Novel mutation in ATP-binding domain of ABCD1 gene in ...

c Indian Academy of Sciences

RESEARCH NOTE

Novel mutation in ATP-binding domain of ABCD1 gene in adrenoleucodystrophy NEERAJ KUMAR1 , KRISHNA K. TANEJA2 , ATUL KUMAR3 , DEEPTI NAYAR3 , BHUPESH TANEJA3 , SATINDRA ANEJA4 , MADHURI BEHARI5 , VEENA KALRA6 and SURENDRA K. BANSAL1∗ 1

Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi 110 007, India 2 Functional Genomics Unit, 3 Structural Biology Unit, Institute of Genomics and Integrative Biology, New Delhi 110 007, India 4 Department of Pediatrics, Lady Hardinge Medical College and Kalawati Saran Childrens’ Hospital, University of Delhi, New Delhi 110 001, India 5 Department of Neurology, 6 Department of Pediatrics, All India Institute of Medical Sciences, New Delhi 110 029, India 6 Present address: Department of Neurology, Indraprastha Apollo Hospital, New Delhi 110 076, India

Introduction In adrenoleucodystorphy, more than one thousand mutations in ABCD1 gene have been reported from all over the world, of which 50% are unique (Moser and Kemp 1999). Recently, we had reported a splice-site mutation in ABCD1 gene. Here, we report the first case of adolescent cerebral adrenoleucodystrophy with a novel missense mutation in exon 8 of ABCD1 gene in Indian population. The diagnosis was based on clinical symptoms, substantial increase in plasma, very long-chain fatty acids and typical MRI pattern. MRI of the patient showed peritrigonal and cerebellar semioval white matter hypodensities and hyperintense areas (T2/fluid attenuated inversion recovery) in bilateral cerebral white matter, predominantly in parieto–occipital region. The molecular analysis by direct sequencing of the ABCD1 gene showed the presence of a novel missense mutation at c.1849C>A / Arg617Ser in the ATP binding domain in the proband and his mother, further establishing the diagnosis of the disease. X-linked adrenoleucodystrophy (X-ALD; OMIM 300100 (Online Mendelian Inheritance In Man) is the most frequently inherited peroxisomal neurodegenerative disorder affecting cerebral white matter, peripheral nerves, adrenal cortex and testis (Moser et al. 2002). The incidence of adrenoleucodystrophy in USA is 1 : 21000 in males *For correspondence. E-mail: [email protected]. [Kumar N., Taneja K. K., Kumar A., Nayar D., Taneja B., Aneja S., Behari M., Kalra V. and Bansal S. K. 2010 Novel mutation in ATP-binding domain of ABCD1 gene in adrenoleucodystrophy. J. Genet. 89, 473–477]

with varying clinical phenotypes (Kemp et al. 2001). Most frequent clinical phenotypes, accounting for 70%–80% of the patients, include severe progressive, inflammatory, demyelinating childhood cerebral form (ccALD) and slowly progressive, noninflammatory, adult adrenomyeloneuropathy (AMN) affecting mainly peripheral nerves and spinal cord (Moser et al. 2007). Other less frequently occurring phenotypes include adolescent cerebral (AdolCALD), adult cerebral (ACALD), olivopontocerebellar, addison-only and asymptomatic patients (Moser et al. 2007). The AdolCALD is similar to ccALD but its onset occurs between 10 and 21 years of age (Van Geel et al. 1997). X-ALD occurs due to alterations in the ATP-binding cassette, subfamily D, member 1 (ABCD1) gene localized at Xq28, which results in accumulation of very long-chain fatty acids (VLCFAs) in affected tissues (Mosser et al. 1993). ABCD1 gene protein product ALDP consists of two important domains, the transmembrane domain (TMD) and nucleotide binding domain (NBD). The NBD is the site for ATP binding (Rees et al. 2009). Currently, all over the world, 1065 mutations are known in ABCD1 gene, of which 522 (49.01%) are unique (nonrecurrent) (http://www.x-ald.nl/). Most of these mutations are missense (Fuchs et al. 1994), of which 24 are present in exon 8 at 13 different codons. Among these, five different missense mutations (four by others and one uploaded by us) are present in a single codon 5 -CGC-3 at position 617 in ALDP, which shows the presence of the largest number of mutations in one codon in this gene (Moser and Kemp 1999).

