An insulin receptor mutant (Asp707 → Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin

We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) α subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to α and β subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I- labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant α subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR α and β subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X. 100-containing buffers, 125I- insulin was efficiently cross-linked to the receptor a subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the a subunit carboxyl terminus to insulin binding

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations

An insulin receptor mutant (Asp(707)->Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin

We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) alpha subunit from a leprechaun patient, changing Asp(707) into Ala, Fibroblasts from this patient had no high affinity insulin binding sites, To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells, Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to alpha and beta subunits, No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of I-125-labeled insulin by di-succinimidyl suberate to these cells failed to label the mutant alpha subunit, Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR alpha and beta subunits, showing its presence on the cell surface, On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, I-125-insulin was efficiently cross-linked to the receptor alpha subunit by disuccinimidyl suberate

Identification and functional assessment of novel and known insulin receptor mutations in five patients with syndromes of severe insulin resistance

We analyzed the insulin receptor gene in four patients with leprechaunism and one with type A insulin resistance. We detected novel and previously reported mutations. The novel mutants were expressed in Chinese hamster ovary cells to evaluate the consequences for insulin receptor function. A type A insulin resistance patient from Morocco was homozygous for Arg252His mutation, similar to a previously described type A patient from Japan. A patient with leprechaunism was homozygous for the Ser323Leu mutation, previously identified in homozygous form in two patients with Rabson-Mendenhall syndrome. Phenotypic expression of this mutation is variable. A patient with leprechaunism is compound heterozygous for the previously described Arg1092Trp mutation and a nonsense mutation in codon 897. Another patient with leprechaunism was homozygous for a novel Asn431Asp mutation, which only partially reduces insulin proreceptor processing and activation of signaling cascades. The novel Leu93Gln mutation that fully disrupts proreceptor processing was found in one allele in a patient with leprechaunism. A nonsense mutation at codon 1122 was in the other allele. These results expand the number of pathogenic insulin receptor mutations and demonstrate the variability in their phenotypic expression. The biochemical analysis of mutant insulin receptors does not reliably predict whether the phenotype will be leprechaunism, the Rabson-Mendenhall syndrome, or type A insulin resistance. The previously reported correlation between fibroblast insulin binding and duration of patient survival was not observed

<i>PIK3CA</i>-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of &lt;i&gt;PIK3CA&lt;/i&gt; have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified &lt;i&gt;PIK3CA&lt;/i&gt; mutations in 60 individuals. Several other individuals ( &lt;i&gt;n&lt;/i&gt; = 12) were identified separately to have mutations in &lt;i&gt;PIK3CA&lt;/i&gt; by clinical targeted-panel testing ( &lt;i&gt;n&lt;/i&gt; = 6), whole-exome sequencing ( &lt;i&gt;n&lt;/i&gt; = 5), or Sanger sequencing ( &lt;i&gt;n&lt;/i&gt; = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 &lt;i&gt;PIK3CA&lt;/i&gt; mutations were novel. We also identified constitutional &lt;i&gt;PIK3CA&lt;/i&gt; mutations in 10 patients. Our molecular data, combined with review of the literature, show that &lt;i&gt;PIK3CA&lt;/i&gt; -related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations