Download Mutations in the mechanotransduction protein

From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
RED CELLS, IRON, AND ERYTHROPOIESIS
Mutations in the mechanotransduction protein PIEZO1 are associated with
hereditary xerocytosis
Ryan Zarychanski,1,2 Vincent P. Schulz,3 Brett L. Houston,4 Yelena Maksimova,3 Donald S. Houston,1,2 Brian Smith,5
Jesse Rinehart,6 and Patrick G. Gallagher3,7,8
1CancerCare
Manitoba, Department of Hematology and Medical Oncology, Winnipeg, MB; 2Department of Internal Medicine, University of Manitoba, Winnipeg,
MB; 3Department of Pediatrics, Yale University School of Medicine, New Haven, CT; 4Faculty of Medicine, University of Manitoba, Winnipeg, MB; 5Department of
Internal Medicine, University of Rochester, Rochester, NY; 6Department of Cellular and Molecular Physiology and Systems Biology Institute, Yale University,
West Haven, CT; 7Department of Pathology, Yale University School of Medicine, New Haven, CT; and 8Department of Genetics, Yale University School of
Medicine, New Haven, CT
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 identi-
fied 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.
(Blood. 2012;120(9):1908-1915)
Introduction
Hereditary xerocytosis (also known as HX or dehydrated stomatocytosis, DHSt; OMIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration.1
HX erythrocytes exhibit decreased total cation and potassium
content that are not accompanied by a proportional net gain of
sodium and water. HX patients typically exhibit mild to moderate,
compensated hemolytic anemia. Erythrocyte mean corpuscular
hemoglobin concentration is increased and erythrocyte osmotic
fragility is decreased, both reflecting cellular dehydration.
A locus for HX on chromosome 16 (16q23-q24) was first
identified in a large, 3-generation Irish family.2 This locus was
refined to D16S511-16qter via study of 10 kindreds with variants of
HX, pseudohyperkalemia, or nonimmune hydrops fetalis.3,4 Recent
studies of one of the original HX families from Rochester, NY,5 and
of a large HX family from Manitoba, Canada6 confirmed the
linkage of the disease phenotype to chromosome 16q, and refined
the candidate region to 16q24.2-16qter, a 2.4 million-bp interval
containing 51 known or predicted genes.6 To identify the HXassociated genetic locus, we performed high-resolution single
nucleotide polymorphism (SNP) typing and whole-exome sequencing on selected persons from both the New York and Canadian HX
kindreds.
In the refined candidate region, no regions of copy number
variation were detected at 16q24.2-16qter. A large region of
homozygosity was detected in this region in DNA from a presumed
homozygote from the New York kindred. Exome sequencing
identified novel mutations affecting PIEZO1 (encoded by the
FAM38A gene) in both HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation
with the disease phenotype in all affected persons in both the New
York and Canadian kindreds. All patients were heterozygous for the
mutations, except for 3 New York patients predicted to be
homozygous for HX 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.
PIEZO1 and PIEZO2 have recently been identified as proteins
involved in mechanosensation and stretch-activated cation channel
activation.7 The ability of a cell to sense mechanical signals is a
fundamental characteristic of all or almost all cells, and this process
has been linked to ion channels, specifically stretch-activated
cation channels.8 Although the molecular identify of these channels
has been well characterized in bacteria for many years, in higher
organisms, the molecular identity of mechanosensory ion channels
has until recently been unknown.9
The identification of PIEZO1 mutations associated with HX is
the first report of human disease associated with mutation in a
mammalian mechanosensory transduction ion channel. PIEZO
proteins may play an important role in maintaining erythrocyte
volume homeostasis.
Submitted April 4, 2012; accepted April 17, 2012. Prepublished online as Blood
First Edition paper, April 23, 2012; DOI 10.1182/blood-2012-04-422253.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2012 by The American Society of Hematology
1908
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
Methods
Patients
Family A: New York pedigree. Miller et al from Rochester, NY described
a Swiss-German pedigree with dominantly inherited congenital hemolytic
anemia associated with stomatocytosis, reticulocytosis, and shortened
erythrocyte survival.5 Intracellular potassium and total monovalent cations
were mildly decreased, and erythrocyte osmotic fragility was decreased.
The pedigree’s lineage was traced to the mid-18th century. In one loop of
the pedigree, paternal and maternal great-great-grandmothers were sisters.
This consanguineous mating produced 3 children predicted to be HX
homozygotes, based on clinical and laboratory data. This research was
approved by the Institutional Review Board at Yale University School of
Medicine.
