Pseudogenes & Mutation analysis
November 22nd, 2010 |Pseudogenes of ABCD1 and the importance for genetic analysis
In most cases, X-ALD is diagnosed biochemically by measuring elevated levels of very long-chain fatty acids (VLCFA) in plasma. This measurement is highly reliable for the diagnosis of male patients. However, care should be taken when VLCFA measurement is used for the identification of females suspected for X-ALD. Approximately 20% of obligate carriers for X-ALD have normal VLCFA levels in plasma. A normal plasma VLCFA level thus does not exclude heterozygosity for X-ALD.
Mutational analysis of the ABCD1 gene has the potential to identify women who are heterozygous for X-ALD with virtually complete accuracy. However, the fact that about half of the mutations are pedigree specific requires screening of the entire gene in an affected male (or obligate carrier) prior to targeted carrier testing for at-risk female relatives.
The gene mutated in X-ALD (ABCD1) is located on the X-chromosome at location Xq28. The ABCD1 gene contains 10 exons and covers approximately 21 kb. Sequence analysis of genomic DNA is made difficult because of the presence of ABCD1 paralogs on four autosomes. At some moment in primate evolution (about 5-10 million years ago), a 9.7 kb DNA segment encompassing exons 7 though 10 of the ABCD1 gene was duplicated from the X-chromosome to the chromosomes 2 (2p11), 10 (10p11), 16 (16p11) and 22 (22q11). In 1997, Eichler and colleagues at the Human Genome Center (Livermore, Ca) performed comparative sequence analysis of this fragment and showed that these four paralogs share 92-96% nucleotide identity. For details, please follow this link.
Picture 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 mutation analysis using genomic DNA is set up. In 1999, Corinne Boehm and colleagues from the Institute of Genetic Medicine at Johns Hopkins University (Baltimore, MD) developed and validated a robust genomic DNA-based diagnostic test for X-ALD. Xq28, ABCD1 gene specific primers were designed that allow accurate mutation analysis without interference of the pseudogenes. For details, please follow this link, or contact us for a copy of the paper.
Below, alignments of the exons 7, 8, 9 and 10 of the ABCD1 gene (Xq28) and the four paralogs on chromosomes 2, 10, 16 and 22 are shown. The asterisks indicate identical nucleotides in all five sequences.
Alignment of exon 7 of the ABCD1 gene with the paralogs.
chr2 GCCCTACATGTCTGTGGGTTCCTTGTGTGACCAGGTGATCTACCCGGACTCAGTGGAGGG 60
chr10 GCCCTACATGTCTGTGGGATCCCTGCGTGACCAGGTGATCTACCCGGACTCAGTGGAGGA 60
chr16 GCCCTACATGTCTGTGGGTTCCCTGCGTGACCAGGTGACCTACCCGGACTCAGTGGAGGA 60
chr22 GCCCTACATGTCTGTGGGTTCCCTGCATGACCAGGTGATCTACCCGGACTCAGTGGAGGA 60
ABCD1 GCCCTACATGTCTGTGGGCTCCCTGCGTGACCAGGTGATCTACCCGGACTCAGTGGAGGA 60
****************** *** ** *********** ********************
chr2 CATGCGAAGGAAGGGCTACCCGGAGCAGGACCTGGAAGCCATCCTGGACATCGTGCACCT 120
chr10 CATGCGAAGGAAGGGCTACTCGGAGCAGGACCTGGAAGCCATCCTGGATATCGTGCACCC 120
chr16 CATGCGAAGGAAGGGCTACTCAGAGCAGGACCTGGAAGCCATCCTGGACATCATGCACCT 120
chr22 CATGCGAAGGAACGGCTACTCGGAGCAGGACCTGGAAGCCATCCTGGACATCGTGCACCC 120
ABCD1 CATGCAAAGGAAGGGCTACTCGGAGCAGGACCTGGAAGCCATCCTGGACGTCGTGCACCT 120
***** ****** ****** * ************************** ** ******
chr2 GCACCACATCCTGCAGTGGGAGGGAG 146
chr10 ACACCACATCCTGCAGTGGGAGGGAG 146
chr16 GCACCACATCCTGCAGCGGGAGGGAG 146
chr22 GCACCACACCCTGCAGCGGGAGGGAG 146
ABCD1 GCACCACATCCTGCAGCGGGAGGGAG 146
******* ******* *********
Alignment of exon 8 of the ABCD1 gene with the paralogs.
chr2 GTTGGGAGGCTATGTGTGACTGGAAAGATGTCCTGCCGGGTGGCGAGAAGCAGAGAATCG 60
chr10 GTTGGGAGGCTATGTGTGACTGGAAGGACATCCTGCCAGGTGGTGAGAAGCAGAGAATCG 60
chr16 GTTGGGAGGCTATGTGTGACTGGAAGGACGTCCTGCCGGGTGGCAAGAAGCAGAGAATCG 60
chr22 GTTGGGAGGCTATGTGTGACTGGAAGGACGTCCTGCCGGGTGGCAAGAAGCAGAGAATCG 60
ABCD1 GTTGGGAGGCTATGTGTGACTGGAAGGACGTCCTGTCGGGTGGCGAGAAGCAGAGAATCG 60
************************* ** ***** * ***** ***************
chr2 GCATGGCCCGCATGTTCTGCCACAG 85
chr10 GCATGGCCCGCATGTTCTACCACAG 85
chr16 GCATGGCCTGCATGTTCTACCACAG 85
chr22 GCATGGCCTGCATGTTCTACCACAG 85
ABCD1 GCATGGCCCGCATGTTCTACCACAG 85
******** ********* ******
Alignment of exon 9 of the ABCD1 gene with the paralogs.
