0021-972X/97/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1997 by The Endocrine Society
Vol. 82, No. 1
Printed in U.S.A.
Olfactory Dysfunction in Type I
Pseudohypoparathyroidism: Dissociation from
Gsa Protein Deficiency*
RICHARD L. DOTY, ALBERTO D. FERNANDEZ, MICHAEL A. LEVINE,
ARNOLD MOSES, AND DONALD A. MCKEOWN
Smell and Taste Center and Department of Otorhinolaryngology: Head and Neck Surgery (R.L.D.,
A.D.F., D.A.M.), School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
Department of Medicine (M.A.L.), Division of Endocrinology and Metabolism, Johns Hopkins
University, Baltimore, Maryland 21205; Department of Medicine (A.M.), College of Medicine, State
University of New York Health Science Center, Syracuse, New York 13210
ABSTRACT
The discovery of variably decreased olfactory ability in Type Ia
pseudohypoparathyroidism (PHP), a syndrome in which generalized
hormone resistance is associated with deficiency of the alpha chain of
the stimulatory guanine nucleotide-binding protein (Gsa) of adenylyl
cyclase, has been used to support the hypothesis that Gsa plays a
major role in human olfactory transduction. However, only a limited
number of olfactory tests have been administered to such patients,
and these patients have other problems that might cause or contribute to their olfactory dysfunction, including an unusual constellation
of skeletal and developmental deficits termed Albright hereditary
osteodystrophy (AHO). In this study, we administered tests of odor
detection, identification, and memory to (i) 13 patients with Type Ia
PHP; (ii) 8 patients with Type Ib PHP; (iii) 7 patients with pseudo-
pseudohypoparathyroidism (PPHP); and (iv) 3 sets of normal controls
matched to these groups on the basis of age, gender, and smoking
history. Although we confirm that PHP Type Ia patients evidence
olfactory dysfunction, we also demonstrate that (i) patients with Type
Ib PHP, who have no AHO, no generalized hormone resistance, and
normal Gsa activity, also evidence olfactory dysfunction relative to
matched controls; and (ii) patients with PPHP, who have AHO, no
generalized hormone resistance, and deficient Gsa protein activity,
have relatively normal olfactory function. These observations do not
support the hypothesis that the olfactory dysfunction associated with
PHP is the result of generalized Gsa protein deficiency and imply that
other mechanisms (e.g. ones associated with PTH or PTHrP resistance) are responsible for the olfactory deficits of this disorder. (J Clin
Endocrinol Metab 82: 247–250, 1997)
N
press generalized hormone resistance, evidence variably decreased olfactory ability relative to non-Gsa deficient Type Ib
PHP patients, whose hormone resistance is specific to parathyroid hormone (PTH) (7, 8). Although strong positive correlations between intensity ratings given to odorants by humans and the propensity of such odorants to stimulate
adenylate cyclase activity in an in vitro frog olfactory ciliary
preparation add further credence to the role for G-proteins
in human olfactory transduction (9), such findings do not
shed light on the specific G proteins involved.
While the decreased olfactory function in PHP Type Ia
patients seems to provide compelling evidence for a role of
Gs in human olfactory processing, few types of olfactory tests
have been administered to these unusual patients, and they
have other problems that might explain or contribute to their
decreased ability to smell. For example, Type Ia PHP patients, unlike Type Ib PHP patients, have Albright hereditary
osteodystrophy (AHO), an unusual constellation of skeletal
and developmental abnormalities that includes obesity,
short stature, brachydactyly, round faces, and subcutaneous
ossifications (10).
To better define the apparent association between Gsa
protein deficiency and olfactory dysfunction, we administered odor identification, detection, and memory tests to
Type Ia and Type Ib PHP patients, as well as to patients with
pseudopseudohypoparathyroidism (PPHP) and to normal
subjects matched to the patients on the basis of age, gender,
UMEROUS hormones, neurotransmitters, and cytokines regulate cell function by stimulating production of the intracellular signalling molecule cAMP (1). These
ligands bind to specific cell surface receptors that are coupled
to membrane-bound adenylyl cyclase molecules by heterotrimeric G proteins. Agonist-bound receptors activate G proteins by promoting exchange of GTP for GDP bound to the
a subunit of the heterotrimer. The a-GTP molecule rapidly
dissociates from the bg complex and can interact with its
effector until its intrinsic GTPase hydrolyzes it to GDP, thus
promoting reassociation of a-GDP with bg. In most cells,
receptors are coupled to activation of one or more forms of
adenylyl cyclase by Gs (2).
It has been suggested that Gs may play an important role
in olfactory transduction, although a genetically distinct G
protein, termed Golf, likely couples olfactory receptors to a
unique form of adenylyl cyclase (3). Support for a role of Gs
in olfactory function comes from reports that Gsa-deficient
Type Ia pseudohypoparathyroidism (PHP) patients, who exReceived August 1, 1995. Revision received July 9, 1996. Accepted
August 27, 1996.