Family B: Canadian pedigree. Houston et al from Winnipeg, MB
described a large Canadian kindred with typical features of HX, including
well-compensated hemolysis associated with elevated mean corpuscular
hemoglobin concentration and decreased erythrocyte osmotic fragility.6
Copy number analyses
DNA from 1 New York heterozygote (M1-24), 1 predicted New York
homozygote (M1-44), and 1 Canadian heterozygote (HA128) was analyzed
using the Illumina Human660W-Quad SNP genotyping array. Genotyping
was performed at the Yale Center for Genome Analysis using standard
Illumina protocols. The HG18 version of the Log R Ratio and B Allele
Frequency values for each sample was exported from the Illumina
GenomeStudio Version 2010.1 software. The PennCNV software (Version
2011-05-03) was used to detect copy number changes using the B Allele
Frequency and Log R Ratio data.10 Copy number change regions were
intersected with RefSeq exonic regions using the BEDtools 2.11.2 software
to identify genes that had copy number changes.11
Targeted exon capture
Targeted regions of genomic DNA were captured using a NimbleGen
SeqCap EZ Exome Version 2.0 solution-based capture system according to
the manufacturer’s protocol. The design targets 329 028 coding exons from
the CCDS database (September 2009) and the RefSeq database (January
2010), as well as 710 microRNAs from miRBase (Version 14, September
2009). Probes were designed against the February 2009 hg19 human
genome sequence with 2.1 million probes capturing approximately 44.1 MB
of targeted sequence.
Exome sequencing
Cluster generation was performed on the Illumina Cluster Station. The
captured, purified, and clonally amplified libraries targeting the exomes
from the HX family members were sequenced on a HiSeq 2000 with
paired-end sequencing at a read length of 75 bp. The Illumina Version
1.7.48 Pipeline was used for image analysis and base calling.
Exome sequence analysis
Fastq sequence reads were aligned to the human genome (hg19build37)
using BWA 0.5.9 software.12 Variant analysis was performed using
GATK analysis Version 1.0.5336 pipeline13,14 as follows: Duplicate
reads were identified using the Picard Version 1.41 MarkDuplicates
software (http://picard.sourceforge.net) and were not used for variant
identification.12 Sequencing reads in the region of insertion/deletions
and clustered SNPs in dbSNP (build 130) and the 1000 genomes (August
4, 2010 release) databases were realigned using the GATK Version
1.1-35 implementation of the Smith-Waterman alignment algorithm to
reduce false positives.13
Base alignment quality scores were determined and used to further
reduce false-positive calls.15 Individual base quality scores were
recalibrated by the GATK software using dbSNP (build 130) and
629 complete human genome sequences from the 1000 genomes project
PIEZO1 AND HEREDITARY XEROCYTOSIS
1909
(August 2010 release)16,17 and machine cycle and dinucleotide covariate. SNPs were called using the GATK Bayesian algorithm for variant
discovery and genotyping, which uses base qualities and allele counts to
determine probabilities for called variants. Indels were called using the
GATK Unified Genotyper implementation of Dindel.18 Indels and novel
SNPs were annotated using the SeattleSeq database (http://snp.gs.washington.edu/SeattleSeqAnnotation131/). The SeattleSeq analysis determines whether an SNP or indel changes a protein sequence or splice site,
whether the nucleotide is conserved,19,20 and provides a Grantham score
and polyphen classification to assess the likely effect of a mutation on
protein function.21,22
mRNA expression analyses
Human CD34-selected stem and progenitor cells were cultured and selected
to yield a population of erythroid cells (R3/R4) as previously described.23
RNA was prepared using the RNeasy kit (QIAGEN) according to the
manufacturer’s instructions. RNA was treated with DNase I and reversetranscribed with oligo(dT) primer using a SuperScript first-strand synthesis
system (Invitrogen). Reverse transcription products were amplified by
real-time PCR with primer pairs tiled across the FAM38A locus (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials
link at the top of the online article).
Sanger sequencing
Sanger sequencing was performed on an Applied Biosystems 3130XL
capillary sequencer. Primers for amplification of genomic DNA are listed in
supplemental Table 2.
