VITAMIN B12-RESPONSIVE NEONATAL MEGALOBLASTIC ANEMIA AND

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Vitamin

B12-Responsive With

Associated

By Lynnette We

present

blastic

findings

anemia,

that

included

There

was

therapy

and

methylmalonic

normal

but

reactions

in the synthesis

ciency

of

tory

the

that

for

and

by

methylmalonic

two end

products

by

cobalamin-dependent

the

Schuh

and

reflect

The

suggested

to

be

methionine

failure

activity

for at

and

vitro

of

methionine

not

We

patient previously concentration but of culture medium. genetic

the Department Australia; the

Immunology,

Monash

Victoria, rics,

Australia;

Queen

ofllematology. Departments Medical

and

Victoria

Monash

Medical

University Centre,

but to

was to

reducing

April

Presented ogy

Meeting,

New

Supported Research

29.

1986;

accepted

in abstractform by Council

Address

Orleans, from

charge

payment.

“advertisement” indicate this fact. (C) 1987 by Grune

requests

(Blood

National MB.

Hospital

never a very smiled

ofthis

article

This

article

must

accordance

Der

with

were

he had

a febrile

week,

after

therefore /8

U.S.C.

Austra-

in part hereby

Inc.

the

hospital

infant,

symmetrical,

during

spasticity

with

at 59

these

ceased.

21%,

MCV

3.5%,

obvious

increased ethanolamine.

liquid

x 109/L.

homocystine

amino

Bone

acid

and

marrow

increased acid

showed analysis

8.6

a

was

not

Three after anemia

101

fl,

x lOt/L,

neutro-

eosinophils

was markedly

cellular

of the urine serine,

was

not

normal performed

showed

alanine,

leucine,

detected,

organic

MCH

anisocytosis,

71%,

electrophoresis

Methylmalonic

chromatography

was

lymphocytes

High voltage

disturbed,

revealed

hematocrit

719

noted.

truncal

phenobarbitone,

admission

WBC

head

present

seizure.

reticulocytes

were

were

when

g/dL,

and

to

extension

g/dL,

The

and

he developed

given

on

On floppy,

were

7.7

2%. Platelets

marked

reflexes

53.2

and megaloblastic.

solely

g,

a generalized

he was

poikilocytosis.

pale,

4,730

weeks

spine

hypersegmentation,

one

3 cm below the costal nodes not enlarged.

hemoglobin and

about

a

pg, MCHC

1985)

by page

was

35,5

with

time

unsmiling.

with

27%

and

At that

abnormalities

he had

Investigations

the breast

lasting

weight

three and

he was

and he

neurologic

retraction

3,180

that

vomiting

weeks cm,

the subsequent

seizures

Medical Depart-

10

no other

head

macrocytosis

Prahran,

and

weight

stated

six weeks.

unresponsive

height

and

However,

phils

of Hematol-

§1734

diarrhea

who had had

birth from

at about

circumference 39.3 cm. The liver was palpable margin, the spleen not palpable, and lymph Despite his generalized hypotonia, deep tendon

and

acid before

gas

pattern. treatment.

Two weeks after the first dose of vitamin B I 2, no homocysteine was detected in the plasma while methionine was 4 zmol/L (n, 5 to 34). Red cell folate was 1,280 nmol/L of packed cells (n, 230 to 2,200), serum

& Stratton,

to

with

parents

fed successfully

he became

which

Road,

be

illness

which

admission

but

to his parents

of Pediat-

Weyden.

defrayed

baby

responded

developmental

parents

gestation, His

at the age of

and

of unrelated

at birth.

until

and

with

described

was referred

at 38 weeks

condition

responsive

and

Plasma

costs in

Van

Commercial

He was born

followed

66.44a,

Health

infant

newly

hypotonia,

He was the first born losses.

greatly to Dr

0006-4971/87/6904-0028$3.OO/O

1 128

1985

the

another

this

infant,

ofanemia,

further

of Australia.

reprint

publication

Society

for investigation

Prahran,

7, 1986.

