ABCD1 mutations and phenotype distribution in Chinese patients with X-linked adrenoleukodystrophy

Gene 522 (2013) 117–120

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ABCD1 mutations and phenotype distribution in Chinese patients with X-linked adrenoleukodystrophy Yan-Fang Niu a, b, c, 1, Wang Ni a, b, c, 1, Zhi-Ying Wu a, b,⁎ a b c

Department of Neurology and Institute of Neurology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, Fuzhou, China

a r t i c l e

i n f o

Article history: Accepted 15 March 2013 Available online 5 April 2013 Keywords: X-linked adrenoleukodystrophy ABCD1 Mutation analysis Chinese

a b s t r a c t X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder resulting from mutations within the ABCD1 gene. Adrenomyeloneuropathy (AMN) and childhood cerebral ALD (CCALD) are most common phenotypes in the Western ALD patients. Here we performed mutation analysis of ABCD1 in 10 Chinese ALD families and identified 8 mutations, including one novel deletion (c.1477_1488 + 11del23) and 7 known mutations. Mutations c.1772G>A and c.1816T>C were first reported in the Chinese patients. Mutations c.1661G>A and c.1679C>T were demonstrated to be de novo mutations. The dinucleotide deletion 1415_16delAG, described as a mutational hotspot in different ethnic groups, was identified in two families. In addition, we performed a retrospective nation-wide mutation study of X-linked ALD in China based on a literature review. The retrospective study further confirmed the hypothesis that exon 6 is a potential mutation cluster region in the Asian populations. Furthermore, it suggested that CCALD is the most common phenotype in China. © 2013 Elsevier B.V. All rights reserved.

1. Introduction X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder resulting from mutations in the ABCD1 gene (NM_000033. 3) localized to Xq28 (Mosser et al., 1993). The incidence of X-ALD in males is estimated at between 1:21,000 and 1:50,000 (Kemp et al., 2001). The clinical manifestation is highly variable, most patients often begin with multifocal demyelination of the central nervous system (CNS) and adrenocortical insufficiency. Diagnosis is usually based on neurological symptoms, such as epilepsy, hypophrenia, impaired hearing, behavior changes, cognition impairment, inflammatory brain demyelination shown on brain magnetic resonance imaging (MRI), adrenal dysfunction, abnormal levels of plasma very long chain fatty acid as well as molecular genetic testing (Kemp et al., 2001; Moser et al., 2007). ALD is mainly classified into cerebral ALD (CER), adrenomyeloneuropathy (AMN), Addison-only (AO), Abbreviations: X-ALD, X-linked adrenoleukodystrophy; CCALD, childhood cerebral adrenoleukodystrophy; ACALD, adult form adrenoleukodystrophy; AMN, adrenomyeloneuropathy; AO, Addison-only; CNS, central nervous system; MRI, magnetic resonance imaging; CER, cerebral; PCR, polymerase chain reaction; TMD, transmembrane domain; NBF, nucleotide binding fold; kb, kilobase; fs, frameshift; del, deletion. ⁎ Corresponding author at: Department of Neurology and Institute of Neurology, Huashan Hospital,Shanghai Medical College, Fudan University, Shanghai, China and Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, 12 Wulumuqi Zhong Road, Shanghai 200040, China. Tel./fax: + 86 21 62483421. E-mail address: [email protected] (Z.-Y. Wu). 1 These two authors contributed equally to this work. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.067

olivopontocerebellar ALD and asymptomatic ALD (Kemp et al., 2001). CER include childhood (CCALD, onset age: 3–10 years old), adolescent (onset age: 11–21 years old) and adult forms (ACALD, onset age: after 21 years old) (Kemp et al., 2001; Moser et al., 2007). CCALD is the most devastating phenotype, which is associated with a rapidly progressive cerebral inflammatory demyelinating reaction. The ABCD1 gene consists of 10 exons spanning 19 kb of genomic DNA and encodes an mRNA of 4.3 kb. The protein product, ALD protein, has been located in a peroxisomal membrane (Mosser et al., 1993, 1994). More than 1300 mutations have been reported and listed in the X-ALD database (http://www.x-ald.nl). In the current study, we analyzed ABCD1 mutations in 10 Chinese ALD families and reviewed all mutations identified in 105 Chinese ALD patients from 83 pedigrees in order to get a mutational spectrum of the ABCD1 gene in China. We also observed all phenotypes, recorded in literature, to obtain a relationship between genotypes and phenotype. 2. Subjects and methods 2.1. Subjects Ten unrelated Chinese Han ALD patients and 8 relatives including 7 parents and 2 siblings were enrolled between May 2008 and December 2012. Medical history and demographic information were collected by a specially-assigned person, and records were reviewed by two senior neurologists. The patients were clinically grouped into 3 phenotypes. Six are CCALD, 2 are adolescent ALD and 2 are ACALD. Except for clinical