Erythrocyte membrane isolation and digestion
Erythrocyte membrane ghosts were prepared as described.24 Protein from
100 ␮L enriched RBC membrane fraction was precipitated with the
methanol/chloroform method as described by Wessel and Flugge.25 The
protein pellet was dissolved in 30 ␮L 8M urea, 0.4M Tris-HCl buffer, pH
8.0 (23°C) by vortex, and an aliquot was used for protein quantitation by
UV-VIS spectrophotometry at 280 nm on a NanoDrop (Thermo Fisher
Scientific). Cysteines were reduced with 4.1mM DTT (10 minutes at 60°C),
and the reaction was quenched on ice. Carbamidomethylation of cysteines
was carried out for 30 minutes at room temperature in the dark using 8.3mM
indole-3-acetic acid IAA. Excess IAA was quenched with DTT and the
reaction diluted to ⬃ 1M urea with 200 ␮L 90mM Tris-HCl buffer, pH
8.0 (23°C) with 2mM CaCl2. Digestion with trypsin was carried out at
37°C for 15 hours using a trypsin/protein ratio of 1:15. The reaction was
quenched with 12 ␮L 20% trifluoroacetic acid. Peptides were desalted
by reversed-phase chromatography using C18 UltraMicroSpin columns.
Peptides were dried in a vacuum centrifuge, dissolved in 20 ␮L
3:8 volume/volume 70% FA/0.1% trifluoroacetic acid and quantified by
UV-Vis on the NanoDrop. The peptide concentration was adjusted to
0.6 ␮g/␮L for liquid chromatography-mass spectrometry (LC-MS).
Mass spectrometry
LC-MS was performed on a triple TOF 5600 (AB Sciex) with nanospray
source connected to a nanoAcquity UPLC (Waters). The mobile phases for
LC separation consisted of water (A) and acetonitrile (B) containing 0.1%
of FA, respectively. A total of 3 ␮g of peptides was injected onto a Waters
Symmetry C18, 5-␮m particle size, 180 ␮m ID ⫻ 20 mm nano Acquity
UPLC trap column, which was connected to a Waters BEH 30 C18, 1.7-␮m
particle size, 75-␮m ID ⫻ 150 mm capillary column operated at 45°C.
Peptides were trapped for 3 minutes at 1% B with a flow rate of 5 ␮L/min.
Gradient elution was carried out using a flow rate of 0.5 ␮L/min with a
2-step gradient from 5% to 40% B in 160 minutes and 40% to 85% B in
3.3 minutes, respectively.
The mass spectrometer was operated in information-dependent aquisition mode with a single 250-ms high-resolution time-of-flight MS survey
scan (m/z ⫽ 400-1250) followed by up to 20 MS/MS scans (m/z ⫽ 1001500) at more than 15 000 resolution and 50-ms accumulation time.
Precursor ions exceeding 125 counts were fragmented with collision energy
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
1910
ZARYCHANSKI et al
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
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.
of 37 eV ⫾ 15 eV using N2 as the collision gas. Fragmented peptides were
set on an exclusion list for 10 seconds.
Results
16q24-16qter HX locus. No regions of copy number variation were
detected in the 16q24.2-16qter candidate region (data not shown).
In one loop of the New York pedigree, the parents were unknowingly
distantly related, and clinical and laboratory data predicted that 3 of their
children were HX homozygotes (see “Mutation detection”). We analyzed the genotyping data in the 16q24.2-16qter candidate region in one
of these children. This patient (III-15; UZ1-34) has an extended block of
homozygosity on chromosome 16 starting after 87445363 (last heterozygous SNP), before 87445839 (first homozygous SNP) and extending to
90109711, the last called SNP at the telomeric end of chromosome 16
(Figure 1). This region contains 100 homozygous SNPs. There are
3 possible heterozygous SNPs at the very end of 16q that could be
miscalls (2 SNPs are in 1000 genomes) or not a variant at all. No other
extended regions of homozygosity are present on chromosome 16.
Copy number analyses
Exome sequencing and novel variant identification
To interrogate the 16q24.2-16qter candidate region for variations in
copy number, high-resolution SNP typing with genomic DNA from
affected persons of both the New York and Canadian HX kindreds was
performed using Illumina Human660W-Quad BeadChip arrays. This
array contains 657 366 markers and includes 3512 markers in the
Whole-exome sequencing was performed using Nimblegen solutionbased exon capture and high throughput sequencing. Paired-end
DNA sequencing data were aligned to the February 2009 GRCh37
hg19 version of the human genome sequence. High mean coverage
of more than 65 was achieved and more than 92.1% of all targeted
Protein database searching
All tandem MS spectra were searched in-house using MASCOT Version 2.2.0
against the human SwissProt database release February 2011. Search criteria
were: Enzyme:Trypsin, allowing 1 missed cleavage. The precursor ion mass
tolerance was set to 20 ppm for the precursor and 0.2 Da for fragment ions,
respectively. Fixed modifications were carbamidomethyl at cysteine, and the
variable modification was oxidation of M. The overall false discovery rate
estimated by MASCOT was better than 3.5%.
