American Journal of Hematology 341121-127 (1990)
Function of Vitamin B,* in the Central Nervous System as Revealed by Congenital Defects Charles A. Hall Research and Medical Services, Veterans Administration Medical Center and Department of Medicine, Albany Medical College, Albany, New York
The 13 cases of methylcobalamin(MeCbl) deficiency presenting in early infancy have all been developmentally delayed, and the majority have had seizures, hypotonia, lethargy, and microcephaly. The CNS injury appears to occur during the first 6 months of postnatal life. The same symptoms are seen in acquired cobalamin (Cbl) deficiency in the same age group. MRI performed at age 18-19 months and after 13-14 months of large amounts of Cbl, in two cases showed delayed myelination, most pronounced in the cerebrum. Isolated MeCbl deficiency is the consequence of cblE and G mutations where the lesion is of a single Cbl-dependent enzyme, the methyltransferase. One effect of a deficiency of MeCbl, and of the associated failure of the methionine synthase reaction, is, therefore, an impairment of myelination of the brain of the newborn. The slow, but usually incomplete, improvement in psychomotor status after years of treatment with Cbl may be related to the eventual myelination. However, the hypotonia, lethargy, and impaired responsiveness react to treatment with Cbl within 24-48 hours, which suggests an expression of MeCbl deficiency on the CNS distinct from the delayed myelination. Although there is much to be learned, it is now clear that a normally functioning Cbl-dependent methyl transferase is required for development and function of the human brain. Key words: cobalamin, nervous system, methyltransferase
The biochemical basis for the central nervous system (CNS) expression of vitamin B,, (cobalamin, Cbl) deficiency in man continues to elude students of the vitamin [ 1,2]. Investigations have been impeded by the absence of suitable models. Although humans share with other mammals the basic metabolism of Cbl, the expression of Cbl deficiency varies greatly among species. Nevertheless, deficient monkeys, pigs, and fruit bats have supplied important information about the function of Cbl in the nervous system. Deficiency has been induced in these species both by dietary means and by inactivation of the Cbl with nitrous oxide. The main theories of Cbl activity are divided along lines defined by the two Cbl-dependent enzyme reactions. The enzymes are the 5-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthase [MS]; EC 2.1.1.13) for which methylcobalamin (MeCbl) is the coenzyme and the methylmalonyl CoA mutase (EC 5.4.99.2) for which adenosylcobalamin (AdoCbl) is the coenzyme. One concept holds that the accumulation of methylmalonate and propionate owing to the impairment 0 1990 Wiley-Liss, Inc.
of mutase activity by Cbl deficiency leads to the synthesis of abnormal fatty acids [ 3 ] . The incorporation of these unusual fatty acids into myelin, especially in the spinal cord, may cause the CNS lesions of Cbl deficiency. The other concept focuses on defective methionine (Met) synthesis, thus reducing the supply of adenosylmethionine (AdoMet), which is synthesized from Met; AdoMet is required for essential methylations in the nervous system [4]. The approach presented here is the application of the study of human congenital defects of Cbl metabolism in the search for an understanding of the function of Cbl in the nervous system. Although there are several pertinent defects, the present discussion will be confined for the most part to the cblE and G mutations. Whereas the principal objective of this specific presentation is a correlation of biochemistry and function for scientific purposes, Adress reprint requests to Charles A. Hall, Nutrition Laboratory for Clinical, Assessment and Research ( I 5 1 E), Veterans Administration Medical Center, Albany, NY 12208.
122
Hall
there are important clinical questions to be answered as well. In reality, the objectives are all the same since a thorough understanding of the disorders are essential to the prompt diagnosis, management, and prognosis. A recent review [ 5 ] and a subsequent report [6] give the details of the reported cases and the pertinent references. There are now 8 known persons with cblG mutation, 6 with cblE, and 1 still unclassified but obviously one of the two disorders. The key abnormality of both groups is a deficiency of MeCbl and reduced in vivo activity of the CbI-dependent MS [ 5 ] . Although formation of MeCbl is impaired by distinct abnormalities in cblE and G, respectively, the biochemical and clinical expressions are the same. The present analysis is based on personal biochemical data from 5 patients and data from the literature for the others. Much information was obtained from the physicians caring for these children and from their parents. There are adequate clinical data for 6 subjects, a modest amount for 4 more, and some for 2. Two are too recent for more than the initial presentation. These 14 presented in infancy, as is typical. The 15th case [7] was quite atypical but may be most important as a bridge between the childhood and adult expression of Cbl deficiency. Although this subject was symptomatic in early infancy, the full expression and diagnosis of cblG came not until the third decade. This case will be analyzed separately. Another child that was treated prior to birth and after without ever becoming symptomatic is excluded from the analysis of symptoms 181. The power of MeCbl deficiency in the derivation and testing of concepts lies in the restriction of the lesion to a single coenzyme that acts in a single enzyme system. All 13 cases presented with anemia, which is known to have been megaloblastic in all but one [ 5 ] . The infants were normal at birth and developed normally for the first few weeks. Symptoms began in at least 70% between 1 and 3 months of age. All were developmentally delayed. Other common symptoms have been lethargy, hypotonia, and seizures. At presentation some have responded poorly to stimuli even to the point of coma. In spite of an immediate initial response and a prompt resolution of the megaloblastic anemia, half of the children experienced new CNS symptoms or worsening of existing ones in the first 3-4 months after treatment was started. Biochemically the expression has been remarkably uniform [5,6]. Homocysteine (Hcy) or its products are increased in the blood and urine, but methylmalonic acid (MMA) is not. The plasma Met is low. The serum Cbl is within the normal range, but in one unreported case MeCbl was low. Serum and erythrocyte folate are normal or high. These data support the presence of a block in the MeCbl dependent MS only, as can be demonstrated in cultured fibroblasts and lymphoblasts. The
cells divide poorly when Met is replaced with Hcy in the medium. They take up labeled methyl folate poorly but take up propionate normally. They take up labeled Cbl bound to transcobalamin 11, internalize it, and bind it to the MS and mutase, respectively. Whereas labeled AdoCbl appears in the cell in the expected amounts, MeCbl is absent or much reduced. In cblE mutation the failure to form MeCbl appears to be in the essential step of reduction of the Co of the Cbl 191. In cblG the defect may be in the step of methylation requiring AdoMet [6]. Regardless, the failure to form functional MeCbl on the MS impairs the activity of the enzyme. To date the study of the Cbl metabolism of cells has been directed toward the establishment of diagnosis. What is now needed are deeper investigations of how and to what extent Met synthesis is impaired and what the biochemical consequences are. All children have responded to treatment. All regimens have included Cbl, and the usual form has been hydroxocobalamin (OH-Cbl), 1 mg i.m. one or more times a week. Five have been treated successfully with cyanocobalamin (CN-Cbl), but 2 of the 9 treated with OH-Cbl responded poorly to CN-Cbl initially and were changed to OH-Cbl. Nine have received either formyl folate or folic acid, and 4 have received methionine. Several have been given betaine, pyridoxine, MeCbl, or carnitine. Trials of different combinations have been common. Because of the heterogeneity