Abstract
We present the first case of triplets with cerebrotendinous xanthomatosis (CTX).A C-to-T base change identified in the genomic DNA and cDNA encoding the sterol 27-hydroxylase led to replacement of arginine by tryptophan at position 441 (Arg441Trp) in the triplets. The triplets were homozygous and their mother was heterozygous for this mutant gene. The triplets exhibited an identical phenotypic expression, which was different from that of a sporadic CTX case with the same mutation.
NEUROLOGY 1996;46: 571-574
Specific defects in the bile acid metabolism constitute the underlying defect in cerebrotendinous xanthomatosis (CTX), a disorder characterized by multiple xanthomas, juvenile cataracts, dementia, cerebellar ataxia, and pyramidal paresis. [1] CTX is transmitted by an autosomal recessive mode and recent studies of the sterol 27-hydroxylase (CYP27) gene have revealed that six different missense mutations are associated with the disease. These missense mutations include the following: a black American with Cys for Arg at position 362 (designated as Arg362Cys), a Canadian (Arg446Cys), a Jewish family (Thr306Met), and Japanese families (Arg104Trp, Arg441Gln, and Arg441Trp). [2-5] Other single base changes resulting in frameshift, splice-junction alteration and premature stop codons were also present in Jewish families. [6,7] It is now possible to perform DNA-based diagnosis in some patients with CTX and to treat the patients at an early stage of the disease. Nevertheless, the clinical presentation of CTX varies considerably, not only between patients with different CYP27 mutations but also between patients who share the same mutation. [6] Here we present the first case of triplets who are homozygous for a mutant gene encoding the CYP27 (Arg441Trp) but who differ in their phenotypic CTX presentation from a sporadic case possessing the same mutation. [5]
Methods.
Patients.
The Japanese triplets were 27-year-old men who showed multiple xanthomas of the foot pads, Achilles tendons, patellas, wrists, and elbows. Mental retardation, cataracts, cerebellar ataxia, and pyramidal paresis also were present. Although they were healthy at birth, neurological symptoms appeared at or before age 5 and were slowly progressive. EEG showed irregular diffuse slow activity. Findings consistent with cardiomegaly and coronary atherosclerosis were demonstrated by ECG recordings. Plasma cholestanol levels were 10 times higher in the triplets than in controls. Clinical and laboratory profiles satisfied the diagnostic criteria for CTX as shown in the Table 1.
Table 1. Clinical and laboratory manifestations of CTX patients with Arg441Trp mutation in the CYP27 gene
Sequence analysis of the CYP27 gene.
Genomic DNA was extracted from peripheral blood leukocytes of the CTX triplets and their mother. The oligonucleotide primers flanking each exon of the CYP27 gene were synthesized based on the published sequence, [6] and all exons were amplified from the genomic DNA by polymerase chain reaction (PCR) using Taq DNA polymerase (Perkin-Elmer Cetus). The PCR products were sequenced directly using a DNA automated fluorescent sequencer (Model 373A, Applied Biosystems).
Restriction fragment length polymorphism (RFLP) analysis.
The DNA fragments containing exon 8 of the CYP27 gene were amplified using oligonucleotide primers (F: 5 prime-TCCTTTCTAGACCCAGTTTGTGTTC-3 prime, R: 5 prime- CCCAGCTCACGCATGCGAGGAGT-3 prime). The fragments were digested with Msp I, electrophoresed on 12% polyacrylamide gels, and stained with ethidium bromide.
Northern blot analysis.
Ten micrograms of total RNA extracted from cultured fibroblasts of the triplets and passage number-matched fibroblasts of five age-matched controls were subjected to Northern blot hybridization with the32P-labeled CYP27 and the beta-actin cDNA probes. The nylon membrane (Hybond-Nplus, Amersham) was exposed to x-ray film (XAR5, Kodak) at minus 80 degrees C for 14 or 3 days and to an imaging plate (Fuji Film, Japan). The ratios of the photostimulated luminescence of the CYP27 bands to those of the beta-actin bands were calculated using an image analyzer (Fujix BAS1000, Fuji Film, Japan).
Results and Discussion.
Direct sequence analysis of both strands of the CYP27 gene fragments revealed a C-to-T transition but no normal sequence within exon 8 in the three triplets with CTX. This mutation corresponded to a codon change from CGG coding for arginine to TGG coding for tryptophan at position 441 of the 498-residue CYP27 molecule and was present in the cDNA fragments reverse-transcribed from fibroblast mRNA of the triplets. The healthy mother had both normal and mutant sequences of the CYP27 gene. To ascertain the linkage of the base change with this type of CTX, we examined PCR products containing exon 8 by RFLP analysis. Msp I digestion of the fragments resulted in three bands in 74 normal subjects, whereas only two bands occurred in the triplets. This result was expected because the new mutation predicted a loss of one Msp I site. The mother showed both restriction patterns (Figure 1, A and B). All the results confirmed that the affected triplets were homozygous and their mother was heterozygous for the mutant CYP27 (Arg441Trp) gene, indicating that their CTX was caused by defects of both alleles of the CYP27 gene. Because a blood sample from the father was not available, we could not exclude the possibility that the mutation might have resulted from the uniparental disomy, reported in several diseases. [8]
Figure 1. Restriction fragment length polymorphism analysis of exon 8 of the CYP27 gene (A) Exon 8 of the CYP27 gene is indicated by the open box. The C-to-T transition results in a loss of one Msp I site in the mutant allele from the triplets. (B) Electrophoretic analysis of polymerase chain reaction products containing exon 8 after digestion with Msp I. Msp I digestion of the fragments resulted in three bands with 123-, 67-, and 43-bp sizes in a normal control but only two bands with 190- and 43-bp sizes in the triplets. The mother showed both restriction patterns.
In this study, we determined expression levels of the CYP27 mRNA in cultured fibroblasts from the triplets. In Northern blot analysis, the triplets showed the same expression level, which was higher than that of control fibroblasts (Figure 2, A and B). This result raised the possibility that some bile acid intermediates had accelerated the transcriptional level of the CYP27 gene in the CTX fibroblasts. Recently, Kim et al [5] reported a sporadic case (designated as Y.S.) who had the same mutation as the one presented here, and this mutation disrupted the CYP27 enzymatic activity.
Figure 2. (A) Northern blot analysis of the CYP27 mRNA. (B) The ratio of CYP27/beta A mRNA expression in fibroblasts from controls and the cerebrotendinous xanthomatosis triplets. Normal controls are indicated by open circles and the triplets by closed circles. beta A equals beta-actin.
Y.S. was well until age 30 when he noticed gait difficulty. [9] He was of normal intelligence without cataracts, and he had married at age 27 and had two healthy children. This clinical expression represents mild CTX, completely different from that of the triplets. All triplets exhibited equally severe neurologic dysfunction with mental retardation and gait disturbance since childhood and multiple xanthomas in adulthood. Laboratory data were almost identical among the three. This uniformity may be attributed to the triplets, who live at the same residence, having essentially the same genetic and environmental background. The clinical differences between the patient Y.S. and our triplets, all of whom were homozygous for the same mutation, indicate that factors in addition to genetics, such as environment, influence the clinical manifestations of CTX.
Acknowledgments
We thank Dr. Yurito Ueda for his critical suggestions and Mr. Yu Tanaka for technical assistance.
- Copyright 1996 by Advanstar Communications Inc.
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