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
PIEZO1 AND HEREDITARY XEROCYTOSIS
1911
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.
bases were read more than 10 times for all the samples, respectively, more than sufficient to determine genotypes in the 16q2416qter region and to identify novel variants in HX family members
with high specificity (supplemental Table 3).
Genotypes for single nucleotide and indel variants in the exome
targeted regions were called for HX and normal samples using the
GATK Unified Genotyper. Variants with quality score less than
50 and that were in dbSNP were filtered out, and the remaining
variants were submitted to the SeattleSeq annotation pipeline. Any
variant that did not change protein sequences or mRNA splicing
was removed.
Candidate gene identification
Assuming a model with dominant inheritance, we searched the
exome data for variants in the 16q24.2-16qter candidate region
present in the New York HX patients, but not in control unaffected
samples. Two genes (FAM38A and TPSAB1) were identified in the
candidate region.
We repeated the same analyses using a subset of patients from
the Canadian HX family. Five genes (FAM38A, ACSM2A, E4F1
CENPBD1, and C16orf55) were identified in the candidate region.
Thus, novel, nonsynonymous variants in the FAM38A gene, which
encodes the protein PIEZO1, were present in both kindreds.
Mutation detection
Sequence analysis of the FAM38A gene in DNA from the New York
HX patients revealed abnormal DNA sequences in the alignment of
multiple short sequencing reads in exon 46 (Figure 2A). The
corresponding DNA sequence revealed a heterozygous T to G
substitution (nucleotide position chromosome 16:88 783 293, hg19)
changing a Met (ATG) to Arg (AGG) at amino acid 2225. Sequence
analysis of the FAM38A gene in DNA from the Canadian HX
patients revealed abnormal DNA sequences in the alignment of
multiple short sequencing reads in exon 51 (Figure 2A). The
corresponding DNA sequence revealed a heterozygous G to A
substitution (nucleotide position chromosome 16:88 782 212, hg19)
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.
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
1912
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
ZARYCHANSKI et al
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.
changing an Arg (CGC) to His (CAC) at amino acid 2456. These
2 variants were not found in dbSNP Version 135, which contains
phase 1 of the 1000 genomes project, and they were not found in
approximately 3200 alleles of the National Heart, Lung, and Blood
Institute exome sequencing project.
No other deleterious variants were detected in the coding
regions of the FAM38A gene. The mutations in exons 46 and 51 of
the FAM38A gene identified by exome sequencing were validated
by conventional Sanger sequencing (Figure 2B).
No mutations were identified in the SLC4A1 gene locus,
previously associated with a subset of HX patients.
Inheritance and mutation analyses
Segregation analyses confirmed transmission of the FAM38A
mutations and cosegregation with the disease phenotype in affected
persons in both the New York and Canadian kindreds (Figure 3).
The mutations were absent in non-HX family members. Heterozygous persons exhibit well-compensated hemolysis, elevated mean
corpuscular hemoglobin concentration, and decreased osmotic
fragility.5,6 In one loop of the New York family, the paternal and
maternal great-great-grandmothers were sisters, and 3 offspring of
this consanguineous mating were identified as homozygotes based
on clinical and laboratory data, including severe hemolytic anemia
with reticulocytosis, shortened red blood cell survival, stomatocytosis, hyperbilirubinemia, and markedly altered erythrocyte sodium
and potassium levels.5 DNA analyses confirmed all 3 patients were
indeed homozygous for the M2225R mutation.
The missense mutations were both predicted to be pathogenic
by PolyPhen, a tool that predicts possible impact of an amino acid
substitution on the structure and function of human proteins using
physical and comparative considerations. The mutations were both
in conserved resides, with highly significant phastCons scores,
M2225R 1.0 and R2456H 0.812, and highly significant conservation scores, M2225R 4.34 and R2456H 3.43. These amino acids are
highly conserved across vertebrate species, including the clades of
placental mammals, the extant Eutherians, and within members of
the PIEZO family of proteins (Figure 4).
The amino acids affected by the FAM38A missense mutations
identified in the HX kindreds are both located in the COOHterminal region of PIEZO1 (Figure 5).
PIEZO1 mRNA expression in erythroid cells
There are limited data regarding expression of PIEZO1 in erythroid
cells. We sought to ensure that both the FAM38A gene and its
protein product were expressed in erythroid cells. mRNA was
prepared from cultured, primary human erythroid progenitor cells,
cDNA prepared by reverse transcription, and then amplified with
4 sets of primers tiled across the FAM38A locus (supplemental
Figure 1A).