American

December

grants

ment ofHematology. Alfred Victoria, 3/81 Australia. The

November

at the

char-

REPORT

male

day

Victoria,

here

characterize

hospital

ha. Submitted

E

may

metabolism.

and on the fifth

Hospital. Department

may

a Caucasian

unresponsive

and

exhibited

assay

Melbourne,

from

cobalamin

anemia and homocystinuria with lymphoblast methionine synthase activity, in contrast to that of the

ofcobalamin

g, and was in good

decreased

which

Alfred

significantly with

described, is independent of assay thiol dependent in part on the cobalamin levels These findings suggest that considerable

heterogeneity

10 weeks

Alfred Hospital. Prahran, of Medicine, Pathology and

School.

reduced

Inc.

CASE

and From Victoria,

cobal-

the

heterogeneity

describe

megaloblastic fibroblast and This reduced

develop-

content

low

conditions.4

regression.

bound

included

at

(thiol)

no fetal

syndrome

synthase,

but

& Stratton,

The patient,

favorably

adenosylCbl

by Grune

Recently

with

cobalamin

findings

not

optimal

man.

this

differ infant

genetic

S 1987

accompa-

homocystinuria

for

mutation.

that

patient’s

increased of

nor-

with

as these

responded

this

patient

described

of both

metabolized

in

reduction

In

are

an infant

basis

of

or

synthesis

normally

who

but

properties

normal

cobalamin,

states

suggest

mutation

obliga-

homocystinuria

anemia,

molecular

methylCbl

unique

and

described

synthase.

fibroblast

11,2 an

reactions

mental delay, megaloblastic not methylmalonic aciduria hydroxoCbl.

to inherited with a defi-

either

latter

intermediates

coworkers3

(meth-

from homocysteine.’

The

and

neonatal reduced activities.

from

consequent

aciduria

acterize

cobal-

synthase

occurs

methylCbl.’

lym-

methylmalonylCoA

uptake of intracellular

cellular

disease

predomi-

These

transcobalamin

to impaired

for

previously

was

but

of the

markedly

characteristics

of our

the

activity

conditions,

culture

with

The

synthase

of

(thiol)

accompanied

medium

adenosylcobalamin

fibroblasts

ofsuccinylCoA

anemia

of activity in

of

synthase

reducing

concentration.

that

meth-

occurs

of methionine

protein

leading

adenosylCbl

by

generation

metabolism

carrier

mediator

mutations nied

catalyzed

megaloblastic

and

(Cbl)

of methionine

of cobalamin

patient’s

levels

that

methionine

of assay

lymphoblasts

Der Weyden

and

reduced

methionine

reduced

of cellular

or methylCbl.

in the

and

Neonatal

by the

cobalamin

Cul-

a

B. Van

reduced

The

Homocystinuria

Activity

Martin

markedly

amin

requirement

proportion

adenosylCbl

methylmalonylCoA

defects

a growth

lymphoblasts

CELLS

(adenosylCbl)

ylCbl)

the

patient’s

as either

mediate

mutase

and

and

development.

and

independent

excretion.

showed

incorporation

normal, in the

nantly

amins

activity

Cobalamin

N HUMAN

neurologic lymphoblasts

Clark,

mal

hematologic

homocystine

C.L.

normal.

seizures.

and

Synthase

were

megalo-

Cyanocobalamin

complete

diminished and

was

ylcobalamin

I

aciduria. by

Arthur

dysfunction tonic

Anemia

Methionine

Sawyer,

neonatal

delay

synthase

methionine.

with

developmental

fibroblasts

phoblasts

infant

neurologic

recovery,

methionine

Margaret

and

subsequently

tured

an

accompanied

neurologic and

J. Hallam,

on

Megaloblastic

Reduced

homocystinuria, no

was

Neonatal

folate

and

renal

tion

showed

41 nmol/L,

function

tests

and were

no abnormality,

Blood,

B12

1 17 pmol/L

normal. and

Cerebral an

EEG

(n,

160 to 500).

ultrasound showed

medium

Liver

examinavoltage

Vol 69. No 4 (April),1987: pp 1128-1133


CONGENITAL

with

METHIONINE

no consistent

SYNTHASE

DEFICIENCY

focal,

or epileptiform

asymmetry, folate

and

antibodies excluded

to intrinsic factor. Transcobalamin II deficiency was by normal findings for patient’s serum mediated 57Couptake elution

after

admission

latter

treatment

as 1,000

peaked

progressive

rise

On treatment

returned

to normal,

no longer

I 2 months.

