Proc. Nati Acad. Sci. USAVol. 80, pp. 2752-2756, May 1983Medical
Sciences
Carbonic anhydrase II deficiency-identified as the primary
defectin the autosomal recessive syndrome of osteopetrosis with
renaltubular acidosis and cerebral calcification
(erythrocytes/bone resorption/parathyroid
hormone/isozymes/heterozygote detection)
WILLIAM S. SLY*, DAVID HEWETT-EMMETTt, MICHAEL P. WHYTEt,
YA-SHIOU L. Yut, ANDRICHARD E. TASHIANt*Departments of Pediatrics,
Medicine, and Genetics, Washington University School of Medicine,
Division of Medical Genetics, St. Louis Children's Hospital,
St.Louis, Missouri 63110; tDivision of Bone and Metabolism,
Departments of Medicine and Pediatrics, Washington University
School of Medicine and the JewishHospital of St. Louis, St. Louis,
Missouri 63110; and tDepartment of Human Genetics, University of
Michigan Medical School, Ann Arbor, Michigan 48109
Communicated by Donald C. Shreffler, January 24, 1983
ABSTRACT The clinical, radiological, and pathological find-ings
in three siblings affected with the autosomal recessive syn-drome
of osteopetrosis with renal tubular acidosis and
cerebralcalcification have been reported. In an effort to explain
the pleio-tropic effects of the mutation producing this disorder,
we pos-tulated a defect in carbonic anhydrase H (CA II), the only
one ofthe three soluble isozymes of carbonic anhydrase that
is.known tobe synthesized in kidney and brain. We report here
biochemicaland immunological evidence for the virtual absence of CA
II inerythrocytes of patients affected with this condition, whereas
CAI level is not reduced. Levels of CA II in erythrocyte
hemolysatesfrom asymptomatic obligate heterozygotes are about half
of nor-mal. These findings: (i) elucidate the basic defect in one
form ofinherited osteopetrosis; (ii) provide genetic evidence
implicatingCA II in osteoclast function and bone resorption; (iii)
explain pre-vious observations that carbonic anhydrase inhibitors
block thenormal parathyroid hormone-induced release of calcium from
bone;(iv) clarify the role of renal CA H in urinary acidification
and bi-carbonate reabsorption; and (v) suggest a method to identify
het-erozygous carriers for the gene for this recessively inherited
syn-drome.
Osteopetrosis is an inherited metabolic bone disease in whicha
generalized accumulation of bone mass prevents normal de-velopment
of marrow cavities and the enlargement of osseousforamena (1-3). It
has been called "marble bone disease" be-cause the bones are very
dense radiographically, although thebones typically have an
increased susceptibility to fracture (2,3). Multiple genetic
defects produce osteopetrosis but themechanism common to all the
known forms of osteopetrosis isa failure of bone resorption (4). In
man, two principal types ofosteopetrosis have been described. One
is a dominantly in-herited, relatively benign condition which is
often detected ra-diologically in asymptomatic adults (2, 3). A
second type is therecessive, lethal, malignant form of
osteopetrosis. In this form,osteopetrosis is usually present at
birth, becomes symptomaticearly in infancy, and leads to death in
infancy or early childhoodfrom infection or bleeding (3). Forms of
osteopetrosis with clin-ical courses of intermediate severity have
also been recognized(3). Various animal models of osteopetrosis
have been identifiedincluding four different mutations that produce
osteopetrosis inmice (4).
In 1972, three separate reports described a distinct form
ofosteopetrosis that occurred in association with renal tubular
aci-dosis (5-7). The pattern of inheritance was autosomal
recessive.
The clinical course was not entirely benign, but the disease
wascompatible with long survival and the hematologic abnormal-ities
that dominate the clinical picture in the recessive lethalform of
osteopetrosis were absent. One of these families wasnot described
in a complete report until 1980 (8), by which timetwo of the three
affected siblings had been found to have cal-cification of the
basal ganglia. In the same year, Ohlsson et al.(9) independently
reported three Saudi Arabian families in-volving first-cousin
marriages that produced offspring with os-teopetrosis, renal
tubular acidosis, and cerebral calcification, asyndrome for which
they proposed the name "marble brain dis-ease.
