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Original Studies |
Medical Research Council Molecular Endocrinology Group, Medical Research Council Clinical Sciences Centre (P.H.D., P.T.C., C.W., D.T., R.V.T.), Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom; Renal Division (M.G.), Department of Molecular Microbiology and Center for Genetics in Medicine (D.S.), and Metabolic Research Unit, Shriners Hospital for Children and Division of Bone and Mineral Diseases (M.P.W.) ,Washington University School of Medicine (M.G.), St. Louis, Missouri 63110; Division of Genetics, Children’s Hospital (I.H.), Boston, Massachusetts 02115; Serono Laboratories, Inc. (J.M.G.), Norwell, Massachusetts 02061; Medizinische Hochschule Hannover, Abteilung Humangenetik, Zentrum Kinderheilkunde und Humangenetik (J.S.), Hannover D-30625, Germany; Childrens Medical Center of Brooklyn, Kings County Hospital Centre, University Hospital of Brooklyn (B.S.), Brooklyn, New York 11203-2098; Department of Growth and Endocrinology, The Birmingham Children’s Hospital National Health Service Trust (N.S.), Ladywood, Birmingham B16 8ET, United Kingdom; Alder Hey Children’s Hospital (C.S.), Liverpool, L12 2AR, United Kingdom; and Hospital de Pediatria Garrahan, Laboratoria de Metabolismo Calccio y Oseo, Endocrinologia (C.T.), Buenos Aeres, Argentina.
Address all correspondence and requests for reprints to: R. V. Thakker, Medical Research Council Molecular Endocrinology Group, Medical Research Council Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: rthakker{at}rpms.ac.uk
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Abstract |
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g) of the 5'untranslated region together with the other mutations
indicates that the dominant XLH phenotype is unlikely to be explained
by haplo-insufficiency or a dominant negative effect. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The XLH gene had been localized by family linkage studies to Xp22.1
(9, 10, 11) within a 500,000-bp region flanked centromerically by
DXS365 and telomerically by DXS1683 (12). The establishment of a YAC
contig of this region (13, 14) facilitated the isolation of candidate
genes and the identification of the PHEX, formerly referred to as PEX
(phosphate-regulating gene with homologies to endopeptidases on the
X-chromosome) gene, which was found to harbor mutations in XLH (15).
The human PHEX gene (Fig. 1
) consists of
22 exons that encode a 749-amino acid protein. PHEX gene expression, as
a 6.6-kilobase (kb) transcript, has been reported by Northern blot
analysis in adult ovary and fetal lung, and to a lesser extent in adult
lung and fetal liver (15, 16, 17), indicating that only 35% of the PHEX
messenger RNA (mRNA) contains the 2247-bp coding sequence with the
remaining 65% representing untranslated regions (UTRs). PHEX has
significant homology to the neutral endopeptidase gene family (18, 19, 20),
which includes neutral endopeptidase, Kell antigen, and
endothelin-converting enzyme 1. Members of this family have a small
amino-terminal intracellular tail, a single transmembrane domain, and a
large carboxy-terminal extracellular domain that contains 10 conserved
cysteine residues and a HEXXH pentapeptide motif that characterizes
many zinc peptidases (21). Neutral endopeptidase and
endothelin-converting enzyme cleave peptide bonds and alter the
activity of angiotensin and vasopressin and big endothelin,
respectively (20, 22), and it is postulated that other family members
may have similar functions. Disorders associated with mutations of
neutral endopeptidase, Kell antigen, or endothelin-converting enzyme
have not yet been identified, but mutations of PHEX, which are likely
to result in a functional loss, have been demonstrated to be associated
with XLH. A characterization of such mutations will help to elucidate
further the important functional domains of PHEX and thereby the role
of this putative endopeptidase in phosphate homeostasis. Therefore,
we performed mutational analysis of the PHEX gene in patients with
familial and nonfamilial (sporadic) forms of hypophosphatemic
rickets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The families of 68 unrelated XLH probands were ascertained and members assessed. The diagnosis of XLH from among the various types of rickets was based on a consistent medical history and physical examination, radiological evidence of rachitic disease, unremarkable serum calcium and electrolyte concentrations, hypophosphatemia caused by selective renal phosphate wasting for which no other etiology was found, and a family history consistent with multigenerational or sporadic (i.e. nonfamilial) occurrence of XLH. Two of the probands suffered from other putative X-linked disorders in addition to XLH; one affected female from the Indian subcontinent suffered from XLH and congenital adrenal hypoplasia (23), and one affected male from Argentina suffered from XLH and Duchenne Muscular Dystrophy. Patients with suspected tumoral rickets had been identified and were excluded from the study. A family history of XLH could be established in 46 of the probands, and there were 172 affected members (62 males and 110 females) and 140 unaffected members (90 males and 50 females). A familial basis for XLH could not be established in 22 of the XLH probands (8 males and 14 females). Venous blood samples were obtained, after informed consent, from 159 affected (62 males and 97 females) and 97 unaffected (52 males and 45 females) members of the families of the 68 XLH probands. Of the 68 probands and their families, 62 were of northern European origin, 3 were of African-American origin, 1 was of Saudi Arabian origin, 1 was of Indian subcontinent origin, and 1 was of southeast Asian origin. These studies had received approval from the Ethical Committee of The Hammersmith Hospital, London and from the Human Studies Committee of the Washington University School of Medicine, St. Louis, MO.