At

assessment

at

good

3 years

100

progress

born

until

Epstein-Barr

fifth

57Co-

One

folate

and and

toward

the daily.

there

was

of red

cell

normal

Anticonvulsants to I ,000

was

discontinued.

normal

limits

2 years at

her

birth.

At

normal cot the

6 weeks

she

a week

had

A

and

a Hb

12.7

She Post

to estab-

lymphoblasts

from

MATERIALS Cell lines.

Human

individuals

were

(CS

medium

this

(FBS

formed

lymphoblast

mother,

father,

lymphoblast

cell

lines

lines

were

were

(15

maintained

mg/L)

10%

from

the

under

normal

vitamin

Bl2

Kansas

City)

with

indicated

studies

the

appropriate

were

in

with

10%

culture

initiated

at

a cell

l05/mL

respectively

with

counts

performed

using

were

trypan

blue

Fibroblasts Laboratories)

condi-

nmol/L

(5

one

density

of

indicated

as the

2 x

culture

index

at

added

at

growth

l03/mL

and

medium

5 x

conditions,

described with

37#{176}C and

assays.

Lymphoblasts

as described,

centrifuged

Enzyme harvested

0.25%

trypsin

subsequent

washed

ofcells

were

thrice

with

in appropriate

performed

by either

Cell

at 4#{176}C and

supernatant

bovine

(Flow

inactivation

serum

of

determined

THF

Kamely

(5-MTHF) in a total

buffer

pH 7.4 (Ajax

methionine

volume

(BDI-I

(SAM)

method

minor

standard

of0.2

mL:

130

Chemicals,

(Boehringer

of Lowry

was

pH

reaction

mmol/L

Australia), Australia),

Mannheim,

Intact

reaction

cell

cellular

but

assayed was

mixtures

assays.

routinely

also

with

under

determined nitrogen,

Incorporation

synthase

cobalamin

of

aerobic

anaerobically which

were

then

into

acid

l4C-5MTHF

activity

in patient

metabolism

in

‘s cells.

the

patient

and

acetic

control

acid.

from

lymphoblasts

Incorporation

l4C-propionate

yITHF defect

was

by patient’s

cells

normal,

that

was of

by trichloroof radioactivity from

14C

meth-

was significantly reduced (Table 1), suggesting a of methionine synthesis without an abnormality in the

function of methylmalonylCoA ties of enzymes of patient’s I .4

cells

of radioactivity TH F into protein

precipitated but

Altered

patient’s

explored by determining the incorporation from l4C-labeled propionate and methyl

synthesis,

nmol/hour/mg

mutase. lymphoblasts

namely

protein

The that

thymidylate

(mean,

level of activiare involved in synthase,

SE)

serine

6.5

±

hydroxyme-

thyltransferase 257 ± 61 nmol/hour/mg protein, or that of 5,10 methylene THF reductase 2.3 ± 0.3 nmol/hour/mg protein were comparable to the corresponding values of those activities in control lymphoblasts: I .8 ± 0.2 nmol/hour/mg protein. measured

by the

14C-5 mixture

was methyl con-

phosphate B-mercap-

.tmol/L

of

in

patient

and

6.7 ± 0.6, Methionine

control

cell

386 ± 36, and synthase was

extracts

with

and

Table

1 . Incorporation Into TCA

of 1 4C-Propionate

Precipitable

Material

and 1 4C-MethylTHF

of Control

and Patient’s

Lymphoblasts 14C-propionate’ 100 imol/L; 14 iCi4imol

Cells

1 4C-methylTHFt 25 imol/L; 25 iCi/mol

measured

sodium

Germany),

that

material was performed according to the method of Mellman et al’2 and incorporation of l4C-propionate (Amersham, England) into acid precipitable material was as described by Willard et al.’3 Incorporation of 57CO-cyanocobalamin (Amersham) 0.1 ng/mL was performed at two hours and 72 hours as described by Mellman et al’4 and for fractionation of intracellular cobalamin cells were extracted according to the method of Linnell et al’5 and cobalamins separated on a SP Sephadex C-25 column (Pharmacia, Sweden) in the dark, as previously described.4

and

200 mmol/L 275

was

7.4

Activity from

of MeCbl

the incubation.

et al.7

measured

modifications.”

The

the

during

was

above

using

reductase’#{176}were

synthase

dark.