Keywords. X-linked adrenoleucodystrophy; neurodegenerative; genotype; phenotype; adolescent cerebral adrenoleucodystrophy; long chain fatty acids; missense mutation. Journal of Genetics, Vol. 89, No. 4, December 2010

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Neeraj Kumar et al. Earlier, we reported different mutations in ABCD1 gene in 20 patients (Moser and Kemp 1999; Kumar et al. 2008). The present report describes a case of an Indian patient whose clinical manifestations matched with AdolCALD, along with a positive family history. In this study, we observed a novel point mutation (Arg617Ser) in exon 8 of the ABCD1 gene both in the proband and his mother.

Materials and methods The patient belonged to a north Indian Hindu family. The healthy controls consisted of 70 age-matched and sexmatched individuals without any neurological and adrenal involvement or family history of the disease and were unrelated to the case. All subjects were north Indians and shared similar ethnicity. The study was approved by the Institutional Ethics Committee. Peripheral blood (5 mL) was drawn and collected in sterile EDTA vacutainers (Greiner Bio-one International, Frickenhausen, Germany) and genomic DNA isolated using phenol–chloroform protocol (Maniatis et al. 1982). All 10 exons of ABCD1 gene, including their flanking regions, were amplified by polymerase chain reaction (PCR). The primers designing and PCR amplification were performed as reported earlier (Kumar et al. 2008). Amplicons were purified using DNA isolation kit (Biological Industries, Beit Haemek, Israel). Direct sequencing of ABCD1 gene was performed using ABI 3100 DNA automated sequencer (Applied Biosystems, Foster City, USA). Multiple protein sequence alignment of ALDP in various eukaryotic organisms was done by constraint-based multiple alignment tool (COBALT) (Papadopoulos and Agarwala 2007). A homology based model for ALDP was obtained using MODWEB modelling server (Pieper et al. 2009). The model encompassed residues 605 to 704 of ALDP. The template used was 1B0U (Hung et al. 1998) and shared 30% sequence identity with ALDP, with an overall model score of 0.39. Two sets of minimization were carried out using AMBER9 (Case et al. 2006) on Silicon Graphics, SGI Altix 450. In the first case, the model was submitted for 80000 cycles of minimization for the native protein. To explore the role of arginine-617 of ALDP, an Arg617Ser substitution was carried out for the model on a graphics workstation and subjected to the same rounds of minimization. Case report

The patient is a 14-year-old male, and was born normal and at full term. The parents are Indians by origin and not related. The age of father is 52 and of mother is 49 years. A positive neurological disease was present in the family history. The proband’s maternal grandfather died at the age of 40 due to undiagnosed neurological disease. Proband’s mother was found to be the carrier of the disorder, adrenoleucodystrophy, but her younger sister is healthy and normal. The patient was normal up to the age of 13 after which the symptoms started appearing. The patient complained of focal deficit of vision 474

(nonresponsive pupils), hearing loss, irritated nature and dementia. He could not feed himself, had difficulty in swallowing and could not walk even 20 steps. No hyperpigmentation was observed. The neurological examination revealed normal bulk and increased tone. The power of muscles was 3/5 and deep tendon reflexes were brisk. Cremasteric and plantar superficial reflexes were present. Computerized tomography (CT) revealed low attenuation lesions in periatrial white matter, abnormalities in corpus callosum and occipital lobes. The MRI showed peritrigonal and cerebellar semioval white matter hypodensities and hyperintense areas (T2/ fluid attenuated inversion recovery) in bilateral cerebral white matter, predominantly in parieto–occipital region (figure 1c). However, motor nerve conduction, F-wave and sensory nerve conduction were obviously slower than the normal in all nerves tested. Plasma VLCFAs levels of the patient showed C26:0 to be 1.44 g/mL (normal level = 0.23±0.09 μg/mL), C24:0 to be 21.65 μg/mL (normal level = 17.59±5.36 μg/mL) and C22:0 to be 19.72 μg/mL (normal level= 20.97±6.27 μg/mL). The ratios of C26:0/C22:0 was 1.098 (normal ratio= 0.84±0.10) and C24:0/C22:0 was 0.073 (normal ratio = 0.01±0.004). The patient was categorized into AdolCALD phenotype. The plasma VLCFAs level of proband’s mother was within the normal range.

