One Novel 2.43Kb Deletion and One Single Nucleotide Mutation of the INSR Gene in a Chinese Neonate with Rabson-Mendenhall Syndrome

Mutations in the insulin receptor (INSR) gene are responsible for Donohue syndrome (DS) and Rabson-Mendenhall syndrome (RMS). Insulin resistance is a feature of both diseases. Our patient was a Chinese neonate suffering from abnormal glucose homeostasis, hyperinsulinemia, dry skin, heavy hair, growth retardation and an elevated testosterone level. To search for candidate point mutations, small insertions or deletions and copy number variants, 2742 inherited disease-gene panel sequencing was performed. One pathogenic mutation (c.3355C>T, p.Arg1119Trp) and a novel 2.43Kb deletion (chr19:7150507-7152938) in INSR were found. The patient was diagnosed as RMS. Sanger sequencing and real-time quantitative polymerase chain reaction (PCR) confirmed the missense variant and microdeletion, respectively. We therefore supposed that these variants were candidate mutations in this case. We report a novel 2.43Kb deletion in INSR gene and provide further proof of the power of next generation sequencing in rare disease diagnosis.

. The inheritance pattern of DS and RMS is autosomal recessive. Typical symptoms of DS include growth retardation, elfin face, gingival hyperplasia, acanthosis nigricans, hypertrichosis and insulin resistance (1). RMS and DS share similar symptoms. The symptoms of DS are more severe, have an infantile onset and may lead to early death. RMS is often encountered as being of childhood-onset and with survival up to adulthood with milder symptoms. The differential diagnosis is based on the onset age and severity of the disease (2).
In this study, we describe a Chinese, male, newborn with hyperinsulinemia and hyperglycemia. Next generation sequencing (NGS) detected a compound heterozygous mutations of INSR, including one known mutation and one novel 2.43Kb deletion.

Case Report
The proband is a male infant and the first child of nonconsanguineous parents. During the fetal period, he was diagnosed with intrauterine growth retardation and oligohydramnios and was delivered by natural labor at 36 weeks of gestation with a low birth weight at 1.7 kg. At age 13 days, he presented with dry skin and heavy hair over his whole body. The plantar grasp, Moro and sucking reflexes were weak. Facial malformation, abnormality of mouth size, acanthosis nigricans and abdominal distention were not observed. Clinical tests showed hyperglycemia (14.7 mmol/L), hyperinsulinemia (>300 IU/mL) and fasting hypoglycemia. C-peptide was 4.05 ng/mL (1.10-4.40 ng/ mL). HbAlc (glycosylated hemoglobin) was normal (5.4%). Other abnormal laboratory test results are shown in Table 1. Insulin auto-antibodies were negative. Routine blood tests, liver function tests and thyroid-stimulating hormone levels were normal. Echocardiography suggested a ventricular septal defect, an atrial septal defect and ultrasonography indicated swelling of both kidneys. Magnetic resonance imaging of the brain indicated an impaired myelination of   Figure 1D). This deletion is not found in HGMD, DECIPHER or DGV. Additionally, it is absent from our internal database. The detected mutation was confirmed using realtime quantitative PCR. PCR-amplified DNA products were subjected to direct automated sequencing (ABI step one plus v.2.0). Both strands of each amplicon were sequenced using the primers 5'-CCTGACCTGGGGACGAAAA-3' and 5'-GTCTCCACCATTCGAGTCTGA-3'. Real-time quantitative PCR indicated the deletion was from the father ( Figure  1E). This region covers part of exon 10 and all of exon 11. The deletion is estimated to cause a truncated protein. We performed a three-dimensional (3D) structural modeling of

Discussion
In this Chinese newborn baby, we identified a novel microdeletion and a known missense mutation within the INSR gene, which caused a compound heterozygous mutation. Another gross deletion contains exon 18. This RMS patient, as a compound heterozygote, also carried a mutation (p.Val66Ala). Nephrocalcinosis was found to be one of the patient's dominant features (7). For the patient we report, the missense mutation (c.3355C>T, p.R1119W) has been reported from a DS patient who had symptoms at birth and died at three months of age (3). Our patient had symptoms 13 days after birth. Some typical RMS features of this patient include abnormal glucose homeostasis, hyperinsulinemia, dry skin, thick hair, elevated testosterone and growth retardation. We diagnosed this patient as RMS. With a microdeletion, our patient presented a mild and atypical phenotype, which may be explained by the unclear genotype-phenotype correlation of mutations in INSR. Different missense mutations in the same codon relate to different phenotypes (8). One DS patient, bearing a homozygous deletion in INSR resulting in inactivation of the INSR lived for 3.5 years (9). The coexistence of modifier genes and compensatory pathways may explain the phenotypic variability (10).
The insulin receptor is a tetramer of two a monomers and two β monomers. It is widely expressed and plays a vital role as a mediator between the extracellular and intracellular insulin signaling pathway. The whole region of the a-subunit is extracellular. The a-subunit contains a Leu-rich-compositionally biased region, Cys-rich-compositionally biased region, a Fibronectin type-III 1-domain and an insulin in-binding-region. The β-subunit extends through the cell membrane into the cytoplasm. The extracellular region of the β-subunit contains a Fibronectin type-III 2-domain and a Fibronectin type-III 3-domain. The cytoplasmic region of the β-subunit consists of several functional domains including a juxtamembrane region, a tyrosine kinase domain and the carboxy-terminal-region (5,11). The a-subunit is responsible for binding affinity to insulin. The Cys-rich-compositionally biased region is the main binding site of insulin. Fibronectin type-III domains 1 and 2 form the secondary insulin-binding site (5). Deficiency of the juxtamembrane region makes the folding of IR unstable and affects its downstream processing (12). The function of the Fibronectin type-III 3-domain remains unknown. A helical transmembrane region follows the Fibronectin type-III 3-domain. Through binding with insulin, IR initiates the phosphorylation of different phosphotyrosine residues in the tyrosine kinase domain (13). Downstream IR substrates bind to phosphotyrosine residues of IR and regulate two main signaling pathways: the phosphatidylinositol 3-kinase-AKT/protein kinase B (PI3K-AKT/PKB) pathway and the Ras-MAPK pathway. The PI3K-AKT/PKB pathway is responsible for controlling cell growth and differentiation. The metabolic action of insulin is mainly regulated by the Ras-MAPK pathway (14). 3D structural modeling indicates that with deficiencies in both a-subunit and β-subunit, IR may be unable to combine with insulin receptor substrates and recruit the downstream signaling molecules (15).
Our patient did not show some of the typical features of RMS including coarse face, gingival hyperplasia and acanthosis nigricans. These three symptoms can be absent in neonates and may develop in adolescence (16). The insulin receptor is expressed in the heart and regulates cardiac cell activity though the PI3K-AKT pathway. Insulin receptor deficiency possibly leads to cardiac dysfunction, as observed in some patients (10,17) and proven using animal models (18). There is no evidence indicating any relationship between heart structural malformation and insulin receptors, so we consider that the ventricle septal defect is not a consequence of insulin receptor deficiency in this case. The long-term prognosis of RMS patients is poor (19). Recombinant human insulin-like growth factor 1 and recombinant leptin are recommended for treatment of severe insulin resistance syndrome (19,20). However, the complications and safety of these drugs remain unknown (21) In summary, we show an RMS patient carrying one known pathogenic mutation and one novel deletion in INSR. Since the presenting clinical features of patients with insulin resistance syndrome can be atypical, when the diagnosis