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Department of Medicine, Rajavithi Hospital (T.S., S.N.), and the Department of Pediatrics, Queen Sirikit National Institute of Child Health (S.C.), Bangkok 10400, Thailand
Address all correspondence and requests for reprints to: Thongkum Sunthornthepvarakul, M.D., Rajavithi Hospital, Bangkok 10400, Thailand. E-mail: thongkum{at}rajavithi.go.th
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Abstract |
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Introduction |
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The human PTH gene contains 3 exons that located on the short arm of chromosome 11 (1). Exon 1 contains the untranslated region. Exon 2 encodes the signal peptide and part of the prohormone sequence. Exon 3 encodes the remainder of the prohormone sequence, the 84-amino acid PTH peptide, and the 3'-untranslated region. PTH is formed as a larger prepro-PTH. This precursor undergoes 2 successive proteolytic cleavages to yield PTH. The signal peptide is cleaved first during cotranslational translocation, releasing the pro-PTH into the lumen of the rough endoplasmic reticulum (RER). Pro-PTH is processed later in the Golgi apparatus to produce the mature PTH (2).
Mutations in the PTH gene have been reported in only 2 families with FIH. The first family had mutation in the hydrophobic core of the signal peptide, producing the autosomal dominant form of FIH (3). The second family had a mutation in the exon 2-intron 2 junction that skipped the next exon and produced the autosomal recessive form of FIH (4). In this paper we described a new autosomal recessive FIH associated with a point mutation at the -3 position (counting from the cleavage site between positions -1 and +1) in the signal peptide of the prepro-PTH gene that leads to an amino acid substitution, Ser to Pro. We have established that the prepro-PTH gene allele bearing the observed mutation is linked to the FIH phenotype.
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Subjects and Methods |
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The proband (subject IV-10) was born in 1975 and presented at 7
days of age with seizures (Fig. 1A
). On
the presumption of epilepsy, she was treated with anticonvulsive agents
for several months, but seizures continued. Subsequent investigation at
the Queen Sirikit National Institute of Child Health revealed
hypocalcemia at 1.5 mmol/L (normal range, 2.0–2.5), with
hyperphosphatemia at 3.6 mmol/L (normal range, 0.9–1.5), and the
diagnosis of isolated hypoparathyroidism was made. Serum calcium levels
were maintained with vitamin D and calcium therapy. She died by
drowning at 10 yr of age. Subsequently, it was learned that the parents
(subjects III-3 and III-4) were consanguineous.
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In 1982, her younger sister (subject IV-13) was born and
presented with neonatal hypocalcemic seizures. Serum calcium was 1.1
mmol/L; phosphate was 3.3 mmol/L. She was also treated with large doses
of vitamin D and calcium. At age 16 yr, serum calcium was 1.2 mmol/L,
phosphate was 2.8 mmol/L, alkaline phosphatase was 105 U/L, and serum
iPTH was undetectable. Her height was 148 cm (10–25th percentile).
Physical examination was normal, except for positive Chvostek’s sign.
Three of 12 siblings of the consanguineous couple, III-3 and III-4, had
FIH (Fig. 1A
). Six of them (subjects IV-4, IV-5, IV-6, IV-8, IV-12, and
IV-14) were examined and found to be unaffected, as was their mother
(subject III-4). Their serum calcium and phosphate levels were normal,
and the iPTH level of their mother (subject III-4) was normal (3.93
pmol/L). We were unable to examine or test 3 of the siblings (subjects
IV-3, IV-7, and IV-9) and their father (subject III-3), but they were
reported to be healthy and had no history of seizures.
In the next generation, the niece of the propositus (subject VI-6),
also born of consanguineous parents (subjects V-I and IV-6), presented
with neonatal hypocalcemic seizures. Serum calcium was 1.2 mmol/L,
phosphate was 2.8 mmol/L, alkaline phosphatase was 178 (normal range,
110–360), and serum iPTH was undetectable. In this generation (Fig. 1), her five siblings (subjects VI-1, VI-2, VI-3, VI-4, and VI-5) as
well as their parents (subjects V-1 and IV-6) were normocalcemia and
had no history of seizures. The serum iPTH level of her mother was
normal (3.88 pmol/L).
