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The Genetic Basis of Human Erythrocyte Pyridoxal Kinase Activity Variation.

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Thirty years ago we reported that erythrocyte pyridoxal kinase activity of African-Americans was strikingly lower than that of persons with European ancestry in a tissue-specific manner. At the time, it was impossible to elucidate the mechanism by which evolution had selectively lowered the enzyme activity in one cell type but not in others. We have now identified a promoter mutation with potential erythroid-specific properties that could be the basis of a novel mechanism of controlling cell-specific decreased activity of an essential enzyme.
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haematologica/the hematolog
y jour
nal | 2006; 91(6) |
801
|
J
onathan M. Flanagan
Ernest Beutler
The genetic basis of human erythrocyte pyridoxal
kinase activity variation
P
yridoxal-5’-phosphate (PLP) is a key co-
factor in many enzymatic reactions of
intermediary metabolism.
1
The synthe-
sis of PLP requires the conversion of dietary
B6 vitamers (pyridoxal, pyridoxine and pyri-
doxamine) into their active co-enzyme
forms. Two enzymes, pyridoxal kinase and
pyridoxine oxidase, are involved in this con-
version.
2
The pyridoxal kinase enzyme
(PdxK; EC 2.7.1.35) phosphorylates all three
B6 vitamers into PLP, while pyridoxine oxi-
dase (PNPO; EC 1.4.3.5) allows interconver-
sion of the three 5'-phosphoforms. The
main site of PdxK activity is in the liver
although it is ubiquitously expressed.
1,3
Thirty years ago we reported that the ery-
throcyte PdxK activity of African-Americans
was strikingly lower than that of persons
with Eur
opean ancestr
y.
4
Mor
eover
, this
racial difference was found to be tissue-spe-
cific, with leukocyte and skin fibroblast
PdxK enzyme activities being the same in
both ethnic groups.
4
Subsequently, these
r
esults wer
e confirmed, and it was suggested
that the selective pr
essur
e of malaria was the
cause of the lowered erythrocyte enzyme
activities.
5
Diet has been shown to influence
PdxK activity
, although this alone is not
enough to explain the difference seen
between African-Americans and Cauca-
sians.
4,6,7
At the time of our original report it
was impossible to elucidate the mechanism
by which evolution had selectively lower
ed
the enzyme activity in one cell type but not
in another. Using more modern methods we
are now able to suggest a plausible mecha-
nism by which ther
e is tissue-specific varia
-
tion in the activity of the human PdxK
enzyme.
Design and Methods
Subject recruitment and genomic DNA
isolation
Blood was obtained with informed con-
sent from normal hospital employees and
from patients attending a Health Appraisal
Clinic in a study approved by the Kaiser
Permanente and Scripps Resear
ch Institute
institutional review boards. The ethnic ori-
gin of each subject was based on self-identi-
fication. DNA was extracted from whole
blood using a GENTRA DNA purification kit
and used directly for polymerase chain reac-
tion (PCR) amplifications. DNA was
obtained for three populations, Caucasian
(n=48), African-American (n=167) and Asian
(n=81).
PCR amplifications and sequencing
The NCBI sequence AP001752 was used
as the r
eference genomic sequence and
NM_003681 for the cDNA sequence for the
human
PdxK gene. The nucleotide immedi
-
ately 5' to the star
t A
TG codon was desig
-
nated -1. Oligonucleotide primers were
designed for the promoter region and the 11
exons of the
PdxK gene. The entir
e PdxK
gene was amplified by PCR amplification
and purified using spin column purification
(Qiagen, CA, USA). Sequencing was then
performed by capillary electrophoresis using
an ABI PRISM 3100 Genetic Analyzer
.
Electrophoretic mobility shift assay
(EMSA)
Har
vested K562 cells wer
e suspended in
cold phosphate-buffered saline and a nuclear
extract was obtained using a Qproteome
Nuclear protein extraction kit (Qiagen, CA,
USA). The protein concentration of the
From the Molecular and
Experimental Medicine, The Scripps
Resear
ch Institute, La Jolla, CA, USA
(JMF, EB).