of both the clinical expression and the mode of treatment it is impossible to offer firm guidelines for treatment. Some form of Cbl is, however, essential. The megaloblastosis responds promptly, and there is an immediate improvement in the responsiveness of the child. In the long term there is a steady gain in psychomotor development, but during the first 3 -4 months after treatment is started there may be both gains and losses in the neurologic status. For example, seizures may first be observed after treatment. Usually the child becomes free of seizures even without suppressive medication. One child, now age 6 years, has a normal IQ and another 5 % year old may have reached that point. The majority, now in the range of 3-9 years, have not caught up with their peers although all continue to improve. Two older children who were treated late may be more severely affected, but little about their status is available. Two others began treatment only recently. Speech has been the most resistant of the impediments. There are only limited data on the biochemical responses. Serum Met normalizes promptly, and serum Hcy falls. We have in two cases found some elevation to persist when total Hcy is measured by a sensitive method. The available data on other amino acids in plasma and spinal fluid are insufficient for meaningful analysis. In one case monitored closely by us serum and erythro-
Function of Vitamin B,,
cyte folate, all 5-methyltetrahydrofolate, remain elevated. We attempted measurement of the MeCbl in the post-treatment serum of two cases, but the astronomical amounts of OH-Cbl or CN-Cbl induced by treatment with the respective vitamin were overwhelming. The increase in serum Met and fall in Hcy suggest that within the cells synthesis of Met via the MeCbl-dependent reaction has been enhanced by the administration of Cbl. In order to correlate the functional and biochemical expressions of MeCbl deficiency the consequences must be better defined. Examination of the affected tissues, other than cultured cells, has been possible only in the one child known to have died with the defect [ 5 ] . The child’s illness was recognized in a general way but at a time when his illness, cblE mutation, had not yet been described. Effective treatment was given only for a short time, and he died at age of 5 % years. The case has not been reported fully, but such information about the anatomical observations as is available does not contradict the hypotheses to be expressed below. The brains of 1 1 children have been examined by computed tomography (CT) scans. Nine were abnormal, and in the 8 where sufficient detail was reported there was atrophy or hypoplasia of the brain. The ventricles were reported as enlarged in 4 patients. Dilatation was marked in one with what appeared to be a communicating hydrocephalus. There was no evidence of obstruction, and although a ventricular shunt was placed, CSF pressure was not recorded. No other abnormalities of the brain have been reported. The earliest, technically satisfactory magnetic resonance imagings (MRI) were performed in two 18 month old children with cblG. They had been treated for 13 and 14 months, respectively. Both were in phases of steady improvement, both continued to improve and are still improving, but both at ages 4 and 5 years, respectively, have impediments. The observations were quite similar. Some structures of the brain were myelinated, but the process was behind the expected at 18 months especially in the cerebral hemispheres. The age of myelination was estimated at 11-14 months. If, as in these cases, myelination was still delayed after a year of treatment, it very likely was delayed before treatment. There has been no recent MRI of one, but the second, now age 5 years, and a third 3 year old have normal MRI but are still impeded, especially in speech. Myelination of the brain undergoes a great burst in the last trimester of uterine life [ 101 and continues at a rapid pace in the year following birth [ 11,121. This is also a time of rapid increase in brain weight. The cblE or G child has a normal birthweight, and limited data show a normal head size. For three cblG children there exist serial measurements of weight, height, and head circumference from birth to age 1 year or more (unpublished).
123
The patterns are identical. Up to 6-8 weeks all measurements increase steadily and are well within the normal range, but then the curves flatten. With treatment, growth as measured by all 3 parameters resumes a normal pace but whether or not the deficit is made up remains to be seen. By the time of diagnosis 8 of 1 1 children for which data are available showed head circumferences in the 2nd to 10th percentile. The remainder were in the 50th to 75th percentile with the largest head in the child with hydrocephalus. Thus, brain growth, as reflected by head size as well as by the CT scans, slows in the early weeks of postnatal life as the MeCbl deficiency is expressed clinically. To this point one must conclude that deficiency of MeCbl with the accompanying reduced synthesis of Met impairs the normal process of the myelination of the brain of the newborn. It is probably significant that injections of OH-Cbl were started in the mother of the child with cblE diagnosed in utero at 25 weeks of gestation [8]. Treatment of the child has been continued since birth. That child, now 5 years old, has only a slight impediment of speech. MRI have not extended to the spinal cord, and for information about myelination of that structure one must await the report of the child that died. There is, however, more to cblE and G than defective myelination. For I 1 of the cases there are complete hospital summaries, literature reports, or personal observations. Immediate responses to treatment are recorded for 7 cases. These have consisted of enhanced responsiveness, alertness, and ability to feed. The improvement, which may be dramatic, occurs in 1-2 days and much too soon for there to be any change in the erythrocyte count. Transfusion, in the few cases where it has been given, has not induced this kind of a response. The change is even more premature to be caused by any structural improvement in the brain. Similar immediate CNS responses are observed in infants of a comparable age with acquired Cbl deficiency [13]. The phenomenon is also seen with the treatment of adult Cbl deficiency, but there are few descriptions in the literature and these casual reports are dismissed as anecdotal. Nevertheless, there is objective evidence of immediate responses, independent of the anemia, in the cerebral metabolism of adult Cbl deficiency [ 14,151. The basis for the immediate response to Cbl could be in the abrupt removal of a toxic substance or from an enhancement of some process such as neurotransmission. Body Hcy is increased in cblE and G [5] and in pernicious anemia [ 161. Serum Hcy falls with treatment of both deficiencies, but there has been no attempt at correlation of this decline with specific neurological manifestations. The possibility of defects of neurotransmission in Cbl deficiency has not been approached
Hall
124
’
formote
-Methylthioribose
//
FomylTHF
5)O
//
// Methenyl THF
k
I
\
Methylthio-
a
0
Adenosine+ Hcy AdoHcy
Pmpionyl CoA t
/acceptor Methylated (S) MethylmabnylCoA
‘fine
It
0
(R) MethylmalonylCo A
Ademylironsfemse
Thymidylato syniheiase
Methyl-transfemse
d TMP
I
dTTP
1
@
3MeIHP-
/
1
MeCbl \
,,kY
\
Cbl(1)
t
\
DNA
rlrcerihrd
nor
, I I
TCII-Cbl i-Txn
’
Succinyl Co A
Reductose
\
I
Cbl (It1
t
Reductose
t
*Cbl(m)
I
I
Cell Membrane
@
Fig. 1. The Cbl metabolism of cells. In MeCbl deficiency the entry of TC Il-Cbl is normal. The pathway from A through E is also unaffected. The defect lies in the failure to form MeCbl required for reaction C. The symptoms of cblE and G mutations could be produced by failures at several points. MeCbl accumulates, C, and could be toxic. Hcy accumu-
lates, C, and could be toxic. Tetrahydrofolate (THF) may be unavailable from reaction C to form the coenzyme for reaction 6.Met may be unavailable, C, for a variety of functions and especially for the synthesis of AdoMet. AdoMet is required for many essential methylations, F. Met is also a source of formate, D.
