Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant hemolytic anemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphedema caused by congenital lymphatic dysplasia. Ex vivo analysis of patients’ erythrocytes as well as in vitro studies in a heterologous system reveal greatly attenuated PIEZO1 function in the affected children. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphedema.
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.MethodsPCR 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).ResultsWe 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.DiscussionLeprechaunism 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.
An integrative diagnostic algorithm for alpha1-antitrypsin (AAT) deficiency testing in the clinical laboratory was developed and evaluated. A novel rapid LightCycler (Roche, Indianapolis, IN) molecular assay was used to detect the common S and Z deficiency allelic variants. However, use of such molecular assays for these variants also can result in the misclassification of significant numbers of "at-risk" patient samples containing other rare AAT deficiency alleles. In the diagnostic algorithm presented herein, patient samples with selected genotypes that exhibit abnormally low AAT concentrations by immunoassay are phenotyped by isoelectric focusing. To test the efficacy of this algorithm, we retrospectively evaluated a data set of 50,020 serum samples for which protein phenotype and AAT concentration had been determined. This algorithm can successfully detect the majority of at-risk samples containing rare deficiency alleles.
Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.
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