Am J Hum Genet 37:326-337, 1985

Heterogeneity of Holocarboxylase Synthetase in Patientswith Biotin-Responsive Multiple Carboxylase Deficiency

BETTY J. BuRRI,1 LAWRENCE SWEETMAN, AND WILLIAM L. NYHAN

SUMMARY

Holocarboxylase synthetase activity has been determined in fibro-blasts of seven patients with the neonatal form of biotin-responsivemultiple carboxylase deficiency. The normal Km for biotin was 15 + 3nmol/l, while in the patients the values ranged from 48 to 1,062 nmolAl.The mean maximum velocity was 27% of normal. Differences amongthe values obtained for the Km for biotin and the heat stability ofholocarboxylase synthetase suggested that the patients studied repre-sented at least four distinct variants at the holocarboxylase synthetaselocus.

INTRODUCTION

The biotin-responsive multiple carboxylase deficiencies are inherited disordersof metabolism in which a characteristic pattern of accumulation of organicacids is caused by decreased activities of the biotin-dependent carboxylases:propionyl-coenzyme A carboxylase (PCC), 3-methylcrotonyl-coenzyme A car-boxylase (MCC), and pyruvate carboxylase (PC) [1-5]. Patients have beenclassified clinically into two groups: a neonatal or early-onset form and a later-infantile form [6]. However, it is not always possible to distinguish between thetwo on the basis of clinical presentation [7]. The two types of patients have alsobeen distinguished by examining the response of the activities of the carboxyl-ases in fibroblasts to the concentration of biotin in the culture medium [6]. Thecells of the neonatal patients have low activities of the carboxylases whencultured in medium low in biotin, and these activities increase in the presence

Received October 24, 1983; revised July 6, 1984.This study was supported by the University of California, San Diego, General Clinical Research

Center, National Institutes of Health, Division of Research Resources, grant RR-00827; and U.S.Public Health Service grant HD04608 from the National Institute of Child Health and HumanDevelopment, National Institutes of Health.

' All authors: Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093.© 1985 by the American Society of Human Genetics. All rights reserved. 0002-9297/85/3702-0007$02.00

326

HOLOCARBOXYLASE SYNTHETASE

of added biotin [8, 9]. In contrast, the cells of the later-infantile patients havenormal activities in either medium [10].

In studies of the patient initially reported with the classic neonatal form [1],we have shown that the primary enzymatic defect is an abnormal holocarboxyl-ase synthetase (HCS) [11]. The Km for biotin was markedly elevated. HCSutilizes ATP to activate biotin to biotinyl adenylate, which then reacts with anE-amino group of a lysine residue in the apocarboxylases to form a biotin amidelinkage in the holocarboxylase. It has recently been shown that patients withthe later-infantile-onset type of multiple carboxylase deficiency have deficientactivity of biotinidase in serum [12, 13].

This study was undertaken to examine the activity of HCS in fibroblastsderived from a number of patients with multiple carboxylase deficiency withvarious patterns of clinical presentation. Kinetic and other properties of theenzyme were studied in seven patients with the neonatal form and six patientswith the later-infantile form of multiple carboxylase deficiency. Defective ac-tivity of HCS was found in each of the infants with the neonatal type of disease.The affected Km's for biotin varied so greatly that it is likely that among theseven patients representing six families at least three distinct variant HCSenzymes were present. HCS activity was normal in each of the later-onsetpatients.

MATERIALS AND METHODS

The clinical information on the 13 patients studied is summarized in table 1. PatientNW was diagnosed prenatally and treated prenatally as well as postnatally with biotinand therefore did not develop clinical symptoms [15]. A previous sibling had presentedin the neonatal period [16]. Patient GB, a sibling of patient BB, was treated prenatallywith biotin but was not treated postnatally with biotin until after symptoms appeared atage 3 months [18, 19].