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features, all patients had typical characteristics of brain MRI, showing that bilateral symmetrical demyelination at the posterior periventricular white matter, parietooccipital and temporal cortex. The protocol was approved by the local ethics committee and the informed consent was signed by each individual or the parents of individuals younger than 18 years old. Genomic DNA was extracted from the sodium citrate treated peripheral blood samples using TIANamp blood DNA Kit (TIANGEN Biotech Co., Ltd., Beijing). 2.2. Mutation screening of ABCD1 The coding region of ABCD1 including the intron–exon boundaries were amplified using the polymerase chain reaction (PCR). Nine pairs of primers were sufficient to cover the exons 1–10 in ABCD1. Primer sequences and amplification conditions are listed in Table 1. PCR products were purified and subjected to direct sequencing; the procedure is as previously reported (Wu et al., 2006). All variants were verified by sequencing with both forward and reverse primers. 3. Results

Table 2 ABCD1 mutations and phenotypes of 10 unrelated Chinese ALD patients. Patient no. Exon

Nucleotide change

Amino acid change

Phenotype

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

None c.1661G>A c.1477_1488 + 11del 23 c.1028G>T c.1553G>A c.1415_16delAG c.1534G>A c.1679C>T c.1772G>A c.1415_16delAG

None p.Arg554His p.Leu493_Arg496del p.Gly343Val p.Arg518Gln p.Gln472fsX83 p.Gly512Ser p.Pro560Leu p.Arg591Gln p.Gln472fsX83

CCALD CCALD Adolescent ALD CCALD CCALD CCALD Adolescent ALD CCALD ACALD ACALD

None 7 5 2 6 5 6 7 7 5

and c.1816T>C causative mutations or polymorphisms? To answer this question, we further screened exon 8 in 25 normal persons and we found three genotypes: 1814TT (1816TT), 1814TC (1816TC) and 1814CC (1816CC). The c.1814T>C and c.1816T>C are linked on the same chromosome. This result indicates that c.1814T>C and c.1816T>C are polymorphisms. Apart from these two polymorphisms, we didn't find any other variation in patient 1.

3.1. Mutation analysis of ABCD1 3.2. Distribution of ABCD1 mutations in the Chinese population A total of 8 different ABCD1 mutations including 6 missense mutations and 2 deletions were identified (Table 2). A deletion, c.1477_1488 + 11del 23 (Fig. 1), resulting from four amino acid deletion in exon 5 of the ABCD1 gene, is novel. The other 7 mutations (Supplementary Fig. 1) have been previously reported as diseasecausing mutations (Barceló et al., 1994; Braun et al., 1995; Feigenbaum et al., 1996; Imamura et al., 1997; Kok et al., 1995; Pan et al., 2005; Smith et al., 1999). The dinucleotide deletion 1415_16delAG (Gln472fsX83) was identified in two patients (P4 and P10). The c.1661G>A carried by patient 2 (P2) and c.1679C>T by patient 8 (P8) were demonstrated to be de novo mutations, as the corresponding mutations were not present in their parents. In addition, c.1772G>A, localized in exon 7 was first reported in the Chinese ALD patients. Interestingly, patient 4 (P4) was identified to have three variations (c.1028G>T, c1814C>T and c.1816T>C) which had been identified as mutations by some experts (http://www.x-ald.nl) long time ago. Further, we found that his mother carried the heterozygous c.1028G>T variation (Supplementary Fig. 2). Thus patient 4 inherited this mutation from his mother. But we found that the mother carried the other two variations as homozygous change (Supplementary Fig. 3). These two variations were also identified in patient 1 and his mother as a heterozygous change (Supplementary Fig. 4). Are c.1814T>C Table 1 Primers designed for the ABCD1 gene and PCR conditions. Primers