through appropriate biochemical experiments. However, in methylenetetrahydrofolate reductase deficiency the neurotransmitter metabolites homovanillic acid and 5hydroxyindoleacetic acid and total biopterins are low in the CSF [ 17,181. A more extensive comparison of this syndrome with cblE and G is given below. In the discussion that follows the reader must keep in mind that Cbl may have several activities within the CNS and that the effects of MeCbl deficiency may fall into two broad categories, structural and functional. Obviously function depends on structure, but MeCbl may affect function within the confines of a fixed structure. Neither are the consequences of treatment clear at this time. Myelination is restored, but the sequence among the many parts of the brain and cord may be distorted. Other anatomical lesions not revealed by imaging may persist. The enzymatic lesion may not be entirely compensated and maintenance processes could be impaired. Psychomotor development must depend upon an orderly sequencc of anatomical development, sensory input, and response by the brain. Compensation for the breakdown during the vital period of 1-6 months of age may never be complete. We do not yet know what can be accom-
plished by training. Finally, if MeCbl is required for day-to-day function, normality can be achieved only if the defect is overridden. The common processes by which all forms of Cbl deficiency disturb the structure and function of the CNS are illustrated in Figure 1 and could include any of the following: 1) toxicity of the accumulated methylfolate, 2) the trapping of folate in a non-functional form, 3) reduced synthesis of formate, 4) failure to synthesize folylpolyglutamate, 5 ) toxicity of the accumulated Hcy, and 6) reduced synthesis of adenosylmethionine (Ado), which would affect essential methylations. We have observed increased methylfolate in both the serum and erythrocytes of cblG. We have observed (unpublished) decreased division of cultured cells when methylfolate is supplied in amounts greater than the basic requirement. There is, however, no evidence of toxicity in other circumstances. The classic theory of the folate trap holds that methylfolate is not demethylated in the absence of a functioning MS and tetrahydrofolate is not available to form the essential folate coenzymes [ 11. The theory is usually applied as an explanation for the disturbed DNA synthesis in the lesion of hematopoiesis
Function of Vitamin B,,
known as megaloblastosis. Such an explanation in its purest form would not seem to apply to non-dividing neurons. However, the concept could be broadened to include processes other than DNA synthesis. Moreover, available folate could be required for the great increase in glial cells that accompany the late gestational burst of myelin synthesis [ 101. The formate starvation hypothesis [ I ] is a closely related explanation for the effects of Cbl deficiency. Substantial supporting evidence has been derived from studies of rats exposed to N,O. A decreased supply of Met from failure of MS activity reduces the available formate. Formate is a source of formyltetrahydrofolate and of methylene in the methylation of deoxyuridine, This hypothesis has not been applied directly to the function of the CNS. A fourth possibility is a disturbance in folate metabolism in Cbl deficiency owing to a failure to form folylpolyglutamate. The percent of erythrocyte folate as polyglutamate was 1% in the cells of one child with cblG treated for 1 1 weeks [6]. Between ages 12 and 24 months it remained at 25-27%. By 3 years it was 63%. The technique consisted of assay of folate by the L . casei method both with and without hydrolysis of the polyglutamates. The difference was the folylpolyglutamate. Scattered, incomplete data derived from the adult suggest that 60-90% of erythrocyte folate is polyglutamate, and by the same technique as we applied to the child’s cells we observed 90% or greater. There are no pediatric reference values. Preliminary data suggest that the fraction may be lower in cells from healthy children ages 3 months to 2 years than in cells from adults but not as low as in the cells of the child with cblG. Before this concept can be extended there must be studies of the ability of cultured cblE and G cells to synthesize folylpolyglutamate. Toxicity for the CNS by Hcy is a realistic possibility. Increased body Hcy characterizes cystathionine p synthase deficiency and is considered to be responsible for the almost universal vascular injury and thromboembolism associated with the syndrome. The story of lymphocyte toxicity from Hcy in adenosine deaminase deficiency is too complex to be told here. Vascular injury, including the blood vessels of the brain and presumably from the increased Hcy, has been present in the majority of those dying of methylenetetrahydrofolate reductase deficiency [ 181. Hcy accumulates in MeCbl deficiency [ 5 ] . Comparison of serum and urinary levels among cases and with levels in other disorders is difficult because of recent changes in methods. By a sensitive measure of total serum Hcy [19] we observed a level of 63 pmol/liter in a child with cblG prior to treatment and a fluctuation between 21 and 46 pmol/liter in the subsequent 3 years. The adult reference range by this technique is 6.9-12.1 pmol/liter, and preliminary data sug-
125
gest a lower range for young children. The cause of death in the one incompletely treated and fatal case of MeCbl deficiency was renal vascular disease [ 5 ] . Thromboembolism has not been observed in the others, but Hcy levels have been at least partly controlled by treatment. Vascular toxicity may increase with age, and most of those with cblE and G are still quite young. Although historically Hcy toxicity has been primarily vascular, there may be injury to other tissues as well. For example in unpublished experiments we have observed Hcy in the culture medium to suppress the formation of MeCbl in normal fibroblasts, a process already defective in cblE and G cells. Hcy toxicity might not be expressed in utero where it can be removed by maternal processes, thus explaining the normal development of infants with MeCbl deficiency before birth. The immediate responses to treatment of MeCbl deficiency could be explained by an abrupt lowering of body Hcy. Perhaps the most attractive theory for the action of Cbl in the CNS and the consequences of MeCbl deficiency is in the need for Met and AdoMet for essential methylations. There are many points of potential action but two could be methylation by AdoMet in the synthesis of proteins and of neurotransmitters. In MeCbl deficiency fibroblasts and lymphoblasts cannot use Hcy, methylfolate, and Cbl to synthesize Met and support cell division [6]. The same cells take up methylfolate poorly and transfer subnormal amounts of methyl groups to Hcy to form Met [5,6]. Serum Met is low but increases promptly with treatment. Missing so far have been any studies of AdoMet levels in cells or body fluids or of the ability of cultured cells to form AdoMet. Since Met is the source, impaired synthesis of AdoMet would be expected. Animal studies have been conflicting. Weir et al. observed suppression of MS activity by N,O to reverse the AdoMet/AdoHcy ratio in the spinal cord of the pig 141. Exogenous Met ameliorated the clinical and biochemical lesions induced by N,O in pigs and monkeys. However, Metz and van der Westhuyzen could demonstrate neither decreased tissue AdoMet nor decreased methylation of myelin basic protein in fruit bats exposed to N,O [2]. The conflict in observations could be explained by differences among species, but the question deserves a thorough reexamination. In any case the role of AdoMet must be examined in human systems before decreased AdoMet can be implicated in the malfunction of the CNS in the Cbl deficiency of man. The thesis of this presentation rests on the validity of extrapolation of data derived from infants with a congenital disorder to Cbl deficiency in general. This is a large step. The underdeveloped infantile CNS does not react to events in the same way as the mature adult CNS. Very different abnormalities in the infant may produce a similar end result such as developmental delay or seizures.