Results The sequencing analysis of coding regions of ABCD1 gene of the patient revealed single mutation ‘C>A’ at position 1849 in exon 8 (figure 1) which is a novel missense point mutation. This mutation was not detected in any of the healthy controls excluding the possibility of any amino acid substitution polymorphism. The multiple protein sequence alignment shows the conserved nature of arginine at position 617 in ALDP (figure 2b). The SIFT score of Arg617Ser is 0.00 sorting intolerant from tolerant (SIFT) score 0.05 is predicted to be deleterious). PolyPhen position-specific independent counts (PSIC) profile score difference on the basis of multiple alignment is 2.846 (PSIC score difference 0.5 is predicted to be deleterious). These scores suggest that Arg617Ser substitution may affect protein structure which may adversely affect its function. Moreover, the SIFT score on substitution of arginine with any other amino-acid at position 617 is worked out to be 0.00, which predicts this change to be deleterious. The asymptomatic carrier mother of the proband also possessed the same mutation in heterozygous condition. Her younger sister did not carry this mutation and her sequence matched the wild-type (GenBank database, http://www.ncbi.nih.gov/entrez, accession no. NG 009022). This mutation was uploaded by us in Human Genetics (accession no. HM 080121) and X-ALD mutation databases (Moser and Kemp 1999; Kumar et al. 2009). In silico analysis was carried out to explore the role of Arg617Ser mutation on binding of ATP. A comparision of homology model of ALDP, generated for the native protein

Journal of Genetics, Vol. 89, No. 4, December 2010

Novel mutation of ABCD1 gene in adrenoleucodystrophy

Figure 1. The criss-cross inheritance of recessive X-linked adrenoleucodystrophy alleles in an Indian family. Upper panel, pedigree; proband (IV-1) inherited allele ‘A’ from his heterozygous carrier mother (III-3) who inherited from her father (died at the age of 40). In inset, uppermost electropherogram shows wild-type reference sequence with allele ‘C’ at position 1849, middle one shows carrier heterozygous mother with allele ‘CA’ and lower one shows proband who inherited allele ‘A’ from his mother. The missense mutation 1849C>A in the ABCD1 gene causes the substitution of arginine with serine residue at 617 position in the ALDP (we used nucleotide numbering system where ‘A’ of the initiator Methionine codon numbered as +1 for reporting X-ALD mutations) (Kok et al. 1995). Lower panel, MRI pictures (a, b and c) show peritrigonal and cerebellar semioval white matter hypodensities and a characteristic pattern of enhanced T-2 signal in the parieto–occipital region with contrast enhancement at the advancing margin.

and arginine 617 substituted with serine, showed major conformational differences in two loops, encompassing residues 629 to 642 and residues 674 to 681 (see figure 1 in electronic supplementary material at http://www.ias.ac.in/jgenet/). Substitution of arginine-617 to serine has an effect on the loop immediately following it includes residues of the Walker B motif and involves in ATP binding (aspartate-629) (figure 2c).

Discussion The proband was diagnosed for AdolCALD. The disease was further established by molecular genetic analysis of the ABCD1 gene. The literature suggests that mutations are present in the entire coding region of the ABCD1 gene. How-

ever, TMD (encoded by exon 1) and NBD (encoded by exons 6 to 9) (figure 2a) contain more missense mutations than the rest of the regions (Smith et al. 1999). The proband possessed a novel missense mutation (c.1849C>A/Arg617Ser) in exon 8 of NBD, which is functionally important domain for binding ATP. In this codon, out of six possible different missense mutations, (three each at position first and second of the codon) four viz. c.1849C>T / Arg617Cys, c.1849C>G / Arg617Gly, c.1850G>A / Arg617His and c.1850G>T / Arg617Leu have already been reported by others (Fanen et al. 1994; Krasemann et al. 1996; Coll et al. 2005). Further, out of these four mutations, in three cases, there was no formation of ALDP in their fibroblasts (Moser and Kemp 1999; Kemp et al. 2001; Coll et al. 2005). In the