Correspondence:
Ernest Beutler, MEM 215,
The Scripps Research Institute,
La Jolla, CA 92037, USA.
E-mail: beutler@scripps.edu
R
ed Cell Disorders • Brief Report
Thirty years ago we reported that erythrocyte pyridoxal kinase activity of African-
Americans was strikingly lower than that of persons with European ancestry in a tis-
sue-specific manner. At the time, it was impossible to elucidate the mechanism by
which evolution had selectively lowered the enzyme activity in one cell type but not in
others. We have now identified a promoter mutation with potential erythroid-specific
properties that could be the basis of a novel mechanism of controlling cell-specific
decreased activity of an essential enzyme.
Key words: pyridoxal kinase, erythrocytes, promoter.
Haematologica 2006; 91:801-804
©2006 Ferrata Storti Foundation
©Ferrata Storti Foundation
| 802 | haematologica/the hematology journal | 2006; 91(6)
nuclear extract was determined by the Bradford assay.
To perform the EMSA, complementary PdxK promoter
oligonucleotides of the PdxK insert or
PdxK wild-type
region (5
µM) were annealed, end-labeled with [γ-
3
2
P]dATP
using polynucleotide kinase (New England Biolabs, MA,
USA) and then purified by G50 Sephadex column purifica-
tion (Pharmacia, NJ, USA).
For each probe to be tested,
5
µg of K562 nuclear extract was incubated for 15 mins
at room temperature, in a binding buffer containing 10
mM Tris-HCL, pH 8.0, 50 mM NaCl, 0.5 mM EDTA, 0.5
mM DTT, 1.0 mM MgCl
2
, 1.5 µg of poly (dI-dC), and
4.0% glycerol. Then exactly 2
×10
3
cpm of a [γ-
32
P]dATP-
labeled
PdxK promoter probe was added and the mix-
ture was incubated for 1 hr at room temperature. The
mixture was subjected to non-denaturing gel elec-
trophoresis at 9 V/cm on a 5% polyacrylamide gel. The
gel was vacuum-dried, and radioactivity was detected
using a Cyclone PhosphorImager (Packard Instruments,
Meridien, CT, USA) equipped with OptiQuant analysis
software (Packard Instruments).
Erythrocyte PdxK enzyme assay
Erythrocyte PdxK activity was measured using the
method of Chern and Beutler.
8
Briefly, fresh venous
blood was collected in acid-citrate dextrose (ACD)
vacutainers
®
(Becton, Dickinson and Co., USA) and
stored at 4°C prior to analysis. The blood was filtered
through a cellulose column to remove white blood cells
and platelets.
9
Hemolysates were prepared by making a
1 to 10 dilution of washed red cells in distilled water
.
PdxK activity of the hemolysates was determined by
measuring the phosphorylation of a [
3
H]pyridoxine sub-
strate, with unreacted [
3
H]pyridoxine being absorbed on
Dowex-50 ion exchange resin.
8
Results and Discussion
A number of polymorphisms were discovered in the
PdxK gene by sequencing (T
able 1). Among these we
found an insertion event (-306_-305InsGCGCGGCG) in
the promoter region. This insert was found to have a
significantly lower gene fr
equency in African-
Americans (43.4%) than in either Caucasians (57.3%;
χ
2
test, p=0.016) or Asians (74.7%; χ
2
test, p=0.001).
Bioinfor
matic analysis using MatInspector softwar
e
10
demonstrated that the insert introduces a putative core
promoter binding protein (CPBP) binding site located at
-306_-293bp to the
PdxK star
t codon. The CPBP tran
-
scription factor is known to enhance transcriptional
activity by at least four-fold.
