LETTER TO THE EDITOR

Homocystinuria due to cystathionineb-synthase de®ciency associatedwith megaloblastic anaemia

DEAREAR SIRIR,

Homocystinuria is an inborn error of amino acidmetabolism in which homocystine, the disulphide of

homocysteine, is excreted in the urine as a conse-

quence of elevated homocysteine levels in the blood.The most common cause of homocystinuria is a

cystathionine b-synthase (CBS) de®ciency, which is

inherited as an autosomal recessive disorder and ischaracterized by mental retardation, lens disloca-

tion, skeletal abnormalities and thrombotic vascular

disease [1]. We encountered a patient with CBS-defective homocystinuria complicated with mega-

loblastic anaemia due to hypovitaminosis of folate

and vitamin B12 and attempted the administrationof folate and vitamin B12. We report the bene®cial

clinical course of the patient, together with thepathogenesis of megaloblastic anaemia in this

patient.

Case report

A 20-year-old man was admitted with dyspnoea oneffort and gingival haemorrhage. He had been

diagnosed with homocystinuria at the age of two,

when his sibling was identi®ed with hypermethio-ninuria due to CBS de®ciency as a result of national

neonatal screening and following CBS assay with

cultured skin ®broblasts. He had been hospitalized inour hospital with sinus thrombosis at the age of 16,

and then administered drugs including pyridoxine

hydrochloride, aspirin and dipyridamole. The clinicalresponse to administration of vitamin B6 was not

satisfactory.

On admission, physical examination revealed alimited IQ (58), funnel chest, ectopia lentis and joint

laxity of the ankle. The initial laboratory studies

showed the following: RBC, 1.11 ´ 106 lL±1; Hb,

4.1 g dL±1; MCV, 111.7 fL; MCH, 36.9 pg; WBC,

5200 lL±1 with normal differential count; PLT,

6.9 ´ 104 lL±1; AST, 61 U L±1 (normal range8±38); ALT, 46 U L±1 (normal range 8±38); LDH,

9088 U L±1 (normal range 106±211). The serum

folate concentration was 1.2 ng mL±1 (normalrange 2.4±9.8) and vitamin B12 level was

157 pg mL±1 (normal range 249±938). Antibody

for intrinsic factor was negative. Bone marrowaspiration showed hypercellular marrow

(31.2 ´ 104lL±1), myeloid/erythroid ratio of 1.24,

and marked megaloblastic changes in both theerythroid and granulocytic series. Upper gastroin-

testinal endoscopy and radiography of the small

intestine revealed no mucosal abnormality. Thefaecal concentrations of a1-antitrypsin and lactoff-

erin were within the normal ranges. Plasma amino

acid analysis revealed a methionine level of0.26 lmol mL±1 (normal range 0.01±0.02) and a

homocystine level of 0.05 lmol mL±1 (normallyundetectable). The urine methionine level was

0.16 lmol mg±1 creatinine (normal range

0.01±0.06), and the urine homocystine level was0.26 lmol mg±1 creatinine (normally undetectable).

Cystathionine was undetectable in either plasma or

urine. The urine methylmalonate level was normal.Treatment with parenteral administration of

mecobalamin 500 lg day±1 for a week resulted in

an effective rise in PLT and a marked decrease inLDH concentration, whilst megaloblastic anaemia

persisted. After the therapy was changed to intra-

venous administration of mecobalamin 500 lg day±

1 and folic acid 15 mg day±1, prompt rises in Hb

concentration were observed. His urine methionine

level elevated further and homocystine decreased.The treatment with oral mecobalamin

1500 lg day±1 and oral folic acid 15 mg day±1 is

continuing, and the Hb level has improved to thenormal range.

In the present case and his sibling with CBS

de®ciency (18-year-old female), examinations were

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ã 2001 Blackwell Science Ltd 453

performed of a complete blood count, serum folic

acid, serum vitamin B12, three urine amino acids,

urine methylmalonate and two urine pyrimidinederivatives (Table 1). Analyses of patients' urine

were performed using a rapid and simple procedure

consisting of urease-treatment of urine, stableisotope dilution and gas chromatography-mass

spectrometry, enabling the simultaneous quanti®ca-

tion of methionine, homocystine, cystine, methyl-malonate, orotate, and uracil [2,3]. The results

of laboratory studies showed that his sibling had

a normal Hb concentration, a normal folicacid concentration, and a low serum concentration

of vitamin B12 (12.7 g dL±1, 4.4 ng mL±1 and 165

pg mL±1, respectively). Urine methionine andhomocystine levels were signi®cantly increased,

whereas methylmalonate was within the normal

range. After the treatment with oral folic acid andmecobalamin, the urine methionine level of his

sibling elevated further and homocystine decreased.

Interestingly, urine orotate concentrations weremarkedly increased in both family members, and

decreased into the normal range after treatmentwith folate and vitamin B12 (Table 1).

Besides homocystinuria due to CBS de®ciency, two

other types of inherited homocystinuria are charac-terized by defective remethylation due to N5,10-

methylenetetrahydrofolate reductase (MTHFR)

de®ciency, and caused by N5-methyltetrahydrofolatehomocysteine methyltransferase de®ciency due to

the defective synthesis of methylcobalamin and

deoxyadenosylcobalamin [4,5]. The major biochemi-cal ®ndings in both of them are moderate homocy-

stinuria with low or relatively normal levels of

plasma methionine, and the condition of the latter

is accompanied by combined homocystinuria and

methylmalonic aciduria. The present patient shouldbe diagnosed as homocystinuria due to CBS de®ci-

ency, because of his clinical symptoms, hypermethi-

oninaemia, undetectable plasma and urinecystathionine and normal urine methylmalonate.