126
Hall
Nevertheless, there may be some processes common to the Cbl deficiency of both the adult and the infant. Cbl deficiency might distrub initial myelin formation in the infant and the repair of myelin in the adult. There is one case of cblG mutation that forms a bridge between the two age groups [7]. The defect was evident in infancy and the diagnosis was later well documented, yet the full expression occurred in the third decade. The defect was in some unknown way attenuated because the usual child with MeCbl deficiency would not survive infancy without treatment. Eventually, however, the patient became severely ill. In infancy and early childhood there were lethargy, poor coordination, developmental delay, possible subnormal intelligence, and difficulty in reading. These are typical of the child with MeCbl deficiency. Progressively during her 20s she developed numbness and paresthesias, disturbed gait and balance, abnormal VEP, decreased vibratory and position senses, decreased pain and temperature senses, and nystagmus. These are the manifestations of Cbl deficiency of the adult. There was a single defect, cblG, but it was expressed at two ages. The analysis of the expression of cblG in the infant and in the adult is paralleled by a comparison of the CNS expression of acquired Cbl deficiency at the two ages. The symptoms of infants born of and nursed by Cbldeficient mothers [ 131 are those of cblE and G , whereas those of adult, acquired Cbl deficiency [I-41 are the same as in “adult” cblG 171. Much is to be learned from several of the other congenital disorders of Cbl, folate, and related enzymes but limits of space permit discussion of only a few. Methylenetetrahydrofolate reductase deficiency [ 181 is germane because it affects the same final pathway as MeCbl deficiency. The lack of MeCbl reduces the synthesis of Met by interfering with the transfer of the methyl from methylfolate to Hcy. The reductase deficiency reduces Met synthesis by failing to provide a supply of methylfolate. The consequences are similar both in the deficiency of Met and in the accumulation of Hcy. The absence of megaloblastosis with reductase deficiency is puzzling because a deficiency of methylfolate should have the same effect as a trapping. The reductase deficiency is seen in a generally older group of children, but the neurologic expression of the two syndromes is similar [ 181. Developmental delay, hypotonia, abnormal EEGiseizures, disturbed breathing and microcephaly are of comparable incidence in the two groups. Upper motor neuron signs, disturbed gait, and coma are more common with reductase deficiency. MeCbl deficiency responds better to treatment. Although demyelination of the brain has been described in the reductase deficiency [ 17,lSJ , comparison with MeCbl deficiency is difficult. Myelinations in the former have not been examined by MRI during life. All data come from autopsy where the
older age of the children and vascular disease complicate the picture. In reductase deficiency there are in some cases neuronal loss and gliosis, but there has been no opportunity to search for these in MeCbl deficiency. Impaired function of the Cbl-dependent mutase is expressed in four inherited syndromes [20]. In two the apoenzyme is defective, and in two the lesion is the synthesis of the cofactor, AdoCbl. Lethargy is as common in the defects of mutase activity as in MeCbl deficiency, whereas hypotonia is somewhat less frequent. Two-thirds of those with no detectable niutase are developmentally retarded as are one-fourth to one-third with the other three defects. This contrasts with the 100% incidence in MeCbl deficiency. The mutase defects are not characterized by a high incidence of seizures. Further comparison is difficult because the mutase defects are complicated by significant acidosis. Moreover, there have been fewer studies of CNS function. To summarize the information derived from the congenital defects of Cbl metabolism, it can be said that Cbl is required for myelination of the developing brain and for other yet-unidentified functions of the CNS. The Cbldependent MS and not the methylmalonyl CoA rnutase is the focus of the CNS activities of Cbl. Whereas the expression of the effects of Cbl deficiency differs between the CNS of the infant and the adult, very likely the function of Cbl is similar at the two ages. ACKNOWLEDGMENTS
I wish to thank Dr. Lynn Van Antwerpen for her assistance in the evaluation of the patients and Mr. James A. Begley and Mrs. Pamela D. Colligan for assistance in the laboratory. REFERENCES I . Chanarin I , Deacon R, Lumb M , Muir M , Perry J: Cobaiamin-Folate Interrelations: A critical review. Blood 66:479-489, 1985. 2. Metz J , van der Westhuyzen J: Mini review: The fruit bat as an experimental model of the neuropathy of cobalamin deficiency. Comp Biochem Physiol [ A ] 88:171-177, 1987. 3. Frenkel EP: Abnormal fatty acid metabolism in peripheral nerves of patients with pernicious anemia. J Clin Invest 52:1237-1245, 1973. 4. Weir DG, Keating S , Molloy A, McPartlin J , Kennedy S, Blanchflower J , Kennedy DC, Rice D, Scott JM: Rapid communication: Methylation deficiency causes vitamin B 12-associated neuropathy in the pig. J Neurochem 51:1949-1952, 1988. 5 . Watkim D, Rosenblatt DS: Functional methionine synthase deficiency (cblE and cblG): Clinical and biochemical heterogeneity. Am J Med Genet, 34:427-434. 1989. 6 . Hall CA, Lindenbaum RH, Arenson E. Begley JA, Chu RC: The nature of the defect in cobalamin G mutation. Clin Invest Med 12: 262-269, 1989. 7. Carmel R , Watkins D, Goodman SI, Rosenblatt DS: Hereditary defect of cobalamin metabolism (cblG mutation) presenting as a neurologic disorder in adulthood. N Engl J Med 318:1738-1741, 1988.
Function of Vitamin B,, 8 . Roaenblatt DS. Cooper BA. Schniutz SM. Zaleski WA, Casey RE: Prenatal vitamin B12 therapy of a fetus with methylcobalamin deficiency (cobalamin E disease). Lancet I : I 127-1 129. 1985. 9. Roaenhlatt S, Cooper BA, Pottier A. Lue-Shing H. Matiasmk N, Grauer K: Altered vitamin B 12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin Invest 74:2149-2 156. 19x4. 10. Gilles FH, Shankle W. Dooling EC: Myelinated tr terns. In Gilles FH. Leviton A , Dooling EC (eds): “The Developing Human Brain.” Boston: John Wright, 1983. p 117-183. I I . Brody BA, Kinney HC. Kloman AS. Gille, FH: Sequence of central nervous syhtem myelination in human infancy. I . An autopsy study of myelination. J Neuropathol Exp Neurol 46:283-301. 1987. 12. Dietrich RB. Bradley WG, Zaragoza EJ, Otto RJ. Taira RK. Wilson G H , Kangarloo H: MR evaluation of early myelination pattern\ in normal and developmentally delayed infants. Am J Neurol Radio1 9:69-76, 1988. 13. Stollhoff K, Schulte FJ: Vitamin B12 and brain development. Eur J Pediatr I46:20 1-205, 1987. 14. Scheinherg P: Cerebral blood flow and metabolism in pernicious anemia. Blood 6213-227. 1951.
127
IS. Samson DC, Swi\her SN. Christian RM, Engel GL: Cerebral nieta-
16.
17.
18.
19. 20.
bolic disturbance and delirium in pernicious anemia. Clinical end electroencephalographic studies. Arch Intern Med 90:4-14, 1952. Lindenhaum J. Healton EB, Savage D C , Brust JCM, Garrett TJ. Podell ER. Marcell PD, Stabler SP, Allen RH: Neuropsychiatric di\orders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med 318:1720-1728, 1988. Hyland K , Smith I. Bottiglieri T , Perry J , Wendel U, Clayton PT. Leonard JV: Demyelination and decreased s-adenosylmethionine in 5.10-methylenetetrahydrofolate reductase deficiency. Neurolosy 38: 459-462. 1988. Erbe RW: Inborn errors of folate metabolism. In Blakley RL. Whitehead VM: “Folates and Pterins.” New York: John Wiley & Sons. 1986. pp 413-465. Chu RC. Hall CA: The tntal aerum homocysteine as an indicator of vitamin 8 1 2 and folate status. Am J Clin Pathol 90:446-449, 1988. Rosenberg LE. Fenton WA: Disorders of propionate and niethylmalonate metabolism. In Scriver CR, Beaudet Al. Sly WS. Valle D (eds): “The Metabolic Basis of Inherited Disease.” New York: McGraw-Hill. 1989. pp 821-844.
