentile for same age and gender), resulting in a BMI of 24.97 kg/m² (over 97^th percentile for same age and gender)(6).
She exhibited thickened and pigmented skin at the neck, armpits, groin, and armpits. There were nipple-like nodules in the neck and armpits with pigmentation, mostly in joint folds, scattered papules on her back like millet grains with normal skin in between the areas of rash, accompanied by itching, increased body hair (Ferriman-Gallwey Score: 9) (7), no striae and no skin tags. She was bilateral Tanner B2 with no lactation. Of note, she exhibited enlarged female external genitalia with an enlarged clitoris (about 2-3 cm) (Figure 1).

OGTT and insulin release test suggested fasting hypoglycemia, diabetes, and hyperinsulinemia (Table 1). Her karyotype was 46,XX. Laboratory testing reported normal liver function, kidney function, blood lipids, and blood uric acid. Glycosylated hemoglobin was 7% and she had weakly positive histone antibody (1:100) but was negative for anti-islet cell antibody and anti-glutamic acid decarboxylase antibody. This patient also exhibited elevated testosterone level, while FSH, LH, estradiol, progesterone, prolactin, androstenediol, and dehydroepiandrosterone were all normal. ACTH and cortisol rhythms were normal, and 24-hours urine free cortisol level was also normal. She had elevated growth hormone and insulin-like growth factor-1, and insulin-like growth factor binding protein-3 was normal. Abdominal ultrasonography suggested fatty liver.

Based on the symptoms of hyperinsulinemia, fasting hypoglycemia, postprandial hyperglycemia, overweight, fatty liver, and hyperandrogenemia in two patients, as well as various degrees of manifestations of AN, a preliminarily diagnosis of insulin resistance syndrome was made.

It is worth noting that these two girls were not related.

Methods

Gene Sequencing

Peripheral venous blood (4 mL) was collected from both patients. Genomic DNA was extracted using the Qiagen FlexiGene DNA Kit (Qiagen, Germany) and stored at -20 ℃ for future use. This study adhered to the ethical principles outlined in the Declaration of Helsinki, and both patients’ guardians/parents signed written informed consent forms approved by the Ethics Committee of the First Hospital of Lanzhou University (approval no.: LDYYLL-2023-487).

The DNA samples were fragmented using an ultrasonic disruptor, resulting in DNA fragments of 150-300 bp. Adapters were added to both ends of the fragmented genome, followed by PCR library amplification and purification to repair the sticky ends. Subsequently, the post-library amplified DNA underwent hybridization and amplification with probes (Agilent, SureSelect probe enrichment system, No.3 Wangjing North Road, Chaoyang District, Beijing, China). The resulting products were purified and quantified. For the gene testing package for diabetes and insulin resistance genes, including INSR, 6q24 region (PLAGL1), 11p15 region (INS, KCNJ11), 6p22 region (ZFP57), HNF4A, GCK, HNF1A, and HNF1B genes using MLPA large fragment detection (Beijing Beijing Kangxu Medical Testing Institute, 2^nd Floor, Building 10, Zone C, Yiyuan Cultural and Creative Industry Base, 65 Xingshikou Road, Haidian District), Illumina’s NextSeq500-amplified products (12^th-13^th floors, Building 23, Science and Technology Oasis, No.1999 Yishan Road, Minhang District, Shanghai, China) were utilized for paired-end sequencing. Modified DNA polymerase and dNTPs with four fluorescent labels were added, and the fluorescence signal results were counted to obtain Fastaq-formatted data. The CASAVA (1.8.2) software converted the raw data into recognizable base sequences, followed by alignment, SNP, and DIP analyses to obtain information on mutation sites in the target region. Finally, SIFT (http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), and Mutation Taster (http://www.mutationtaster.org) were employed for protein functional change analysis to qualitatively predict the probability of the results. This process helped identify mutation sites requiring further validation. Gene sequences for the identified mutation sites were retrieved from the GenBank human genome database, and primers were designed and synthesized using the Primer Z website (http://genepipe.ncgm.sinica.edu.tw/primerz/primerz4.do). PCR amplification was performed on the mutation sites, and the obtained sequences were aligned with previous sequences to exclude false-positive sites in second-generation sequencing.

