A Novel Compound Heterozygous CYP27A1 Variant in Cerebrotendinous Xanthomatosis: A Case Report from a Non-Consanguineous Family

Abstract

Introduction

Cerebrotendinous xanthomatosis (CTX) is a rare, autosomal recessive lipid storage disorder characterized by the accumulation of cholesterol and cholestanol in various tissues. It is caused by pathogenic variants in the CYP27A1 gene, which encodes the mitochondrial enzyme sterol 27-hydroxylase.

Case Presentation

Here, we present an 8-year-old boy with attention-deficit/hyperactivity disorder, born to non-consanguineous parents. He was referred to our center for CYP27A1 gene analysis and genetic counseling, following the identification of a homozygous deletion in exon 6 of the CYP27A1 gene in his mother. His plasma cholestanol levels were also elevated, supporting a diagnosis of CTX. The proband’s father had a history of epilepsy and mild intellectual disability. Genetic analysis of the father revealed a novel heterozygous p.(Glu170Valfs*16) variant in the CYP27A1 gene. Based on these findings, the proband was found to carry a compound heterozygous variant in CYP27A1, confirming the molecular diagnosis of CTX. After genetic counseling, treatment with chenodeoxycholic acid (CDCA) was initiated. Plasma cholestanol levels normalized, and some clinical symptoms showed improvement after 2 months of treatment.

Conclusions

Early genetic screening of presymptomatic family members is critical, as timely initiation of CDCA therapy can prevent or significantly attenuate the clinical progression of CTX.

Established Facts

• Cerebrotendinous xanthomatosis (CTX; OMIM 213700) is a rare lipid storage disease characterized by the accumulation of cholesterol and cholestanol in various tissues. The clinical manifestations of CTX include infantile-onset diarrhea, childhood-onset cataracts, premature atherosclerosis, tendon xanthomas developing in young adulthood, and progressive neurological dysfunction.

• CTX is inherited in an autosomal recessive manner and is caused by biallelic pathogenic variants in the CYP27A1 gene, which is located on chromosome 2q35.

Novel Insights

• We identified a previously unreported compound heterozygous variant in the CYP27A1 gene, consisting of an exon 6 deletion and a frameshift insertion: c.508_509ins16 (p.Glu170Valfs*16). Both variants were classified as likely pathogenic. These findings were associated with markedly elevated plasma cholestanol levels, consistent with the clinical diagnosis of cerebrotendinous xanthomatosis (CTX) in the patient.

• In this paper, we highlight the importance of considering autosomal recessive diseases even in patients born to non-consanguineous parents.

• Although CTX is a rare disorder, it is thought to be underdiagnosed. Therefore, genetic testing of presymptomatic family members of affected individuals is recommended to enable early initiation of chenodeoxycholic acid therapy, which can prevent or mitigate disease manifestations.

Introduction

Cerebrotendinous xanthomatosis (CTX) is a rare lipid storage disorder caused by sterol 27-hydroxylase deficiency. CTX is characterized by cholestanol accumulation in tissues and increased urinary excretion of bile alcohols. The disease is inherited in an autosomal recessive manner and is caused by variants in the CYP27A1 gene, which is located at 2q35 and contains nine exons [1]. This gene encodes a cytochrome P450 oxidase, commonly known as sterol 27-hydroxylase, a mitochondrial enzyme that is widely expressed in most tissues and macrophages. This enzyme catalyzes a key step in the biosynthesis of bile acids from cholesterol and plays a critical role in maintaining cholesterol homeostasis [2–4] (Fig. 1). Deficiency of this enzyme leads to reduced synthesis of bile acids, including chenodeoxycholic acid and cholic acid and results in the accumulation of cholestanol and bile alcohols in various tissues, including the brain [5]. Patients with CTX usually exhibit normal or low plasma cholesterol levels, in contrast to elevated plasma cholestanol levels. The accumulation of sterols is associated with systemic manifestations, including infantile-onset chronic diarrhea, juvenile-onset cataracts, tendon xanthomas developing in young adulthood, premature atherosclerosis, and progressive neurological dysfunction in adulthood. Neurological findings may include dementia, epilepsy, psychiatric disorders, extrapyramidal symptoms, peripheral neuropathy, myopathy, and cerebellar and pyramidal signs. Magnetic resonance imaging (MRI) of the central nervous system typically reveals bilateral hyperintensities in the white matter tracts and dentate nuclei, along with diffuse cerebral and cerebellar atrophy [6].