Function of Vitamin B,* in the Central Nervous System as Revealed by Congenital Defects Charles A. Hall Research and Medical Services, Veterans Administration Medical Center and Department of Medicine, Albany Medical College, Albany, New York
The 13 cases of methylcobalamin(MeCbl) deficiency presenting in early infancy have all been developmentally delayed, and the majority have had seizures, hypotonia, lethargy, and microcephaly. The CNS injury appears to occur during the first 6 months of postnatal life. The same symptoms are seen in acquired cobalamin (Cbl) deficiency in the same age group. MRI performed at age 18-19 months and after 13-14 months of large amounts of Cbl, in two cases showed delayed myelination, most pronounced in the cerebrum. Isolated MeCbl deficiency is the consequence of cblE and G mutations where the lesion is of a single Cbl-dependent enzyme, the methyltransferase. One effect of a deficiency of MeCbl, and of the associated failure of the methionine synthase reaction, is, therefore, an impairment of myelination of the brain of the newborn. The slow, but usually incomplete, improvement in psychomotor status after years of treatment with Cbl may be related to the eventual myelination. However, the hypotonia, lethargy, and impaired responsiveness react to treatment with Cbl within 24-48 hours, which suggests an expression of MeCbl deficiency on the CNS distinct from the delayed myelination. Although there is much to be learned, it is now clear that a normally functioning Cbl-dependent methyl transferase is required for development and function of the human brain. Key words: cobalamin, nervous system, methyltransferase
The biochemical basis for the central nervous system (CNS) expression of vitamin B,, (cobalamin, Cbl) deficiency in man continues to elude students of the vitamin [ 1,2]. Investigations have been impeded by the absence of suitable models. Although humans share with other mammals the basic metabolism of Cbl, the expression of Cbl deficiency varies greatly among species. Nevertheless, deficient monkeys, pigs, and fruit bats have supplied important information about the function of Cbl in the nervous system. Deficiency has been induced in these species both by dietary means and by inactivation of the Cbl with nitrous oxide. The main theories of Cbl activity are divided along lines defined by the two Cbl-dependent enzyme reactions. The enzymes are the 5-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthase [MS]; EC 2.1.1.13) for which methylcobalamin (MeCbl) is the coenzyme and the methylmalonyl CoA mutase (EC 5.4.99.2) for which adenosylcobalamin (AdoCbl) is the coenzyme. One concept holds that the accumulation of methylmalonate and propionate owing to the impairment 0 1990 Wiley-Liss, Inc.
of mutase activity by Cbl deficiency leads to the synthesis of abnormal fatty acids [ 3 ] . The incorporation of these unusual fatty acids into myelin, especially in the spinal cord, may cause the CNS lesions of Cbl deficiency. The other concept focuses on defective methionine (Met) synthesis, thus reducing the supply of adenosylmethionine (AdoMet), which is synthesized from Met; AdoMet is required for essential methylations in the nervous system [4]. The approach presented here is the application of the study of human congenital defects of Cbl metabolism in the search for an understanding of the function of Cbl in the nervous system. Although there are several pertinent defects, the present discussion will be confined for the most part to the cblE and G mutations. Whereas the principal objective of this specific presentation is a correlation of biochemistry and function for scientific purposes, Adress reprint requests to Charles A. Hall, Nutrition Laboratory for Clinical, Assessment and Research ( I 5 1 E), Veterans Administration Medical Center, Albany, NY 12208.
122
Hall
there are important clinical questions to be answered as well. In reality, the objectives are all the same since a thorough understanding of the disorders are essential to the prompt diagnosis, management, and prognosis. A recent review [ 5 ] and a subsequent report [6] give the details of the reported cases and the pertinent references. There are now 8 known persons with cblG mutation, 6 with cblE, and 1 still unclassified but obviously one of the two disorders. The key abnormality of both groups is a deficiency of MeCbl and reduced in vivo activity of the CbI-dependent MS [ 5 ] . Although formation of MeCbl is impaired by distinct abnormalities in cblE and G, respectively, the biochemical and clinical expressions are the same. The present analysis is based on personal biochemical data from 5 patients and data from the literature for the others. Much information was obtained from the physicians caring for these children and from their parents. There are adequate clinical data for 6 subjects, a modest amount for 4 more, and some for 2. Two are too recent for more than the initial presentation. These 14 presented in infancy, as is typical. The 15th case [7] was quite atypical but may be most important as a bridge between the childhood and adult expression of Cbl deficiency. Although this subject was symptomatic in early infancy, the full expression and diagnosis of cblG came not until the third decade. This case will be analyzed separately. Another child that was treated prior to birth and after without ever becoming symptomatic is excluded from the analysis of symptoms 181. The power of MeCbl deficiency in the derivation and testing of concepts lies in the restriction of the lesion to a single coenzyme that acts in a single enzyme system. All 13 cases presented with anemia, which is known to have been megaloblastic in all but one [ 5 ] . The infants were normal at birth and developed normally for the first few weeks. Symptoms began in at least 70% between 1 and 3 months of age. All were developmentally delayed. Other common symptoms have been lethargy, hypotonia, and seizures. At presentation some have responded poorly to stimuli even to the point of coma. In spite of an immediate initial response and a prompt resolution of the megaloblastic anemia, half of the children experienced new CNS symptoms or worsening of existing ones in the first 3-4 months after treatment was started. Biochemically the expression has been remarkably uniform [5,6]. Homocysteine (Hcy) or its products are increased in the blood and urine, but methylmalonic acid (MMA) is not. The plasma Met is low. The serum Cbl is within the normal range, but in one unreported case MeCbl was low. Serum and erythrocyte folate are normal or high. These data support the presence of a block in the MeCbl dependent MS only, as can be demonstrated in cultured fibroblasts and lymphoblasts. The
cells divide poorly when Met is replaced with Hcy in the medium. They take up labeled methyl folate poorly but take up propionate normally. They take up labeled Cbl bound to transcobalamin 11, internalize it, and bind it to the MS and mutase, respectively. Whereas labeled AdoCbl appears in the cell in the expected amounts, MeCbl is absent or much reduced. In cblE mutation the failure to form MeCbl appears to be in the essential step of reduction of the Co of the Cbl 191. In cblG the defect may be in the step of methylation requiring AdoMet [6]. Regardless, the failure to form functional MeCbl on the MS impairs the activity of the enzyme. To date the study of the Cbl metabolism of cells has been directed toward the establishment of diagnosis. What is now needed are deeper investigations of how and to what extent Met synthesis is impaired and what the biochemical consequences are. All children have responded to treatment. All regimens have included Cbl, and the usual form has been hydroxocobalamin (OH-Cbl), 1 mg i.m. one or more times a week. Five have been treated successfully with cyanocobalamin (CN-Cbl), but 2 of the 9 treated with OH-Cbl responded poorly to CN-Cbl initially and were changed to OH-Cbl. Nine have received either formyl folate or folic acid, and 4 have received methionine. Several have been given betaine, pyridoxine, MeCbl, or carnitine. Trials of different combinations have been common. Because of the heterogeneity of both the clinical