A Novel Mutation in the Sterol 27-hydroxylase Gene of a Pakistani Family with Autosomal Recessive Cerebrotendinous Xanthomatosis

Cerebrotendinous xanthomatosis (CTX) is a rare neurometabolic disease, inherited as an autosomal recessive trait. ^[1] The main clinical manifestations include tendon xanthomas, juvenile cataracts, pyramidal tract signs, cerebellar ataxia, progressive dementia, peripheral neuropathy, and premature atherosclerosis. ^[2] The biochemical pathogenesis is linked to an increase in levels of plasma and tissue cholestanol arising from defective bile acid synthesis. ^[3,4] The abnormal neurologic features may result from replacement of cholesterol by cholestanol in myelin sheath of the central and peripheral nervous system. The precise mechanism by which cholestanol is deposited is not known. ^[5]

The mitochondrial sterol 27-hydroxylase gene (CYP27) maps to chromosome 2q33-qter and is defective in CTX. ^[6] CYP27 encodes a mitochondrial enzyme that catalyzes the oxidation of side chains of cholesterol and other sterol intermediates in bile acid biosynthesis. ^[7] The absence or inactive form of this enzyme leads to an accumulation of cholestanol, a derivative of cholesterol in multiple tissues. The biochemical pathogenesis is complex because the relation between the deficient oxidation of cholesterol side chain and the increased biosynthesis of cholestanol in CTX is not yet understood. ^[8,9]

Following the molecular cloning of the sterol 27-hydroxylase cDNA and characterization of gene structure, different laboratories reported eight distinct mutations in CYP27. ^[7,10-14] Japan may have a higher frequency of CTX as more cases have been reported from that country. ^[14] In this paper we describe a novel nonsense mutation in the CYP27 gene in a Pakistani family with CTX and discuss the genotype associated with this mutation. This is the first report of a mutation in CYP27 in a south Asian family.

Methods.

Single stranded conformational polymorphism (SSCP) genotyping and DNA sequencing analysis.

The pedigree of this Pakistani family is given in Figure 1a. The family was screened for mutations in the CYP27 gene. The genomic DNA of family members was isolated from blood lymphocytes. The splice junctions and all coding regions of the sterol 27-hydroxylase gene were amplified with oligonucleotide primers derived from previously published sequences ^[13] followed by SSCP analysis ^[15] and polymerase chain reaction (PCR) sequencing using fmol DNA sequencing system (Promega Corporation) according to the dideoxynucleotide chain termination method. ^[16] Amplified products were analyzed on 1.5% agarose gels. The appropriate band was excised and purified, and then sequenced.

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Figure 1. Pedigree structure and single-stranded conformational polymorphism (SSCP) analysis in the family. (A) The proband is marked with an arrow. Darkened pedigree symbols designate clinically affected individuals. Half-darkened pedigree symbols designate heterozygotes. Males are designated by squares and females by circles. (B) PCR-SSCP analysis of exon 4. Lane C, normal control DNA; lanes 1-8, members of the Pakistani family. ND = not detected. (C) CTX genotype determined according to band migration pattern of the control and family members. The presence of the mutant allele is indicated by a plus sign. + / + = homozygotes normal; + / - = heterozygotes; - / - = homozygous normal.

Case report.

The proband was a 22-year-old Pakistani man who presented with abnormal movements of his head as well as with gait disturbances. He was born full term to consanguineous parents. His early psychomotor development was normal. At age 12 years, he was noted to have bilateral cataracts and mild mental retardation; cataract extraction was performed on his right eye at age 16 years. He could not continue schooling beyond the fourth grade because of poor scholastic performance. Three of his nine siblings (one elder sister and two younger brothers) have similar symptoms. The symptoms include tendon xanthomas, bilateral cataracts, and mental subnormality. Three maternal first cousins (two females and one male) also have a similar disease.

The proband was a thin, ill-looking young man of average height with dysmorphic facial features. He had a high arched palate and feet, hypermobility of proximal interphalangeal joints, xanthomas of both Achilles tendons, dense cataract in his left eye, and aphakia of the right eye. He was well oriented, but his short-term memory was poor. His speech was slow and slurred, and he had a staggering gait and could not walk in tandem. He also had gross truncal ataxia with mild dysmetria and past pointing. Bulk of the muscles was reduced, but the power and tone were normal. Deep tendon reflexes were exaggerated on the right side with an extensor plantar response. Vibration and position sense were impaired in the lower limbs, and there was Romberg sign. The right fundus was normal; the left could not be visualized. Examination of the abdomen and cardiovascular and respiratory systems was unremarkable.