There have been no previous reports of homocystin-

uria due to CBS de®ciency associated with megalob-lastic anaemia. Some patients with CBS de®ciency

have had mild folate de®ciency at the time of

diagnosis [6±9]. In no instance was the folatede®ciency severe enough to cause clinical

manifestations, with the single exception of moderate

macrocytic anaemia in a patient who was receivingphenytoin [6]. The cause of the mild folate de®ciency

seen in some of these untreated CBS-de®cient

patients has been proposed to be excessive utilizationof 5-methyltetrahydrofolate in the methylation of

homocysteine to form methionine. This patient had

severe de®ciency of both folate and vitamin B12. Noresponse to pyridoxine in this patient had been

observed until megaloblastic anaemia occurred. Hehad been receiving a normal diet and had not

received anticonvulsants until admission. Moreover,

he had no gastrointestinal lesions, and serum anti-body for intrinsic factor was negative.

After treatment with folate and vitamin B12, the

megaloblastic anaemia improved dramatically, theurine methionine level elevated further and homo-

cystine decreased. Thus, we speculate that the

severe folate and vitamin B12 de®ciency and meg-aloblastic anaemia of our patient had the following

cause: the excessive utilization of folate and vitamin

Table 1 Urinary metabolite levels in patients with homocystinuria

Case Hcys Met Cys MMA Orotate Uracil Hb Vitamin B12 Folate

1

Before 26.36 11.82 0.97 1.03 9.60 8.85 4.1 157 1.2

After 4.35 30.73 2.07 0.48 0.43 4.66 12.6 533 41.5

2

Before 21.09 12.55 1.35 2.04 2.16 3.38 12.7 165 4.4

After 12.55 17.32 1.02 1.57 0.21 8.83 13.4 214 5.8

Control (n � 27) (range)

Mean UD 3.00 7.7 1.83 0.72 8.25 M16.0 � 2.0 233±914 2.4±9.8

SD UD 1.58 4.84 0.94 0.33 4.6 F 14.0 � 2.0

Hcys, homocystine; Met, methionine; Cys, cystine; MMA, methylmalonate; UD, undetectable.

Values are expressed as mmol mol)1 creatinine except for Hb, folate and vitamin B12; Hb, folate and vitamin B12 were determined by

routine laboratory test (g dL)1, ng mL)1 and pg mL)1, respectively).

Case 1, the present case; Case 2, the sibling of case 1. Data for both cases are presented before and after treatment.

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ã 2001 Blackwell Science Ltd Journal of Internal Medicine 250: 453±456

B12 increased, and subsequently the accumulation

of homocysteine accelerated re-methylation of

homocysteine to methionine. We believe that folatewas highly involved in the pathogenesis of mega-

loblastic anaemia, primarily because no prompt rise

in Hb level of our patient was observed by thetreatment with vitamin B12 alone, and the serum

folate concentration of his sibling without anaemia

was normal.The mechanism by which vitamin B12 de®ciency

causes megaloblastic anaemia remains controversial.

There is, however, general agreement that DNAsynthesis is impaired due to interference with the

folate metabolism. Vitamin B12 acts as a cofactor in

the methylation reaction of homocysteine to methio-nine, in which 5-methyltetrahydrofolate (methyl-

THF) is converted to tetrahydrofolate (THF) (Fig. 1).

Methyl-THF is the form of folate which all body cells,including those of the bone marrow, receive from

plasma. It was originally postulated that vitamin B12

de®ciency produced a block in the folate metabolismby trapping folate as methyl-THF, thus depriving

cells of THF and therefore of all the other coenzymeforms of folate derived from THF [10]. A more recent

theory suggested that the reduced supply of methio-

nine leads to reduced availability of `activatedformate' and hence of formyl THF, and that it is

this defect that results in the failure of folate

coenzyme synthesis [11].

In our study, the patient and his sibling showedincreased urinary concentrations of orotate, which

decreased into the normal range after treatment

with folate and vitamin B12. Orotate was the onlyintermediate in pyrimidine biosynthesis, which was

analysable by the procedure using urease-treatment

of urine, isotope-dilution and gas chromatography[2]. Conversion of dUMP to dTMP, catalysed by

thymidylate synthase, is folate-dependent, and pyr-

imidine biosynthesis is regulated by end-productinhibition (Fig. 1). Therefore, it was suggested that

folate and vitamin B12 de®ciency caused impaired

DNA synthesis and enhanced pyrimidine biosynthe-sis, together with orotic aciduria and megaloblastic

anaemia. Analysing the urinary excretion of orotate

may be useful for monitoring the biochemicalconditions of impaired DNA synthesis in patients

with megaloblastic anaemia.

S . I S H I D AS H I D A1 , H . I S O T A N IS O T A N I

1 ,

K . F U R U K A W AU R U K A W A1 & T . K U H A R AU H A R A

2

1Department of Internal Medicine,

Hirakata City Hospital, Osaka, Japan2Division of Human Genetics,

Medical Research Institute,

Kanazawa Medical University, Ishikawa, Japan

Fig. 1 Metabolism of methionine, homocysteine and cystathionine, and its relationship to folate metabolism, a cofactor vitamin B12 and the

DNA synthesis. Glu, glutamic acid; Asp, aspartic acid; THF, tetrahydrofolate; SAM, S-adenosyl methionine; SAH, S-adenosylhomocysteine;CBS, cystathionine b-synthase.

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Received 21 September 2000; revision received 7 November

2000; accepted 20 December 2000.

Correspondence: Dr Shimon Ishida, Department of Internal Medi-

cine, Hirakata City Hospital, 2-14-1 Kinya-honmachi, Hirakata,

Osaka 573-1013, Japan (fax: +81-72-847-2825).

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