expression and the mode of treatment it is impossible to offer firm guidelines for treatment. Some form of Cbl is, however, essential. The megaloblastosis responds promptly, and there is an immediate improvement in the responsiveness of the child. In the long term there is a steady gain in psychomotor development, but during the first 3 -4 months after treatment is started there may be both gains and losses in the neurologic status. For example, seizures may first be observed after treatment. Usually the child becomes free of seizures even without suppressive medication. One child, now age 6 years, has a normal IQ and another 5 % year old may have reached that point. The majority, now in the range of 3-9 years, have not caught up with their peers although all continue to improve. Two older children who were treated late may be more severely affected, but little about their status is available. Two others began treatment only recently. Speech has been the most resistant of the impediments. There are only limited data on the biochemical responses. Serum Met normalizes promptly, and serum Hcy falls. We have in two cases found some elevation to persist when total Hcy is measured by a sensitive method. The available data on other amino acids in plasma and spinal fluid are insufficient for meaningful analysis. In one case monitored closely by us serum and erythro-
Function of Vitamin B,,
cyte folate, all 5-methyltetrahydrofolate, remain elevated. We attempted measurement of the MeCbl in the post-treatment serum of two cases, but the astronomical amounts of OH-Cbl or CN-Cbl induced by treatment with the respective vitamin were overwhelming. The increase in serum Met and fall in Hcy suggest that within the cells synthesis of Met via the MeCbl-dependent reaction has been enhanced by the administration of Cbl. In order to correlate the functional and biochemical expressions of MeCbl deficiency the consequences must be better defined. Examination of the affected tissues, other than cultured cells, has been possible only in the one child known to have died with the defect [ 5 ] . The child’s illness was recognized in a general way but at a time when his illness, cblE mutation, had not yet been described. Effective treatment was given only for a short time, and he died at age of 5 % years. The case has not been reported fully, but such information about the anatomical observations as is available does not contradict the hypotheses to be expressed below. The brains of 1 1 children have been examined by computed tomography (CT) scans. Nine were abnormal, and in the 8 where sufficient detail was reported there was atrophy or hypoplasia of the brain. The ventricles were reported as enlarged in 4 patients. Dilatation was marked in one with what appeared to be a communicating hydrocephalus. There was no evidence of obstruction, and although a ventricular shunt was placed, CSF pressure was not recorded. No other abnormalities of the brain have been reported. The earliest, technically satisfactory magnetic resonance imagings (MRI) were performed in two 18 month old children with cblG. They had been treated for 13 and 14 months, respectively. Both were in phases of steady improvement, both continued to improve and are still improving, but both at ages 4 and 5 years, respectively, have impediments. The observations were quite similar. Some structures of the brain were myelinated, but the process was behind the expected at 18 months especially in the cerebral hemispheres. The age of myelination was estimated at 11-14 months. If, as in these cases, myelination was still delayed after a year of treatment, it very likely was delayed before treatment. There has been no recent MRI of one, but the second, now age 5 years, and a third 3 year old have normal MRI but are still impeded, especially in speech. Myelination of the brain undergoes a great burst in the last trimester of uterine life [ 101 and continues at a rapid pace in the year following birth [ 11,121. This is also a time of rapid increase in brain weight. The cblE or G child has a normal birthweight, and limited data show a normal head size. For three cblG children there exist serial measurements of weight, height, and head circumference from birth to age 1 year or more (unpublished).
123
The patterns are identical. Up to 6-8 weeks all measurements increase steadily and are well within the normal range, but then the curves flatten. With treatment, growth as measured by all 3 parameters resumes a normal pace but whether or not the deficit is made up remains to be seen. By the time of diagnosis 8 of 1 1 children for which data are available showed head circumferences in the 2nd to 10th percentile. The remainder were in the 50th to 75th percentile with the largest head in the child with hydrocephalus. Thus, brain growth, as reflected by head size as well as by the CT scans, slows in the early weeks of postnatal life as the MeCbl deficiency is expressed clinically. To this point one must conclude that deficiency of MeCbl with the accompanying reduced synthesis of Met impairs the normal process of the myelination of the brain of the newborn. It is probably significant that injections of OH-Cbl were started in the mother of the child with cblE diagnosed in utero at 25 weeks of gestation [8]. Treatment of the child has been continued since birth. That child, now 5 years old, has only a slight impediment of speech. MRI have not extended to the spinal cord, and for information about myelination of that structure one must await the report of the child that died. There is, however, more to cblE and G than defective myelination. For I 1 of the cases there are complete hospital summaries, literature reports, or personal observations. Immediate responses to treatment are recorded for 7 cases. These have consisted of enhanced responsiveness, alertness, and ability to feed. The improvement, which may be dramatic, occurs in 1-2 days and much too soon for there to be any change in the erythrocyte count. Transfusion, in the few cases where it has been given, has not induced this kind of a response. The change is even more premature to be caused by any structural improvement in the brain. Similar immediate CNS responses are observed in infants of a comparable age with acquired Cbl deficiency [13]. The phenomenon is also seen with the treatment of adult Cbl deficiency, but there are few descriptions in the literature and these casual reports are dismissed as anecdotal. Nevertheless, there is objective evidence of immediate responses, independent of the anemia, in the cerebral metabolism of adult Cbl deficiency [ 14,151. The basis for the immediate response to Cbl could be in the abrupt removal of a toxic substance or from an enhancement of some process such as neurotransmission. Body Hcy is increased in cblE and G [5] and in pernicious anemia [ 161. Serum Hcy falls with treatment of both deficiencies, but there has been no attempt at correlation of this decline with specific neurological manifestations. The possibility of defects of neurotransmission in Cbl deficiency has not been approached
Hall
124
’
formote
-Methylthioribose
//
FomylTHF
5)O
//
// Methenyl THF
k
I
\
Methylthio-
a
0
Adenosine+ Hcy AdoHcy
Pmpionyl CoA t
/acceptor Methylated (S) MethylmabnylCoA
‘fine
It
0
(R) MethylmalonylCo A
Ademylironsfemse
Thymidylato syniheiase
Methyl-transfemse
d TMP
I
dTTP
1
@
3MeIHP-
/
1
MeCbl \
,,kY
\
Cbl(1)
t
\
DNA
rlrcerihrd
nor
, I I
TCII-Cbl i-Txn
’
Succinyl Co A
Reductose
\
I
Cbl (It1
t
Reductose
t
*Cbl(m)
I
I
Cell Membrane
@
Fig. 1. The Cbl metabolism of cells. In MeCbl deficiency the entry of TC Il-Cbl is normal. The pathway from A through E is also unaffected. The defect lies in the failure to form MeCbl required for reaction C. The symptoms of cblE and G mutations could be produced by failures at several points. MeCbl accumulates, C, and could be toxic. Hcy accumu-
lates, C, and could be toxic. Tetrahydrofolate (THF) may be unavailable from reaction C to form the coenzyme for reaction 6.Met may be unavailable, C, for a variety of functions and especially for the synthesis of AdoMet. AdoMet is required for many essential methylations, F. Met is also a source of formate, D.
