Methylenetetrahydrofolate Reductase Deficiency ina Patient With Phenotypic Findings ofAngelman Syndrome

Pamela H. Arn,1* Charles A. Williams,2 Roberto T. Zori,2 Daniel J. Driscoll,2 andDavid S. Rosenblatt3

1Department of Pediatrics, Division of Genetics, Nemours Children’s Clinic, Jacksonville, Florida2R.C. Philips Research Unit, Gainesville, Florida3Division of Medical Genetics, Department of Medicine, McGill University, Montreal, Canada

Deficiency of methylenetetrahydrofolate re-ductase (MTHFR) is associated with a vari-able phenotype that includes mental retar-dation, gait abnormalities, and seizures.Many of the same clinical findings are alsoseen in patients with Angelman syndrome.We report on a patient with MTHFR defi-ciency who was initially diagnosed as hav-ing Angelman syndrome. This case illus-trates that MTHFR deficiency can mimic thephenotype of Angelman syndrome and thatMTHFR deficiency should be excluded inpatients with manifestations of Angelmansyndrome whose molecular studies of chro-mosome 15 are normal. Am. J. Med. Genet.77:198–200, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: methylenetetrahydrofolatereductase deficiency; methy-lenetetrahydrofolate reduc-tase; Angelman syndrome

INTRODUCTION

Deficiency of methylenetetrahydrofolate reductase(MTHFR), an autosomal recessive disorder, is the mostcommon inborn error of folic acid metabolism. Methy-lenetetrahydrofolate reductase forms 5-methyltetra-hydrofolate, which is a methyl donor for the remethyl-ation of homocysteine to methionine. Patients withsevere deficiencies of MTHFR (0–20% activity in fibro-blasts) can have variable phenotypes with symptomsincluding developmental delay, mental retardation,motor and gait abnormalities, seizures, and psychiatricdisturbances [Goyette et al., 1994].

Angelman syndrome is characterized by severe men-tal retardation, lack of speech, gait abnormalities, sei-

zures, and a characteristic happy demeanor. Micro-brachycephaly, midface hypoplasia, and mandibularprognathism are usually present. Sporadic microdele-tions in chromosome region 15q11–15q13 occur in 60–80%, but other genetic mechanisms such as paternaluniparental disomy also occur [Williams et al., 1995b].Although a gene for Angelman syndrome has recentlybeen identified and mutations in the gene have beencharacterized, routine laboratory diagnosis by muta-tion analysis is not available [Kishino et al., 1997; Mat-suura et al., 1997]. Therefore, 10–20% of Angelmansyndrome cases are diagnosed solely based on clinicalcriteria.

We describe a child with MTHFR deficiency whoseclinical features initially appeared identical to thoseseen with Angelman syndrome.

CLINICAL REPORT

The patient was evaluated at 10 years of age for achronic seizure disorder and long-standing mental re-tardation. He was noted to have a happy affect andabsent speech. Physical examination revealed brachy-cephaly, protrusion of the tongue, prognathism, anataxic wide-based gait, and an excitable personalitywith uplifted arms and hand-waving behavior (Fig. 1).Birth history was unremarkable. Developmental delaywas noted by age 6 months. Family history was unre-markable. Other relatives did not have prognathism orsimilar facial changes. MRI of the brain showed mod-erate cortical atrophy and compensatory ventriculo-megaly. Plasma amino acids assayed by ion exchangechromatography with ninhydrin detection and urineorganic acids assayed by gas chromatography were re-ported to be normal. High-resolution karyotype wasnormal. A clinical diagnosis of Angelman syndromewas made. Leukocyte DNA methylation patterns (DN34,DW71, and 58SNRPN) were normal, and loci D1559,D15510, GABRB3, and D15512 were intact by densi-tometry.

At 12 years of age, the patient began to deteriorateand became anorexic, somnolent, and nonambulatory.Examination upon admission to the hospital showed

*Correspondence to: Pamela H. Arn, M.D., Nemours Children’sClinic, 807 Nira Street, Jacksonville, FL 32207.

Received 29 May 1997; Accepted 12 November 1997

American Journal of Medical Genetics 77:198–200 (1998)

© 1998 Wiley-Liss, Inc.

extreme lethargy. Muscle tone was increased in thelower limbs with mildly increased deep tendon reflexes.Results of routine blood chemistry studies, includinglactic acid and ammonia, and cerebrospinal fluid chem-istry studies were normal. An electroencephalogramshowed no obvious seizure focus; MRI showed cerebralatrophy similar to that seen in previous studies.Plasma amino acids assayed by ion exchange chroma-tography [Smith, 1989] demonstrated elevated homo-cystine levels with low methionine levels (Table I). Spoturine amino acids showed a free homocystine level of389 mmol/L (normal undetectable); urine nitroprussidescreen was positive. Urine organic acids and plasmacarnitine were normal. Serum folate level was 3.6nmol/L (1.6 ng/mL) (normal 4–22 nmol/L). Hema-tocrit, hemoglobin, and mean corpuscular volume wereunremarkable.