In an effort to explain the pleiotropic effects of the
mutationunderlying this disorder by a single enzyme defect, we
pos-tulated a defect in, one of the three isozymes of carbonic
an-hydrase (CA I, CA II, CA III) which are known to be under
sep-arate genetic control in humans (10-14). This hypothesis
seemedattractive for two reasons: (i) metabolic acidosis can be
pro-duced by sulfonamide inhibitors of CA (12), and (ii) several
re-ports have shown that CA inhibitors can block the
parathyroidhormone-induced release of calcium from bone, suggesting
arole for CA in bone resorption (15-17).
The relationship of CA deficiency to cerebral calcification
wasless apparent, although it is known that CA II is present in
brain(18) and that CA inhibitors inhibit cerebral spinal fluid
pro-duction (19) and affect electrical activity of the brain (20).
A de-fect in the CA II isozyme seemed most likely because this is
themost widely distributed of the three known soluble isozymesof CA
in human tissues (10, 11) and CA II is the only solubleisozyme so
far identified in renal and brain tissue (18, 21, 22).In addition,
a genetically determined, virtually complete ab-sence of CA I in
mature erythrocytes has been found to haveno clinical consequences
(23). Because both CA I and CA II areexpressed in human
erythrocytes, it was possible to test thishypothesis by examining
these isozymes in hemolysates of pe-ripheral blood from the family
we reported previously (8).
In this report, we describe studies showing what appears tobe an
almost complete absence of CA II in erythrocytes of pa-tients
affected with the syndrome of osteopetrosis, renal tu-bular
acidosis, and cerebral calcification. Furthermore, we re-port that
in asymptomatic normal parents of affected patientsthe levels ofCA
II are half of normal, which supports the inter-pretation that the
CA II deficiency is the basic defect under-lying this clinical
disorder and suggests a means to identify het-erozygote
carriers.
Abbreviations: CA, carbonic anhydrase; PTH, parathyroid
hormone.
2752
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Proc. Natl. Acad. Sci. USA 80 (1983) 2755
kidney also contains a membrane-bound CA that is an
intrinsiccomponent of the brush border of the proximal tubule
(30-32).Genetic and structural evidence suggests that at least the
sol-uble isozymes comprise a multilocus enzyme family derived froma
common ancestral gene by gene duplications (11). However,the
kinetic parameters of these isozymes and their sensitivityto
inhibitors can differ markedly (33, 34). These findings
havesuggested that the physiological roles for the different
isozymesare diverse, which is also suggested by their different
tissuedistributions.The human CA II isozyme, whose turnover number
for the
CO2 hydration reaction under physiological conditions (1.3-1.9X
10' sec') is the highest known for any enzyme (35, 36), hasbeen
identified (immunologically or by purification) in a widevariety of
cells, tissues, and organs including erythrocytes, brain,eye,
kidney, cartilage, liver, lung, skeletal muscle, pancreas,gastric
mucosa, and anterior pituitary body (10, 11). The otherisozymes,
whose activities toward CO2 and HCO3 are lowerthan those of CA II
in the order CA II > CA IV > CA I> CAIII (29, 33, 34),
appear to have a more limited distribution. CAI is found primarily
in erythrocytes, CA III mainly in red skel-etal muscle, and CA IV
in lung.