Fibroblasts were cultured in Eagle's minimal essential medium with 10% fetal-calfserum as the low biotin medium (6 nmol/l of biotin) and in Hamm's F-10 medium with10% fetal-calf serum, a high biotin medium (220 nmol/l of biotin).The activities of PCC and MCC in fibroblasts were measured by methods previously

described with the modifications that the assay volume was decreased to 100 [L1, and theconcentrations of propionyl-CoA and 3-methylcrotonyl-CoA were increased to 2.8 and1.0 mmol/l, respectively [9].Activity of HCS in fibroblasts was assayed using the method we previously adopted

[11]. Extracts of human fibroblasts containing HCS were passed through avidin affinitycolumns to remove biotin and holocarboxylases and then incubated with apo-PCC par-tially purified from rat liver, biotin, and ATP to generate active holo-PCC. The holo-PCC formed was measured in a second incubation by its ability to incorporate radioac-tive bicarbonate into methylmalonyl-CoA, with propionyl-CoA as substrate. HCSactivity in lymphocytes was assayed using a similar method. Lymphocytes were pre-pared from freshly drawn blood by layering blood diluted 1 to 1 with Tris-buffered saline(TBS: 15 mM Tris, 3 mM EDTA, 0.9% NaCl, pH 7.4) over 15 ml of Isolymph (Phar-macia, Piscataway, N.J.) and centrifuging for 30 min at 400 g. The lymphocyte layer waswashed three times with TBS, centrifuged, resuspended in 0.5 ml lysis buffer, sonicated,and passed through an avidin affinity column [11]. HCS activity was determined bymixing 50 pul of lymphocyte preparation (7 x 104 cells, 0.01 mg protein) with apo-PCC,ATP, and various concentrations of biotin in 300 ,ul assays. After a 30-min incubation at30°C, the reaction was stopped by placing the assay vials in an ice bath, and the holo-

327

BURRI ET AL.

Cs

00

4 -

"t W) - - - 00 CD=_ _-'t_

_-- _ _- _-

"it

_4 C4_: e*aeNJeNWI *) C

++ ++

+ I+ + I++ I

+I++I++ ++I++I

+ II+ II+ Z+ + + + +

+

+I+ I++I

+ II

+I+++++ ++++++

+I+++++ ++I+++

+IZ++++ X++I++

ao co 0 0 0 0

r 3 r e E_40_o}o Z en Ze £ 00

.. . . . . . . . .. . . . . . .

_._, _ m _r- ° m m.~ ~~~*N zC9L,

328

4a)C)

0

be

._

CtO~

x

C)

CO

C)

0.0

.0

EC)Cdca)

N

a)

oCO

00Q

OCO0._2

C)CO

COi

C)aI~ ._

= 0r-

.S

CO

a)o

uz

0

a)

m

x

0

on

04

.4

z

U

zF-

Lu

6

00.

E

0

L

0.0

0.- 0

r

( E0CV

CO

Cd

oaCa

.0 0.

C 0a

a)

0C

a)

CCC- C

Ec o .0

0C0_ D

Z * 4- 402*t CO Q

Oon 0

CO ;eXnC

0CO *0.VFc

HOLOCARBOXYLASE SYNTHETASE

PCC formed was measured by the incorporation of radioactive bicarbonate with pro-pionyl-CoA as substrate.The activity of holo-PCC produced by HCS in fibroblast or lymphocyte lysates was

linear with amount of lysate protein to 80 pug [11]. It was also linear with the time ofincubation of the lysate with apo-PCC, biotin, and ATP for 90 min [ 1]. A small amountof holo-PCC continued to be formed during the subsequent incubation for the assay ofholo-PCC in spite of the dilution involved. The linearity of holo-PCC activity with thetime of the first incubation indicates that the holo-PCC activity from a fixed incubationtime of 60 min is a valid measure of the velocity of the HCS reaction. There wasconsiderable experimental variation, particularly in the maximum velocities of HCS,which may be due to the two-step assay and to differences in the proportion of nativeand denatured apo-PCC as a result of the instability of the apo-PCC.