1
1
Interestingly,
MatInspector analysis revealed that the putative CPBP
site was adjacent to a binding site for an erythrocyte
specific transcription factor
, erythroid Krüppel like fac-
tor (EKLF). This led to the hypothesis that the pr
esence
of the insert would lead to enhanced transcription of
PdxK in erythrocytes only, influencing increased PdxK
enzyme activity
. T
o deter
mine whether the
PdxK inser
t
caused changes in transcription factor binding near the
EKLF site, an EMSA was performed. A [
γ-
32
P]dATP
labeled
PdxK promoter probe was made for the PdxK
wild-type and the PdxK insert variants of the PdxK pro-
moter. These two probes contained the EKLF binding
site with or without the putative CPBP binding site,
respectively. Probes were also constructed that con-
tained only the CPBP binding site region. Equal
amounts of these probes (2
×10
3
cpm) were then incu-
bated with nuclear extracts from the erythroid cell line
K562. There was 3.2-fold greater transcription factor
binding to the
PdxK insert probe than to the PdxK wild-
type probe (Figure 1). Probes specific for the
PdxK CPBP
site showed no transcription factor binding (Figure 1A).
These
in vitro data suggested that the PdxK insert causes
increased transcription factor binding at the EKLF site
and that this increase requires both the CPBP and the
EKLF sites. As observations made
in vitro may not
always reflect the entire situation
in vivo, the in vivo
effect of the insert was determined by measuring the
erythrocyte PdxK enzyme activity of 8 African-
American, 16 Caucasian and 5 Asian subjects. In agree-
ment with the original finding, the majority of the
African-American samples had a PdxK activity at the
lower range of activity documented in either
Caucasians or Asians (Figur
e 2A). The pr
esence of the
PdxK insert was found to correlate with increased ery-
throcyte PdxK enzyme activity, regardless of ethnic ori-
gin (Figur
e 2B). Homozygotes for the insert (n=11;
1.05±0.24 mU/gHb) had a significantly higher PdxK
activity than had either heter
ozygotes (n=11; 0.69
±0.23
mU/gHb) or homozygote
wildtype individuals (n=7;
0.74±0.32 mU/gHb),
p=0.0018 and p=0.0319, respec-
tively, by
t-test analysis. Overall there was a significant
dif
fer
ence between the PdxK activity of individuals
homozygous for the insert (n=11; 1.05±0.24 mU/gHb)
and that of individuals either homozygous wildtype or
heterozygous (n=18; 0.71±0.26 mU/gHb),
t-test,
p=0.0016. However, variability was observed in the
association between genotype and PdxK enzyme activ
-
ity
. It had pr
eviously been shown that cer
tain dr
ugs or
vitamin B6 supplementation and deficiency can cause
changes in erythrocyte PdxK activity.
6,7,12
Retrospective
analysis of the assayed individuals r
evealed that two
out of the three individuals who were homozygous
wildtype yet had a high PdxK enzyme activity, had
been taking a high dose vitamin B6 supplement
J.M. Flanagan et al.
Table 1. DNA sequencing screening results of the PdxK gene. The
number of chromosomes (n) included in the analysis for each pop-
ulation is given in brackets.
P
dxK Polymorphism African-American Caucasian Asian
R
egion Allele Allele Allele
F
requency Frequency Frequency
(
n=334) (n=96) (n=162)
P
romoter -306_-305InsGCGCGGCG 43.4% 57.3%* 74.7%°
E
xon 9 nt837C
T
(S213S) 18% 0% 0%
3
'UTR *13T
C
36% 30% 32%
*
χ
2
test, p=0.016 difference compared to African-Americans; °
χ
2
test, p=0.001 dif-
ference compared to African-Americans.
©Ferrata Storti Foundation
Human erythrocyte pyridoxal kinase activity variation
haematologica/the hematology journal | 2006; 91(6) | 803 |
(>20mg/day) for at least 6 months prior to the assay
(Figure 2B). All other individuals assayed had been tak-
ing a low dose (<5 mg/day) or no vitamin B
6
supple
-
ment.
Thus, we have identified an 8bp PdxK promoter inser-
tion that is less common in persons of African origin
than in those of European or Asian ancestry. Through
combined
in silico and in vitro experiments, this insert is
predicted to cause increased transcription factor binding
of EKLF through the transcriptional enhancer CPBP. The
CPBP transcription factor is ubiquitously expressed in
humans, while EKLF expression is r
estricted to fetal liver
and bone marrow.