Chemicals,

Extracts

HCL

was determined

of I4C-methionine

in the

tamed toethanol

formation

(PBS). Tris

hydroxymethyltransferaset

Methionine et al with

by the

Protein

(THF)

tetrahydrofolate described.

at 4#{176}C,

in liquid nitrogen or by 1,500 g for 10 minutes

by the

serine

flushing

sealed

at

for assay.

as standard

synthase,t

5,lO-methylene

thawing

were

I 0 minutes

10 mmol/L

centrifuged

used

albumin

as previously

method

were

fibroblasts

g for saline

usually

freeze

extracts

Thymidylate

confluent

phosphate-buffered buffers,

sonication.

or

at I ,000

by

synthase

as described

thymidylate

at 96 hours

of viability.6

by treatment

minute

acid

lymphoblast

as previously

harvested

for

and

FBS.

with

and

the

exclusion

were

or amino

Fibroblast

and

cell

vitamin

concentration.

Methionine

Methionine All

growth

3.7

omission

RESULTS

zg/L). In experiments with modifications of concentration of vitamm B12, folate, or methionine in the culture medium, cells were grown in a modified RPMI 1640 medium (K-C Laboratories, the

protein.

patient,

supplemented

the

fetal

previously.5

in exponential

and

with

EBV-trans-

as described

Laboratories)

normal

essential

with

established

of methionine

mmol/L

and

Australia).

individuals

(Flow

patient

minimum

Australia)

Laboratories,

and normal cell

the

Eagle’s

in

Parkville,

Flow

RPMI 1640 medium FBS. The concentration

from

grown

Laboratories,

serum

METHODS

fibroblasts

routinely

bovine

is 0.1

AND

skin

obtained

precipitable

made mortem

Attempts

and activity

the holoenzyme. Incubation was in the dark at 37#{176}C for 60 minutes. The reaction was terminated by adding 0.8 mL of ice-cold water. The mixture was passed through Bio-Rad AG-1X8 columns (BioRad Laboratories, Richmond, CA), and the columns were washed with an additional I .0 mL of water. The radioactivity in the pooled effluent was determined in a liquid scintillation counter. Enzymespecific activities are expressed as nmol of product formed/hour/mg conditions

modalities.

at 9 weeks.

of death.

(EBV)-transformed

at

physically

morphology.

death

cause

discontin-

in all

was

cell

were twice

Developmental

She

red

sudden

to reveal virus

later.

g

(MeCbl) (Sigma Chemicals, St Louis), 500 mol/ L DL-homocysteine (Sigma Chemicals, St Louis), prepared just before use from the thiolactone derivative, 600 smol/L (14C) 5-MTHF 2 MCi/mol (Amersham, England) and 150 to 160 zg of cell extract. The assay control was minus the addition of MeCbl and methylcobalamin

SAM,

week

B12,

(IM)

day,

no

were not successful.

individual

tions

the return

acid

fI, with

failed

both

reduced

is within

was

examination

on and

folic

normal

MCV

serum

intramuscularly

23%

B I 2 was

2 years

hematologically g/dL,

with

in his urine.

and

sibling

and

had

his general health improved, his muscle tone and there were no further seizures. Homocystine

detected

ued at six months,

lish

at

she

gel chromatography.

begun

in hemoglobin

changes.

female

was

and

lymphoblasts

with

of cyanocobalamin

Reticulocytes

was

by normal pattern

normal,

His

serum

cyanocobalamin

were

features.

mother’s

cyanocobalamin

BI2

1129

S-adenosyl

50 zmol/L

Control

825

±

163

479

±

31

Patient

788

±

190

154

±

26

‘cpm/

106 cells/4

hours.

Value

given

is mean

±

SE for three

separate

determinations. tcpm/ separate

106

cells/24

hours.

determinations.

1:P not significant. §P < 0.01, Student’s

ttest.

Value

given

is mean

±

SE for

seven


Table

2.

Methionine

Patient’s

Synthase

and Control

Activity

in Extracts

Fibroblasts

of Cultured

and Lymphoblasts Specific

2.