Kinetic constants were calculated from duplicate determinations of HCS activity atsix to 11 different concentrations of substrate (ATP or biotin) bracketing the observedKm values and with the other substrate at saturating concentrations. Provisional kineticconstants were determined by weighted linear regression of the double reciprocal plot[26]. These constants were refined by unweighted reiterative fitting of the data to thehyperbolic Michaelis-Menten equation with an Apple computer program based on themethod of Wilkinson [26].The heat stability of HCS was determined by adding 30 ixl of fibroblast extract (0.075

mg protein) to 150 pul of buffer T [I1] and incubating at 480C for 0, 10, 20, 30, and 60 min.Then apo-PCC, biotin, and ATP were added, and the HCS activity assayed at 30TC withthe usual two-step assay.

RESULTS

The activities of PCC and MCC in fibroblasts of the patients studied aresummarized in table 2. Normal fibroblasts displayed similar activities in bothmedia and so did those listed as biotinidase deficiency or later-onset multiplecarboxylase deficiency. In contrast, in the fibroblasts of patients listed as HCSdeficiency, the activities of both carboxylases were distinctly deficient whengrown in medium containing 6 nmol/l of biotin. In this low biotin medium, thelevels of activity of both of the carboxylases were similar. The mean value ofPCC was 11% of normal (range 0%-30%) and that of MCC was 8% of normal(range 0%-29%). When the cells were cultured in medium containing 220 nmol/1 of biotin, the activities were about fivefold higher. The means were 47% ofnormal for PCC (range 19%-92%) and 48% of normal for MCC (range 10%-96%). There was some variation among the patients in this assay. Fibroblastsof patient AF had low activities of the carboxylases even in the high biotinmedium, and no activity could be detected in the medium low in biotin. Whencultured in medium containing 8,000 nmol/l of biotin, the activity of PCC in thispatient's cells became 30% of normal and that of MCC 28% of normal. Theactivities of the carboxylases in fibroblasts of the parents of patients JRi and thetwo siblings BB and GB ranged from 29% to 89% of normal in the low biotinmedium.The kinetic properties of HCS in fibroblasts with biotin as the variable sub-

strate are shown in table 3. In fibroblasts from four normal individuals and onepatient with propionic acidemia (SZ), the mean Km for biotin was 15 + 3 nmol/land the maximum velocity was 345 + 145 pmol/min per mg of protein for theproduct PCC. The Km values for biotin of the HCS in the HCS deficiency

329

BURRI ET AL.

N_

_ N- 1- 'i-0s rz o

c 0

'oo ) mN ON

_ _ ___--,--, ,-,00_0 %0 C )m0 - m W) C m -II00 00 " 1-

-00

" 0 WO 0%_ 4

e. t- en

014

000()0 rn%0N 00<

.

00 No

oo cn e

el WI 00W

" " in " _

100

t--en

00o - W)

Cl l

et Coo o CN

"- N

~e

_0

_C

00 *

enI%,0 0)

,R O0 V, .. .

eq en 'Itl

00 en 00

W) OntN e4 _-

Wl0%q WCfo 00N

%0u No lb NnCD00 Nt 00

-

W m............. ....... ._C) .....

.C--* Q ---

...C-V C.0 --

C.... Q C *-. -

330

u

u

U

Cu3

z

W

0

C.-

U

z

zru0

CE0

z0

U

Cu

z

0

U

x

z

az0

V)

0z.

go

Cu4

.-Im

S0

C>

C

S0

40

C

Ec

~c

z

z

+I Ced-1 ,

r Ao0

73

.6

C Cu

j;- EL

C>

OEa-C)

o~ 2

E.2

Cu

00

X C

00

-N_

* C1

Cu

o~ *2

C Cu

o cc 01

C 0.C

Cu Cu e

*0 V

-.U4)rQ+l.

