Pseudogenes & Genetic Testing

Pseudogenes of ABCD1 and the importance for genetic analysis

Stephan Kemp, Ph.D

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 link Figure 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