Fig. 1.

Schematic representation of bile acid biosynthesis, showing both the classic and alternative pathways. In patients with CTX, a deficiency in sterol 27-hydroxylase (CYP27A1) impairs both pathways, as indicated by the red cross. CDCA therapy inhibits bile acid synthesis via negative feedback on cholesterol 7α-hydroxylase in the classic pathway.

Although more than 300 patients have been diagnosed with CTX worldwide, its true prevalence remains unclear. A recent epidemiological study based on the ExAC cohort suggests that the disorder is likely underdiagnosed and potentially misdiagnosed [7–9].

Long-term treatment with chenodeoxycholic acid (CDCA) normalizes plasma and cerebrospinal fluid cholestanol levels and improves both neurological and non-neurological manifestations, thereby slowing the progression of disability [10, 11]. Another potential alternative treatment involves the use of HMG-CoA reductase inhibitors (statins), either as monotherapy or in combination with CDCA [12–14].

Case Report

Here, we present the case of an 8-year-old boy with attention-deficit/hyperactivity disorder, learning difficulties, introverted behavior, and aggression. He is the first-born child of non-consanguineous parents. He was referred to our center for CYP27A1 gene analysis and genetic counseling following the identification of a homozygous deletion in exon 6 of the CYP27A1 gene in his mother. He was born at term via cesarean section after an uneventful pregnancy, with a birth weight of 3,400 g, whereas birth length was unknown. His developmental milestones were within normal limits.

On physical examination, his height measured 130 cm (50th–75th percentile), weight 25 kg (25th–50th percentile), and head circumference 51 cm (10th–25th percentile). The patient showed no dysmorphic features or tendon xanthomas. Ophthalmologic and neurological examinations revealed no signs of cataract or peripheral neuropathy, respectively. Sleep electroencephalography was also within normal limits. MRI was not performed to evaluate for Achilles tendon xanthomas. Although the patient experienced diarrhea in infancy, there was no history of cholestatic jaundice. In addition, the patient’s medical history included bilateral postaxial polydactyly of the hands, and his father had bilateral postaxial polydactyly both the hands and feet. Elevated plasma cholestanol (78.12 μmol/L; reference range: 3–16 μmol/L) and 7-dehydrocholesterol levels (65.78 μmol/L; reference range: 0–2 μmol/L) were observed. The proband’s mother, now 35 years old, has a medical history of bilateral cataracts, tendon xanthomas, severe cognitive impairment, spastic paraparesis, and severe dysarthria. Her symptoms first appeared at the age of 15, initially presenting with ataxic gait and dysarthria. The proband’s mother was found to have a homozygous deletion in exon 6 of the CYP27A1 gene at age 34. His father also had a history of epilepsy and mild intellectual disability.

In our patient, CDCA treatment was initiated at a dose of 10–15 mg/kg/day following confirmation of the CTX diagnosis. Two months after treatment initiation, the patient showed improvement in cognitive function. Routine cranial and diffusion-weighted MRI scans, performed regularly over the 6 years following the diagnosis and during CDCA therapy, revealed no abnormalities or signs of disease progression (Fig. 2).

Fig. 2.

Brain MRI before (a) and after (b) CDCA treatment, obtained 4 years apart.

Materials and Methods

Genomic DNA was isolated from the peripheral blood samples of the proband and his father using the MagNA Pure LC DNA Isolation Kit and the MagNA Pure LC instrument (Roche Applied Science, Mannheim, Germany). Because the patient’s mother was known to have a homozygous variant in the CYP27A1 gene, targeted next-generation sequencing (NGS) was first performed on the father using the MiSeq platform (Illumina, San Diego, CA, USA) to determine whether he also carried a pathogenic variant. Subsequently, the proband was analyzed. The variant identified by NGS was confirmed in both the proband and the father by Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). To investigate the exon 6 deletion previously identified in the mother, multiplex ligation-dependent probe amplification (MLPA) targeting the CYP27A1 gene was subsequently performed for the proband, using the SALSA MLPA Probemix P300 Reference-1 kit (MRC Holland, Amsterdam, The Netherlands). Later, Sanger sequencing was performed in the proband’s paternal grandmother to detect the known familial variant in the CYP27A1 gene, while targeted NGS was used to analyze the entire CYP27A1 gene in his father’s brother. In addition, cytogenetic analysis was performed on the proband’s father using standard peripheral blood culture and Giemsa-trypsin-G banding (GTG banding).