These cDNA transcripts were amplified (supplemental Figure
1B), subcloned, and sequenced. These overlapping cDNA transcripts corresponded to the human full-length FAM38A cDNA (GI:
257196141), which is homologous to the murine Fam38A transcript of Piezo1 associated with induction of mechanically activated cellular currents.7 Microarray analyses performed by Keller
et al demonstrated moderate levels of FAM38A mRNA (GEO
accession no. GDS2431)27 in cultured human erythroid progenitor
cells differentiated in vitro, confirming our observations.
Unbiased discovery proteomics
Western blot analyses of wild-type R3/R4 cell proteins and human
erythrocyte ghosts using 3 different commercial antibodies gave
inconsistent and irreproducible results. Thus, we performed a series
of mass spectrometry-based, unbiased proteomic analyses of
human erythrocyte ghosts to search for PIEZO1-derived peptides.
This discovery proteomics strategy yielded 4 peptides corresponding to PIEZO1: GFALYNAANLK (peptide 1), QHQLAPLPAQAVFASGTR (peptide 2), YLLTQELLQGGEVHR (peptide 3), and
IPELEEAELFAEGQGR (peptide 4). These peptides were aligned
to the predicted human PIEZO1 amino acid sequence. These
peptides are spread across the PIEZO1 protein (Figure 5; supplemental Figure 2). The identification of peptide 1 indicates that the
long isoform of PIEZO1, similar to murine Piezo1 associated with
a unitary conductance approximately 25 pS current in transfected
HEK293 cells on membrane deformation,7 is present in human
erythrocyte membranes.
Discussion
Mechanotransduction, the conversion of mechanical force into
biologic signals, is a fundamental physiologic process of mammalian cells. It influences many critical processes, including embryonic development, tactile, pain, and auditory sensation, regulation
of vascular tone, flow sensing in the kidney, and muscle and tendon
stretch.8 The primary mechanism for mechanosensation is linked to
calcium permeable, stretch-activated cation channels. In bacteria,
the molecular identity of mechanosensitive ion channels has been
understood at the molecular level for many years. However, in
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
PIEZO1 AND HEREDITARY XEROCYTOSIS
1913
Figure 5. A model of human PIEZO1. The predicted transmembrane regions of PIEZO1 (UniProt accession Q92508) were displayed using TMRPres2D software.26 UniProt
predictions are based on the regions that have ␣-helical transmembrane potential in at least 2 of the predictive tools, TMHMM, Memsat, Phobius, and the hydrophobic moment
plot method of Eisenberg et al.28 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.
higher organisms, the molecular identity of mechanosensory ion
channels has been unknown.9
Recently, PIEZO proteins have been identified as mechanically
activated ion channels in eukaryotes.7 Originally identified in the
murine neuroblastoma cell line N2A, PIEZO proteins induce
mechanically activated cationic currents in various cell types.29
PIEZO-induced currents are blocked by gadolinium, ruthenium
red, and the peptide GsMTx4 (Grammostola spatulata mechanotoxin 4),30,31 commonly used to inhibit mechanosensitive currents.
Murine Piezo1 assembles into a homo-multimeric complex that is
fully active without other proteins. Similar to murine Piezo protein,
the single Piezo protein in Drosophila induces mechanically
activated cation currents.32
Protein structure prediction programs indicate that PIEZO proteins
are very large integral membrane proteins with 24 to 40 transmembrane
domains located across the protein (Figure 5).7 What is the role of
PIEZO1 in erythroid cells? Does it regulate a stretch-activated calcium
pathway? On deformation, calcium enters the erythrocyte through an as
yet unidentified pathway.33,34 Recent studies indicate that local deformations in the erythrocyte membrane can transiently activate a calciumpermeability pathway leading to increased intracellular calcium, activation of potassium currents through the calcium-sensitive Gardos channel,
and secondary anion currents through 5-nitro-2-(3-phenylpropylamino)
benzoic acid and diisothiocyanostilbene-2,2⬘-disulfonate-inhibitable
channels.35 However, this and other previous work provides no evidence
for mechanically induced PIEZO-associated currents through a 10- to
25-pS channel in wild-type mature erythrocytes either in whole cell
configuration or in single-channel recordings after deformation.35
Identification of this stretch-induced calcium-activated cation
pathway and determination of whether PIEZO channels participate
is of great physiologic relevance to erythrocyte biology. Circulatory shear stress has been suggested to cause reversible increases in
erythrocyte calcium permeability.34,36 Erythrocyte aging has been
associated with alterations in membrane permeability for
calcium.37-39 Localized membrane deformation has been associated
with increased calcium in the process of apical alignment in the
initial steps of malaria invasion.38 Finally, in sickle cell disease,
deoxygenated, circulating erythrocytes exhibit reversible increases
in membrane permeability to cations through an as yet unidentified
pathway known as Psickle,40,41 and membrane deformation is
necessary for this increase in sickling-induced cation permeability.42 Increased calcium levels mediated by Psickle lead to activation
of the Gardos channel, a critical step in the dehydration of sickle
erythrocytes.43-46 A recent study shows similarities between erythrocyte nonspecific cation conductance pathways and Psickle and
demonstrate sensitivity of the pathway to GsMTx4, indicating
properties of a mechanosensitive ion channel.47
PIEZO1 may serve as a sensor for cell swelling, as GsMTx4
blockade inhibits both mechanically activated currents and whole-cell
regulatory volume decrease in NRK-49 cells.31,48 Perhaps PIEZO
proteins play a previously unrecognized role in erythrocyte volume
regulation, with PIEZO1-mutant erythrocytes gradually becoming dehydrated during their repeated cycles of travel through the microcirculation, associated with changes in oxygenation/deoxygenation.