11,13
Therefore, any effect of the insert
through CPBP would be tissue-specific for erythrocytes.
The insert had a significantly higher gene frequency in
Caucasians or Asians than in African-Americans, and
this could account for the observed ethnic variation in
erythrocyte PdxK activity. We attempted to confirm this
hypothesis by assaying the erythrocyte PdxK enzyme
activity of individuals genotyped for the
PdxK insert
polymorphism. Individuals homozygous for the insert
had significantly higher PdxK activities than had either
homozygous wildtype or heterozygous individuals.
Some additional variation was observed in the correla-
tion between inser
t genotype and erythrocyte PdxK
activity, which is likely due to dietary or drug influences.
These r
esults suggest an explanation for the original dif
-
fer
ence obser
ved between the er
ythr
ocyte Pdxk enzyme
activity of African-Americans and Caucasians,
4
with
Nature supplying a novel way of selecting for a mecha-
nism that allows cell-specific decr
eased activity of an
essential enzyme.
JF performed the experimental research, intrepreted the data and
drafted of the article; EB instigated the initial concept and experi-
mental design, revised the drafted article and gave final approval
of the submitted manuscript.
This is manuscript number 16691-MEM from The Scripps
Research Institute. This work was supported by the Skaggs
Institute for Chemical Biology and by the Stein Endowment Fund.
The authors declare that they have no potential conflicts of interest.
Manuscript received November 17, 2005. Accepted March 30,
2006.
Figure 1. A. DNA-binding activity of the PdxK pro-
moter insert. Equal amounts of probe (2×10
3
cpm)
were added to 5
µg of K562 nuclear extract.
Transcription factor binding was compared
between 1, free
Pdxk wild-type probe; 2, duplicate
lanes of
Pdxk wild-type binding; 3, free Pdxk insert
probe; 4, duplicate lanes of
Pdxk insert binding; 5,
empty lane; 6, free Pdxk wild-type short probe; 7,
Pdxk wild-type short probe binding; 8, free Pdxk
insert short probe; 9, Pdxk insert short probe bind-
ing; 10, free Pdxk mutated CPBP insert short
probe; 11,
Pdxk mutated insert probe binding.
Duplicate lanes represent independent binding
assays performed simultaneously. The short probes
have no EKLF site. Binding at the CPBP/EKLF
region is indicated by the upper arrow, and free
probe is shown by the lower arrow. B.
Q
uantification of relative transcription factor bind-
ing. Data are presented as percent density units
(%DU) versus
PdxK wild-type; n=3 experiments in
each group (mean ± SD); **p<0.004 by t-test
analysis.
F
igure 2. A.
Comparison of erythrocyte PdxK enzyme activity
between African-Americans, Caucasians and Asians. B. Overall
erythrocyte activity of the PdxK enzyme compared to
PdxK pro-
moter genotype. Open diamonds indicate that the individual was
taking >20mg/day pyridoxal supplement. Ins/Ins denotes PdxK
inser
tion homozygotes,
Het signifies heterozygotes and Wt/Wt
indicates
PdxK wild-type individuals. The mean and standard de
vi
-
ation (SD) of erythrocyte PdxK activity (mU/gHb) of each group is
given.
A
B
A
B
1
2 3 4 567891011
P
dxK PdxK
Wild-type Insert
A
frican- Caucasian Asian
American
Mean 0.66 Mean 0.88 Mean 1.02
SD
±0.32 SD±0.27 SD±0.20
Wt/Wt het Ins/Ins
Mean 0.74 Mean 0.69 Mean 1.05
SD±0.32 SD±0.23 SD±0.24
Mean
S
D
% Density Units
%DU %DU
1
00 321
0
63
(
**
p=
0.004)
450
400
350
300
250
200
150
100
50
0
Erythrocyte PdxK activity
(mU/gHb)
Erythrocyte PdxK activity
(mU/gHb)
1.6
1
.4
1.2
1.0
0.8
0.6
0
.4
0
.2
0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Bound
P
robe
F
ree
Probe
©Ferrata Storti Foundation
J.M. Flanagan et al.