Results

A novel heterozygous NM_000784.4:c.508_509ins16 (p.Glu170Valfs*16) variant, located in exon 3 of the CYP27A1 gene, was identified in the patient and his father by NGS. This variant was not present in either the 1000 Genomes or ExAC databases. According to the ACMG guidelines, the variant is classified as likely pathogenic based on the following criteria: PVS1 (null variant in a gene with known loss-of-function mechanism) and PM2 (variant not found in gnomAD genomes). In addition, while the proband’s paternal grandmother [I.2] had the wild-type genotype, whereas his paternal uncle [II.1] was found to be heterozygous for the same variant (Fig. 3). Furthermore, a previously unreported heterozygous deletion in exon 6 of the CYP27A1 gene was identified in the proband by MLPA and was shown to be maternally inherited. This deletion corresponds to rsa 2q35 (CYP27A1 exon 6) x1. This variant was not listed in either the HGMD or ClinVar databases. According to the ACMG/ClinGen guidelines for structural variant interpretation, the variant is classified as likely pathogenic based on the following criteria: 1A (the variant affects protein coding or other functionally important genomic elements) and 2E (both breakpoints are located within the coding region of the same gene). Consequently, a compound heterozygous variant was identified in the proband (Fig. 4). MLPA analysis performed on the proband’s father revealed no pathogenic copy number variants. Chromosomal analysis of the proband’s father revealed a normal male karyotype (46,XY).

Fig. 3.

Pedigree of the family. The mother (II.4) is homozygous for a deletion in exon 6 of the CYP27A1 gene. The father (II.3) and the paternal uncle (II.1) are heterozygous for the p.(Glu170Valfs*16) variant in the CYP27A1 gene. The proband (III.2, indicated by the arrow) is compound heterozygous for an exon 6 deletion and the p.(Glu170Valfs*16) frameshift variant. The maternal aunt, who showed symptoms consistent with CTX, was not genetically analyzed.

Fig. 4.

a MLPA result of the proband showing a heterozygous deletion of exon 6 in the CYP27A1 gene. b Sanger sequencing demonstrating the heterozygous insertion c.508_509ins16 (p.Glu170Valfs*16) in the proband. The arrow indicates the insertion start site.

Discussion

In the proband, a novel compound heterozygous variant in the CYP27A1 gene was identified. The first variant, p.(Glu170Valfs*16), was detected in both the proband and his father and has not been previously reported in the literature. While this variant is located in exon 3, approximately 50% of the reported variants in the CYP27A1 gene are clustered within exons 6 to 8 [15] (Fig. 5). The second variant segregating from the mother was a heterozygous deletion of exon 6 of the CYP27A1 gene, a region where variants are most frequently detected.

Fig. 5.

CYP27A1 variants reported in the Human Gene Mutation Database (HGMD).

In a study evaluating cholestanol levels for the diagnosis and follow-up of CTX, higher levels were observed in patients with xanthomas, cataracts, and polyneuropathy. High cholestanol levels were considered useful for diagnosing CTX; however, they did not correlate with disease severity. They may also be helpful in monitoring adherence to CDCA treatment and guiding dose adjustment [10, 16].

CTX is often missed at initial presentation due to the lack of classical clinical features. Duell et al. [10] reviewed the diagnosis, treatment, and clinical outcomes in 43 patients with CTX. The mean age at diagnosis was 32 years (range: 8–55 years), with the following frequencies of clinical manifestations: chronic diarrhea (53%), cognitive impairment (74%), premature cataracts (70%), tendon xanthomas (77%), neurologic disease (81%), and premature cardiovascular disease (7%). Published case series on CTX have shown a significant diagnostic delay between symptom onset and diagnosis, with an average lag of 20–25 years [17]. In addition, cataracts have been reported in 70–90% of patients [10, 18, 19]. Although early-onset cataracts are rare, they may serve as an important clue for the early diagnosis of CTX [20].

