Pseudogenes & Genetic Testing
Pseudogenes of ABCD1 and the importance for genetic analysis
Genetic testing of the ABCD1 gene has the potential to identify women who are heterozygous for adrenoleukodystrophy with virtually complete accuracy. However, sequencing is complicated by the presence of autosomal paralogs.
The ALD gene is located on the X chromosome at location Xq28. The gene contains 10 exons and approximately 20 kb in size. Sequence analysis of genomic DNA is complicated by the presence of ABCD1 paralogs on four autosomes (Figure). At some point in primate evolution (approximately 5 to 10 million years ago), a 9.7 kb DNA segment encompassing exons 7 through 10 of the ABCD1 gene was duplicated from the X chromosome to chromosomes 2 (2p11), 10 (10p11), 16 (16p11), and 22 (22q11). In 1997, Eichler and colleagues at the Human Genome Center (Livermore, Ca) performed a comparative sequence analysis of this fragment and showed that these four paralogs share 92-96% nucleotide identity. Please follow this linkFigure taken from Eichler et al. Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity (1997) Hum Mol Genet 6: 991-1002.
Because of this very high homology between the pseudogenes and the ABCD1 gene, great care should be taken when performing genetic testing using genomic DNA. In 1999, Corinne Boehm and colleagues at the Institute of Genetic Medicine at Johns Hopkins University (Baltimore, MD) developed and validated a robust genomic DNA-based diagnostic test for adrenoleukodystrophy. Xq28, ABCD1 gene-specific primers (see list below) were designed to allow accurate mutation analysis without interference from the pseudogenes. Please follow this link for details or contact us for a copy of the paper.
The alignments of exons 7, 8, 9 and 10 of the ABCD1 gene (Xq28) and its four paralogs on chromosomes 2, 10, 16 and 22 are shown below. The asterisks indicate identical nucleotides in all five sequences.
Alignment of exon 7 of the ABCD1 gene with the paralogs.
ABCD1 acactgcctgggaggcgcagagtatcttgggggaggcagagccggcccttccctccgtgg Chr2 acactgcctgggaggcacagagtatcttgggggaggcagggccagcccttccctccgtgg Chr10 acactgcctgggaggcgcagagtatctcgggggaggcagggccggcccttccctccgtgg Chr16 acactgcctgggaggcgcagagtgtcctgggggaggcagggccggcccttccctccatgg Chr22 acactgcctgggaggcgcagagtatcctgggggaggcagggccggcccttccctccatgg **************** ****** ** *********** *** ************ *** 1635 . . . . ABCD1 acacccagctttcccacagGCCCTACATGTCTGTGGGCTCCCTGCGTGACCAGGTGATCT 1675 Chr2 acacccagctttcccacagGCCCTACATGTCTGTGGGTTCCTTGTGTGACCAGGTGATCT Chr10 acacccaggtttcccacagGCCCTACATGTCTGTGGGATCCCTGCGTGACCAGGTGATCT Chr16 acacccagctttcccacagGCCCTACATGTCTGTGGGTTCCCTGCATGACCAGGTGATCT Chr22 acacccagctttcccacagGCCCTACATGTCTGTGGGTTCCCTGCATGACCAGGTGATCT ******** **************************** *** ** ************** . . . . . . ABCD1 ACCCGGACTCAGTGGAGGACATGCAAAGGAAGGGCTACTCGGAGCAGGACCTGGAAGCCA 1735 Chr2 ACCCGGACTCAGTGGAGGGCATGCGAAGGAAGGGCTACCCGGAGCAGGACCTGGAAGCCA Chr10 ACCCGGACTCAGTGGAGGACATGCGAAGGAAGGGCTACTCGGAGCAGGACCTGGAAGCCA Chr16 ACCCGGACTCAGTGGAGGACATGCGAAGGAACGGCTACTCGGAGCAGGACCTGGAAGCCA Chr22 ACCCGGACTCAGTGGAGGACATGCGAAGGAACGGCTACTCGGAGCAGGACCTGGAAGCCA ****************** ***** ****** ****** ********************* . . . . . ABCD1 TCCTGGACGTCGTGCACCTGCACCACATCCTGCAGCGGGAGGGAGgtaggaggcctgggg 1780 Chr2 TCCTGGACATCGTGCACCTGCACCACATCCTGCAGTGGGAGGGAGgtaggaggcctgggg Chr10 TCCTGGATATCGTGCACCCACACCACATCCTGCAGTGGGAGGGAGgtaggaggcctgggg Chr16 TCCTGGACATCGTGCACCCGCACCACACCCTGCAGCGGGAGGGAGgtaggaggcctgggg Chr22 TCCTGGACATCGTGCACCCGCACCACACCCTGCAGCGGGAGGGAGgtaggaggcctgggg ******* ********* ******* ******* ************************ ABCD1 ctggcagccaccctttgtcccaccctggcctctcccttggcctccagggagtgaagatta Chr2 ctggcagccgccctttgtcccaccctggcctctcccttggcctccagggagtgaagatta Chr10 ctggcagccgccctttgtcccaccctggcctctcccttggcctccagggagtgaagatta Chr16 ctggcagccgccctttgtcccaccctggcctctcccttggcctccagggagtgaagatta Chr22 ctggcagccgccctttgtcccaccctggcctctcccttggcctccagggagtgaagatta ********* **************************************************
Alignment of exon 8 of the ABCD1 gene with the paralogs.