|
804 | haematologica/the hematology journal | 2006; 91(6)
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©Ferrata Storti Foundation
... To date epilepsy or seizures due to mutations in the gene pdxK that codes for PL Kinase has not been reported. The genetic basis of variation among human erythrocyte PL kinase activity was reported by Flanagan and Beutler in 2006 [77]. Erythrocyte PL kinase activity of African Americans was found to be lower than persons with European or Asian ancestry and was attributed to 8bp pdxK promoter insertion [77]. ...
... The genetic basis of variation among human erythrocyte PL kinase activity was reported by Flanagan and Beutler in 2006 [77]. Erythrocyte PL kinase activity of African Americans was found to be lower than persons with European or Asian ancestry and was attributed to 8bp pdxK promoter insertion [77]. Flanagan and Beutler also suggested that tissue specific variations in the activity of human PL kinase enzyme could be attributed to polymorphisms in the PdxK gene promoter region [77]. ...
... Erythrocyte PL kinase activity of African Americans was found to be lower than persons with European or Asian ancestry and was attributed to 8bp pdxK promoter insertion [77]. Flanagan and Beutler also suggested that tissue specific variations in the activity of human PL kinase enzyme could be attributed to polymorphisms in the PdxK gene promoter region [77]. ...
Article
Pyridoxal 5′-phosphate (PLP), the active cofactor form of vitamin B6 is required by over 160 PLP-dependent (vitamin B6) enzymes serving diverse biological roles, such as carbohydrates, amino acids, hemes, and neurotransmitters metabolism. Three key enzymes, pyridoxal kinase (PL kinase), pyridoxine 5′-phosphate oxidase (PNPO), and phosphatases metabolize and supply PLP to PLP-dependent enzymes through the salvage pathway. In born errors in the salvage enzymes are known to cause inadequate levels of PLP in the cell, particularly in neuronal cells. The resulting PLP deficiency is known to cause or implicated in several pathologies, most notably seizures. One such disorder, PNPO-dependent neonatal epileptic encephalopathy (NEE) results from natural mutations in PNPO and leads to null or reduced enzymatic activity. NEE does not respond to conventional antiepileptic drugs but may respond to treatment with the B6 vitamers PLP and/or pyridoxine (PN). In born errors that lead to PLP deficiency in cells have also been reported in PL kinase, however, to date none has been associated with epilepsy or seizure. One such pathology is polyneuropathy that responds to PLP therapy. Phosphatase deficiency or hypophosphatasia disorder due to pathogenic mutations in alkaline phosphatase is known to cause seizures that respond to PN therapy. In this article, we review the biochemical features of in born errors pertaining to the salvage enzyme's deficiency that leads to NEE and other pathologies. We also present perspective on vitamin B6 treatment for these disorders, along with attempts to develop zebrafish model to study the NEE syndrome in vivo.
... 39 In PK these differences have been ascribed to an erythroid specific promoter. 40 PK is also known to be under the control of temporally regulated PAR bZip transcription factors. Knockout of these transcription factors in mice leads to seizures and low brain concentrations of dopamine, serotonin and PLP. ...
... The absence of this variant has been suggested to be the cause of reduced PK activity in the erythrocytes of Black Americans. 40 It is possible that the reduced dried blood spot PK activity identified in the two p.A228T homozygous individuals and/or the lowest values amongst the control samples was caused by the absence of this insertion. The PDXK promoter region was therefore sequenced for each of the samples. ...
... ]pyridoxine38,40,221 as substrates has reported activities of 30 -120 nmol PLP/gHb/h. If each DBS is assumed to contain 3.2 µL of whole blood at 14 gHb/dL (individual patient haematocrits were unavailable), results in pmol/DBS/h can be converted to nmol/gHb/h using a conversion factor of x2.2; resulting in activities of 5.7 ...