Activity’ protein) Holoensyme

Total

(nmol/hotx/mg Enzyme

Control

2.6

±

0.3

0.6

±

0.1

Patient

0.8

± 0.2

0.4

±

0.1

2.7

± 0.2

0.9

±

0.01

±

0.06

Cells

.i

Fibroblastst

2.0

Lymphoblasts Control

p

Mother

1.9

±

0.2

0.8

Father

2.5

±

0.3

1.3 ± 0.04

Patient

0.8

±

0.1

0.4

‘Values

given

are the mean

SE, in excess

±

1.5’

0.03

±

of six separate

I

determina-

tions.

tFibroblasts supplemented assayed

were cultured in Eagle’s minimum essential medium with 1 0% FBS. Cells were harvested upon confluency and

for

methionine

Lymphoblasts 1 0%

were

FBS and

assayed

cells

48

synthase

as

1 640 hours

described

medium

in the

initiation

control

levels

added

total

enzyme

and

lymphoblasts.

was

evident

with

of culture

and

from

cobalamin

up to 96 hours.

no

significant

at

P

Assay

methionine

to

synthase

synthase

that

conditions

under

of control

showed

aerobic

protein

were

2.2

(aerobic)

and

and

2.3

0.6 (anaerobic). Corresponding values for patient lymphoblasts were 0.36 and 0.21 (holoenzyme) nmol/hour/mg protein (aerobic) and 0.40 and 0.30 nmol/hour/mg protein

and

(anaerobic).

In data

not shown,

lymphoblast

extracts

yielded

synthase

activity,

alternatively,

the

excluding

activity

Effect synthase

of alteration ofpatient’s

the

by constituents

of cells.

strated

that

patient normal

with megaloblastic at conventional

methionine

varying

assay

mixing control and patient expected levels of methiopossibility

of patient’s

activation

synthase

methionine

in the

previously

increasing

cell

Effect

described

activities

25

either

mmol/L) and B mercaptoethanol, (7.5 to 200 mmol/L) were determined with lymphoblast extracts of the current patient. In both instances at all concentrations of thiol assayed the

shown

patient’s

fold with

methionine

significantly

synthase those

lower

patient

and

activity

than

control at

the

activities of control

ing conditions

produced nor

a unique

Methionine were

also

with

extracts, concentration

by these interaction

synthase assayed

(Figs 1 and lymphoblasts.

lymphoblast maximal

mmol/L) was approximately four mercaptoethanol (200 mmol/L), equivalent synthase.

(DTT

ofdithiothreitol

times that suggesting thiol of

of patient varying

DTT

are

with

and

control

assay

In

mmol/L.

With

methionine

these

synthase

concentration

addition,

of B

culture

synthase for

in

thiol activ-

of methylcobal-

substitution

and

of

methyl

3.

of

375

used

lymphoblasts

showed

a twofold

and

activity

this

Accumulation of and

and

control

in

methionine

now

distribution culture

of

in the

label

lymphoblasts

(0.5

sg/L, a five-

and the enzyme higher cobalamin

oflabeled

3).

conditions activity,

synthase

normal

levels cul-

(Table

identical

deprivation from

are with

exhibited

comparable under

was

methionine

accumulation

were

with

cultured

nmol/L)

cultured increase

level

cultured

cyanocobalamin

g/L,

on

synthase

or concentration

increase in methionine synthase, hydroxoCbl or cyanoCbl at the concentration

patient

lymphoblasts

lymphoblasts

concentration to 500

methionine

compounds Control

in the

of cobalamin

The

patient

cobalamin

nmol/L

Patient’s

The

concentration

activity.

control

Table

increasing

effect

fibro-

of varying

varying

0.375

neither

methionine

concentrations

the

extracts.

methionine

ture (25

observed with B that the reduc-

reagents DTT

2) were For

methionine of

200

fibroblast

cobalamin by hydroxocobalamin or cyanocobalamin assay system produced no significant differences.