Methylenetetrahydrofolate reductase deficiency wassuspected, and the patient began a regimen of dailypyridoxine 250 mg orally, cyanocobalamin 1 mg IM,and folinic acid 3 mg IM. Within 72 hr, the patientbecame less somnolent, but he continued to have poormotor control. Betaine monohydrate 6 g per day orallywas begun and resulted in a decrease in plasma homo-cystine levels. Over the next month, the patient re-

gained his baseline mental status and had improvedmotor control, but he remained nonambulatory.

Uptake of [14C]methyltetrahydrofolate and that of[57Co]cyanocobalamin in cultured fibroblasts werewithin the control range. The level of methylcobalaminwas relatively low (Table II). Measurement of the ac-tivity of methylenetetrahydrofolate reductase in cul-tured fibroblasts confirmed that the patient was se-verely deficient with activity of 0.757 nmol of formal-dehyde/(mg of protein ? hr) (control range 13.3 ± 4.6)[Rosenblatt et al., 1979, 1992; Rosenblatt and Erbe,1977].

DISCUSSION

Although homocystinuria is seen consistently in allpatients with MTHFR deficiency, the excretion of ho-mocystine is much less than the level seen in patientswith homocystinuria due to cystathionine b-synthasedeficiency. Only free homocystine is normally detectedin plasma amino acid profiles, and if the plasmasample is not processed and run promptly, low levels ofhomocystine may not be detected, since protein-boundhomocysteine is precipitated prior to measurement.There was a delay between collection of the amino acidsample and receipt by the laboratory when the initialstudy was done at age 10 years. This may explain theearly report of normal plasma amino acids.

Urine homocystine levels were more elevated thanthose seen in plasma, and the urine nitroprusside testwas positive. Neither of these tests were performedduring the initial diagnostic workup. Total plasma ho-mocysteine (both free and protein-bound) determina-tion by high pressure liquid chromatography (HPLC)should be sensitive to minor elevations in homocyste-ine but is not readily available in most labs. Cliniciansshould be aware of the potential for problems in thedetection of low levels of free homocystine, especiallywhen samples must be shipped to reference labs. Thecombination of a urine nitroprusside and urine aminoacids should be performed in cases in which MTHFR issuspected. Definitive diagnosis of MTHFR deficiencyrequires enzyme analysis on cultured skin fibroblastsor peripheral leukocytes.

Treatment of MTHFR deficiency has been only par-tially successful [Nishimura et al., 1985; Harpey et al.,1981; Allen et al., 1980]. Betaine has the theoreticaladvantage of lowering homocysteine levels and supple-menting methionine levels; it may prevent the progres-sion of neurological symptoms in patients with this dis-order [Kishi et al., 1994; Wendel and Bremer, 1984].Our patient showed clinical improvement with the ad-dition of betaine to the treatment regimen.

Identification of patients with MTHFR deficiencywill allow earlier treatment of the disease and accurategenetic counseling for the family. A human cDNA forthe MTHFR gene has been isolated, and mutationshave been characterized in several patients. The corre-lation between the genotype, enzyme activity, and phe-notype in cases of severe deficiency has, thus far, beenconsistent; however, the numbers of patients studied issmall [Goyette et al., 1994, 1995, 1996].

TABLE I. Plasma Amino Acid Results (in mmol/L)*

Amino acid Normal range A B C

Cystine (44–96) 21 11 26Methionine (7–43) 11 8 12Homocystine (free) (Undetectable) 23 41 3

*A 4 Prior to treatment; B 4 Treatment with folinic acid, B6, and B12;C 4 Treatment B with betaine added.

Fig. 1. The patient at 12 years of age.

MTHFR With Angelman Syndrome Manifestations 199

Diagnostic criteria for Angelman syndrome havebeen published, and clinical awareness of Angelmansyndrome has increased over the past few years. Ge-netic laboratory studies of chromosome 15 will be nor-mal in 10–20% of individuals whose clinical presenta-tion is characteristic of Angelman syndrome [Williamset al., 1995a]. When initially evaluated, our patientexhibited both a developmental history and physicalsigns compatible with Angelman syndrome. Plasmaamino acids were initially reported to be normal, mak-ing an inborn error of amino acid metabolism seemunlikely. Our case illustrates that other diagnoses, par-ticularly MTHFR deficiency, should be excluded in thisgroup.

REFERENCESAllen RJ, Wong P, Rothenberg SP, Dimauro S, Headington JT (1980):

Progressive neonatal leukoencephalomyopathy due to absent methy-lenetetrahydrofolate reductase, responsive to treatment. Ann Neurol8:211.