The inheritance of FIH in this family is autosomal recessive. No family member had evidence of mucocutaneous candidiasis, autoimmune endocrine disease, or somatic features consistent with a developmental or embryological disorder. This family was referred to Rajavithi Hospital for further investigations.
Preparation of genomic DNA and DNA sequencing
Genomic DNA of affected subject IV-11 was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). The genomic DNA was used as a template for PCR amplification of exon 1 of the prepro-PTH gene and a region extending from exon 2 through exon 3 covering the coding regions and splice junctions. Primer sequences for PCR amplification of exon 1 were 5'-ctctcttggtaagcagaaga-3' (sense) and 5'-ccttgaagaaacaacatggt-3' (antisense). Primer sequences for exons 2–3 were 5'-gcttctcgtgaaaaccaacc-3' (sense) and 5'-ccctacactgtctagagcag-3' (antisense). The conditions for amplification by PCR were 100 µL containing 0.2 µg genomic DNA, 100 pmol of each primer, 200 µmol/L of each deoxy-NTP, 2.5 mmol/L MgCl2, 5 mmol/L Tris-HCl (pH 8.0), 10 mmol/L NaCl, 10 µmol/L ethylenediamine tetraacetate, 0.5 mmol/L dithiothreitol, 5% glycerol, 0.1% Triton X-100, and 0.8 U Taq DNA polymerase (Promega Corp.). Initial denaturation was performed at 94 C for 5 min, followed by 35 cycles of 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min and a final extension at 72 C for 15 min. The amplified DNA fragment was sequenced using a 373 DNA Sequencer (PE Applied Biosystems, Perkin Elmer Corp., Foster City, CA).
Confirmation of the mutation
To confirm the presence of the mutant nucleotide in genomic DNA
and to identify family members who harbored the mutation, we amplified
exons 2 and 3 of the prepro-PTH gene of the subjects’ genomic DNA as
described above. As the mutation in position 23 of the signal peptide,
a replacement of thymine by cytosine, creates a new recognition site
for MspI (CCGG), this endonuclease was used to digest the
amplified 607-bp fragment. The presence of the mutant cytosine
generates two fragments of 430 and 177 bp detected by electrophoresis
on a 2% agarose gel (Fig. 1B). Partial or complete cleavage of the DNA
fragment indicated that the mutant nucleotide was present in one or
both alleles, respectively. The experiments were performed at least
twice, and the results were reproducible. Each experiment had affected
patient’s DNA that was homozygous for the mutation to ensure that all
samples were completely digested by restriction enzyme.
A total of 16 individuals were tested for genotyping (Fig. 1A). Subject
IV-10 was the only affected patient who was given a biochemical test,
but she died before genotyping. In generation IV, there were 12
siblings: 8 siblings received physical examination, biochemical test,
and genotyping; 1 sibling (subject IV-10) received physical examination
and biochemical test, except genotyping; and 3 siblings received
neither physical examination nor any test. In generation VI, all
members were tested for genotyping.
To predict the signal sequence cleavage site probability
We applied the method of von Heijne (5) to predict the locations of signal peptide cleavage sites and alternative cleavage sites by comparing wild-type prepro-PTH sequence with the sequence of the prepro-PTH mutant. On a weight-matrix approach, this method can identify the correct cleavage site about 75–80% of the time when applied to new sequences. This -3,-1 rule combined with the expected distribution of other amino acids within the cleavage domain (-13 to +2) have been used to construct a weight matrix to calculate the probability of cleavage at a specific site.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Affected family subjects had severe hypocalcemia and hyperphosphatemia,
whereas the unaffected members were healthy, and their serum calcium
and phosphate levels were normal. The serum iPTH levels of all
symptomatic patients were undetectable despite severe hypocalcemia. The
data explain why the severe hypocalcemia was very difficult to correct
with calcium and vitamin D supplementation.