Thesis
The active form of vitamin B6, pyridoxal 5’-phosphate (PLP), is a cofactor required for many essential functions such as the metabolism of amino acids and neurotransmitters, the one-carbon cycle, haem biosynthesis, glycogenolysis, and sphingolipid metabolism. Humans are not capable of de novo PLP synthesis but do have a pathway for the interconversion of B6 vitamers. Several inborn errors of metabolism (IEMs) can lead to an insufficient supply of available PLP (e.g. pyridox(am)ine 5’-phosphate oxidase [PNPO], aldehyde dehydrogenase 7 family member A1 [ALDH7A1] and pyridoxal 5’-phosphate homeostasis protein [PLPHP] deficiencies). These disorders are typically characterised by neonatal/infantile-onset seizures refractive to standard anti-epileptic drugs but responsive to vitamin B6 supplementation. This thesis describes the investigation of B6 vitamer measurement from dried blood spots (DBS) as a diagnostic method for the B6-responsive epilepsies with a focus on PNPO deficiency. In addition, a diagnostic LC-MS/MS-based enzyme assay was developed for the measurement of PNPO activity from DBS. The biochemical effect of a novel IEM leading to pyridoxal kinase deficiency was also characterised using LC-MS/MS-based enzyme assays. Some PNPO deficient individuals receiving high-dose PLP for seizure treatment develop signs of liver damage leading eventually to cirrhosis. The photodegradation profile of PLP was characterised in order to help elucidate the mechanism causing liver damage in these patients; hypotheses as to the cause of this phenomenon are discussed. Vitamin B6 has been reported as an effective anticonvulsant in genetic epilepsies other than those known to directly affect vitamin B6 metabolism. Whole exome and whole genome sequencing data was used in order to investigate a genetic basis for vitamin B6-responsive seizures in 5 children. In two of these individuals, variants affecting the ion channel KCNQ2 were identified. A response to B6 supplementation in cases of KCNQ2-related epilepsy has also been documented in the literature. The mechanism behind this was investigated using electrophysiological techniques.
... 21 Sequencing this region showed that the reduced activity of all affected individuals was not attributable to this insertion (Supplementary Table 4). 42 Finally, we measured plasma PLP concentrations in all affected individuals. Plasma PLP was greatly reduced in cases carrying p.Arg220Gln and p.Ala228Thr compared to age-matched controls (see Fig 3G, H). ...
Article
Full-text available
Objective To identify disease‐causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. Methods We performed genome‐wide sequencing, homozygosity mapping, and segregation analysis for novel disease‐causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient‐derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. Results We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair‐bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP‐binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5′‐phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. Interpretation We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels.
... 21 Sequencing this region showed that the reduced activity of all affected individuals was not attributable to this insertion (Supplementary Table 4). 42 Finally, we measured plasma PLP concentrations in all affected individuals. Plasma PLP was greatly reduced in cases carrying p.Arg220Gln and p.Ala228Thr compared to age-matched controls (see Fig 3G, H). ...
Article
Full-text available
Objective: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. Methods: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. Results: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATPbinding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 50 phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. Interpretation: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels.
... 21 Sequencing this region showed that the reduced activity of all affected individuals was not attributable to this insertion (Supplementary Table 4). 42 Finally, we measured plasma PLP concentrations in all affected individuals. Plasma PLP was greatly reduced in cases carrying p.Arg220Gln and p.Ala228Thr compared to age-matched controls (see Fig 3G, H). ...
Preprint
Objective: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. Methods: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. Results: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATPbinding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 50- phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. Interpretation: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels.
... 21 Sequencing this region showed that the reduced activity of all affected individuals was not attributable to this insertion (Supplementary Table 4). 42 Finally, we measured plasma PLP concentrations in all affected individuals. Plasma PLP was greatly reduced in cases carrying p.Arg220Gln and p.Ala228Thr compared to age-matched controls (see Fig 3G, H). ...