Address correspondence to Richard L. Doty, Ph.D., Director, Smell
and Taste Center, University of Pennsylvania Medical Center, 3400
Spruce Street, Philadelphia, Pennsylvania 19104.
* Supported by NIDCD Grant P01 00161 (R.L.D.), NIH Grants DK
34281 and DK 34281 (M.A.L.) and NIH-5M01 RR00722 to the Johns
Hopkins Outpatient GCRC.
247
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DOTY ET AL.
and smoking habits. PPHP is a condition sometimes found
in relatives of patients with PHP Type Ia and shares with
Type Ia PHP the presence of AHO and decreased Gsa protein
activity. However, PPHP is unaccompanied by generalized
end organ insensitivity to hormones. Our results confirm that
PHP Type Ia patients score lower on an odor identification
test than do patients with PHP Type Ib. However, we also
demonstrate (i) that PHP Type Ib patients, relative to
matched controls, evidence deficits in olfactory function and
(ii) that Gsa protein deficient PPHP patients have relatively
normal olfactory function. These observations do not support the hypothesis that the olfactory dysfunction associated
with PHP is the result of a generalized deficiency of Gsa
protein.
Materials and Methods
Subjects
Thirteen patients with Type Ia PHP, 8 patients with Type Ib PHP, and
7 patients with PPHP served as subjects, along with 3 groups of normal
controls whose members were matched individually to each of the
patients on the basis of age, gender, and smoking history (Table 1). One
Type Ia PHP patient did not receive the odor detection threshold test,
and another did not receive the odor memory test (see Olfactory Test
Procedure section). Most of the patients were recruited from families with
multiple affected members who were followed by M.A.L. and A.M. at
Johns Hopkins University and the State University of New York, Syracuse, respectively. Two were patients of Robert Rosenfeld, M.D., of
Wyler Children’s Hospital in Chicago, and one of Andrew Stewart,
M.D., of the Veteran’s Affairs Medical Center, West Haven, Connecticut.
Four were patients of Luis Aparicio, M.D., of Metabolic Associates, Erie,
Pennsylvania. All of the patients provided informed consent to be tested
and were tested in their homes by A.D.F. or D.A.M. The diagnoses of
Type Ia PHP, Type Ib PHP, and PPHP were based on determination of
hormone responsiveness, Gsa activity, and the presence or absence of
AHO, as previously described (11, 12). Erythrocyte membrane Gsa was
analyzed using either a bioassay to measure activity (12) or by a quantitative immunoblot technique to determine the relative level of immunoactive protein (13), as described below. Results for many of the patients evaluated in this study were reported previously (12–15).
Demographic information, as well as mean (sd) Gsa erythrocyte levels
and scores on the Mini-Mental State Examination (16), a measure of
cognitive function, and the Picture Identification Test (17), a visual test
designed to control for nonolfactory elements of the odor identification
test used in this study, are presented in Table 1.
Measurement of membrane Gsa bioactivity
Assays of membrane Gsa bioactivity were performed as previously
described (12), based on activation of adenylyl cyclase in membranes
from the cyc̄ clone of the S49 murine lymphoma cell line, which genetically lacks Gsa (18). Membranes from S49 cyc2 cells were prepared as
previously described (19).
Quantitative immunoblot analysis
Membrane proteins (50 –100 mg protein) were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis using the system of
Laemmli (20). In order to optimize resolution among G proteins, membrane samples were alkylated with N-ethylmaleimide (21) before electrophoresis through 11% acrylamide gels. The separated proteins were
transferred from the gel to PVDF membranes (Millipore) using a Transblot chamber (Bio-Rad). We performed immunoblot analysis using specific polyclonal rabbit antisera that had been raised against synthetic
peptides corresponding to defined regions of the Gsa protein (i.e. antiserum C584 was raised against a peptide [CTPEPGEDPRVTRAKY]
consisting of residues 325–339 of Gsa, and antiserum RM was raised
against the c-terminal decapeptide [RMHLRQYELL] corresponding to
residues 385–394). Both antisera recognize the 45 kDa and 52 kDa forms
of Gsa. Each immunoblot was incubated with a saturating concentration
of the appropriate antiserum, and antibody binding was detected by
subsequent incubation with I125-labeled protein A and autoradiography.
The optical density of each band was measured by two-dimensional
scanning densitometry, and regions of the blot that corresponded to the
bands were excised and the amount of radioactivity was quantitated by
g-spectrophotometry. Over the range of protein concentrations used in
these studies, optical density was proportional to the amount of radioactivity associated with each antigen band. In all cases presented, there
was a linear relationship between the amount of membrane protein
loaded onto the gel and the amount of immunoreactive signal detected.