DNA sequence analysis of PHEX gene
DNA from leukocytes was prepared by standard methods, and RNA was extracted from Epstein-Barr virus transformed lymphoblastoid cell lines obtained from the peripheral blood cells of affected individuals from each family, using methods previously described (24, 25, 26). DNA sequence abnormalities were initially sought in each of 36 probands (30 familial and 6 sporadic) by RT-PCR amplification using 12 pairs of nested PHEX-specific primers (our unpublished observations; details available on request, from R.V.T.) and lymphoblastoid RNA, as described (26). The PCR products were then gel purified, and the DNA sequences of both strands were determined by Taq polymerase cycle sequencing and a semiautomated detection system (ABI 373A sequencer, PE Applied Biosystems, Foster City, CA) (27, 28). DNA sequence abnormalities were confirmed either by restriction endonuclease analysis of genomic PCR products obtained by the use of appropriate primers, or by sequence-specific oligonucleotide (SSO) hybridization analysis or by agarose gel electrophoresis (27, 28). In addition, the DNA sequence abnormalities were confirmed and demonstrated to cosegregate with the disorder and to be absent as common polymorphisms in the DNA obtained from 72 unrelated normal individuals (34 males, 38 females). Microsatellite polymorphism analysis at D11S533, D1S422, D13S260, D3S1303, and a variable number tandem repeat (VNTR) at the PTH-related peptide (PTHrP) locus were used to exclude nonpaternity as described previously (28). Southern blot hybridization analysis (29) was used to investigate the genomic deletions (data not shown).
The sensitivity and specificity of single-stranded conformational polymorphism (SSCP) analysis for the detection of mutations was initially investigated by assessing the detection rate of the identified abnormalities in the 36 XLH probands. Genomic DNA from XLH probands and 6 unrelated normal individuals was used with the appropriate primers (our unpublished observations; details available on request, from R.V.T.) for PCR amplification, and the PCR products analyzed by SSCP using the Phast electrophoresis system (Pharmacia Biotech, Uppsala, Sweden) and silver staining, as previously described (28). In addition, another 32 XLH probands (16 familial and 16 sporadic) were investigated solely by SSCP analysis for PHEX mutations. The DNA sequence of abnormal SSCPs was determined and confirmed by restriction endonuclease and SSO analysis as described above.
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Results |
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An analysis of the 2247-bp coding sequence of PHEX and of the
508-bp 5'UTR in the 68 XLH probands revealed 31 mutations (Table 1). Thus, 7 nonsense, 6 deletions, 2
deletional insertions, 2 insertions, 1 duplication, 4 splice site, 1
5'UTR, and 8 missense mutations were detected (Fig. 1
). The nonsense,
insertional, duplication, and majority of the deletional mutations were
associated with premature termination codons, and the splice site
mutations were either associated with exon skipping or with use of
cryptic splice sites, which resulted in frameshifts that included
premature termination codons (Table 1). Approximately 70% of these
mutations are likely to result in a truncated PHEX protein and thus be
inactivating. Twenty four (77%) of the PHEX mutations were from the 46
probands with familial XLH, and 7 (23%) of the PHEX mutations were
from the 22 probands with sporadic XLH. Thus, PHEX mutations were
likely to be observed more often in probands with established X-linked
dominant inheritance rather than the nonfamilial forms. However, PHEX
mutations were not observed in 22 of the probands, and in 5 of these
families cosegregation of XLH and PHEX had been established by studies
using X-linked flanking markers (10, 30). Each of the mutations in the
31 XLH probands was confirmed, and in the 24 familial XLH patients was
demonstrated to cosegregate with the disease, either by restriction
enzyme analysis (Figs. 2 and 3), or SSO hybridization analysis (Fig. 4) or gel electrophoresis (Table 1). In
addition, the absence of the DNA sequence abnormalities in 110 alleles
from 72 unrelated normal individuals (34 males and 38 females)
established that these abnormalities were mutations and unlikely to be
polymorphisms, which would be expected to occur at a frequency of more
than 1% in the general population. The 31 PHEX mutations, two
of which occurred more than once, were observed in different ethnic
groups. Thus 26 of the PHEX mutations were observed in families of
northern European origin, 2 in African-American families, 1 in a
Saudi Arabian family, 1 in a southeast Asian family, and 1 in a family
from the Indian subcontinent (Table 1). A more detailed examination of
the mutations revealed several interesting findings.