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Objective To identify disease‐causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. Methods We performed genome‐wide sequencing, homozygosity mapping and segregation analysis for novel disease‐causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on ATP‐binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient‐derived fibroblasts, plasma and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology and biochemical quantification. Results We identified bi‐allelic mutations in PDXK in five individuals from two unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair‐bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP‐binding pocket. Low PDXK ATP‐binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'‐phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in one family, improvement in power, pain and fatigue contributing to patients regaining their ability to ambulate during the first year of PLP normalization. Interpretation We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown aetiology characterised by reduced PLP levels. This article is protected by copyright. All rights reserved.
... Americans of Afro-Caribbean origin have a high incidence of a polymorphism in the erythroid-specific promoter of pyridoxal kinase. 10 This leads to low activity of pyridoxal kinase and this is thought to confer resistance to malaria. Sickle hemoglobin binds PLP more avidly than normal b-hemoglobin. ...
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Many micronutrients or cofactors derived from micronutrients are highly reactive, hence their role in catalysis of reactions by enzymes. The concentration of cofactors has to be kept low to avoid unwanted reactions while allowing them to bind to the (apo)enzymes that need them. A new disorder causing B6-responsive epilepsy (proline synthetase cotranscribed bacterial homologue deficiency) is probably due to the absence of an important intracellular pyridoxal phosphate chaperone. The availability of some micronutrients varies by orders of magnitude in different geographical areas. Selenium is both essential and toxic, and during evolution, different populations have had to adapt to this differing availability. An “inborn error of metabolism (IEM)” in a low selenium area of China may be a selective advantage in a high selenium area and vice versa; the concept of nutrigenomics is an important one for micronutrients. The gut flora may make an important contribution to vitamin synthesis. This is difficult to study, but experiments can be undertaken with the nematode, Caenorhabditis elegans (with or without an IEM) and a single clone of Escherichia coli (with or without an IEM) as food and gut flora. This model shows that the gut microbiome can have profound influences on the folate cycle and associated vitamins. Our innate immune system makes use of the micronutrient requirements of pathogens and can deprive a pathogen of essential micronutrient(s) or expose it to toxic levels. It is not surprising, therefore, that some mutations affecting the way the host handles micronutrients can confer an advantage in resistance to infection and this may have acted as a selective advantage during evolution. This will be discussed by reference to the relationship of inborn errors to resistance to malaria. Conversely, other inborn errors of micronutrient metabolism are likely to make it more difficult for the host to use nutritional immunity to fight infection; this probably accounts for the list of infections that are more serious in patients with hereditary haemochromatosis.
... A second enzyme important in maintaining high levels of PLP in the red cell is pyridoxal kinase. Mean red blood cell pyridoxal kinase activity in black Americans is 40% that of white Americans (Chern and Beutler, 1976) and it has been suggested that this lower activity was favoured by natural selection because it conferred resistance to malaria (Martin et al., 1978;Flanagan and Beutler, 2006). Plasmodium produces enzymes for de novo synthesis of PLP and for phosphorylation of pyridoxal, but it is possible that erythrocyte schizogony requires some PLP to be provided by the host. ...
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PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5’-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5’-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5’-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin mononucleotide or pyridoxal 5’-phosphate and many of them showed residual enzyme activity. One sequence change (R116Q), predicted to affect flavin mononucleotide binding and binding of the two PNPO dimers, and with high residual activity was found in Groups (ii) and (iii). This sequence change has been reported in the 1000 Genomes project suggesting it could be a polymorphism but alternatively it could be a common mutation, perhaps responsible for the susceptibility locus for genetic generalized epilepsy on 17q21.32 (close to rs72823592). We believe the reduction in PNPO activity and B6-responsive epilepsy in the patients reported here indicates that it contributes to the pathogenesis of epilepsy.
