Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy

Abstract

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.

Figure 1

Pedigree of XL-SMA Family #2 As described by Kobayashi et al. Affected males are indicated by black boxes, obligate carriers with a dotted circle. The index patient is marked with an arrow. The synonymous C→T substitution (c.1731 C→T, p.Asn577Asn) was shown by direct sequencing approaches to segregate with disease in the family.

Figure 2

Sequence Alterations Detected in Exon 15 of the UBE1 Gene (A) The synonymous c.1731 C→T substitution. Top, wild-type sequence with the “C” highlighted in blue; center, heterozygote status in an obligate carrier; bottom, C→T substitution in a patient with the “T” highlighted in gray. (B) The c.1617 G→T substitution, p.Met539Ile in family #5. Top, wild-type sequence; center, heterozygote status of an obligate carrier; bottom, G→T substitution in a patient.

Figure 3

Genomic Structure of the UBE1 Gene and Amino Acid Conservation of Exon 15 in Several Species (A) Yellow boxes represent the 26 exons of UBE1 with an alternative exon 1a. The gene structure was obtained from publicly available databases (UCSC Genome Bioinformatics). Translation start in exon 2 is indicated with a black arrow. Five exonic CpG islands were detected by using in silico CpG island prediction tools (MethPrimer). (B) Position of CpG island and the methylated CpG dinucleotides in exon 15 are represented by green boxes. In index patients of three XL-SMA families, one C methylation less is observed resulting from the C→T exchange (red box). For bisulphite genomic sequencing, genomic DNA was isolated from cell lines or whole blood by means of the DNA Extraction Mini Kit (QIAGEN). The bisulfite modification reaction was performed with the EZ DNA Methylation Kit (HISS Diagnostics), and the bisulfite-treated DNA was amplified with primer pair (1f, 5′-GGT GAA TGT ATA AAT AAG TGA GT-3′; 1r, 5′-ACA CCC CTC TTA ATA TAT ACA C-3′) and subsequently sequenced with Big Dye kits (Perkin Elmer). The positions of the three detected mutations in exon 15 are marked with red stars. (C) Exon 15 represents parts of the transcript that codes for a highly conserved protein domain that form interactions with gigaxonin. Amino acid exchanges in the different species are highlighted in blue.

Figure 4

Model for Suggested Transcription Regulatory Element in Exon 15 of UBE1 (A) In the wild-type, the CpG island in exon 15 is fully methylated (symbolized by four green cylinders), which prevents the binding of proteins (red diamond) that block a specific transcription enhancer element, thus allowing a putative enhancer to stimulate the transcription. (B) In the XL-SMA patient harboring the synonymous C→T exchange, the CpG island in exon 15 is only partly methylated (symbolized by a white cylinder among three green cylinders), thus allowing the proteins to bind and as a consequence the transcription enhancer is blocked.