Goyette P, Christensen B, Rosenblatt DS, Rozen R (1996): Severe and mildmutations in cis for the methylenetetrahydrofolate reductase gene(MTHFR) gene, and description of five novel mutations in MTHFR. AmJ Hum Genet 59:1268–75.

Goyette P, Frosst P, Rosenblatt DS, Rozen R (1995): Seven novel mutationsin the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductasedeficiency. Am J Hum Genet 56:1052–9.

Goyette P, Sumner JS, Milos R, Duncan AM, Rosenblatt DS, Matthews RG,Rozen R. (1994): Human methylenetetrahydrofolate reductase: Isola-tion of cDNA mapping and mutation identification. Nat Genet 7:195–200.

Harpey JP, Rosenblatt DS, Cooper BA, Moel GL, Roy C, Lafourcade J(1981): Homocystinuria caused by 5,10-methylenetetrahydrofolate re-ductase deficiency: A case in an infant responding to methionine, folinicacid, pyridoxine, and vitamin B12 therapy. J Pediatr 98:275–8.

Kishi T, Kawamura I, Harada Y, Eguchi T, Sakura N, Ueda K, NarisawaK, Rosenblatt DS (1994): Effect of betaine on S-adenosylmethioninelevels in the cerebrospinal fluid in a patient with ethylenetetrahydro-folate reductase deficiency and peripheral neuropathy. J Inherit MetabDis 17:560–5.

Kishino T, Lalande M, Wagstaff J (1997): UBE3A/E6-AP mutations causeAngelman syndrome. Nat Genet 15:70–4.

Matsuura T, Sutcliffe JS, Fang P, Galiaard R, Jiang Y, Benton CS, Rom-mens JM, Beaudet AL (1997): De novo truncating mutations in E6-APubiquitin–protein ligase gene (UBE3A) in Angelman syndrome. NatGenet 15:74–7.

Nishimura M, Yoshino K, Tomita Y, Takashima S, Tanaka J, Narisawa K,Kurobane I (1985): Central and peripheral nervous system pathology ofhomocystinuria due to 5,10-methylenetetrahydrofolate reductase defi-ciency. Pediatr Neurol 1:375–8.

Rosenblatt DS, Cooper BA, Lue-Shing S, Wong PW, Berlow S, Narisawa K,Baumgart Nev R (1979): Folate distribution in cultured human cells:Studies on 5,10-CH2-H4PteGlu reductase deficiency. J Clin Invest 63:1019–25.

Rosenblatt DS, Erbe RW (1977): Methylenetetrahydrofolate reductase incultured human cells. II. Genetic and biochemical studies of methy-lenetetrahydrofolate reductase deficiency. Pediatr Res 11:1141–3.

Rosenblatt DS, Lue-Shing H, Arzoumanian A, Low-Nang L, Matiaszuk N(1992): Methylenetetrahydrofolate reductase (MR) deficiency: Thermo-lability of residual MR activity, methionine synthase activity, andmethylcobalamin levels in cultured fibroblasts. Biochem Med MetabBiol 47:221–5.

Smith JA (1989): Amino acid analysis techniques. In Hugli TE (ed): ‘‘Tech-niques in Protein Chemistry.’’ San Diego: Academic Press, pp 251–4.

Wendel U, Bremer HJ (1984): Betaine in the treatment of homocystinuriadue to 5,10-methylenetetrahydrofolate reductase deficiency. Eur J Pe-diatr 142:147–50.

Williams CA, Angelman H, Clayton-Smith J, Driscoll DJ, Hendrickson JE,Knoll JH, Magenis E, Schinzel A, Wagstaff J, Whidden EM, and ZoriRT (1995a): Angelman syndrome: Consensus for diagnostic criteria.Am J Med Genet 56:237–8.

Williams CA, Zori RT, Hendrickson J, Stalker H, Marum T, Whidden E,Driscoll DJ (1995b): Angelman syndrome. Curr Probl Pediatr 25:216–31.

TABLE II. Cobalamin Uptake and Distribution*

Cell lineCbl uptakea

(pg/106 cell)

Cbl distributiona (%)

AqCbl CNCbl AdoCbl MeCbl Other

Patient 16.6 29.2 6.4 24.6 34.0 5.816.4

Control 9.8 10.1 7.7 15.1 63.9 3.29.8

Control rangeb 4.6 ± 2.0 8.3 ± 3.9 11.3 ± 6.9 15.3 ± 4.2 57.9 ± 6.7 7.2 ± 2.7

*Cbl 4 cobalamin; AqCbl 4 aquacobalamin; CNCbl 4 cyanocobalamin; AdoCbl 4 adenosylcobalamin; MeCbl 4 methylcobalamin.a[57Co]CN ? Cbl 25 pg/mL, 4 days incubation.bBased on 12 determinations (three different controls).

200 Arn et al.