Thesis
Die Phosphatase PDXP (auch bekannt als Chronophin) gehört zur Familie der HAD Phosphatasen, einer ubiquitär exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivität gegenüber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente Mäuse (Knockout-Mäuse) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen. Hierzu wurden HPLC-basierte Analysen der erythrozytären PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten Mäusen durchgeführt. Dabei ließen sich die rund doppelt so hohen erythrozytären PLP-Level in den KO-Mäusen bestätigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivität von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozytäre PDXP-Aktivität nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozytärem PLP während Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozytären PLP-Konzentrationen aus gefasteten mit normal gefütterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. Während Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter“ von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp (über die Zwischenprodukte PNP und PLP) eine PDXP-abhängige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach höhere PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell für die erythrozytäre Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilität, die ihn bis zu einem gewissen Grad unabhängig von der Nahrungsauswahl macht. Zudem können Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht möglich ist, mit PL versorgt werden. Aus der hohen Reaktivität von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik für die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozytären PLPs gebunden an Proteine (vor allem Hämoglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien“ und der „gebundenen“ Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich höhere Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-Mäuse ~1/3 höhere freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte Änderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen ältere Wildtypen im Hippocampus eine fünffach erhöhte PDXP-Expression verglichen mit jüngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten ältere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegenüber jüngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozytären PNPO-Expression würde somit für den Organismus einen Verlust seiner metabolischen Flexibilität bedeuten, die mit der Konversion von PN zu PL einhergeht.
Article
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Previous reports indicated that in growing rats the vitamin B-6 pool in muscle was relatively stable during deficiency but increased in response to increased vitamin B-6 intake. To determine whether human muscle would show a similar response 10 college-aged males received a low vitamin B-6 diet (1.76 mumol/d) for 6 wk followed by 6 wk on a self-selected diet supplemented with 0.98 mmol pyridoxine HCl/d. During depletion, excretion of pyridoxic acid rapidly adjusted to approximate the intake. Plasma pyridoxal phosphate concentrations at the end of the baseline, depletion, and supplementation periods were 81 +/- 51, 9 +/- 3, and 455 +/- 129 nmol/L, respectively, whereas muscle concentrations were 21 +/- 9, 20 +/- 4, and 25 +/- 7 nmol/g, respectively and total vitamin B-6 in muscle was 28 +/- 10, 27 +/- 4, and 35 +/- 10 nmol/g, respectively. These data provide further confirmation that the vitamin B-6 pools in skeletal muscle are resistant to depletion. They also demonstrate that in humans with constant body weight, vitamin B-6 supplementation is not associated with marked increases in vitamin B-6 in muscle.
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Asthmatics treated with theophylline, a potent inhibitor of pyridoxal kinase and therefore a vitamin B6 antagonist, demonstrated a significant correlation (r = 0.71; p < 0.001) between drug plasma levels and erythrocyte pyridoxal kinase activities. A cross-over, placebo controlled study was completed on 15 healthy volunteers to investigate the mechanism by which theophylline induces pyridoxal kinase activity. The subjects were supplemented with vitamin B6 or placebo for two weeks before theophylline therapy was started. Vitamin B6 supplementation resulted in a four-fold increase in circulating pyridoxal 5'-phosphate levels, while placebo had no effect. When theophylline therapy was commenced, erythrocyte pyridoxal kinase activities increased significantly (p < 0.001) irrespective of whether vitamin B6 or placebo was supplemented. It is concluded that a depressed vitamin B6 status is not responsible for higher erythrocyte pyridoxal kinase activities encountered during theophylline therapy, but that the drug is directly responsible for elevated enzyme levels.
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The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.
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The red-cell glucose-6-phosphate dehydrogenase (G.-6-P.D.) activity and red-cell pyridoxal-kinase (P.L.K.) activity of 27 Nigerian children with severe Plasmodium falciparum parasitaemia were compared with those of 26 healthy Nigerian children and 6 White adults. The mean P.L.K. activity of the malaria patients was similar to that of the Whites but significantly higher than that of the Nigerian controls. Correction for reduced mean red-cell age in patients was made by comparing the P.L.K.: G.-6-P.D. ratio for those subjects with stable G.-6-P.D. phenotypes. The mean P.L.K.:G.-6-P.D. ratio was the same for malaria patients and adult White but significantly higher than that for the Nigerian controls. These results suggest that the relatively high frequency of low red-cell P.L.K. activity among Blacks may have been selected for by falciparum malaria.
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