HOLOCARBOXYLASE SYNTHETASE 331

TABLE 3

Km VALUES FOR BIOTIN OF HOLOCARBOXYLASE SYNTHETASE (HCS) IN FIBROBLASTS

Biotin Km* Vmax*Fibroblasts (nmol/1) (PCC, pmollmin mg protein)

Controls:FS1 .18 (4-25) 3 291 (240-350) 3FS2 ....................... 13 (12-14) 2 585 (477-693) 2FS3 ....................... 12 (7-18) 3 349 (320-375) 3FS5 ....................... 17 ... 1 300 ... ISZ ........................ 16 ... 1 197 ... 1Mean ± I SD (no. = 5)... 15 ± 3 ... ... 345 ± 145 ... ...

HCS deficiency:AF ........................ 1062 (789-1335) 2 42 (20-63) 2NW ....................... 718 (458-977) 2 102 (100-105) 2JRi ........................ 346 (325-367) 2 119 (100-138) 2JT ........................ 394 (372-417) 2 124 (25-223) 2BB ........................ 322 (35-595) 4 70 (26-121) 4GB ........................ 281 1 95 ... IMC ....................... 48 (48-49) 2 96 (29-163) 2

Biotinidase deficiency/late-onsetmultiple carboxylase deficiencyDH ....................... 22 (8-35) 2 371 (199-543) 2JRo ....................... 18 (16-25) 3 385 (343-461) 3SK ........................ 9 (7-11) 2 502 (391-612) 2SB ........................ 11 (4-18) 2 80 (71-88) 2CR ........................ 10 (7-13) 2 204 (155-253) 2DB ........................ 20 ... 1 87 ... IMean ± I SD (no. = 6)... 15 ± 6 210 ± 196

* Mean, (range of values), no. determinations.

patients ranged from 48 to 1062 nmol/l. These elevations were three- to 70-foldthe normal mean. The maximum velocities ofHCS in these patients were lowerthan normal; the mean value was 27% of normal. The lowest Vmax was observedin AF, the patient with the highest Km. In the other patients, the values of Vmaxwere similar, but the variations among Km values suggested that the patientsunder study represented at least three different genetic variants of HCS.The Km values for biotin ofHCS in the biotinidase or late-onset patients were

normal. The mean value of 15 + 6 nmol/l was identical to the normal mean. Themean maximum velocity was 61% of the normal mean. However, in all but twopatients, the Vmax values were indistinguishable.The Km values for ATP of HCS were determined in fibroblasts from some of

the patients. The mean Km for ATP of two controls and three biotinidasedeficiency or late-onset patients was 0.25 mmol/l (range 0.13-0.35). The Kmvalues for ATP were similar to normal for HCS deficiency patients (0. 13, 0.30),but one patient (AF) had a moderately elevated Km of 1.32 mmol/l). This wasthe patient who also had the most highly elevated Km for biotin.The Km values for apo-PCC as substrate for HCS may be elevated in HCS

deficiency. Three apo-PCC preparations at a concentration that gave maximalactivities for HCS of two normals (FS2 and FS3) did not give maximal activitiesfor HCS for patients JRi and NW. The activity of HCS of these two patients

increased when higher concentrations of apo-PCC were used. Km values forapo-PCC could not be determined because of the instability of apo-PCC anddifficulties in quantifying the amount of native apo-PCC in the different prepa-rations.The predicted activity of HCS as percent of the mean normal maximum

velocity was calculated from the kinetic data of table 3 for the HCS deficiencypatients, using the Michaelis-Menten equation for biotin concentrations of 6and 220 nmol/l. These concentrations of biotin were the high and low biotinconcentrations in the fibroblast cultures. Assuming that biotin is not concen-trated in cultured fibroblasts, these would be the approximate intracellularconcentrations in the fibroblasts that led to the carboxylase activities shown intable 2. In figure 1, the observed activities of PCC (as percent of normal) infibroblasts of these HCS deficiency patients cultured in high and low biotinmedia were plotted against the calculated activities of HCS at these concentra-tions of biotin. There was a linear relationship in which full carboxylase activitywas achieved at about 30% of the maximum velocity of normal HCS. Thus, thekinetic properties of HCS determined in vitro are consistent with the functionalactivity of HCS in intact fibroblasts in the synthesis of holopropionyl-CoAcarboxylase.