Oligonucleotide of primers

Size of PCR products

Annealing temperatures

Exon 1.1

F 5′-AGGGTCAGAGCAACAATCC-3′ R 5′-AGGTAACGGATGGCACTG-3′ F 5′-TTTTGGCTGGCAGCTGCTG-3′ R 5′-CCACACCTTTGGCATCAGC-3 F5′-ACTGGGAGACCCTGACCATC-3′ R5′-ACCTCTGAAAGCATCAAGGC-3′ F 5′-ATTTGCAGAAGAGCCTCGC-3′ R5′-AAGCACATGGAGGTCCCTG-3′ F5′-TGCTGGTCAGGAACCAGCTG-3′ R 5′-GGTCTCTCACCTTGACCTTG-3′ F5′-AGAGATCAAGAATGGCCTGC-3′ R5′-CCAAAACAGGGCAGTGGATC-3′ F 5′- GATCCACTGCCCTGTTTTGG-3′ R5′-TGGCACTTTAGACTCTGGATG-3′ F 5′-CTGTCGTCACAGCTAGCTC-3′ R 5′-TGTGTGTGGTATTTCCTGGC-3′ F 5′-ATTGCCCTGCTCTCCATCAC-3′ R 5′-GTGCTGCTGTCTCCTTCATG-3′

552 bp

60 °C

633 bp

60 °C

420 bp

64 °C

623 bp

68 °C

366 bp

68 °C

460 bp

64 °C

393 bp

64 °C

620 bp

68 °C

490 bp

68 °C

Exon 1.2 Exon 2 Exons 3(4) Exon 5 Exon 6 Exon 7 Exons 8(9) Exon 10

We reviewed all references (Chiu et al., 2006; Lan et al., 2011; Li et al., 2010; Liu et al., 2007; Mak et al., 2005; Pan et al., 2005; Ping et al., 2007) about mutation analysis of ABCD1 in Chinese X-ALD patients and showed all mutations reported previously in Supplementary Tables 1 and 2. As shown in Fig. 2, in the Chinese ALD patients, mutations have been found throughout the entire gene except exon 10.To our surprise, statistical analysis showed that mutations localized in exon 6 accounted for 19.3%, apparently higher than that (11%) listed in the worldwide database. This result is consistent with the hypothesis that exon 6 is another potential mutation cluster region in the Asian populations (Chiu et al., 2006). In addition, the deletion c.1415_1416delAG (p.Gln472fsX83), which had been identified as a hotspot (10.3%) in Western countries and Japan (Kemp et al., 2001; Takano et al., 1999), also existed in Chinese patients (Lan et al., 2011; Pan et al., 2005). The finding indicates that it is a most common mutation in different populations. 3.3. Clinical features of the patients carrying different ABCD1 mutations and the phenotype in Chinese ALD patients In the current study, most patients manifested hypophrenia and memory impairment as initial symptoms, and gradually presented behavior changes, epilepsy, impaired vision and hearing. Patients 4, 6 and 9 had epilepsy. Patient 2 with c.1661G>A suffered from poor academic performance since age 10, then progressive behavioral, cognitive, auditory and visual deficit within 1 year. However, patient 3 with

Fig. 1. Chromatogram of c.1477_1488 + 11del23. The upper panel is the normal sequence whereas the lower panel is the mutated sequence.

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Fig. 2. Distribution of the ABCD1 mutations in the Chinese population and worldwide.

c.1477_1488 + 11del 23 only presented memory impairment at 15 years old without significant progression. His brain MRI showed a bilateral symmetric high signal in the posterior periventricular white matter (Fig. 3A). Patient 6, a childhood cerebral ALD with c.1415_1416delAG mutation presented poor memory and epilepsy. As shown in Fig. 3B, a wide range of hyperintensity lesions, involving parieto-occipital and temporal lobes were observed on his MRI scanning. While another patient (P10) with that deletion was an adult, his clinical finding was progressive mental disorder, unresponsive in 4 years. According to the relevant features of different phenotypes described in several reviews (Kemp et al., 2001; Moser et al., 2007), we summarized the phenotype of Chinese patients. 6 patients in our study manifested the childhood cerebral form; the onset ages were between 4 and 10 years. Phenotypes of 105 Chinese X-ALD patients from 83 pedigrees were as follows: 79 patients with the child cerebral form, 10 with the adult cerebral form, 1 with the adolescent form, 10 with AMN, 3 with ADO, 1 presenting as spinocerebellar ataxia and 1 asymptomatic patient (Chiu et al., 2006; Lan et al., 2011; Li et al., 2010; Liu et al., 2007; Mak et al., 2005; Pan et al., 2005; Ping et al., 2007). 4. Discussion Up to date, 1301 mutations have been identified and 47% of them are non-recurrent (http://www.x-ald.nl). Therefore a detailed mutation analysis of ABCD1 for each new ALD family is necessary. Direct sequencing of PCR products is a simple and reliable approach for mutation analysis. As we know, ABCD1 protein is a peroxisomal membrane protein. It has a half-transporter structure that consists of a transmembrane domain