Routine laboratory tests such as hemoglobin, complete blood count, urea, creatinine, electrolytes, fasting blood sugar, liver function tests, serum proteins, prothrombin time, uric acid, chest radiograph, ECG, urine examination, and ultrasound of the abdomen were within normal limits. Erythrocyte sedimentation rate was 40 mm/1st hour (Wesergreen). Serum cholesterol was very low (15 mg/dL), and triglycerides were 85 mg/dL. CSF examination revealed increased protein (142 mg/dL). EEG showed a background activity of a mixture of irregular 8-10 Hz and runs of generalized higher voltage 2-3 Hz rhythmic delta waves. MRI of the head showed an area of high intensity signal on T₂-weighted images in the cerebellum, which was hypointense on T₁-weighted images, indicating encephalomalacia. The fourth ventricle was enlarged, and cerebellar folia and cortical sulci were widened. This appearance was consistent with diffuse atrophic changes, more prominent in the cerebellum. Microscopic examination of a tendon biopsy revealed fibrotendinous tissue with marked chronic inflammation, foamy macrophages, and foreign body giant cell reaction to needle like crystalline material resembling cholestanol.

Results.

DNA sequence analysis.

Sequence analysis of the CYP27 mutant allele in CTX revealed a single base substitution of C to T in codon 237 that would alter the codon for arginine (CGA) to a stop codon (TGA). Heterozygous individuals showed both the normal and mutant sequences. Normal individuals homozygous for the normal exon 4 SSCP migrating pattern had only the wild type sequence (Figure 2). This mutation introduces an early termination codon in the message, which would result in a truncated protein. The possibility that this family may carry another CYP27 mutation or a compound mutation was ruled out by both SSCP examination and sequencing of all exons and their splice junctions (results not shown).

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Figure 2. DNA sequence analysis for mutations in the CYP27 gene of the family. Exon 4 and its flanking sequences were PCR amplified using genomic DNA and sequenced. The experimental conditions are given in Methods.

Discussion.

We found a novel CYP27 nonsense mutation in a Pakistani family with CTX. Cytosine (237) was substituted by thymidine in exon 4, which results in a stop codon. The mRNA of the mutant allele is therefore expected to translate a truncated protein with virtually complete absence of sterol 27-hydroxylase activity.

Most of the CTX mutations have been reported from the southern Mediterranean-in Jews from north Africa and in Arabs of the Druze community. In all cases, there was consanguinity. All eight mutations reported in CYP27 occurred in CTX families. Cali et al. ^[7] reported a mutation of a single base pair that converted arginine codons (CGP) to cysteine codons (TGP) at codon 362 and 446 in two CTX patients from Canada and the United States. Leitersdorf et al. ^[10] described a deletion of cytosine 376 that led to a termination codon at residue 109 in a Druze family. Reshef et al. ^[12] found a C to T transition at cDNA codon 1037, which predicted a threonine to methionine substitution at residue 306 in Jews of Algerian origin. In addition, two different mutations were found by Leitersdorf et al. ^[13] in Moroccan Jews-a deletion of T in exon 4 that results in a stop codon 35 nucleotide downstream and a G to A substitution at the 3 prime splice acceptor site of intron 4.

CTX may be more common in Japan, where three mutations in CYP27 have been identified. Nakashima et al. ^[11] noted a point mutation in CYP27 at codon 104, resulting in an arginine (CGG) to tryptophan (TGG) substitution in a Japanese family. Kim et al. ^[14] identified two different mutations in Japanese patients with CTX-A to G at codon 441 [CGG (Arg) to CAG (Gln)] and C to T at codon 441 [CGG (Arg) to TGG (Trp)].

Thus, CTX families from several ethnic origins are associated with mutations in CYP27 in CTX. The novel mutation described here is the first mutation discovered in a south Asian family and is different from those previously reported. The conventional method for diagnosis of CTX is usually based on the presence of certain clinical characteristics and the demonstration of an increased plasma concentration of bile alcohol and cholestanol. However, some homozygous unaffected individuals in CTX families typed to have wild type CYP27 have moderately elevated plasma cholestanol concentrations. ^[10] As mutations in CYP27 are casually associated with CTX, molecular diagnosis of the CYP27 gene by mutational analysis will assist in the confirmation of CTX and could be used to detect carriers of the mutation for genetic counseling in communities with high consanguinity. Experiments to test the stability of the mutant CYP27 mRNA translation of the truncated protein and possible gain of another novel function gained by a such truncated protein is being explored.