Properties of HCS in addition to the kinetic constants were studied for pa-tients with multiple carboxylase deficiency. Two HCS deficiency patients, JRi

100/ AMC* Normal

Mean90 /

> 80-

70

*JT

i 40

z 30L / *~~~GB

cr 20 - / *NW0 MC

BBJT1

JR I° AFNW

AF 5 10 15 20 25 30 35CALCULATED VELOCITY OF HOLOCARBOXLASE SYNTHETASE

Percent of Normal Mean Maximum Velocity

FIG. 1.-Propionyl-CoA carboxylase activity in fibroblasts of patients with HCS deficiency as afunction of the velocity of HCS calculated from the individual Km values for biotin and the concen-trations of biotin in the culture media. Open symbols: cells cultured with 6 nmol/l of biotin; closedsymbols: cells cultured with 220 nmol/l of biotin.

332 BURRI ET AL.

HOLOCARBOXYLASE SYNTHETASE

>20 /

0 1 2 30 XBB

Z 80JWKC Ri0 60 \

20 MC

-NW

10 20' 30 40 50 60TIME AT 480C min.)

FIG. 2.-Stability of HCS at 480C. At the indicated time points, residual enzyme activity wasassayed at 30TC. Shaded area indicates the range of activities of three normal cell lines.

and BB, and one biotinidase deficiency patient had the same pH optimum forHCS activity as the normal HCS. The heat stability at 480C of HCS infibroblasts from five patients is shown in figure 2. The HCS of two patients, MCand NW, were more labile than the normal enzyme, while the HCS of threepatients (JRi, BB, and AF) were more stable than the normal. The considerablydifferent heat-stability curve in patient AF further characterized this very dif-ferent enzyme in which the Km for ATP was somewhat high and the Km forbiotin very markedly greater than any of the others studied.The Km values of HCS for biotin were determined in the lymphocytes of

normal subjects and two patients with HCS deficiency (table 4). The Km forbiotin of HCS of normal lymphocytes of 4.4 nmol/l was similar to the Km of 15nmol/l for fibroblasts. The maximum activity of HCS in lymphocytes was 55pmol/min per mg, which approximated the maximum activity in lymphoblastsof one early-onset patient, MC, but was over twice that of patient AF.

DISCUSSION

These observations indicate that the early-onset or neonatal form of biotin-responsive multiple carboxylase deficiency is caused by deficiency of HCS.Our observations indicate further that the later-infantile disease is not. Abnor-mality of HCS has been shown for the fibroblasts [27] or lymphoblasts [17] oftwo other patients using endogenous apocarboxylase as substrate. The discov-ery of a deficiency of biotinidase by Wolf et al. [12, 13] indicates that there areat least two different genetic defects that cause multiple carboxylase

333

334 BURRI ET AL.

TABLE 4

KINETIC PROPERTIES OF HOLOCARBOXYLASE SYNTHETASE (HCS) IN LYMPHOCYTES

BIOTIN

Km VmaxSOURCE (nmol/1) (PCC, pmol/min/mg protein)

Controls:A ....................................... 4.4 55B........................................ 2.7 59C........................................ 4.3 165D ................................. .... 4.7 50Mean ± SD (no. = 4) 4.0 ± 0.9 82 ± 55

HCS deficiency:AF ....................................... 2,551 23MC ....................................... 1122 51