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(TMD) with 6 putative membrane spanning segments and a putativehydrophilic ATP-binding cassette domain (designated the nucleotide binding fold, NBF) (Moser et al., 2007; Mosser et al., 1994). According to the X-ALD database (www.x-ald.nl); mutations have been documented throughout the entire ABCD1 gene, and the distribution is not evenly. Some mutations were observed to cluster in the transmembrane (40%) and NBF domains (30%) (Kemp et al., 2001). As shown in Fig. 2, the mutations identified in Chinese ALD patients were mainly distributed in exon 1 and exons 6–9, containing the ATP-binding domain. In the current study, we have identified 9 mutations and 6 of them are within exons 6–8. According to the estimation of X-ALD database (www.x-ald.nl), sporadic patients with X-ALD due to de novo mutations account for at most 7% of the total. Two de novo mutation, c.1661G>A and c.1679C>T, identified in the present study, had been detected in other pedigrees (Coll et al., 2005; Feigenbaum et al., 1996). Gonadal mosaicism probably results in sporadic patients with X-ALD, but it has not been studied systematically (Bowling et al., 1999; Kemp et al., 2001). It is well-known that the phenotype of X-ALD is wide and even different phenotypes can appear within a pedigree (Berger et al., 1994; Sobue et al., 1994). After going through all medical records, no consistent correlation between phenotype and the mutation was found, as previously observed (Kemp et al., 2001; Moser et al., 2007). A high frequency of disease-causing mutations was found in functionally important regions (Kemp et al., 2001). But it didn't mean that the severity of disease was associated with the location of mutation. In the current study, patient 6 whose mutation was located in exon 5, had mental decline, impaired visual and seizure while mild presentation was related with 23 nucleotides deletion in exon 5 (patient 3). In our retrospective study, more than 75% of Chinese X-ALD patients had CCALD phenotype, while only 10% of patients had AMN phenotype (Chiu et al., 2006; Lan et al., 2011; Li et al., 2010; Liu et al., 2007; Mak et al., 2005; Pan et al., 2005; Ping et al., 2007). This distribution is in disagreement with other populations. The data from Kennedy Krieger Institute and the Mayo Clinic showed that AMN was the most common phenotype (40–46%) in the USA, and CCALD was another common phenotype (31–35%) (Moser et al., 2000, 2007). The phenotype distribution of 80 Spanish X-ALD patients was CCALD (34%), AMN (27%), ACALD (14%), adolescent cerebral ALD (7%), and Addison-only (7%) (Coll et al., 2005). While in an epidemiological survey of X-ALD in 142 Japanese patients, the CCALD plus adolescent form accounted for about 69.5%, and 8.5% of patients had AMN phenotype (Shimozawa et al., 2010). Our results seem to be consistent with the data of Japanese ALD patients. The phenotypic variability makes it difficult to explore the pathogenesis of the disease. Moser et al. and Barbier et al hypothesized that ABCD1 mutation might not be the only cause of ALD, some modifier genes might involve in the pathogenesis of X-ALD (Barbier et al., 2012; Moser et al., 2007). Sobue et al. postulated that clinical phenotypes might be involved by environmental factors after describing various phenotypes in monozygotic twins (Sobue et al., 1994). And the discordant phenotype distribution among different populations as stated above suggested that the phenotype might be also related with the ethnic factor. In conclusion, the present study extended the spectrum of mutation in X-ALD. The retrospective study suggested that CCALD is the most common phenotype in China while the frequency of AMN is low. Further studies are needed to elucidate it. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.03.067. Acknowledgments

Fig. 3. Brain MRI of patient 3 (A) and patient 6 (B).

The authors sincerely thank the participants for their help and willingness to participate in this study and the anonymous reviewers

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for helping to improve this manuscript. This work was supported by a grant from the National Natural Science Foundation of China (81125009, Beijing) to Zhi-Ying Wu.

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