The finding of a quantitative defect in CA II in these
patientsprovides us with an unusual opportunity to assess the
impor-tance and function of this isozyme. In view of the high CO2
hy-drase activity of CA II and its wide tissue distribution, one
mightexpect widespread effects of this deficiency in organs in
whichCAII plays an important role. However, it should be noted
thatthe finding of a virtual absence of CA II in erythrocytes
doesnot necessarily imply a comparable deficiency in other
tissuesand organs in which CA II has been reported. CA II levels
incells with considerably more rapid turnover than
erythrocytescould be appreciably higher. Also, there may be
additional, stillunidentified, CA genes contributing to enzyme
levels in tissuesspared by this mutation. However, what is clear
from these pa-tients is that the quantitative deficiency of CA II
that we havedemonstrated in erythrocytes has important clinical
conse-quences for bone, for kidney, and for brain that merit
discus-sion.Bone Metabolism. All known forms of osteopetrosis are
as-
sociated with failure to resorb bone (4). Studies of several
pa-tients with osteopetrosis have demonstrated impaired
hyper-calcemic responses to infused parathyroid hormone (PTH)
(37,38). The cause for this impairment might differ in the
differentforms of osteopetrosis. Studies showing inhibition of
PTH-in-duced release of calcium from bone by CA inhibitors have
sug-gested a role for CA in bone resorption (15-17). Also, CA
hasbeen demonstrated histochemically in chick and hen
osteoclasts(39). On the basis of these and other observations (40,
41), ithas been suggested that PTH activates CA in certain bone
cellswhere it might aid the resorptive process by mediating
secre-tion of H' (16, 39). The genetic evidence presented here
pro-vides strong support for a role of CA in bone resorption,
whichwas suspected from the pharmacological and histochemical
evi-dence cited above, and specifically implicates the CA II
iso-zyme in bone resorption.
Renal Tubular Acidosis. There is general agreement that
renalreabsorption of bicarbonate is a major factor in the
maintenanceof acid-base homeostasis (42). Most of the bicarbonate
recla-mation takes place in the proximal tubule and depends on
CA(43). Only recently has it become clear how both a soluble
(cy-tosolic) and a membrane-bound (luminal) CA play separate
rolesin the proximal tubule (21, 42-46). Bicarbonate reclamation
de-pends on H+ secretion, the major mechanism for proximal tu-bular
acidification (42). The H' secreted into the lumen of theproximal
tubule is titrated by the HCO3 in the glomerular fil-
trate to produce H2CO3 which is in contact with the
membrane-bound CA. The luminal CA then catalyzes the dehydration
ofH2CO3 to CO2 and H20 (42, 43). The CO2 diffuses freely intothe
proximal tubular cell, where it can be hydrated by the cy-tosolic
CA to H2CO3. Dissociation of this product into H' andHCO3 allows
HCO3 to be transported by unknown mecha-nisms into interstitial
fluid or the peritubular capillary, com-pleting the reclamation of
filtered bicarbonate (42, 43). The re-generated H' can be secreted
in exchange for Na+ to initiateanother cycle (42). From the above,
it is clear how two differentCAs operate at different sites to
participate in bicarbonate rec-lamation.The enzymatic dehydration
of H2CO3 in the lumen appears
to be mediated entirely by the membrane-bound CA presentin the
brush border of proximal tubular cells (42, 45, 46). Al-though we
have no direct information on the status of thisenzyme in the
patients described here, we have no reason tosuspect that this
enzyme, which is biochemically and im-munologically distinct from
CA II and the other soluble iso-zymes (21, 22, 31, 32), is
defective in these patients. On theother hand, the enzymatic
hydration of intracellular CO2 is pre-sumably mediated entirely by
the soluble enzyme CA II, forwhich these patients are deficient.
The renal tubular acidosispresent in patients with this syndrome
must be explained inthis context.
Although the renal tubular acidosis in the different pedi-grees
has been variable in severity, and somewhat heteroge-neous in type,
most of the patients reported have a significantdistal defect (47).
In those cases in which HCO3 reabsorptionhas been adequately
studied, a proximal component, evidencedby HCO3 wasting at normal
plasma HCO3 concentrations,has also been found (47). Thus, the
patients appear to have amixed or hybrid type of renal tubular
acidosis which includesboth a proximal component and a distal
component. The hy-dration of CO2 in cells of the proximal tubule
presumably ismediated by CA II which appears to be the major (and
perhapsonly) soluble isozyme present in the kidney (21, 22, 48).