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), and no mutations were
observed in the putative transmembrane and intracellular domains. In
addition, point mutations altering the 10 conserved cysteine residues
and the zinc-binding motif were not observed. Second, 2 different
mutations (R747X and P534L) were found to occur more than once and in
unrelated families. The R747X mutation was observed in a Saudi Arabian
family (31) and in a nonfamilial XLH, northern European patient from
the United States. The P534L mutation was observed in a northern
European XLH family and in a nonfamilial XLH patient of
African-American origin (Table 1). Third, 2 of the 7 PHEX mutations
observed in the sporadic XLH patients were confirmed to have arisen
de novo (Fig. 3 and Table 1). Haplotype analysis using
microsatellite polymorphisms (data not shown) had excluded nonpaternity
and nonmaternity in these families. Fourth, a mutation in the 5'UTR,
located 429 bp upstream from the initiation (ATG) site, was detected in
one family. This mutation involved an ag transversion and was found
to cosegregate with the disease and to be absent in 247 alleles (110
from the 72 unrelated normal individuals, 33 from 20 unrelated
individuals from the Indian subcontinent, and 104 alleles from the
other XLH probands). In addition, this nucleotide (a) is evolutionarily
conserved in man, rat, and mouse (17). These findings indicate that
this 5'UTR ag transversion is not likely to be a benign
polymorphism, but is likely to be a significant mutation that may alter
translation efficiency (32) (Table 1). Polymorphisms in PHEX gene
Six polymorphisms that were all detected by SSCP analysis and
confirmed by DNA sequence analysis, together with restriction
endonuclease analysis and SSO hybridization analysis, were observed
(Table 1 and Fig. 1) in the PHEX gene. Two of these polymorphisms
occurred in introns 17 and 18, two occurred in the 5'UTR, and the other
two, which occurred in exon 5, involved the third base of a codon that
did not lead to an alteration of the encoded amino acid. The
heterozygosity frequencies of these polymorphisms ranged from less than
1% to 43%. The polymorphisms in intron 17, which involved a poly T
tract, and the polymorphism in intron 18 have been previously observed
(33, 34). The importance of these polymorphisms lies in their
recognition and distinction from the SSCP and DNA sequence
abnormalities that represent PHEX mutations. In addition, the g/a
polymorphism in the 5'UTR, which has a heterozygosity frequency of
43%, may be of potential help in segregation studies in the 55% of
XLH families in whom a PHEX mutation cannot be identified, but who may
require presymptomatic diagnosis for some family members.
Mutation detection by SSCP analysis
Eleven (>60%) of the 18 PHEX mutations in the XLH
probands detected by DNA sequence analysis of RT-PCR products that
encompassed the 2247 bp of the coding sequence were correctly
identified by SSCP analysis. In addition, SSCP analysis helped to
identify 11 further mutations (Table 1), and the detection of 5 of
these is demonstrated in Fig. 5. SSCP
analysis has been reported to be more reliable for the detection of
mutations in PCR products that are smaller than 250 bp, and 15 of the
24 total pairs of primers used amplified products less than 250 bp.
However, the primers used for exon 22, which harbored 6 of the PHEX
mutations (Table 1), yielded fragments of 343 bp, and this may
particularly account for the lower (<66%) detection rate for
mutations in this exon. SSCP proved to be reliable in the detection of
more than 60% of all the PHEX mutations, and a redesigning of primers
to amplify smaller fragments may help to improve this.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1) in 68 unrelated XLH patients and their
families are comparable with the combined results from three recent
studies (33, 34, 35) that consisted of nonsense [23% vs. 25%;
this report vs. combined studies (33, 34, 35)], deletions (19%
vs. 33%), duplications (3% vs. 2%), insertions
(6% vs. 4%), deletional insertions (6% vs.