chr2 GCCCAAGTACGCCCTCCTGGATGAATGCACCAGTGCTGTGAGCATCGACGTGGAAGGCAA 60
chr10 GCCCAAGTACGCCCTCCTGGATGAAGGCACCAGTGCCGTGAGCATCGACGTGGAAGGCAA 60
chr16 GCCCAAGTACACCCTCCTGGATGAATGCACCAGTGCCATGAGCATCGACGTGGAAGGCAA 60
chr22 GCCCAAGTACACCCTCCTGGATGAATGCACCAGTGCCATGAACATCGACGTGGAAGGCAA 60
ABCD1 GCCCAAGTACGCCCTCCTGGATGAATGCACCAGCGCCGTGAGCATCGACGTGGAAGGCAA 60
********** ************** ******* ** *** ******************
chr2 GATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCAGCCCTC 120
chr10 GATCTTCCAGGTGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCTCCAGCCCTC 120
chr16 GATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTC 120
chr22 GATCTTCCAGGCGGCCAAGGACGCAGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTC 120
ABCD1 GATCTTCCAGGCGGCCAAGGACGCGGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTC 120
*********** ************ ************************* ** ******
chr2 C-TGTG 125
chr10 CCTGTG 126
chr16 CCTGTG 126
chr22 CCTGTG 126
ABCD1 CCTGTG 126
* ****
Alignment of exon 10 of the ABCD1 gene with the paralogs.
chr2 GGAGTACCACACACACTTGCTACAGTTCGATGGGGAGGGTGGCTGGAAGTTCGAGAAGCT 60
chr10 GGAGTACCACACACACTTGCTACAGTTCGATGGGGAGGGTGGCTGGAAGTTCGAGAAGCT 60
chr16 GGAGTACCACACACACTTGCTACAGTTCGATGGGGAGGGCGGCTGGAAGTTCGAGAAGCT 60
chr22 GGAGTACCACACACACTTGCTACAGTTCGATGGGGAGGGCGGCTGGAAGTTCGAGAAGCT 60
ABCD1 GAAATACCACACACACTTGCTACAGTTCGATGGGGAGGGCGGCTGGAAGTTCGAGAAGCT 60
* * *********************************** ********************
chr2 AGGCTCGGCTGCCCGCCTGAGCCTGACAGAGGAGAAGCAGCGGCTGGAGCAGCAGCTGGC 120
chr10 GGACTCAGCTGCCCACCTGAGCCTGACAGAGGAGAAGCAGCGGCTGGAGCAGCAGCTGGC 120
chr16 GGACTCAGCGGCCAGCCTGAGTCTGACAGAGGAGAAACAGCGGCTGGAGCAGCAGCTGGC 120
chr22 GGACTCAGCGGCCCGCCTGAGTCTGACAGAGGAGAAGCAGCGGCTGGAGCAGCAGCTGGC 120
ABCD1 GGACTCAGCTGCCCGCCTGAGCCTGACGGAGGAGAAGCAGCGGCTGGAGCAGCAGCTGGC 120
* *** ** *** ****** ***** ******** ***********************
chr2 GGGCATTCCCAAGATGCAGCGGCACCTCCAGGAGCTCTGCCAAATCCTGGGCGAGGCCGT 180
chr10 GGGCATTCCCAAGATGCAGCGGCACCTCCAGGAGCTCTGCCAAATCCTGGGCGAGGCCGT 180
chr16 AGGCATTCCCAAGATGCAGCGGCACCTCCAGGAGCTCTGCCAAATCCTGGGCGAGGCCGT 180
chr22 AGGCATTCCCAAGATGCAGCGGCACCTCCAGGAGCTCTGCCAAATCCTGGGCGAGGCCGT 180
ABCD1 GGGCATTCCCAAGATGCAGCGGCGCCTCCAGGAGCTCTGCCAGATCCTGGGCGAGGCCGT 180
********************** ****************** *****************
chr2 GGCCCCAGCGCACGTGCCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTC 240
chr10 GGCCCCAGCGCACGTGCCGGCACCTAGCCTGCAAGGCCCTGGTGGACTCCAGGGTGCCTC 240
chr16 GGCCCCAGCGCATGTGCCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTC 240
chr22 GGCCCCAGCGCATGTGCCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTC 240
ABCD1 GGCCCCAGCGCATGTGCCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTC 240
************ **************** *************** **************
chr2 CACCTGA 247
chr10 CACCTGA 247
chr16 CACCTGA 247
chr22 CACCTGA 247
ABCD1 CACCTGA 247
*******
ABCD1 gene specific primers
| Exon/s | Primer name | 5′ -> 3′ Sequence | Primer length | 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 and 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 and 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.
