Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis

Abstract

Hereditary xerocytosis (HX, MIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis.

Figure 1

Copy number analyses via single nucleotide polymorphism microarray. Whole-genome SNP typing was performed using DNA from the patients indicated and the Illumina HumanHap 550 Bead Chip array. The figure shows SNPs from 3 patients on chromosome 16. For each patient, the top panel represents the intensity ratio, and the bottom panel represents the allele ratio. Intensity ratio is a measure of patient SNP log signal intensity compared with reference sample SNP intensity; 2 copies = intensity ratio of 0; 1 copy = intensity ratio of −1. Most values are centered around zero, indicating diploid copy number. Allele ratio is the ratio of the signal for one of 2 genotypes over the total signal. Homozygous SNPs localize to 0 or 1; heterozygous SNPs localize to 0.5. In Family A homozygote III-15, genotyping data in the 16q24.2-16qter candidate region demonstrate an extended block of homozygosity extending to the telomeric end of chromosome 16 (red bar). Note that the intensity ratio in this region is unchanged, indicating the homozygosity is not caused by a large deletion. The bottom panel shows, from top to bottom, (+) strand genes, cytogenetic bands, chromosome position, and (−) strand genes.

Figure 2

Exome sequencing. (A-B) Sequence tags. (A) New York HX patient II-10 (exon 46). Eight of 19 full-length reads have a C instead of an A (G replacing T on opposite strand), leading to a missense mutation, Met to Arg at amino acid 2225. (B) Canadian HX patient IV-4 (exon 51). Ten of 20 full-length reads have a T instead of a C (A replacing G on opposite strand), leading to a missense mutation, Arg to His at amino acid 2456. (C-D) Sanger sequencing confirmation of FAM38A gene mutations. (C) New York kindred. Top: Partial exon 46 wild-type sequence. Middle: corresponding sequence from a New York family HX heterozygote. Bottom: corresponding from a New York family HX homozygote. (D) Canadian kindred. Top: partial exon 51 wild-type sequence. Bottom: corresponding sequence from a Canadian HX heterozygote. The .bam file yielding the sequence traces from exome sequencing in panels A and B places the FAM38A gene in reverse complement orientation. The letters K and R are designations in the IUPAC code where K represents the nucleotides G or T and R represents the nucleotides A or G.

Figure 3

Cosegregation of FAM38A gene mutations with HX phenotype. The FAM38A gene mutations detected by exome sequencing in affected HX persons cosegregated with disease phenotype in all affected persons in both kindreds. Family A indicates New York pedigree. As predicted by clinical, genetic, and biochemical studies, the parents and affected HX persons are heterozygous for the FAM38A mutation and persons III-14, III-15, and III-21 are homozygous for the FAM38A mutation. Family B indicates Canadian pedigree. Inheritance of the FAM38A mutation is heterozygous. “NT” in the symbols indicates persons who were “not tested” and clinical, laboratory, biochemical, and genetic data are not available.

Figure 4

Conservation of mutations across vertebrate species. The mutant amino acid residues identified in HX patients are conserved across vertebrate species, including the clades of placental mammals, the extant Eutherians, and within members of the Piezo family of proteins.

Figure 5

A model of human PIEZO1. The predicted transmembrane regions of PIEZO1 (UniProt accession Q92508) were displayed using TMRPres2D software. UniProt predictions are based on the regions that have α-helical transmembrane potential in at least 2 of the predictive tools, TMHMM, Memsat, Phobius, and the hydrophobic moment plot method of Eisenberg et al. Arrows indicate the location of HX-associated mutations; red line, the location of PIEZO1 peptides identified in erythrocyte membranes; and green, the locations of peptides identified in discovery proteomics of erythrocyte membrane ghosts.