through appropriate biochemical experiments. However, in methylenetetrahydrofolate reductase deficiency the neurotransmitter metabolites homovanillic acid and 5hydroxyindoleacetic acid and total biopterins are low in the CSF [ 17,181. A more extensive comparison of this syndrome with cblE and G is given below. In the discussion that follows the reader must keep in mind that Cbl may have several activities within the CNS and that the effects of MeCbl deficiency may fall into two broad categories, structural and functional. Obviously function depends on structure, but MeCbl may affect function within the confines of a fixed structure. Neither are the consequences of treatment clear at this time. Myelination is restored, but the sequence among the many parts of the brain and cord may be distorted. Other anatomical lesions not revealed by imaging may persist. The enzymatic lesion may not be entirely compensated and maintenance processes could be impaired. Psychomotor development must depend upon an orderly sequencc of anatomical development, sensory input, and response by the brain. Compensation for the breakdown during the vital period of 1-6 months of age may never be complete. We do not yet know what can be accom-
plished by training. Finally, if MeCbl is required for day-to-day function, normality can be achieved only if the defect is overridden. The common processes by which all forms of Cbl deficiency disturb the structure and function of the CNS are illustrated in Figure 1 and could include any of the following: 1) toxicity of the accumulated methylfolate, 2) the trapping of folate in a non-functional form, 3) reduced synthesis of formate, 4) failure to synthesize folylpolyglutamate, 5 ) toxicity of the accumulated Hcy, and 6) reduced synthesis of adenosylmethionine (Ado), which would affect essential methylations. We have observed increased methylfolate in both the serum and erythrocytes of cblG. We have observed (unpublished) decreased division of cultured cells when methylfolate is supplied in amounts greater than the basic requirement. There is, however, no evidence of toxicity in other circumstances. The classic theory of the folate trap holds that methylfolate is not demethylated in the absence of a functioning MS and tetrahydrofolate is not available to form the essential folate coenzymes [ 11. The theory is usually applied as an explanation for the disturbed DNA synthesis in the lesion of hematopoiesis
Function of Vitamin B,,
known as megaloblastosis. Such an explanation in its purest form would not seem to apply to non-dividing neurons. However, the concept could be broadened to include processes other than DNA synthesis. Moreover, available folate could be required for the great increase in glial cells that accompany the late gestational burst of myelin synthesis [ 101. The formate starvation hypothesis [ I ] is a closely related explanation for the effects of Cbl deficiency. Substantial supporting evidence has been derived from studies of rats exposed to N,O. A decreased supply of Met from failure of MS activity reduces the available formate. Formate is a source of formyltetrahydrofolate and of methylene in the methylation of deoxyuridine, This hypothesis has not been applied directly to the function of the CNS. A fourth possibility is a disturbance in folate metabolism in Cbl deficiency owing to a failure to form folylpolyglutamate. The percent of erythrocyte folate as polyglutamate was 1% in the cells of one child with cblG treated for 1 1 weeks [6]. Between ages 12 and 24 months it remained at 25-27%. By 3 years it was 63%. The technique consisted of assay of folate by the L . casei method both with and without hydrolysis of the polyglutamates. The difference was the folylpolyglutamate. Scattered, incomplete data derived from the adult suggest that 60-90% of erythrocyte folate is polyglutamate, and by the same technique as we applied to the child’s cells we observed 90% or greater. There are no pediatric reference values. Preliminary data suggest that the fraction may be lower in cells from healthy children ages 3 months to 2 years than in cells from adults but not as low as in the cells of the child with cblG. Before this concept can be extended there must be studies of the ability of cultured cblE and G cells to synthesize folylpolyglutamate. Toxicity for the CNS by Hcy is a realistic possibility. Increased body Hcy characterizes cystathionine p synthase deficiency and is considered to be responsible for the almost universal vascular injury and thromboembolism associated with the syndrome. The story of lymphocyte toxicity from Hcy in adenosine deaminase deficiency is too complex to be told here. Vascular injury, including the blood vessels of the brain and presumably from the increased Hcy, has been present in the majority of those dying of methylenetetrahydrofolate reductase deficiency [ 181. Hcy accumulates in MeCbl deficiency [ 5 ] . Comparison of serum and urinary levels among cases and with levels in other disorders is difficult because of recent changes in methods. By a sensitive measure of total serum Hcy [19] we observed a level of 63 pmol/liter in a child with cblG prior to treatment and a fluctuation between 21 and 46 pmol/liter in the subsequent 3 years. The adult reference range by this technique is 6.9-12.1 pmol/liter, and preliminary data sug-
125
gest a lower range for young children. The cause of death in the one incompletely treated and fatal case of MeCbl deficiency was renal vascular disease [ 5 ] . Thromboembolism has not been observed in the others, but Hcy levels have been at least partly controlled by treatment. Vascular toxicity may increase with age, and most of those with cblE and G are still quite young. Although historically Hcy toxicity has been primarily vascular, there may be injury to other tissues as well. For example in unpublished experiments we have observed Hcy in the culture medium to suppress the formation of MeCbl in normal fibroblasts, a process already defective in cblE and G cells. Hcy toxicity might not be expressed in utero where it can be removed by maternal processes, thus explaining the normal development of infants with MeCbl deficiency before birth. The immediate responses to treatment of MeCbl deficiency could be explained by an abrupt lowering of body Hcy. Perhaps the most attractive theory for the action of Cbl in the CNS and the consequences of MeCbl deficiency is in the need for Met and AdoMet for essential methylations. There are many points of potential action but two could be methylation by AdoMet in the synthesis of proteins and of neurotransmitters. In MeCbl deficiency fibroblasts and lymphoblasts cannot use Hcy, methylfolate, and Cbl to synthesize Met and support cell division [6]. The same cells take up methylfolate poorly and transfer subnormal amounts of methyl groups to Hcy to form Met [5,6]. Serum Met is low but increases promptly with treatment. Missing so far have been any studies of AdoMet levels in cells or body fluids or of the ability of cultured cells to form AdoMet. Since Met is the source, impaired synthesis of AdoMet would be expected. Animal studies have been conflicting. Weir et al. observed suppression of MS activity by N,O to reverse the AdoMet/AdoHcy ratio in the spinal cord of the pig 141. Exogenous Met ameliorated the clinical and biochemical lesions induced by N,O in pigs and monkeys. However, Metz and van der Westhuyzen could demonstrate neither decreased tissue AdoMet nor decreased methylation of myelin basic protein in fruit bats exposed to N,O [2]. The conflict in observations could be explained by differences among species, but the question deserves a thorough reexamination. In any case the role of AdoMet must be examined in human systems before decreased AdoMet can be implicated in the malfunction of the CNS in the Cbl deficiency of man. The thesis of this presentation rests on the validity of extrapolation of data derived from infants with a congenital disorder to Cbl deficiency in general. This is a large step. The underdeveloped infantile CNS does not react to events in the same way as the mature adult CNS. Very different abnormalities in the infant may produce a similar end result such as developmental delay or seizures.