0.2 to

concentrations

and

75

ities were 0. 1 7, 1 .45, and 2.5 nmol/hour/mg protein. Corresponding values for patient fibroblast methionine synthase were 0.04, 0.39, and 0.8 nmol/hour/mg protein. In data not

patient

extracts.

anemia and homocystinuria thiol concentrations. The

5,

control

amin (up to 50-fold), 5-adenosyl methionine (up to fivefold), and 5 methylTHF (up to fivefold) did not significantly alter the reduced methionine synthase activity exhibited by

assay conditions on methionine Rosenblatt et a14 have demon-

synthase

mercaptoethanol: concentrations,

shown,

of inhibition

reduced

of control

Fig 1 . Levels of methionine synthase of patient and control lymphoblasts assayed with varying B mercaptoethanol concentrations. Lymphoblasts were cultured in RPMI 1 640 medium supplemented with 1 0% fetal bovine serum, cells were harvested and assayed for methionine synthase as described in Materials and Methods, with B mercaptoethanol concentration ranging from 7.5 to 200 mmol/L. The values given are the mean of duplicate determinations. , Control; A, patient.

was effect of

blasts

200

mM

conditions.

lymphoblasts

nmol/hour/mg

activity interof patient

at all

synthase

anaerobic

and

fibroblasts

in culture

of methionine under

difference

(holoenzyme)

synthase

150

( 13 Mercaptoethanol)

0.01,

<

holoenzyme

in patient’s

lymphoblasts

lymphoblasts

Methionine

reduced

reduced

This

control

2). Both

(Table

were

in patient’s

and

both

100

50

activity.

different

activities

vals

nine

0.5

text.

supplemented

after

1.0

test.

t

without

activity

in RPMI

harvested

significantly

Student’s

0.9

synthase

cultured

for methionine

Values

or,

ET AL

HALLAM

1 130

range. cobalamin on

cell

and growth.

57Co-cyanocobalamin after

two

hours

in or 24 hours


CONGENITAL

METHIONINE

SYNTHASE

DEFICIENCY

1131

markedly reduced

C

reduced (Table 4). The growth rates when cultured

medium When

or when methionine 2 x l05/mL control

was replaced and patient

plated

in complete

growth

cells

was

9

5

0.2

x

±

z 0 I ‘U.

I05/mL.

105/mL

for the

studied.

Both

initiated E

When

in methionine for

patient 15

ing

(DITHIOTHREITOL)mM

no increase

homocysteine

0.1

x

at 96

culture

3.2

x

was cpm,

l0

patient

19

x

distribution

at

comparable:

control l0

2.4

cpm,

x

control

showed

and

x

cells;

l0

3.

Methionine

Lymphoblasts

Synthase

Cultered

Conditions’

the concentration

and

fibroblasts

Varying

Cobalamin

lymmajor of was

CyanoB

Conditions

12

HydroxoB

12

nmol/L

0.375

3.75

Methionine

187 synthase

375

Control

1.8

2.8(0.8)

8.1

9.0(1.3)

13.0

Patient

0.8

0.9(0.4)

1.7

1.7(0.7)

21

Culture conditionst MethylTHF

1

Methionine

2

HydroxoB

-

synthase

(nmol/hr/mg

4.8

2.1

Patient

1.5

0.6

‘Lymphoblasts with

1 0%

fetal

were calf

hydroxocobalamin

assayed

with

Cells

activity

were

cultured

at

2.tmol/L,

at 48 hours,

described Figures

concentration

RPMI

concentrations

synthase

hydroxocobalamin harvested

in standard varying

as indicated.

for methionine

tLymphoblasts

cultured

serum

were

in RPMI

of

1 640 3.75

and assayed

medium nmol/L

these

disease

genetic

entities

values.

to

0.9

±

lymphoblast

holoenzyme methionine

represent

CbIC

+3

to

enters

+2,

in

Distribu

12

two

different

mutations,

which

tion

include

anemia of our

forma-

not

and

confined

to mdi(I ) were

patient

CN-B12

a

exhibited

content,

and Adenosylc

(2)

obalamin

Extracts Ado-B12

CH3-B12

Cell Line

but

adenosylCbl

and Fibroblast

and

methylma-

disorders,

cobalamin

of Methylcobalamin

S03-B12

occurs

the

mutase

findings neurologic

but

in I ymphoblast

normally

precedes

methylmalonylCoA

Clinical

Cbl

that

and

reduccobalt of

methylCbl. Thus common to of affected cells to accumuand methylCbl and reduced

accumulating

methyl

4.

of

cell

of megaloblastic mutation.’ Cells

CbIC

defective

decreased

least

CbID

event

the

homocystinuria,

incidence with

complementation

at

and

an

into

activities synthase.’

aciduria,

not

genetic

an assumed defect in Cob(lll)alamin activity reduces the charge of the

cobalamin from after cobalamin

OH-B12

% of Total Cobalamin’

Lymphoblast

hours

and

without or with and methylTHF synthase

as

Control

3

2

25

60

7

Patient

4

0

3

87

7

Control

3

0

58

31

8

Patient

3

0

10

76

11

Fibroblast

in the text.

for methionine

control

increased

aciduria-homocystinuria. and

states

termed

both involve tase.’’7 This

‘Results owth balamin

are holoenzyme

methylmalonic

studies,

supplemented at 48

exhibit-

of methionine.

the patient’s x 106/mL.