HCS deficiency heterozygote:OF ....................................... 937 35

deficiency. Most, but not all, of the patients in whom we found a normalactivity of HCS have now been shown to have defective biotinidase activity[24, 28]. This enzyme releases biotin from biocytin (biotinyl-e-N-lysine) andbiotinyl peptides and is involved in the degradation of the holocarboxylases[29]. The patients with an abnormal HCS usually present at an earlier age thanthose with a deficiency of biotinidase, but the two defects cannot always bereliably distinguished from the age of onset and the clinical features [7]. How-ever, none of the patients in whom we found a normal HCS presented in thenewborn period (table 1). The clinical phenotype in patients with HCSdeficiency is usually that of a presentation in the neonatal period with vomiting,acidosis, ketosis, hyperammonemia, and lactic acidemia (table 1). The sister ofone patient (AF) had died in an early neonatal episode of hypothermia, pro-found acidosis, and coma [30]. A few HCS deficiency patients and all of thebiotinidase deficiency patients present later, usually between 3 and 10 monthsof age. Usual features are hypotonia, alopecia, and an erythematous rash rem-iniscent of acrodermatitis enteropathica, progressive to keratoconjunctivitis,seizures, ataxia, and developmental regression [3, 23, 25]. Episodes of meta-bolic acidosis and lactic acidosis may ensue [3, 31].The activities of the carboxylases in fibroblasts cultured in medium contain-

ing a low concentration of biotin of about 6 nmol/l represent useful means ofidentifying those patients with an abnormal HCS and distinguishing them frombiotinidase deficiency. All of the seven patients, in whom Km values for biotinranged from three to 70 times normal, had activities of PCC and MCC in thisassay less than 30% of normal. In contrast, all six patients in whom HCS wasnormal had PCC and MCC activities greater than 37% of normal. The activitiesof the carboxylases of the patients with HCS deficiency were increased from10% to 96% of normal when the fibroblasts were cultured in medium with a highconcentration of biotin (220 nmol/l). The extent of the increase was dependenton the individual Km for biotin. The excellent correlation between PCC activity

HOLOCARBOXYLASE SYNTHETASE

in fibroblasts of HCS deficiency patients cultured in media with different con-centrations of biotin with the calculated activity of HCS as percent of max-imum with the experimentally determined Km values for biotin (fig. 1) indicatesclearly that the elevated Km values for biotin of HCS are primarily responsiblefor the decreased activity of the carboxylases.

Similarly, among patients with HCS deficiency, clinical responsiveness tobiotin could broadly be related to the kinetics for biotin of the synthetase. TheKm values of HCS for biotin in the majority of HCS deficiency patients were20-25 times normal, but one patient had a value only three times normal andanother 70 times normal. The patient, MC, with only a threefold normal Km forbiotin, had a later onset at age 2 months [20]. Patient AF, with a 70-fold normalKm presented on the first day of life, had a sibling that died of the disease [30]and never responded completely to therapy with biotin [14]. While receiving 20mg of biotin per day, the concentration of biotin in plasma was 4,830 nmol/l, orabout 4.5 times the Km for biotin of his HCS, but the elevated excretions oforganic acids in urine indicated that the carboxylases were not fully active.Even while receiving 60 mg of biotin per day, he continued to have elevatedexcretions of abnormal metabolites characteristic of low activities of PCC,MCC, and PC and to have activities of these carboxylases in lymphocytes ofapproximately 40% of normal. Furthermore, his skin lesions have never com-pletely resolved. These observations are consistent with activities of PCC andMCC of 30% of normal in his fibroblasts cultured with 8,000 nmol/l of biotin, aconcentration approximately eight times the Km for biotin of his HCS. It wouldappear that the maximum velocity of HCS of about 12% of normal isinsufficient for full activation of the carboxylases. Treatment of an infant with10 mg of oral biotin per day produces concentrations of biotin in plasma of1,300 nmol/l, well above the Km for biotin of HCS of most patients with HCSdeficiency. Determinations of the Km values for biotin of the HCS in patientsshould permit the development of rational treatment with doses of biotinsufficient to maintain the plasma levels of biotin at some small multiple of theKm value of each patient.

ACKNOWLEDGMENTSWe express our appreciation to the following for providing fibroblasts of their pa-

tients: Drs. J. V. Leonard, S. Packman, Y. E. Hsia, R. A. Gravel, K. S. Roth, H. Cann,J. Thoene, C. Bachmann, J. M. Saudubray, and R. Baumgartner.