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Figure 2. (a) Schematic presentation of ALDP showing transmembrane domain (TMD) and nucleotide binding domain (NBD). (b) Multiple protein sequence alignment of ALDP of various species indicating conserved nature of arginine at position 617 in Homo sapiens. (c) Comparisons of native and mutated models of ALDP. Superposition of native (black) and Arg617Ser substituted model (gray) of ALDP showed an overall rmsd (root mean square deviation) of 1.4 Å for 100 atoms (residues 605–704). While overall structure of the two models is very similar, major differences were observed at the N-termini and C-termini and loops L1 (residues 629–642) and L2 (residues 674–681). The residues at the termini of the models (605 at N-terminal and 704 at the C-terminal) are indicated. Arginine 617 and aspartate 629 are shown in ball and stick.

fourth case (c.1849C>G/Arg617Gly) the status of formation of ALDP is not reported (Krasemann et al. 1996; Pan et al. 2005). In the present study, we identified the third novel missense mutation at the first position of the codon (1849). This mutation c.1849C>A in exon 8 of ABCD1 gene leads to exchange of amino acid arginine to serine at position 617 in the ALDP (Arg617Ser). Mutations involving this codon have been reported in many populations from various countries viz. USA, France, Spain, Germany, The Netherlands, PR China, Japan and Taiwan (Moser and Kemp 1999), suggests that this codon to be the most at risk for missense mutations and therefore a candidate for further studies. Position 617 in ALDP is highly conserved in most of the eukaryotic organism (figure 2b). Based on SIFT and PolyPhen scores, it can be predicted that any amino acid substitution at this position will have deleterious effect on the ALDP. It further gets support from the literature which shows that substitution of argnine by any other amino acid (Fanen et al. 1994; Krasemann et al. 1996; Coll et al. 2005) was deleterious as it led to the absence of ALDP. Therefore, here, we also propose that this mutation will cause the simi-

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lar effects on ALDP as reported by the studies as above. The status of ALDP could not be determined by us, the patient died before fibroblast collection. However, in silico analysis suggests that Arg617Ser mutation located in the Walker B region in the ATP binding domain of ALDP may change pocket size of NBD which may affect ATP binding. We had reported a de novo point mutation −2A>G at the 3 splice-site in intron 4 of the ABCD1 gene which was the first report to the best of our knowledge (Kumar et al. 2008) on the genetic features for X-ALD in Indian population. However, recently three more novel variants in three unrelated Indian families were also reported by Shukla et al. (2009). The possibility of identifying new mutations in Indian population is enormous due to its size, and genetic and ethnic variations that make it a crucial population for survey. Moreover, almost half of the total mutations observed in the ABCD1 gene are unique all over the world. Therefore, large scale family screenings are essential to identify new disease causing mutations in the Indian population that would help to understand the genetic basis of this disorder by enriching the existing X-ALD database.


Novel mutation of ABCD1 gene in adrenoleucodystrophy Acknowledgements Authors thankfully acknowledge Prof. Madhulika Kabra, Genetics Division, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India, and Ann Moser (Neurogenetics, Kennedy Krieger Institute, Baltimore, USA) for VLCFA analysis, and to the Council of Scientific and Industrial Research, India, for Senior Research Fellowship to Dr N. Kumar.