± 0.1

require-

methionine,

had

and

of cobalamin metabolism resulting in and homocystinuria include the muta-

of biochemical

of cyanocobalamin

harvested

in the text. in parenthesis

1640

as described

defects anemia

protein)

Control

0.4

basis

Table + HydroxoB

was

of congenital the

variable viduals

protein)

for

hours

whereas

hours

Inherited megaloblastic

lonic

375

(nmol/hour/mg

96

x

control

growth

absence

106

0.1

±

lymphoblasts

substituted

at

tion of both adenosylCbl and these mutations is the inability late cobalamin as adenosylCbl

and Control

of

Cobalamin

Added

The

the

of Patient’s

Concentration

Culture

cells.

in human

Activity

Under

hours

was

2.4

the

at 96 x

0.3

±

DISCUSSION

On

24

for fibroblasts

cellular cobalamin is adenosylCbl, but methylCbl in patient’s lymphoblasts Table

patient at

cpm/106

adenosylCbl

in contrast

that

hours

cpm/106

17

of methylCbl

phoblasts

I0

two

Both

in the

also were

Growth 3.2

a preferential

numbers

density

were

and

the patient’s

106 (SEM)/mL,

density

tions in

showed

in cell

lymphoblasts’

was

lymphoblasts

with

was

cultures

x I 06 cells/mL.

lymphoblasts.

for methionine

When

Fig 2. Levels of methionine synthase of patient and control lymphoblasts assayed with varying dithiotreitol concentrations. Lymphoblasts were cultured in RPMI 1 640 medium supplemented with 1 0% fetal bovine serum. and cells were harvested and assayed for methionine synthase as described in Materials and Methods, with dithiothreitol ranging from 0.2 to 25 mmol/L. The values given are the mean of duplicate determinations. #{149}. Control; A. patient.

patient’s

cells

mmol/L)

Lymphoblasts

medium

control

lymphoblasts

ment 10

the

(0.2

lymphoblasts

ofO.5

for control

for the patient’s

media, growth at 96 hours fibroblasts and 3 ± 0.2 x

patient

replete

for

l06/mL

at 96 hours and

fibroblasts. and

at a cell density

hours

by homocysteine. fibroblasts were

homocysteine

patient

control

(SEM)/mL

or

medium,

x l0 (SEM)/mL

± 0.3

substituted for methionine in the was 8 ± 0.5 x 105/mL for control

0 ‘U

patient’s cells exhibited in methionine-replete

tions

expressed

as % of total

for 72 hours

in RPMI

(0. 1 ng/mL).

Values

in duplicate.

1640

cobalamin medium

given are means

in cell extracts

containing

following

57Co-cyanoco-

of two separate

determina-


utilized

the

methyl

group

of methylTHF

normal cells, and (3) incorporated propionate into protein at normal patient

exhibited

megaloblastic

less effectively

than

the radio label from rates. In addition,

anemia

with

patient’s

B12

on

magnitude increased

homocystinuria

and

activity,

serum

l4C our

it is of some

presentation

of increase cobalamin

that exhibited by control activity achieved by the cells

grown

the differing

This finding homocystinuria

may

characteristics

cally, the reduced patient’s lymphoblasts with

optimal

reducing

B-mercaptoethanol.