Gene Sequencing Results

Patient 1, harboring a mutation in exon 13 of the INSR gene, was found to have a c.2663A>G mutation. This was a heterozygous missense mutation where the A nucleotide at position 2663 is substituted with a G nucleotide, resulting in the amino acid at position 888 of the β-subunit of INSR being converted from a tyrosine to a cysteine (p.Tyr888Cys) (Figure 2). Protein functional analysis using SIFT, PolyPhen, and MutationTaster indicated that the p.Tyr888Cys mutation was predicted to be “pathogenic” by SIFT (score: 0, disease prediction: Deleterious), PolyPhen2_HVAR software (score: 1, disease prediction: Deleterious), and MutationTaster (score: 1, disease prediction: Deleterious).

Patient 2 manifested an in-frame mutation in exon 1 of the INSR gene, resulting in a 24-base pair deletion between the 38^th and 61^st bases (c.38_61del). This deletion leads to the fusion of the original 37^th base C with the 62^nd base T, causing the loss of eight amino acids: Pro13 (P13), Leu14 (L14), Leu15 (L15), Val16 (V16), Ala17 (A17), Val18 (V18), Ala19 (A19), and Ala20 (A20) (p.Pro13_Ala20del). Notably, the translation of the amino acids preceding and following the deletion, Ala12 (A12) and Leu21 (L21), respectively, remained unaltered (Figure 3).

Treatment and Follow-up

Two patients received metformin at a dosage of 500 mg twice daily to address insulin resistance and facilitate weight control. Subsequently, blood sugar levels were effectively managed and maintained within the normal range. Additionally, a noticeable reduction in skin pigmentation was observed (Table 3).

Discussion

Here, we present findings from two unrelated female pediatric patients who exhibited hyperinsulinemia, fasting hypoglycemia, and postprandial hyperglycemia during OGTT and insulin release tests. Both patients displayed characteristic symptoms of AN. Both also presented with hyperandrogenism, overweight, and fatty liver. Notably, our genetic sequencing revealed specific heterozygous mutations in the INSR gene for each patient.

In patient 1, a heterozygous missense mutation, c.2663A>G, in exon 13 of INSR was identified, resulting in the amino acid at position 888 of the β-subunit of the INSR being changed from a tyrosine to a cysteine (p.Tyr888Cys) (Figure 2). Patient 2, in contrast, exhibited a deletion of 24 base pairs between positions 38 and 61 in exon 1 of INSR, leading deletion of eight amino acids between proline at position 13 and alanine at position 20 in the α-subunit of INSR (p.Pro13_Ala20del) (Figure 3). Consequently, considering the clinical phenotypes and laboratory results of both cases, we diagnosed these patients with H-SIRS.

Most mutations causing H-SIRS have been identified in the INSR gene. Ardon et al. (8) summarized 132 pathogenic variants of INSR mutations, including missense, non-sense, insertion, deletion, and complex rearrangements. Recently, new mutation sites have been identified. You et al. (9) recently reported a patient with hyperinsulinemia associated with AN, and gene sequencing revealed a novel variant, c.3472C>T (p.Arg1158Trp), in the index case and his father’s INSR gene. Poon et al. (10) reported a case of hyperinsulinism and hypoglycemia in an infant who did not respond to diazoxide treatment. It was later found that she carried a heterozygous INSR gene mutation, c.1246C>T, leading to the replacement of the arginine codon at position 416 with a stop codon.

Different types of INSR gene mutations can affect the molecular structure of the INSR, leading to varying pathotypes. Zhou et al. (11) reported two cases of A-IR and one case of DS: the proband with A-IR and his sister had compound heterozygous mutations c.3670G>A and c.3614C>T in the INSR gene, while the patient with DS had mutations c.749_751del and c.3355C>T. The impact of these new variants on INSR function was determined by expressing the mutant receptors in Chinese hamster ovary (CHO) cells. The results showed that Thr250 and Val1224 are located in the cysteine-rich region and tyrosine kinase domain of INSR, respectively. The new variant c.749_751del (p.Thr250del) in the α subunit reduced the expression of mature INSR protein and severely impaired INSR function. In contrast, although protein function analysis suggested that the c.3670G>A (p.Val1224Met) mutation was pathogenic, the new variant in the β subunit did not affect the expression and phosphorylation of INSR. The tyrosine kinase activity of INSR is crucial for insulin action in vivo, with the α subunit containing the insulin binding site and the β subunit containing the tyrosine kinase domain. Phosphorylation of the INSR β subunit is necessary for mediating insulin action. The translation products of INSR mutations lacking kinase activity do not mediate the promotion of glycogen synthesis, glucose uptake, cell proliferation, or gene transcription by insulin (12).