Unraveling the effects of these HX-associated mutations will
provide insight into the structure and function of PIEZO1 and will
shed additional insight into the pathogenesis of primary and
secondary disorders of erythrocyte hydration. The mutations may
disrupt critical protein-protein interactions, may influence PIEZO1
function in erythroid cells, or may impair PIEZO1 trafficking to the
erythrocyte membrane. Juxtamembrane mutations of highly conserved arginine residues have been shown to disrupt transporter
membrane trafficking, such as occurs with anion exchanger 1 in
hereditary spherocytosis and distal renal tubular acidosis,49,50
perhaps because of misfolding or mutation of arginine-based
endoplasmic reticulum retention/retrieval signals.51
Another major unsolved puzzle in PIEZO1-associated HX is the
great phenotypic variability exhibited between patients. Associated
clinical syndromes include familial pseudohyperkalemia with or
without hematologic abnormalities. Some patients exhibit in utero
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
1914
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
ZARYCHANSKI et al
hydrops fetalis, not associated with fetal anemia that typically
resolves spontaneously before or shortly after birth without any
subsequent sequelae. Many patients exhibit marked iron overload,
suggesting either a pattern of abnormal iron homeostasis or marked
dyserythropoiesis.
In conclusion, PIEZO proteins are the first mammalian mechanosensory signal transduction proteins identified to date. When
mutated, PIEZO1 is associated with the phenotype of hereditary
xerocytosis. Determination of the structure and function of PIEZO
proteins will shed light on the erythrocyte hydration pathways
mediated by PIEZO proteins in mature erythrocytes.
Acknowledgments
The authors thank the patients for their participation and Dr Joseph
Hoffman for critical manuscript review.
This work was supported in part by the Doris Duke Foundation
(P.G.G.), and the Manitoba Medical Services Foundation (R.Z.).
B.L.H. was a recipient of an American Society of Hematology
Trainee Award.
Authorship
Contribution: R.Z., B.L.H., D.S.H., Y.M., J.R., V.P.S., and P.G.G.
participated in experimental design, execution, and interpretation;
B.S. participated in patient phenotyping; and all authors wrote and
reviewed the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Patrick G. Gallagher, Department of Pediatrics, Yale University School of Medicine, 333 Cedar St, PO Box
208064, New Haven, CT 06520-8064; e-mail: patrick.
[email protected].
References
1. Bruce LJ. Hereditary stomatocytosis and cationleaky red cells: recent developments. Blood Cells
Mol Dis. 2009;42(3):216-222.
2. Carella M, Stewart G, Ajetunmobi JF, et al.
Genomewide search for dehydrated hereditary
stomatocytosis (hereditary xerocytosis): mapping
of locus to chromosome 16 (16q23-qter). Am J
Hum Genet. 1998;63(3):810-816.
3. Grootenboer S, Schischmanoff PO, Laurendeau I,
et al. Pleiotropic syndrome of dehydrated hereditary
stomatocytosis, pseudohyperkalemia, and perinatal
edema maps to 16q23-q24. Blood. 2000;96(7):25992605.
4. Iolascon A, Stewart GW, Ajetunmobi JF, et al. Familial pseudohyperkalemia maps to the same locus as dehydrated hereditary stomatocytosis (hereditary xerocytosis). Blood. 1999;93(9):31203123.
5. Miller DR, Rickles FR, Lichtman MA, La Celle PL,
Bates J, Weed RI. A new variant of hereditary hemolytic anemia with stomatocytosis and erythrocyte cation abnormality. Blood. 1971;38(2):184204.