Olfactory test procedures
The patients and matched controls were administered standardized
tests of odor identification, detection, and odor memory. The order of
presentation of the tests was randomized across subjects. The test session
lasted approximately an hour and a half, and the subjects were allowed
to take rest breaks between the administration of the tests.
The first test that we administered was the University of Pennsylvania
Smell Identification Test (UPSIT; commercially available as the Smell
Identification Test, Sensonics, Inc., Haddon Hts., NJ). This 40-item microencapsulated test is the most widely-administered test of olfactory
function in North America (having been administered to an estimated
30,000 patients) and is described in detail elsewhere (22, 23). Briefly, a
subject is required to identify, in a 4-alternative multiple choice format,
each of 40 odorants presented on microencapsulated “scratch and sniff”
labels. For example, one of the test items reads, “This odor smells most
like: (a) chocolate; (b) banana; (c) onion; or (d) fruit punch.” The subject
must provide a response even if no odor is perceived (i.e. the test is
forced-choice). The number of items out of 40 that were answered
correctly served as the dependent measure.
The second test we administered was the phenyl ethyl alcohol (PEA)
odor detection threshold test. In this test, detection threshold values for
PEA, a rose-like smelling odorant with minimal intranasal trigeminal
nerve activity, were determined using a modified single staircase procedure described in detail elsewhere (22). In the present study, the
staircase was begun at the 26.50 log concentration step of a half-log step
(vol/vol) dilution series extending from 210.00 log concentration to
22.00 log concentration and was moved upward in full log steps until
correct detection occurred on five sets of consecutive trials at a given
TABLE 1. Description and demographics of study population
Males/Females
(no.)
Age
Current, Previous, &
Never Smokers (no.)
Mini-Mental
State
Examination
Picture
Identification
Test
Gs1
(% of normal)
I. Disease group
PHP-Ia
PHP-Ib
PPHP
10/3
6/2
0/7
25.42 (14.85)
44.63 (14.94)
40.29 (11.80)
2, 1, 10
1, 0, 7
4, 2, 1
26.50 (3.40)
29.38 (1.06)
29.00 (0.82)
38.17 (2.04)
40.00 (0)
40.00 (0)
46.42 (8.86)
94.50 (9.40)
53.67 (7.61)
II. Normal controls
PHP-Ia
PHP-Ib
PPHP
10/3
6/2
0/7
25.39 (13.17)
43.50 (13.65)
40.71 (12.51)
2, 1, 10
1, 0, 7
4, 2, 1
29.50 (0.84)
29.67 (0.58)
28.75 (2.50)
39.77 (0.60)
40.00 (0)
39.86 (0.38)
ND
ND
ND
Group
Numbers in parentheses reflect standard deviations. Gs is erythrocyte Gs protein activity. See text for details. ND, no data.
OLFACTORY DYSFUNCTION IN TYPE I PHP
249
concentration. If an incorrect response was given on any trial, the staircase was moved upward a full log step. When a correct response was
made on all five trials, the staircase was reversed and subsequently
moved up or down in 0.50 log increments or decrements, depending
upon the subject’s performance on two pairs of trials (each pair consisting of a choice between a blank and an odorant) at each concentration
step. The geometric mean of the four staircase reversal points following
the third staircase reversal was used as the threshold measure.
The third test of the test battery was one of odor recognition memory.
On a given trial of this 12-trial test (see ref. 24 for details), a microencapsulated “target odorant” is presented to a subject, followed by four
odorants from which the subject is instructed to select the one identical
to the target stimulus. On four trials, a 10-second interval is interspersed
between the sampling of the target stimulus and the presentation of the
first of the four alternatives. On four others, a 30-second interval is
enforced, whereas on the other four trials a 60-second period intervenes.
In the present study the number of trials in which the target odor was
correctly identified, irrespective of delay interval, served as the dependent measure.
Results
The mean (sem) test scores for the three olfactory tests for
the PHP Type Ia, PHP Type Ib, PPHP, and matched control
subjects are presented in Figs. 1–3. Matched-pairs t-tests revealed that the Type Ia PHP subjects performed significantly
more poorly than their matched controls on all three tests
[t-tests with Bonferroni-corrected a levels: UPSIT t (12) 5
11.08, P , 0.0001; PEA t (11) 5 3.46, P , 0.05; memory t (11)
5 6.20, P , 0.0001]. The Type Ib PHP patients were also less
sensitive than the controls on the PEA odor detection threshold test [PEA t (7) 5 6.64, P 5 0.005] and on the odor memory
test [t (7) 5 3.48, P , 0.05], but not on the UPSIT [t (7) 5 1.49,
P . 0.20]. None of the scores of the PPHP patients differed
significantly from those of their matched controls (all Ps .
0.50).