DNA sequencing revealed normal DNA sequence of exon 1 of the prepro-PTH
gene. We found a mutation in exon 2 located at the first nucleotide of
position 23 in the 25-amino acid signal peptide (Fig. 2B). A thymine (TCG) was substituted by a
cytosine (CCG), resulting in the replacement of the normal Ser by Pro
(Fig. 2B). This mutation in the prepro-PTH gene was confirmed by
digestion with MspI, a new endonuclease recognition site
created by the mutation. The result showed that all affected members
were homozygous for the mutant allele, and their parents were
heterozygotes in agreement with the autosomal recessive mode of
inheritance. In generation IV, we found the mutation in only 6 of the 8
subjects tested: 2 were affected homozygotes, and 4 were heterozygotes
and clinically normal. Two normal subjects had no mutation in either
allele. In generation VI, there were 6 children: 1 affected homozygote
for the mutant allele, 2 heterozygotes for the mutation, and 3 with
both normal alleles. The latter as well as the heterozygotes had normal
serum calcium and phosphate levels.
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), which
suggested that signal peptidase is unlikely to cleave the mutant
prepro-PTH protein.
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Discussion |
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Signal peptides typically are made up of three domains, consisting of a positively charged NH2-terminal region, a central hydrophobic region, and a polar COOH-terminal region (9, 10). The NH2-terminal region may have something to do with the docking protein and is important for translocation. The central hydrophobic region is believed to be the target for the SRP (11). The COOH-terminal region influences the efficiency and fidelity of signal peptidase cleavage (9, 10).
Prepro-PTH has a 25-residue signal sequence, followed by a 6-residue
propeptide sequence and an 84-residue of mature hormone (Fig. 2A).
Structural features of signal peptide are critical for the
translocation of secretory proteins and their cleavage by signal
peptidase (12). The COOH-terminal region, which could introduce
flexibility into the molecule, may allow signal peptidases to adopt a
loop or hairpin structure near the signal cleavage site in the
membrane. The hairpin configuration has been proposed as the structure
appropriate for insertion of the precursor into the membrane (13, 14)
and for presenting an appropriate substrate to the signal peptidase
(15). Alteration near the cleavage site can disrupt signal peptidase
cleavage (15, 16). The human propeptide (Lys-Ser-Val-Lys-Lys-Arg) is
cleaved from pro-PTH just before secretion, presumably in the
trans-Golgi tubular network. Processing occurs after the
dibasic residues Lys-Arg. Pro-PTH is not secreted from cells, and
neither the pro-specific fragment nor any of the its possible
degradation products accumulate in the cell (2). Thus, any role for the
propeptide must be an intracellular one.
We described herein a point mutation at the first nucleotide of
position 23 in the signal peptide-encoding region of a prepro-PTH gene
in FIH. A thymine was substituted by a cytosine, resulting in
replacement of the normal Ser (TCG) by Pro (CCG). Consanguinity and
family size establish the autosomal recessive mode of inheritance. This
mutation is conceivably the cause of the hypoparathyroidism in affected
members of this family, because genotyping shows that inheritance of
PTH deficiency is tightly linked to the mutant allele. Furthermore, the
mutation is located in the crucial position for signal peptidase
cleavage site. The prepro-PTH 23 (Ser
Pro) mutation was found in both
alleles of the affected patients, and their parents were heterozygous
for the mutation.
The mutation corresponds to the -3 position of the prepro-PTH protein
cleavage site. According to the -3,-1 rule of the signal peptidase
recognition site (10, 12), the region around the cleavage site shows
strong preferences for specific amino acids in particular positions.