6. Houston BL, Zelinski T, Israels SJ, et al. Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred. Blood
Cells Mol Dis. 2011;47(4):226-231.
7. Coste B, Mathur J, Schmidt M, et al. Piezo1 and
Piezo2 are essential components of distinct mechanically activated cation channels. Science.
2010;330(6000):55-60.
8. Nilius B. Pressing and squeezing with Piezos.
EMBO Rep. 2010;11(12):902-903.
9. Tsunozaki M, Bautista DM. Mammalian somatosensory mechanotransduction. Curr Opin Neurobiol. 2009;19(4):362-369.
10. Wang K, Li M, Hadley D, et al. PennCNV: an integrated hidden Markov model designed for highresolution copy number variation detection in
whole-genome SNP genotyping data. Genome
Res. 2007;17(11):1665-1674.
11. Quinlan AR, Hall IM. BEDTools: a flexible suite of
utilities for comparing genomic features. Bioinformatics. 2010;26(6):841-842.
12. Li H, Durbin R. Fast and accurate short read
alignment with Burrows-Wheeler transform.
Bioinformatics. 2009;25(14):1754-1760.
for analyzing next-generation DNA sequencing
data. Genome Res. 2010;20(9):1297-1303.
15. Li H. Improving SNP discovery by base alignment
quality. Bioinformatics. 2011;27(8):1157-1158.
16. Durbin RM, Abecasis GR, Altshuler DL, et al. A
map of human genome variation from populationscale sequencing. Nature. 2010;467(7319):10611073.
17. Sherry ST, Ward MH, Kholodov M, et al. dbSNP:
the NCBI database of genetic variation. Nucleic
Acids Res. 2001;29(1):308-311.
18. Albers CA, Lunter G, Macarthur DG, McVean G,
Ouwehand WH, Durbin R. Dindel: accurate indel
calls from short-read data. Genome Res. 2011;
21(6):961-973.
19. Cooper GM, Stone EA, Asimenos G, Green ED,
Batzoglou S, Sidow A. Distribution and intensity
of constraint in mammalian genomic sequence.
Genome Res. 2005;15(7):901-913.
20. Siepel A, Bejerano G, Pedersen JS, et al. Evolutionarily conserved elements in vertebrate, insect,
worm, and yeast genomes. Genome Res. 2005;
15(8):1034-1050.
dimensional profiles. Methods Enzymol. 1997;
277:396-404.
29. Coste B, Xiao B, Santos JS, et al. Piezo proteins
are pore-forming subunits of mechanically activated channels. Nature. 2012;483(7388):176181.
30. Bae C, Sachs F, Gottlieb PA. The mechanosensitive ion channel Piezo1 is inhibited by the peptide
GsMTx4. Biochemistry. 2011;50(29):6295-6300.
31. Delmas P, Hao J, Rodat-Despoix L. Molecular
mechanisms of mechanotransduction in mammalian sensory neurons. Nat Rev. 2011;12(3):139153.
32. Kim SE, Coste B, Chadha A, Cook B, Patapoutian A.
The role of Drosophila Piezo in mechanical nociception. Nature. 2012;483(7388):209-212.
33. Brain MC, Pihl C, Robertson L, Brown CB. Evidence for a mechanosensitive calcium influx into
red cells. Blood Cells Mol Dis. 2004;32(3):349352.
34. Johnson RM. Membrane stress increases cation
permeability in red cells. Biophys J. 1994;67(5):
1876-1881.
21. Adzhubei IA, Schmidt S, Peshkin L, et al. A
method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248249.
35. Dyrda A, Cytlak U, Ciuraszkiewicz A, et al. Local
membrane deformations activate Ca2⫹-dependent
K⫹ and anionic currents in intact human red
blood cells. PLoS One. 2010;5(2):e9447.
22. Grantham R. Amino acid difference formula to
help explain protein evolution. Science. 1974;
185(4154):862-864.
36. Larsen FL, Katz S, Roufogalis BD, Brooks DE.
Physiological shear stresses enhance the Ca2⫹
permeability of human erythrocytes. Nature.
1981;294(5842):667-668.
23. Steiner LA, Maksimova Y, Schulz V, et al. Chromatin architecture and transcription factor binding
regulate expression of erythrocyte membrane
protein genes. Mol Cell Biol. 2009;29(20):53995412.
37. Lew VL, Daw N, Perdomo D, Etzion Z,
Bookchin RM, Tiffert T. Distribution of plasma
membrane Ca2⫹ pump activity in normal human
red blood cells. Blood. 2003;102(12):4206-4213.