Be-cause this reaction generates some of the H' secreted by
theproximal tubule, and bicarbonate reclamation in the
proximaltubule depends almost entirely on H' secretion (42-44),
wecan understand why patients with CA II deficiency might havea
renal tubular acidosis that includes a proximal component.The
prominent distal component of the renal tubular acidosis
in CA II-deficient patients, evidenced by inappropriately
highurine pH values when patients were quite acidotic (47),
initiallywas more difficult to understand. A possible explanation
wasprovided recently by immunohistochemical evidence showingmuch
more intense reaction for CA II in the distal tubules (andeven in
collecting ducts) than in proximal tubules of human kid-neys (48).
These results suggest that CA II plays a more im-portant role in
the distal tubule than was previously suspected(22), either in
generating H+ or in titrating OH- produced bythe
proton-translocating ATPase.The report (23) that a genetically
determined, virtually com-
plete, absence of erythrocyte CA I in man is not associated
withrenal tubular acidosis is consistent with biochemical and
im-munological evidence that CA II is the only soluble
isozymepresent in kidney (21, 22, 48). In view of this evidence,
and ofthe findings presented here, it is difficult to understand
the sig-nificance of the abnormalities in erythrocyte CA I that
have beenreported in a few patients with distal type renal tubular
acidosis(49, 50).
Brain Metabolism. The function of CA II in brain and thereasons
for brain calcification in patients with defects in CA IIare less
well understood. In the central nervous system, CA IIis primarily a
glial enzyme and occurs predominantly in oh-
Medical Sciences: Sly et al.
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2756 Medical Sciences: Sly et al.
godendrocytes (18). CA II has been identified in brain
homog-enates, with up to 50% of the activity in a
membrane-boundform (51). Although the functions of CA in brain are
still spec-ulative, it is worth noting that many of the patients
with thesyndrome of osteopetrosis with renal tubular acidosis and
ce-rebral calcification have significant mental retardation (9).
Thepatients in the family reported here are exceptional in this
re-gard because their IQ scores are in the low normal range
(8).Like the mechanism of the cerebral calcification in this
syn-drome, the mechanism of the mental retardation is not yet
clear.
Erythrocyte Function. One might expect some
secondaryconsequences of CA II deficiency in tissues in which the
CO2produced must be delivered to circulating erythrocytes and
dis-charged from the lungs. This transport depends on the abilityof
the CA activity in erythrocytes to convert metabolic CO2 toHCO3
rapidly in the tissues and to catalyze the reverse re-action in
lung capillaries. Although CA II normally accounts foronly 14-17%
of the CA in erythrocytes (CA I accounts for therest), it has been
estimated that CA II accounts for about 90%of the CA activity of
erythrocytes in vivo (36, 52). This estimateis based on the much
greater specific activity ofCA II comparedto CA I and on the much
greater sensitivity ofCA I to inhibitionby the normal chloride
concentration of erythrocytes (52). Thisestimate is in general
agreement with the findings by W. R.Chegwidden (personal
communication), in a preliminary ex-periment on the family
described here, that the relative CA ac-tivities for the HCO3
dehydration reaction in hemolysates froman affected homozygote
(III-1), an obligate heterozygote (II-14),and an unrelated control
were 0.17, 0.43, and 1.0, respectively,after correction for the
nonenzymatic control reaction. Thus, allof the available evidence
indicates that CA II is more importantthan CA I for the CO2 hydrase
reaction in the erythrocyte.However, Wistrand (36) has estimated
that only 2% of normallevels of CA activity in erythrocytes would
be required for un-loading CO2 in lung capillaries at rest, and 4%
would be re-quired at work. If all of these estimates are correct,
CA I ac-tivity alone may be sufficient for this erythrocyte
function. Thefact that the patients described here have no
disability that wecan ascribe to the virtual absence of CA II in
their erythrocytessupports this conclusion.We thank Patrick J.
Venta and Dr. Alan Robson for helpful sugges-
tions, Mrs. Sabra Lovejoy for typing the manuscript, and Mrs.
DorothyChesnut, RN, for obtaining blood samples. This research was
supportedby National Institutes of Health Grants GM 21096, GM 31988
(to W.S. S.),and GM 24681 (to R.E.T.) and by the National
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