Acceptable cleavage domains conform to the following rules: the residue
at position -1 from the cleavage site must be small (Ala, Ser, Gly,
Cys, Thr, or Gln); the residue at position -3 must not be aromatic
(Phe, His, Tyr, and Trp), charged (Asp, Glu, Lys, and Arg), or large
polar residues (Asn and Gln); and there must be no Pro residue in the
region between -3 and +1 position (10, 12). The -3,-1 positions of
signal sequence are crucial for signal peptidase to cleave prepro-PTH
to pro-PTH protein in the RER. The change at position -3 of Ser for
Pro has never been encountered, and Pro is a strong helix-breaking
residue. We, therefore, hypothesize that the prepro-PTH 23 (SerPro)
mutation might exert its dramatic effect on signal function by
interfering with signal peptidase cleavage. If the signal sequence is
not cleaved, the prepro-PTH mutant may anchor in the microsomal
membrane, and eventually it might be degraded in the RER (13, 17, 18),
it may pass completely through the membrane (19), or it could have a
new signal peptidase cleavage site downstream and make new pro-PTH
protein. The process might impair the release of PTH molecules from the
parathyroid gland to circulation because of the absence of PTH in the
affected patients during hypocalcemia. Unfortunately, we were
unsuccessful in expressing the prepro-PTH gene, either wild type or
mutant, to support our hypothesis.
The mutation in signal peptide that closely relates to our mutation is
coagulation factor XSanto Domingo (FXsd) (21).
FXsd is a mutant form of human factor X in which a point mutation
results in the substitution of Arg for Gly at the critical -3 position
of the signal peptide (21). The patient bearing the mutation exhibits a
severe bleeding diathesis associated with less than 1% FX enzymatic
activity and less than 5% circulating FX protein. The mutation does
not interfere with targeting and translocation to the RER, but cleavage
by signal peptidase is dramatically impaired (22). It should be noted
that this mutation does not induce a shift in the signal peptidase
cleavage site, an effect that has been observed in other cases. Signal
peptidase appears to have some degree of flexibility in its selection
of cleavage site if a suitable alternative site is present. In the case
of prepro-FXsd, it appears that a suitable alternative cleavage site is
not available, so the result of the mutation is to block cleavage
completely. Similarly, in the case of the prepro-PTH-23 (SerPro)
mutation, we hypothesized that this mutation can block signal peptide
cleavage completely. To support this hypothesis, we used von Heijne’s
probabilistic method (5) to define the alternate cleavage site of the
mutant precursor peptide. The method allows comparison of the mutant
precursor to sequences of other characterized precursor proteins to
predict appropriate cleavage sites. The probability of alternative
cleavage site is extremely low in mutant peptide. It appears that a
suitable alternative cleavage site is not available, so the result of
the mutation is to block cleavage completely.
In the family reported herein, the inheritance is autosomal recessive, which contrasts with previous reports of mutations in the signal sequence of human secreted proteins that appear to have dominant inheritance (3, 20, 22). The affected patients containing the mutation in both alleles of prepro-PTH gene had no detectable PTH in the circulation that favors lack of PTH secretion from parathyroid glands. Their parents and heterozygous siblings, who had one mutant and one normal prepro-PTH allele, were clinically normal and normocalcemic and had normal levels of serum iPTH. The apparent ability of only one normal prepro-PTH allele to maintain PTH secretion is sufficient amount to prevent hypocalcemia and maintain calcium homeostasis, which is compatible with other case of FIH reported by Parkinson et al. (4). Their mutation involved a donor splice site mutation at the exon 2-intron 2 boundary that caused exon skipping, and the inheritance is autosomal recessive (4). In contrast, Arnold et al. (3) reported a dominantly inherited FIH associated with the substitution of Arg for Cys within the hydrophobic core of prepro-PTH. The mutation causes a disruption of the core that leads to impair interaction of the nascent protein with SRP, the translocation machinery, and signal peptidase cleavage (23). Hypoparathyroidism in the presence of one normal PTH allele would therefore suggest that the mutant gene product exerts a dominant negative effect in vivo. Although our mutation is in signal peptide of prepro-PTH gene, the mutation might interfere only with signal peptidase cleavage, and the mutant gene product might not interfere with normal PTH production from the normal prepro-PTH allele.
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Acknowledgments |
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Footnotes |
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Received March 17, 1999.
Revised May 19, 1999.
Accepted July 2, 1999.
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