The proband did not present with juvenile cataracts or developmental delay, both of which are typical childhood-onset symptoms of CTX. Instead, his clinical features were limited to neurobehavioral symptoms, including attention-deficit/hyperactivity disorder, learning difficulties, introversion, and aggression. Following genetic counseling, his paternal grandmother, who was also his legal guardian, provided consent for CDCA treatment. CDCA inhibits bile acid synthesis through negative feedback on both the cholesterol 7α-hydroxylase pathway and overall cholesterol biosynthesis, thereby preventing the accumulation of cholestanol and cholesterol (Fig. 1).

In our patient, CDCA treatment was initiated at a dose of 10–15 mg/kg/day, administered in three divided doses. After 2 months of follow-up, cholestanol levels normalized and mental functions improved. No complications or clinical regression were observed during the 6-year follow-up period.

According to the literature, while most patients with CTX receiving CDCA therapy show clinical improvement or slowed disease progression, some patients experience only stabilization, depending on their age and the presence of neurological symptoms at the time of diagnosis. Similar findings have also been reported for cognitive function, as assessed by IQ scores [21]. Without treatment, the life expectancy of patients with CTX is typically limited to the fifth or sixth decade of life; however, treatment is associated with improved survival [22].

Unfortunately, no clinical improvement was observed in the proband’s mother during 6 years of bile acid replacement therapy, which was initiated when she was 34 years old. She is currently being fed via a nasogastric tube due to severe progressive neurological impairment.

The etiology of epilepsy and intellectual disability in the father is unknown. Although epileptic seizures are a recognized clinical manifestation of CTX, they have not been described in individuals carrying a heterozygous CYP27A1 variant. The potential relationship between the heterozygous CYP27A1 variant detected in the father and his epilepsy remains unclear and warrants further investigation. Alternatively, the father’s symptoms might be attributable to complications during delivery. The proband’s 38-year-old paternal uncle, who was found to carry the same heterozygous variant (p.Glu170Valfs*16), was evaluated and found to be clinically unaffected, with no neurological, cognitive, or systemic findings suggestive of CTX. This observation suggests that the variant alone may not be sufficient to cause clinical disease.

A study reported a heterozygous carrier with reduced sterol 27-hydroxylase activity (∼10% of normal), normal serum cholestanol levels, and no clinical symptoms. Although reduced sterol 27-hydroxylase activity plays a central role in the pathogenesis of CTX, a significant increase in cholestanol levels may not occur even when enzyme activity is reduced by up to 90% [23].

Additionally, when the clinical features of the patient were evaluated according to the suspicion index (SI) proposed by Mignarri et al. [17], our patient yielded a low score because only psychiatric symptoms were present. Although his mother was diagnosed with CTX, this factor did not contribute to the suspicion index score, as he did not have an affected sibling. Likewise, a history of diarrhea in infancy did not influence the score. Although there was no consanguinity between the parents, the patient was evaluated due to his psychiatric symptoms and his mother’s diagnosis of CTX. An early diagnosis was established through molecular analysis following the detection of elevated cholestanol levels. These findings suggest that in cases where a parent with CTX has a child with a low suspicion index score and no consanguinity, measuring the child’s cholestanol levels may still be warranted.

This paper emphasizes that autosomal recessive disorders should be considered even in the absence of consanguinity. CTX should be suspected in patients presenting with common clinical features such as infantile-onset diarrhea, childhood-onset cataracts, adolescent- or young adult-onset tendon xanthomas, and progressive neurologic symptoms in adulthood. Although CTX is a rare disorder, it is believed to be underdiagnosed. Therefore, genetic testing of presymptomatic family members of affected individuals is recommended to enable early intervention with CDCA therapy, which may prevent or mitigate disease manifestations.

Acknowledgments

We gratefully acknowledge the participation of the patient and his family in this study.

Statement of Ethics

Ethical approval was not required for this study in accordance with local and national guidelines. Written informed consent was obtained from the patient’s legal guardian for the publication of this case report and any accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was not supported by any sponsor or funder.

Author Contributions

Hande Nur Cesur Baltacı wrote the first draft of the manuscript. Nüket Yürür Kutlay, Hande Nur Cesur Baltacı, Serap Tıraş Teber, and Turgay Coşkun performed the clinical evaluation of the patient. Hande Nur Cesur Baltacı and Burcu Sağlam Ada performed the laboratory tests. Nüket Yürür Kutlay and Ajlan Tükün revised the manuscript and contributed to its final version.

Funding Statement

This study was not supported by any sponsor or funder.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon request. The CARE Checklist is provided in the online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000547016).