It should be noted that, while Type Ib PHP patients typically outperformed the Type Ia PHP patients on the olfactory tests, their matched controls did likewise. This suggests
the possibility that the seemingly better performance of the
Type Ib patients could be the result of nondisease-related
variables. To establish whether the relative olfactory function
of the Type Ib PHP patients was, in fact, superior to that of
FIG. 2. Mean (SEM) scores on the phenyl ethyl alcohol (PEA) single
staircase odor detection threshold test in the three patient groups and
their individually matched age, gender, and smoking-habit controls.
See text for details.
FIG. 3. Mean (SEM) scores on the 12-item odor memory test in the
three patient groups and their individually matched age, gender, and
smoking-habit controls. The test scores were combined for the three
retention intervals, which did not differ significantly from one another. See text for details.
the Type Ia PHP patients, we performed t-tests across the
patient test scores after they had been subtracted from those
of their matched controls. These analyses revealed no significant differences between the Type Ia and Ib PHP patients
for the PEA odor detection and odor memory tests [PEA t (18)
5 0.99; memory t (18) 5 1.17; all Ps . 0.20]. However, a
significant difference did emerge for the UPSIT [t (19) 5 6.29,
P , 0.0001], implying that odor identification is more markedly altered in Type Ia patients than in Type Ib patients.
Discussion
FIG. 1. Mean (SEM) scores on the University of Pennsylvania Smell
Identification Test (UPSIT) in the three patient groups and their
individually matched age, gender, and smoking-habit controls. See
text for details.
The present study confirms earlier observations that Type
Ia PHP patients evidence olfactory dysfunction (7, 8). However, this study also demonstrates (i) that patients with Type
Ib PHP have some olfactory dysfunction relative to matched
controls, and (ii) that Gsa protein deficient PPHP patients
have relatively normal olfactory function. Taken together,
250
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DOTY ET AL.
these observations throw into question the hypothesis that
the olfactory dysfunction associated with PHP Type Ia is
caused by generalized Gsa protein deficiency. Furthermore,
these data clearly demonstrate that the loss is not the result
of AHO, per se.
Although the basis for the olfactory deficits in Type Ia and
Type Ib PHP patients is unknown, several possibilities are
worthy of consideration. First, it is conceivable that these
types of PHP patients have, perhaps to differing degrees,
decreased function in the primary olfactory G protein, Golf,
or in the type III olfactory adenylyl cyclase that is coupled by
Golf to odorant receptors (5, 6). Second, in as much as the
olfactory adenylyl cyclase behaves as a coincidence detector
that can be stimulated by both Golf and Ca21 (5), it is possible
that mild hypocalcemia, which is common in patients with
Type Ia and Type Ib PHP, may reduce generation of cAMP
in olfactory neuroepithelium and thereby impair olfaction.
Third, given that PHP Type Ia patients (i) seem to have
greater odor identification dysfunction than PHP Type Ib
patients and (ii) exhibit a wider range of hormone resistance
than do PHP Type Ib patients, the difference in odor identification ability may be related to the degree of target tissue
resistance to hormones that act via stimulation of the cAMP
second messenger system. The failure to find this degree of
difference on olfactory tests other than odor identification
may reflect their relatively lower reliability and sensitivity,
rather than the sampling of different neural substrates (25).
Fourth, given the normal olfactory function of the PPHP
patients, who do not exhibit PTH resistance, the decreased
olfactory function in the Type Ia and Ib PHP patients could
reflect PTH resistance, per se. However, since no olfactory
disturbance has been reported in other patients with PTH
deficiency, such as patients with hypoparathyroidism, it is
possible that PTRrP resistance, rather than PTH resistance, is
the mechanism responsible for the decreased olfactory
function.
Whatever the basis for the olfactory alterations observed
in Type I PHP, the present study clearly demonstrates that
such losses are observed on several types of olfactory tests.
While the influence of Type I PHP on nominally disparate
olfactory tests could reflect the involvement of cAMP-related
biochemical changes at multiple levels within the nervous
system, alterations within limited sectors of the olfactory
pathway could also explain this result. For example, if detection ability is altered as a result of biochemical changes
within the olfactory epithelium, poor performance would
also occur on tests of odor memory and identification, because no odor sensation would be available for encoding the
percept needed for such tasks (26). Thus, in such cases, underlying neural circuits needed for odor memory or identification could still be intact. Clearly, research is needed to
determine if the PHP-related changes in olfactory function
reflect, in fact, alterations in the olfactory receptor cells themselves, or if alterations in other elements of the olfactory
pathway are also present.
Acknowledgments
We thank Robert Rosenfield, M.D., of Wyler Children’s Hospital in
Chicago, Illinois for locating and arranging for the testing of two of the
PHP and PPHP patients evaluated in this study. We also thank Luis
Aparicio, M.D., of Metabolic Associates, Erie, Pennsylvania, for allowing
us to test four of his patients.
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