In

of

suggest activity

of

previously

the

addition,

either

normal

Specifi-

on

described

pletely

or

with

at the

patient

with

as

locus

congenital

homocystinuria and abnormal methionine synthase activity,3 only genetic complementation studies will resolve this question. That this altered methionine synthase is of significance for intact cells is suggested by the reduced methylCbl content

of the

impaired

patient’s

growth

when

rate

methionine

the

cell

absence

cells

of patient

was

fibroblasts

fibroblasts

substituted

of extracellular

is generated

methionine

and

and

in

methionine,

in human

synthase

cells

or

methylthio-containing

by

from

either

portion

this

activity

of

activity

to the

culture cobalamin the patient’s and activity.

these

An

cells

and

by

defects

homocysteine from

the a

cellular

pool.

increase

in

the

differing methionine

concentration

of

added to the culture medium was accompanied increase in this activity of control lymphoblasts with

a twofold

identical medium

change

for

patient’s

cells

changes

decreased

pattern

anemia,

of vitamin

pernicious

anemia

syndromes

are

B12

such

CbIE

homocystin-

or the infants

their

II acquired

of vegan

accompanying

by

of the described

as transcobalamin

mutation

the

characterized

for mutant

activity exhibited and developmental metabolism on the some comment.

in breast-fed

mothers,5’22

of

concentra-

characterization As the two

disorders now

enzyme

difference

and hypothesis. megaloblastic

and

in

of response

to cobalamin

altered methionine synthase features, including seizures impact of altered cobalamin neurologic system deserves CblC,

this

in vivo.

or

neurologic

diversity

if one

sets

aside the background of failure to thrive, listlessness, and irritability. Possibly the specific neurologic syndromes depend primarily on the developmental stage of the infant at the onset of vitamin deficiency. Whatever the manifesta-

Changing effects for synthase

our

is associated

nition

with

a favorable

outcome

as illustrated

in

patient.

cobalamin by a fivefold compared

cultured

conditions. Limiting cobalamin was accompanied by greater degrees

a fundamental

in hereditary

that

activity

tions, failure to diagnose the specific defect and to institute appropriate therapy can have dire consequences for the infant’s ultimate neurologic status,22’24 whereas early recog-

contribution

methionine

it is feasible

reduced

with

suggests

neonatal

for cobalamin.

cells is not entirely clear. It is and probably reflects stabi-

synthase

with

uria, and neurologic delay, the developing

with than

since both the responded com-

cobalamin-related

methionine

patients

Whether

and

cobalamin

Only purification will satisfy this

deficiency,

relative

conditions produced control lymphoblasts’

in culture

in

methylthioadenosine,

the

tion

the

synthase was less

conditions

is so, the differing

activity. enzyme

In

acid

byproduct of polyamine metabolism.20’2’ Methionine for cell growth is potentially a summation of the contributions of these two pathways. The differing growth capacity of our patient’s cells in culture when methionine has been replaced by homocysteine reflects in part the reduced methionine synthase

patient’s

by

amino

by

If this

the

medium

of reactions

of

an

for

patient’s

the absolute level of of the same order as in

of the

basis

our

Although

significance anemia

of cyanoCbl,

augmentation

of enzyme

turnover.”

in methionineevolving.’8”9

this

a series

and

lymphoblasts

culture

The mechanisms involved growth are controversial and

homocysteine.

dependent

lymphoblasts

standard

synthase ofculture of protein synthesis

lization

methio-

same

lymphoblasts, patient was

under

molecular

that

suboptimal.

have in vivo and megaloblastic

doses

by

methionine independent

Although

altered

to large

is mediated

The

con-

5-adenosylme-

activity.

our patient have a defect

DTT

substrate

or

reduced

the

that may

with

increasing

methylCbl,

effect

no

these findings nine synthase that

conditions,

methylTHF,

had

synthase.

methionine synthase activities of our and fibroblasts were not normalized

assay

centrations thionine

of methionine

interest

was

of patient’s methionine in the culture medium

not methylmalonic aciduria. These findings exclude CbIC or CbID mutations and are identical with those reported for the patient with CbIE disease. At variance are

but

of

ET AL

HALLAM

1132

under

in the culture of reduction of

ACKNOWLEDGMENT

We would the fibroblast continuing ration

like to thank I. Jack and Dr G. Young for establishing and lymphoblast cell lines of the patient and for their help

of the

and

advice

and

Maureen

Cross

for excellent

prepa-

manuscript.

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1987 69: 1128-1133

Vitamin B12-responsive neonatal megaloblastic anemia and homocystinuria with associated reduced methionine synthase activity LJ Hallam, M Sawyer, AC Clark and MB Van der Weyden

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VITAMIN B12-RESPONSIVE NEONATAL MEGALOBLASTIC ANEMIA AND

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