24. Marchesi SL, Knowles WJ, Morrow JS, Bologna M,
Marchesi VT. Abnormal spectrin in hereditary elliptocytosis. Blood. 1986;67(1):141-151.
38. Lew VL, Tiffert T. Is invasion efficiency in malaria
controlled by pre-invasion events? Trends Parasitol. 2007;23(10):481-484.
25. Wessel D, Flugge UI. A method for the quantitative recovery of protein in dilute-solution in the
presence of detergents and lipids. Anal Biochem.
1984;138(1):141-143.
39. Romero PJ, Romero EA. Differences in Ca2⫹
pumping activity between sub-populations of
human red cells. Cell Calcium. 1997;21(5):353358.
26. Spyropoulos IC, Liakopoulos TD, Bagos PG,
Hamodrakas SJ. TMRPres2D: high quality visual
representation of transmembrane protein models.
Bioinformatics. 2004;20(17):3258-3260.
40. Lew VL, Bookchin RM. Ion transport pathology in
the mechanism of sickle cell dehydration. Physiol
Rev. 2005;85(1):179-200.
13. Depristo MA, Banks E, Poplin R, et al. A framework for variation discovery and genotyping using
next-generation DNA sequencing data. Nat
Genet. 2011;43(5):491-498.
27. Keller MA, Addya S, Vadigepalli R, et al. Transcriptional regulatory network analysis of developing human erythroid progenitors reveals patterns of coregulation and potential transcriptional
regulators. Physiol Genom. 2006;28(1):114-128.
14. McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework
28. Eisenberg D, Luthy R, Bowie JU. VERIFY3D:
assessment of protein models with three-
41. Lew VL, Ortiz OE, Bookchin RM. Stochastic nature and red cell population distribution of the
sickling-induced Ca2⫹ permeability. J Clin Invest.
1997;99(11):2727-2735.
42. Mohandas N, Rossi ME, Clark MR. Association
between morphologic distortion of sickle cells and
deoxygenation-induced cation permeability increase. Blood. 1986;68(2):450-454.
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
BLOOD, 30 AUGUST 2012 䡠 VOLUME 120, NUMBER 9
43. Browning JA, Ellory JC, Gibson JS. Pathophysiology of red cell volume. Contrib Nephrol. 2006;
152:241-268.
44. Browning JA, Staines HM, Robinson HC, Powell T,
Ellory JC, Gibson JS. The effect of deoxygenation
on whole-cell conductance of red blood cells from
normal individuals and sickle cell patients. Blood.
2007;109(6):2622-2629.
45. Brugnara C. Sickle cell disease: from membrane
pathophysiology to novel therapies for prevention
of erythrocyte dehydration. J Pediatr Hematol Oncol. 2003;25(12):927-933.
46. Joiner CH. Gardos pathway to sickle cell therapies? Blood. 2008;111(8):3918-3919.
47. Ma YL, Rees DC, Gibson JS, Ellory JC. The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors. J Physiol.
2012;590(Pt 9):2095-2105.
48. Hua SZ, Gottlieb PA, Heo J, Sachs F. A mechanosensitive ion channel regulating cell volume.
Am J Physiol Cell Physiol. 2010;298(6):C1424C1430.
49. Quilty JA, Li J, Reithmeier RA. Impaired trafficking of distal renal tubular acidosis mutants
PIEZO1 AND HEREDITARY XEROCYTOSIS
1915
of the human kidney anion exchanger kAE1.
Am J Physiol Renal Physiol. 2002;282(5):F810F820.
50. Quilty JA, Reithmeier RA. Trafficking and folding
defects in hereditary spherocytosis mutants of the
human red cell anion exchanger. Traffic. 2000;
1(12):987-998.
51. Michelsen K, Yuan H, Schwappach B. Hide and
run: arginine-based endoplasmic-reticulumsorting motifs in the assembly of heteromultimeric
membrane proteins. EMBO Rep. 2005;6(8):717722.
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
2012 120: 1908-1915
doi:10.1182/blood-2012-04-422253 originally published
online April 23, 2012
Mutations in the mechanotransduction protein PIEZO1 are associated
with hereditary xerocytosis
Ryan Zarychanski, Vincent P. Schulz, Brett L. Houston, Yelena Maksimova, Donald S. Houston,
Brian Smith, Jesse Rinehart and Patrick G. Gallagher
Updated information and services can be found at:
http://www.bloodjournal.org/content/120/9/1908.full.html
Articles on similar topics can be found in the following Blood collections
Red Cells, Iron, and Erythropoiesis (793 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.