The potential consequences of bidirectional promoter methylation on GLA and HNRNPH2 expression in Fabry disease phenotypes in a family of patients carrying a GLA deletion variant

Abstract

Fabry disease (FD) is a rare inherited disease characterized by a wide range of symptoms attributed to GLA mutations resulting in defective α-galactosidase A (α-Gal A) and accumulation of glycosphingolipids. The GLA locus is paired in a divergent manner with the heterogeneous nuclear ribonucleoprotein HNRNPH2 locus mapped in the RPL36A-HNRNPH2 readthrough locus. As a follow-up to our recent finding of the co-regulation of GLA and HNRNPH2 via a bidirectional promoter (BDP) in normal kidney and skin cells, the potential accumulative influence of BDP methylation and GLA mutation on the severity of FD in patients from the same family, two males and two females carrying a GLA deletion mutation, c.1033_1034delTC (p.Ser345Argfs) was addressed in the present study. The molecular analyses of the FD patients compared with the control revealed that the expression of GLA was significantly low (P<0.05), and HNRNPH2 showed a tendency of low expression (P=0.1) when BDP methylation was elevated in FD patients, compared with low BDP methylation and high GLA expression (P<0.05), and a high trend of HNRNPH2 expression in normal individuals. The accumulative effects of the mutation and BDP methylation with the severity of the disease were observed in three patients. One male FD patient, a member of the FD family diagnosed with progressive loss of kidney function, hypertension, and eventually a stroke, and the lowest level of α-Gal A enzyme activity showed the highest BDP DNA methylation level. It is concluded that the DNA methylation of GLA-HNRNPH2 BDP may serve a role in diagnosing and treating FD.

Keywords: DNA methylation; Fabry disease; GLA; HNRNPH2; bidirectional promoter.

Figure 1

The pedigree of the FD family. Circle, female; square, male; white, not examined subjects or individuals who showed no symptoms; black diagonal line, FD deceased family member; red diagonal line, carrier; arrow, the first patient diagnosed in the study. FD1-FD4: patients participated in the study. FD, Fabry disease.

Figure 2

The GLA and HNRNPH2 genes in the RPL36A-HNRNPH2 readthrough region. (A) GLA and HNRNPH2 loci are divergently paired, mapped at forward and reverse strands respectively. (B) The BDP shares a sequence of 991 nucleotides at 5' ends of GLA and HNRNPH2 loci. CGI: CpG Island. BDP, bidirectional promoter.

Figure 3

Genomic features of the c.1033_1034delTC mutation in the GLA locus. (A) The GLA transcript NM_000169.2 is composed of seven exons; the location of the deletion mutation is in the translated sequence of exon 7. (B) The position of the TC deletion (circled) in the normally translated sequence of GLA-exon 7. At the end of GLA exon 7 is the normal termination codon, TAA (circled). Underlined sequence shows the TC repeat. (C) A TC deletion generated a new sequence of codons for new amino acids that are shown in the box. The mutation generated a premature termination codon, TAA (circled), and two more termination codons were generated, TAA and TGA. Brown boxes, translated sequences of exons; gray boxes, un-translated sequences of exons 1 and 7.

Figure 4

The generated amino acid sequence of the α-Gal A polypeptide at the site of the deletion mutation. (A) The affected amino acid Serine (S) at position 345 along the normal α-Gal A polypeptide sequence. (B) The deletion mutation generated an arginine (R), and the new amino acid sequence is terminated by a premature nonsense codon (END). α-Gal A, α-galactosidase A.

Figure 5

Association between BDP methylation and the expression of GLA and HNRNPH2 in the normal individuals and FD patients. (A) DNA methylation of the BDP in the males in the normal group and FD group. (B and C) Expression of GLA and HNRNPH2 in the males in the healthy group and FD group. (D) DNA methylation of the BDP in the females in the normal group and FD group. (E and F) Expression of GLA and HNRNPH2 in the females in the healthy group and FD group. FD, Fabry disease patients; BDP, bidirectional promoter.