126
Hall
Nevertheless, there may be some processes common to the Cbl deficiency of both the adult and the infant. Cbl deficiency might distrub initial myelin formation in the infant and the repair of myelin in the adult. There is one case of cblG mutation that forms a bridge between the two age groups [7]. The defect was evident in infancy and the diagnosis was later well documented, yet the full expression occurred in the third decade. The defect was in some unknown way attenuated because the usual child with MeCbl deficiency would not survive infancy without treatment. Eventually, however, the patient became severely ill. In infancy and early childhood there were lethargy, poor coordination, developmental delay, possible subnormal intelligence, and difficulty in reading. These are typical of the child with MeCbl deficiency. Progressively during her 20s she developed numbness and paresthesias, disturbed gait and balance, abnormal VEP, decreased vibratory and position senses, decreased pain and temperature senses, and nystagmus. These are the manifestations of Cbl deficiency of the adult. There was a single defect, cblG, but it was expressed at two ages. The analysis of the expression of cblG in the infant and in the adult is paralleled by a comparison of the CNS expression of acquired Cbl deficiency at the two ages. The symptoms of infants born of and nursed by Cbldeficient mothers [ 131 are those of cblE and G , whereas those of adult, acquired Cbl deficiency [I-41 are the same as in “adult” cblG 171. Much is to be learned from several of the other congenital disorders of Cbl, folate, and related enzymes but limits of space permit discussion of only a few. Methylenetetrahydrofolate reductase deficiency [ 181 is germane because it affects the same final pathway as MeCbl deficiency. The lack of MeCbl reduces the synthesis of Met by interfering with the transfer of the methyl from methylfolate to Hcy. The reductase deficiency reduces Met synthesis by failing to provide a supply of methylfolate. The consequences are similar both in the deficiency of Met and in the accumulation of Hcy. The absence of megaloblastosis with reductase deficiency is puzzling because a deficiency of methylfolate should have the same effect as a trapping. The reductase deficiency is seen in a generally older group of children, but the neurologic expression of the two syndromes is similar [ 181. Developmental delay, hypotonia, abnormal EEGiseizures, disturbed breathing and microcephaly are of comparable incidence in the two groups. Upper motor neuron signs, disturbed gait, and coma are more common with reductase deficiency. MeCbl deficiency responds better to treatment. Although demyelination of the brain has been described in the reductase deficiency [ 17,lSJ , comparison with MeCbl deficiency is difficult. Myelinations in the former have not been examined by MRI during life. All data come from autopsy where the
older age of the children and vascular disease complicate the picture. In reductase deficiency there are in some cases neuronal loss and gliosis, but there has been no opportunity to search for these in MeCbl deficiency. Impaired function of the Cbl-dependent mutase is expressed in four inherited syndromes [20]. In two the apoenzyme is defective, and in two the lesion is the synthesis of the cofactor, AdoCbl. Lethargy is as common in the defects of mutase activity as in MeCbl deficiency, whereas hypotonia is somewhat less frequent. Two-thirds of those with no detectable niutase are developmentally retarded as are one-fourth to one-third with the other three defects. This contrasts with the 100% incidence in MeCbl deficiency. The mutase defects are not characterized by a high incidence of seizures. Further comparison is difficult because the mutase defects are complicated by significant acidosis. Moreover, there have been fewer studies of CNS function. To summarize the information derived from the congenital defects of Cbl metabolism, it can be said that Cbl is required for myelination of the developing brain and for other yet-unidentified functions of the CNS. The Cbldependent MS and not the methylmalonyl CoA rnutase is the focus of the CNS activities of Cbl. Whereas the expression of the effects of Cbl deficiency differs between the CNS of the infant and the adult, very likely the function of Cbl is similar at the two ages. ACKNOWLEDGMENTS
I wish to thank Dr. Lynn Van Antwerpen for her assistance in the evaluation of the patients and Mr. James A. Begley and Mrs. Pamela D. Colligan for assistance in the laboratory. REFERENCES I . Chanarin I , Deacon R, Lumb M , Muir M , Perry J: Cobaiamin-Folate Interrelations: A critical review. Blood 66:479-489, 1985. 2. Metz J , van der Westhuyzen J: Mini review: The fruit bat as an experimental model of the neuropathy of cobalamin deficiency. Comp Biochem Physiol [ A ] 88:171-177, 1987. 3. Frenkel EP: Abnormal fatty acid metabolism in peripheral nerves of patients with pernicious anemia. J Clin Invest 52:1237-1245, 1973. 4. Weir DG, Keating S , Molloy A, McPartlin J , Kennedy S, Blanchflower J , Kennedy DC, Rice D, Scott JM: Rapid communication: Methylation deficiency causes vitamin B 12-associated neuropathy in the pig. J Neurochem 51:1949-1952, 1988. 5 . Watkim D, Rosenblatt DS: Functional methionine synthase deficiency (cblE and cblG): Clinical and biochemical heterogeneity. Am J Med Genet, 34:427-434. 1989. 6 . Hall CA, Lindenbaum RH, Arenson E. Begley JA, Chu RC: The nature of the defect in cobalamin G mutation. Clin Invest Med 12: 262-269, 1989. 7. Carmel R , Watkins D, Goodman SI, Rosenblatt DS: Hereditary defect of cobalamin metabolism (cblG mutation) presenting as a neurologic disorder in adulthood. N Engl J Med 318:1738-1741, 1988.
Function of Vitamin B,, 8 . Roaenblatt DS. Cooper BA. Schniutz SM. Zaleski WA, Casey RE: Prenatal vitamin B12 therapy of a fetus with methylcobalamin deficiency (cobalamin E disease). Lancet I : I 127-1 129. 1985. 9. Roaenhlatt S, Cooper BA, Pottier A. Lue-Shing H. Matiasmk N, Grauer K: Altered vitamin B 12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin Invest 74:2149-2 156. 19x4. 10. Gilles FH, Shankle W. Dooling EC: Myelinated tr terns. In Gilles FH. Leviton A , Dooling EC (eds): “The Developing Human Brain.” Boston: John Wright, 1983. p 117-183. I I . Brody BA, Kinney HC. Kloman AS. Gille, FH: Sequence of central nervous syhtem myelination in human infancy. I . An autopsy study of myelination. J Neuropathol Exp Neurol 46:283-301. 1987. 12. Dietrich RB. Bradley WG, Zaragoza EJ, Otto RJ. Taira RK. Wilson G H , Kangarloo H: MR evaluation of early myelination pattern\ in normal and developmentally delayed infants. Am J Neurol Radio1 9:69-76, 1988. 13. Stollhoff K, Schulte FJ: Vitamin B12 and brain development. Eur J Pediatr I46:20 1-205, 1987. 14. Scheinherg P: Cerebral blood flow and metabolism in pernicious anemia. Blood 6213-227. 1951.
127
IS. Samson DC, Swi\her SN. Christian RM, Engel GL: Cerebral nieta-
16.
17.
18.
19. 20.
bolic disturbance and delirium in pernicious anemia. Clinical end electroencephalographic studies. Arch Intern Med 90:4-14, 1952. Lindenhaum J. Healton EB, Savage D C , Brust JCM, Garrett TJ. Podell ER. Marcell PD, Stabler SP, Allen RH: Neuropsychiatric di\orders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med 318:1720-1728, 1988. Hyland K , Smith I. Bottiglieri T , Perry J , Wendel U, Clayton PT. Leonard JV: Demyelination and decreased s-adenosylmethionine in 5.10-methylenetetrahydrofolate reductase deficiency. Neurolosy 38: 459-462. 1988. Erbe RW: Inborn errors of folate metabolism. In Blakley RL. Whitehead VM: “Folates and Pterins.” New York: John Wiley & Sons. 1986. pp 413-465. Chu RC. Hall CA: The tntal aerum homocysteine as an indicator of vitamin 8 1 2 and folate status. Am J Clin Pathol 90:446-449, 1988. Rosenberg LE. Fenton WA: Disorders of propionate and niethylmalonate metabolism. In Scriver CR, Beaudet Al. Sly WS. Valle D (eds): “The Metabolic Basis of Inherited Disease.” New York: McGraw-Hill. 1989. pp 821-844.