ABCD1 ctccccggctggcccccgggtctgggtgctggtggaactgagccaagaccattgcccccg Chr2 ctccccggctggaccccaggtctgggtactggtggaactgagccaagaccattgcccctg Chr10 ctccccggctggcccccaggtctgggtactggtggaactgagccaagaccattgcccctt Chr16 ctccccggctggcccccaggtctgggtactggtggaactgagccaagaccattgcccctg Chr22 ctccccggctggcccccaggtctgggtactggtggaactgagccaagaccattgcccctg ************ **** ********* ****************************** 1781 . . . . . ABCD1 cctagGTTGGGAGGCTATGTGTGACTGGAAGGACGTCCTGTCGGGTGGCGAGAAGCAGAG 1835 Chr2 cctagGTTGGGAGGCTATGTGTGACTGGAAAGATGTCCTGCCGGGTGGCGAGAAGCAGAG Chr10 cctagGTTGGGAGGCTATGTGTGACTGGAAGGACATCCTGCCAGGTGGTGAGAAGCAGAG Chr16 cctagGTTGGGAGGCTATGTGTGACTGGAAGGACGTCCTGCCGGGTGGCAAGAAGCAGAG Chr22 cctagGTTGGGAGGCTATGTGTGACTGGAAGGATGTCCTGCCGGGTGGCAAGAAGCAGAG ****************************** ** ***** * ***** ********** . . . ABCD1 AATCGGCATGGCCCGCATGTTCTACCACAGgtgagcactccgggccggcaggctccctgg 1865 Chr2 AATCGGCATGGCCCGCATGTTCTACCACAGgtgagcactccgggccggcaggctccctgg Chr10 AATCGGCATGGCCCGCAAGTTCTACCACAGgtgagcactccaggctggcagcctccctgg Chr16 AATCGGCATGGCCTGCATGTTCTACCACAGgtgagcactccaggctggcaggctccctgg Chr22 AATCGGCATGGCCTGCATGTTCTACCACAGgtgagcactccaggctggcaggctccctgg ************* *** *********************** *** ***** ********
Alignment of exon 9 of the ABCD1 gene with the paralogs.