REFERENCES

1. GOMPERTZ D, DRAFFAN GH, WATTS JL, HULL D: Biotin-responsive 13-methylcrotonylglycinuria. Lancet ii:22-24, 1971

2. SWEETMAN L, BATES SP, HULL D, NYHAN WL: Propionyl-CoA carboxylasedeficiency in a patient with biotin-responsive 3-methyl-crotonylglycinuria. PediatrRes 11:1144-1147, 1977

3. COWAN MJ, PACKMAN S, WARA DW, ET AL.: Multiple biotin-dependent carboxylasedeficiencies associated with defects in T-cell and B-cell immunity. Lancet ii: 115-118, 1979

4. SAUNDERS M, SWEETMAN L, ROBINSON B, ROTH K, COHN R, GRAVEL RA: Biotinresponse organicaciduria-multiple carboxylase defects and complementation stud-

335

ies with propionicacidemia in cultured fibroblasts. J Clin Invest 64:1695-1702, 19795. ROTH K, COHN R, YANDRASITZ J, PRETI G, DODD P, SEGAL S: Beta-methylcrotonic

aciduria associated with lactic acidosis. J Pediatr 88:229-235, 19766. SWEETMAN L: Two forms of biotin-responsive multiple carboxylase deficiency. J

Inher Metab Dis 4:53-54, 19817. SHERWOOD WG, SAUNDERS M, ROBINSON BH, BREWSTER T. GRAVEL RA: Lactic

acidosis in biotin-responsive multiple carboxylase deficiency caused by holocar-boxylase synthetase deficiency of early and late onset. J Pediatr 101:546-550, 1982

8. BARTLETT K, GOMPERTZ D: Combined carboxylase defect: biotin-responsiveness incultured fibroblasts. Lancet ii:804, 1976

9. WEYLER W, SWEETMAN L, MAGGIO DC, NYHAN WL: Deficiency of propionyl-CoAcarboxylase in a patient with methylcrotonylglycinuria. Clin Chim Acta 76:321-328,1977

10. PACKMAN S, SWEETMAN L, YOSHINO M, BAKER H, COWAN M: Biotin-responsivemultiple carboxylase deficiency of infantile onset. J Pediatr 99:421-423, 1981

11. BuRRi BJ, SWEETMAN L, NYHAN WL: Mutant holocarboxylase synthetase: evidencefor the enzyme defect in early infantile biotin-responsive multiple carboxylasedeficiency. J Clin Invest 68:1491-1495, 1981

12. WOLF B, GRIER RE, PARKER WD JR, GOODMAN SI, ALLEN RJ: Deficient biotinidaseactivity in late-onset multiple carboxylase deficiency. N Engl J Med 208:161, 1983

13. WOLF B, GRIER RE, ALLEN RJ, GOODMAN SI, KIEN CL: Biotinidase deficiency: theenzymatic defect in late-onset multiple carboxylase deficiency. Clin Chim Acta131:273-281, 1983

14. WOLF B, HSIA YE, SWEETMAN L, ET AL.: Multiple carboxylase deficiency: clinicaland biochemical improvement following neonatal biotin treatment. Pediatrics68:113-118, 1981

15. PACKMAN S, COWAN MJ, GOLBUS MS, ET AL.: Prenatal treatment of biotin-responsivemultiple carboxylase deficiency. Lancet i: 1435-1439, 1982

16. PACKMAN S, SWEETMAN L, BAKER H, WALL S: The neonatal form of biotin-responsive multiple carboxylase deficiency. J Pediatr 99:418-420, 1981

17. SAUNDERS ME, SHERWOOD WG, DUTHIE M, SURH L, GRAVEL RA: Evidence for adefect in holocarboxylase synthetase activity in cultured lymphoblasts from a pa-tient with biotin-responsive multiple carboxylase deficiency. Am J Hum Genet34:590-601, 1982

18. ROTH KS, YANG W, ALLAN L, SAUNDERS M, GRAVEL RA, DAKSHINAMURTI K: Pre-natal administration of biotin in biotin-responsive multiple carboxylase deficiency.Pediatr Res 16:126-129, 1982