0%), splice site (13% vs. 14%), and missense (26%
vs. 22%) mutations. The mutations are scattered throughout
the PHEX gene, and the majority (>70%) of the 31 mutations are
nonsense or frameshift deletions, duplications, insertions, or splice
site abnormalities that are likely, if translated, to result in a
functional loss of the PHEX protein. Interestingly, of all the
PHEX abnormalities reported to date, including this study, only one has
involved the putative intracellular domain (34), and none has involved
the putative transmembrane region (33, 34, 35). In addition, the
independent and multiple occurrences of the P534L and R747X mutations,
which have also been observed four and three times, respectively, in
other studies (33, 34), indicate that these may represent PHEX codons
or regions that are particularly prone to mutations. Our findings of
two de novo mutations (Fig. 3) among the 7 sporadic XLH
patients confirms the occurrence of such mutations together with their
inheritance in subsequent generations. Five of the 7 mutations
identified in the sporadic XLH patients occurred in exons 21 and 22 and
2 of them represented the only deletional insertional mutations to be
observed. These results indicate that 22% of PHEX mutations may have
arisen de novo, and this is in agreement with the previously
reported 18% estimate for de novo mutations derived from
clinical observations (8).
Our study is the first to report a PHEX mutation of the 5'UTR, which
was observed in a female patient from the Indian subcontinent with XLH
(Table 1) and congenital adrenal hypoplasia (23). Such a mutation of
the 5'UTR may lead to an alteration in the binding sites for ribosomal
and other translation factors, such as tissue-specific regulatory
proteins (32). Thus, PHEX expression may be reduced and
haplo-insufficiency may represent a possible mechanism in the etiology
of XLH in this female patient. However, the R747X and W749R mutations,
which, if translated, result in almost intact PHEX proteins, suggest
that haplo-insufficiency is unlikely to be an explanation for the
dominant nature of PHEX mutations in XLH females. An alternative
possibility is that these mutations may have a dominant negative
effect, caused by dimerization. However, this possibility is equally
unlikely, because X-chromosome inactivation in each female cell is
likely to lead to the expression of only a wild-type or a mutant form
of PHEX, and not both, thereby precluding any interaction between
wild-type and mutant PHEX proteins. The role of such PHEX mutations in
the dominant XLH phenotype in females remains to be elucidated.
The mutational diversity within the 2247 bp of the PHEX coding region, splice site regions and 5'UTR sequences makes mutational screening by a direct DNA sequencing approach in patients considered to suffer from XLH time consuming and impractical. We have therefore explored the use of the SSCP technique for the more rapid screening of PHEX mutations. Our results demonstrate that SSCP was successful in the detection of more than 60% of the PHEX mutations, and that redesigning of some primers to amplify DNA fragments less than 250 bp in exons 1, 5, 9, 11, 12, 18, 22, and the 5'UTR may help to increase this detection rate. However, our DNA sequence analysis of the RT-PCR products from the coding regions and the 5'UTR did not detect PHEX mutations in 16 of the 36 probands. With regard to this, it is important to note that only 2.25 kb of the approximately 6.6-kb PHEX mRNA transcript has been investigated, and that a more likely explanation for the lack of mutations in these XLH patients is that they may harbor mutations within the remaining 4.4-kb mRNA that contains the 3'UTR and probable additional 5'UTR. In addition, this failure to detect PHEX mutations in XLH patients could also partly stem from genetic heterogeneity with the possible involvement of other X-linked genes, for example, the voltage-gated chloride channel, CLCN5, that is located on Xp11.2 and mutated in some patients with X-linked recessive hypophosphatemic rickets (26), or alternatively, some of the sporadic cases may represent autosomal forms of hypophosphatemia (4, 5, 6).
Expression of the PHEX gene has been detected by Northern blot analysis
in mouse osteoblasts (16, 36) and in human lung and ovary (17). Its
expression in other tissues appears to be low and detected only by
RT-PCR (15, 36, 37). The manner in which a functional loss of the
putative PHEX enzyme in these tissues leads to the anatomically remote
renal tubular defects of phosphate transport together with the other
defects seen in XLH remains to be elucidated. PHEX function may be
analogous to neutral endopeptidase, which cleaves peptide bonds to
inactivate a wide range of hormones (18, 22, 38), or to that of
endothelin-converting enzyme, which activates its substrate, big
endothelin (20). It has been postulated that the substrate for PHEX may
be phosphatonin, which is the putative tumour-derived phosphaturic
factor from mixed mesenchymal tumours (39, 40, 41, 42). Thus, PHEX may
inactivate phosphatonin by a possible paracrine action (43), and a loss
of PHEX function caused by mutation may lead to increased phosphatonin
activity appearing in the circulation that may alter the activity of
the sodium-phosphate cotransporter (NPT2) (44) and hence lead to
phosphaturia and hypophosphatemia in XLH (45, 46). The identification
of a substrate for PHEX together with the functional expression of PHEX
mutants (Table 1 and Fig. 1) will help to elucidate further the role of
PHEX in phosphate homeostasis.
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Footnotes |
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Received April 16, 1998.
Revised June 29, 1998.
Accepted July 6, 1998.
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),
affected males (
), unaffected females (
), and affected females
(•). DNA size standard (s) markers, which were a 1-kb ladder, are
indicated.