ABCD1 gggctggggtgttgggccctggagggtgcacagactctcctctcggcccggacccccagG 1866 Chr2 ggactggggtgttgggccctggagggtgcacagactctcctctcggcccggacccccagG Chr10 gggctggggtgttgggccctggagggtgcacagactcttctctcggaccggacccacagG Chr16 gggttggggtgttgggccctggagggtgcacagactctcctctcggcccggacccccagG Chr22 gggttggggtgttgggccctggagggtgcacagactctcctctaggcccggacccccagG ** ********************************** **** ** ******** **** . . . . . . ABCD1 CCCAAGTACGCCCTCCTGGATGAATGCACCAGCGCCGTGAGCATCGACGTGGAAGGCAAG 1926 Chr2 CCCAAGTACGCCCTCCTGGATGAATGCACCAGTGCTGTGAGCATCGACGTGGAAGGCAAG Chr10 CCCAAGTACGCCCTCCTGGATGAAGGCACCAGTGCCGTGAGCATCGACGTGGAAGGCAAG Chr16 CCCAAGTACACCCTCCTGGATGAATGCACCAGTGCCATGAGCATCGACGTGGAAGGCAAG Chr22 CCCAAGTACACCCTCCTGGATGAATGCACCAGTGCCGTGAGCATCGACGTGGAAGGCAAG ********* ************** ******* ** *********************** . . . . . . ABCD1 ATCTTCCAGGCGGCCAAGGACGCGGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTCC 1986 Chr2 ATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCAGCCC-TC Chr10 ATCTTCCAGGTGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCTCCAGCCCTCC Chr16 ATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTCC Chr22 ATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTCC ********** ************ ************************* ** **** * . ABCD1 CTGTGgtaggtgccctgtctccctgcctggggtcggtgggagtggctgcctgaggggagg 1991 Chr2 CTGTGgtaggtgccctgtctccctgcctggggtcagtgggagtggctgcctgaggggagg Chr10 CTGTGgtaggtgccctgtctccctgcctggggtcagtgggagtggctgcctgaggggagg Chr16 CTGTGgtaggtgccctgtctcccttcctggggtgagtgggagtggctgcctgaggggagg Chr22 CTGTGgtaggtgccctgtctcccttcctggggtgagtgggagtggctgcctgaggggagg ************************ ******** *************************
Alignment of exon 10 of the ABCD1 gene with the paralogs.
ABCD1 tgcccctgaccctgtccctctcctggccagGAAATACCACACACACTTGCTACAGTTCGA 2021 Chr2 tgcccctgaccctgtccctctcctggccagGGAGTACCACACACACTTGCTACAGTTCGA Chr10 tgcccctgaccccgtccctctcctggccagGGAGTACCACACACACTTGCTACAGTTCGA Chr16 tgcccctgaccctgtccctctcctggccagGGAGTACCACACACACTTGCTACAGTTCGA Chr22 tgcccctgaccctgtccctctcctggccagGGAGTACCACACACACTTGCTACAGTTCGA ************ ****************** * ************************** . . . . . . ABCD1 TGGGGAGGGCGGCTGGAAGTTCGAGAAGCTGGACTCAGCTGCCCGCCTGAGCCTGACGGA 2081 Chr2 TGGGGAGGGTGGCTGGAAGTTCGAGAAGCTAGGCTCGGCTGCCCGCCTGAGCCTGACAGA Chr10 TGGGGAGGGTGGCTGGAAGTTCGAGAAGCTGGACTCAGCTGCCCACCTGAGCCTGACAGA Chr16 TGGGGAGGGCGGCTGGAAGTTCGAGAAGCTGGACTCAGCGGCCAGCCTGAGTCTGACAGA Chr22 TGGGGAGGGCGGCTGGAAGTTCGAGAAGCTGGACTCAGCGGCCCGCCTGAGTCTGACAGA ********* ******************** * *** ** *** ****** ***** ** . . . . . . ABCD1 GGAGAAGCAGCGGCTGGAGCAGCAGCTGGCGGGCATTCCCAAGATGCAGCGGCGCCTCCA 2141 Chr2 GGAGAAGCAGCGGCTGGAGCAGCAGCTGGCGGGCATTCCCAAGATGCAGCGGCACCTCCA Chr10 GGAGAAGCAGCGGCTGGAGCAGCAGCTGGCGGGCATTCCCAAGATGCAGCAGCACCTCCA Chr16 GGAGAAACAGCGGCTGGAGCAGCAGCTGGCAGGCATTCCCAAGATGCAGCGGCACCTCCA