19. ROTH KS, YANG W, FOREMAN JW, ROTHMAN R, SEGAL S: Holocarboxylase synthe-tase deficiency: a biotin-responsive organic aciduria. J Pediatr 96:845-849, 1980

20. PACKMAN S, CASWELL N, GONZALEz-RIos MDC, ET AL.: Acetyl-CoA carboxylase incultured fibroblasts: differential biotin dependence in the two types of biotin-responsive multiple carboxylase deficiency. Am J Hum Genet 36:80-92, 1984

21. BAUMGARTNER R, SUORMALA T, WICK H, BACHMANN C, JAGGI KH: Biotin depen-dency causing multiple carboxylase deficiency in vivo. Pediatr Res 15:1189, 1981

22. BAUMGARTNER R, WICK H, BACHMANN C, LEHNERT W, NEIDERHOFF H: Biotin depen-dency in 3 patients suggesting a defect in biotin resorption or transport, in Advancesin the Treatment ofInborn Errors ofMetabolism, edited by CRAWFURD MD'A, GIBBSDA, WATTS RWE, Proc. 2nd Clinical Research Centre Symposium, Sept. 1981,New York, John Wiley, 1982, p 310

23. THOENE J, BAKER H, YOSHINO M, SWEETMAN L: Biotin-responsive carboxylasedeficiency associated with subnormal plasma and urinary biotin. N Engl J Med304:817-820, 1981

24. THOENE J, WOLF B: Biotinidase deficiency in juvenile multiple carboxylasedeficiency. Lancet ii:398, 1983

25. MUNNICH A, SAUDUBRAY JM, COTISSON A, ET AL.: Biotin dependent multiple car-

336 BURRI ET AL.

HOLOCARBOXYLASE SYNTHETASE

boxylase deficiency presenting as a congenital lactic acidosis. Eur J Pediatr137:203-206, 1981

26. WILKINSON GN: Statistical estimation of enzyme kinetics. Biochem J 80:324-332,1961

27. GHNEIM HK, BARTLETT K: Mechanism of biotin-responsive combined carboxylasedeficiency. Lancet i:1187-1188, 1982

28. GAUDRY M, MUNNICH A, OGIER H, ET AL.: Deficient liver biotinidase activity inmultiple carboxylase deficiency. Lancet ii:397, 1983

29. WRIGHT LW, DRISCOLL CH, BOGER WP: Biocytinase, an enzyme concerned with thehydrolytic cleavage of biocytin. Proc Soc Exp Biol Med 86:335-337, 1954

30. SWEETMAN L, NYHAN WL, SAKATI NA, ET AL.: Organic aciduria in neonatal multiplecarboxylase deficiency. J Inher Metab Dis 5:49-53, 1982

31. BARTLETT K, NG H, LEONARD JV: A combined defect of three mitochondrial car-boxylases presenting as biotin-responsive 3-methylcrotonyl glycinuria and 3-hydroxyisovaleric aciduria. Clin Chim Acta 100: 183-186, 1980

The Hereditary Cancer Institute of Creighton University is sponsoring an IN-TERNATIONAL MEETING ON HEREDITARY GYNECOLOGIC ANDBREAST CANCER on June 23-25, 1985, at the Red Lion Inn in Omaha,Nebraska. This meeting will be devoted to the genetics of gynecologic andbreast carcinoma. In addition to focusing upon primary genetic factors, majorattention will be given to epidemiology, biomarkers, pathology, cancer control,and cancer registries as well as to patient and physician education. Thesesessions will also coincide with the inaugural meeting of the International Soci-ety for Hereditary Gynecologic and Breast Cancer, which is committed toeducational programs involving both patients and physicians and will serve as abasis for interchange of scientific knowledge, including sharing of biologicalsamples, through a network of national and international registries of cancer-prone families. For information, contact: Henry T. Lynch, M.D., HereditaryCancer Institute, Creighton University, Omaha, NE 68178.

337