The two INSR gene mutations we report have not been presented in previous studies, therefore, the molecular structure and function resulting from these two mutations are unknown. However, mutant sites at adjacent positions in the INSR gene have been reported previously. Qin et al. (13) reported a case of c.62T > G (p.L21R) and c.2563G > T (p.V855F) mutations in the INSR gene, in which the patient presented with thickening of the skin of the neck and trunk accompanied by hyperpigmentation, roughness of the face, enlargement of the head, thickness of the lips, generalized hirsuitism, reduction of subcutaneous fat, and a severe speech disorder. Molecular dynamics simulations showed that the c.62T > G missense mutation located in the α-subunit led to functional defects in the signal peptide, and the c.2563G > T missense mutation was located in the cysteine-rich structural domain of the β-subunit, which completely altered the tertiary conformation of INSR, led to inactivation of the INSR, and interfered with INSR binding to the ligand. In addition, Brierley et al. (14) evaluated the impact of INSR gene mutations using a cell culture model. The results indicated that when the INSR mutation site is located on the cell surface, the binding of INSR to insulin and signal transduction are impaired. When the aspartic acid placement at position 707 on the β-subunit of the INSR is replaced by an alanine, this mutation is located near the cysteine residue, which may affect disulfide bond formation as well as the autophosphorylation of the INSR and its binding to substrates.

The two INSR gene mutations identified in this study, with the c.2663A>G (p.Tyr888Cys) mutation site located at the junction between the α and β subunits, may affect transmembrane signaling of insulin. In contrast, the c.38_61del (p.Pro13_Ala20del) mutation site located in the α subunit may affect insulin binding to the receptor, but the specific mechanism still needs further basic research confirmation.

Gene mutations severely impaired the sensitivity of the INSR to insulin, leading to hyperinsulinemia and reduced affinity for peripheral tissue insulin receptors, further promoting insulin resistance. This results in pancreatic beta cells secreting more insulin compensatively, resulting in a vicious cycle. Over time, pancreatic cell function eventually declined, increasing the risk of chronic complications, such as diabetes (3). The main histological features of AN included hyperkeratosis and epidermoid cyst disease, along with mild or absent acanthosis and excessive basal pigmentation (15). Insulin promotes cell proliferation, and hyperinsulinemia should lead to elevated circulating levels of insulin-like growth factor 1 (IGF-1), causing overactivation of IGF-1 receptors on fibroblasts and keratinocytes and driving excessive cell proliferation and differentiation. Therefore, AN could be considered a cutaneous manifestation of insulin resistance (16). In addition, both patients had hyperandrogenemia. The cause might be the cross-reactivity between high concentrations of insulin and IGF-1 receptors in the ovaries, leading to excessive secretion of androgens (17).

The treatment of AN depends on the underlying conditions. In cases of insulin resistance, weight control or weight loss surgery can improve symptoms. In addition, the indications for metformin in T2DM have been extended to include PCOS and AN. Limited data are available regarding cosmetic interventions such as melatonin, urea cream, vitamin D analogs, or topical tretinoin. In the early stages, lifestyle changes and improvement of insulin resistance should be started, along with the use of keratolytic agents (such as α hydroxy acids and salicylic acid) in combination with depigmenting agents (such as hydroquinone or azelaic acid). Topical tretinoin can be administered when velvety skin changes are observed (15).

There are limitations to this study. The present study did not investigate the effect of the mutant site on the molecular structure and function of the INSR. These changes in INSR remain to be further investigated by in vivo or in vitro experiments in the future.

Conclusion

Our study reports two cases of H-SIRS in female children, both presenting with hyperinsulinemia, fasting hypoglycemia, postprandial hyperglycemia, fatty liver disease, hyperandrogenism, and varying degrees of hirsutism. Patient 1 exhibited suspected intellectual disability. Genetic testing revealed the presence of a c.2663A>G (p.Tyr888Cys) missense mutation in Patient 1 and a c.38_61del (p.Pro13_Ala20del) frameshift mutation in INSR in Patient 2. These mutations have the potential to impact the binding of INSR to its ligand, insulin, thereby disrupting insulin receptor binding and resulting in abnormal insulin signaling. This disruption leads to insulin resistance and other associated clinical manifestations. Hyperinsulinemia and insulin resistance are relatively common in clinical practice, but their causes are diverse. Therefore, genetic testing is important to determine the etiology of insulin resistance.