Chr22 GGAGAAGCAGCGGCTGGAGCAGCAGCTGGCAGGCATTCCCAAGATGCAGCGGCACCTCCA ****** *********************** ******************* ** ****** . . . . . . ABCD1 GGAGCTCTGCCAGATCCTGGGCGAGGCCGTGGCCCCAGCGCATGTGCCGGCACCTAGCCC 2201 Chr2 GGAGCTCTGCCAAATCCTGGGCGAGGCCGTGGCCCCAGCGCACGTGCCGGCACCTAGCCC Chr10 GGAGCTCTGCCAAATCCTGGGCGAGGCCGTGGCCCCAGCGCACGTGCCGGCACCTAGCCT Chr16 GGAGCTCTGCCAAATCCTGGGCGAGGCCGTGGCCCCAGCGCATGTGCCGGCACCTAGCCC Chr22 GGAGCTCTGCCAAATCCTGGGCGAGGCCGTGGCCCCAGCGCATGTGCCGGCACCTAGCCC ************ ***************************** **************** . . . ABCD1 GCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACCTGAcacaaccgtccccggcccctgcc 2238 Chr2 GCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACCTGAcgccaccctccccagcccctgcc Chr10 GCAAGGCCCTGGTGGACTCCAGGGTGCCTCCACCTGAccccaccctctccagccccttcc Chr16 GCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACCTGAcgccaccctccccagcccctgcc Chr22 GCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACCTGAcgccaccctccccagcccctgcc *************** ********************** * *** ** ** ****** **
ABCD1 gene specific primers
Exon/s | Primer name | 5′ -> 3′ Sequence | nt | Amplicon size (incl M13 tails) |
1a | ALDe1A-F | TGTAAAACGACGGCCAGTACAACAGGCCCAGGGTCAGA | 38 | 458 bp |
ALDe1A-R | CAGGAAACAGCTATGACCAGGAAGGTGCGGCTCACCA | 37 | ||
1b | ALDe1B-F | TGTAAAACGACGGCCAGTAACCGGGTATTCCTGCAGCG | 38 | 421 bp |
ALDe1B-R | CAGGAAACAGCTATGACCACTGGTCAGGGTTGCGAAGC | 38 | ||
1c | ALDe1C-F | TGTAAAACGACGGCCAGTCCACGCCTACCGCCTCTACTT | 39 | 520 bp |
ALDe1C-R | CAGGAAACAGCTATGACCAGACTGTCCCCACCGCTC | 36 | ||
2 | ALDe2-F | TGTAAAACGACGGCCAGTGGCACTGGGAGACCCTG | 35 | 368 bp |
ALDe2-R | CAGGAAACAGCTATGACCTCAGCACCCAGCGGTATGG | 37 | ||
3 & 4 | ALDe3/4-F | TGTAAAACGACGGCCAGTGCAGAAGAGCCTCGCCTTTC | 38 | 606 bp |
ALDe3/4-R | CAGGAAACAGCTATGACCGCAGCAGGTCAGCACCTGCA | 38 | ||
5 | ALDe5-F | TGTAAAACGACGGCCAGTCTGCCAGGGATGGGAATGAG | 38 | 373 bp |
ALDe5-R | CAGGAAACAGCTATGACCTCTCACCTTGACCTTGGCCC | 38 | ||
6 | ALDe6-F | TGTAAAACGACGGCCAGTGCCATAGGGTACGGGAAGGG | 38 | 312 bp |
ALDe6-R | CAGGAAACAGCTATGACCGCCTCTGCAGGAAGCCATGT | 38 | ||
7 | ALDe7-F | TGTAAAACGACGGCCAGTCGATCCACTGCCCTGTTTTGG | 39 | 527 bp |
ALDe7-R | CAGGAAACAGCTATGACCCTTCCCTAGAGCACCTGG | 36 | ||
8 & 9 | ALDe8/9-F | TGTAAAACGACGGCCAGTCTGAGCCAAGACCATTGCCCCCG | 41 | 507 bp |
ALDe8/9-R | CAGGAAACAGCTATGACCTGCTGCTGCCGGGCCCGC | 36 | ||
10 | ALDe10-F | TGTAAAACGACGGCCAGTGAGGGGAGGAGGTGGCCTGGC | 39 | 463 bp |
ALDe10-R | CAGGAAACAGCTATGACCGCGGGGTGCGTGCATGGGTGG | 39 |
Primer sequences in black are M13F and M13R sequences that are used for sequencing. Nucleotides in green are Xq28, ABCD1 gene, specific. Primers were taken from Boehm et al.: Accurate DNA-based Diagnostic and Carrier Testing for X-linked Adrenoleukodystrophy (1999) Mol Genet Metab 66: 128-136.
Last modified | 2024-06-25