References Case D. A., Darden T. A., Cheatham III T. E., Simmerling C. L., Wang J. and Duke R. E. 2006 AMBER9. University of California, San Francisco, USA. Coll M. J., Palau N., Camps C., Ruiz M., Pa’mpols T. and Girós M. 2005 X-linked adrenoleukodystrophy in Spain. Identification of 26 novel mutations in the ABCD1 gene in 80 patients. Improvement of genetic counseling in 162 relative females. Clin. Genet. 67, 418–424. Fanen P., Guidoux S., Sarde C. O., Mandel J. L., Goossens M. and Aubourgt P. 1994 Identification of mutations in the putative ATPbinding domain of the adrenoleukodystrophy gene. J. Clin. Invest. 94, 516–520. Fuchs S., Sarde C. O., Wedemann H., Schwinger E., Mandel J. L. and Gal A. 1994 Missense mutations are frequent in the gene for X-chromosomal adrenoleukodystrophy (ALD). Hum. Mol. Genet. 3, 1903–1905. Hung L. W., Wang I. X., Nikaido K., Liu P. Q., Ames G. F. and Kim S. H. 1998 Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396, 703–707. Kemp S., Pujol A., Waterham H. R., Van Geel B. M., Boehm C. D., Raymond G. V. et al. 2001 ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations. Hum. Mutat. 18, 499–515. Kok F., Neumann S., Sarde C. O., Zheng S., Wu K. H., Wei H. M. et al. 1995 Mutational analysis of patients with X-linked adrenoleukodystrophy. Hum. Mutat. 6, 104–115. Krasemann E. W., Meier V., Korenke G. C., Hunneman D. H. and Hanefeld F. 1996 Identification of mutations in the ALD-gene of 20 families with adrenoleukodystrophy/adrenomyeloneuropathy. Hum. Genet. 97, 194–197. Kumar N., Shukla P., Taneja K. K., Kalra V. and Bansal S. K. 2008 De novo ABCD1 gene mutation in an Indian patient with adrenoleukodystrophy. Pediatr. Neurol. 39, 289–292.

Kumar N., Taneja K. K., Kalra V., Behari M., Aneja S. and Bansal S. K. 2009 Novel human pathological mutations. gene symbol: ABCD1. disease: adrenoleucodystrophy. Hum. Genet. 126, 344. Maniatis T., Fritsch E. F. and Sambrook J. 1982 Molecular cloning: a laboratory manual, 1st edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Moser H. W. and Kemp S. 1999 Open database: Kennedy Krieger Institute, USA and Laboratory of Genetic Metabolic Diseases, Department of Clinical Chemical and Pediatrics, Academic Medical Centre/Emma Childrens Hospital, Amsterdam, The Netherlands (cited 2010, March 12). Available from: http://www.x-ald. nl. Moser H. W., Mahmood A. and Raymond G. V. 2007 X-linked adrenoleukodystrophy. Nat. Clin. Pract. Neurol. 3, 140–151. Moser H. W., Smith K. D., Watkins P. A., Powers J. and Moser A. B. 2002 X-linked adrenoleukodystrophy. In The metabolic and molecular bases of inherited disease, 8th edition (ed. C. R. Seriver, A. L. Beaudet, W. S. Sly and D. Valle), pp. 3257–3301. McGraw Hill, New York, USA. Mosser J., Douar A. M., Sarde C. O., Kioschis P., Feil R. and Moser H. 1993 Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361, 726– 730. Pan H., Xiong H., Wu Y., Zhang Y. H., Bao X. H., Jiang Y. W. and Wu X. R. 2005 ABCD1 gene mutations in Chinese patients with X-linked adrenoleukodystrophy. Pediatr. Neurol. 33, 114–120. Papadopoulos J. S. and Agarwala R. 2007 COBALT: constraintbased alignment tool for multiple protein sequences. Bioinformatics 23, 1073–1079. Pieper U., Eswar N., Webb B. M., Eramian D., Kelly L., Barkan D. T. et al. 2009 MODBASE, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 37, 347–354. Rees D. C., Johnson E. and Lewinson O. 2009 ABC transporters: the power to change. Nat. Rev. Mol. Cell Biol. 10, 218–227. Shukla P., Gupta N., Kabra M., Ghosh M., Sharma R., Gupta A. K. et al. 2009 Three novel variants in X-linked adrenoleukodystrophy. J. Child Neurol. 24, 857–860. Smith K. D., Kemp S., Braiterman L. T., Lu J. F., Wei H. M., Geraghty M. et al. 1999 X-linked adrenoleukodystrophy: genes, mutations, and phenotypes. Neurochem. Res. 24, 521–535. Van Geel B. M., Assies J., Wanders R. J. A. and Barth P. G. 1997 X-linked adrenoleukodystrophy: clinical presentation, diagnosis and therapy. J. Neurol. Neurosurg. Psychiatr. 63, 4–14.

Received 21 December 2009, in revised form 12 March 2010; accepted 2 June 2010 Published on the Web: 23 November 2010


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