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Identification of Cryptic Novel α-Galactosidase A Gene Mutations: Abnormal mRNA Splicing and Large Deletions

  • Takashi HiguchiEmail author
  • Masahisa Kobayashi
  • Jin Ogata
  • Eiko Kaneshiro
  • Yohta Shimada
  • Hiroshi Kobayashi
  • Yoshikatsu Eto
  • Shiro Maeda
  • Akira Ohtake
  • Hiroyuki Ida
  • Toya OhashiEmail author
Research Report
Part of the JIMD Reports book series (JIMD, volume 30)

Abstract

Anderson-Fabry (FD) disease is an inborn error of metabolism caused by a deficiency of α-galactosidase A (GLA), a lysosomal enzyme. Many male FD patients display a classic FD phenotype; however, some female patients have neither reduced leukocyte GLA enzyme activity level nor FD symptoms. Thus, GLA gene analysis is especially important for diagnosing suspected FD in female subjects. In this study, we revealed 4 novel GLA gene mutations in 5 independent families using GLA cDNA analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. These distinct mutations included a large deletion mutation from intron 1 to exon 5 (c.195-471_c.691del5.5k, corresponding to g.8508_g.14069del5.5k), an insertion mutation of splicing enhancer sequence in intron 4 (c.639+329_c.639+330ins113, corresponding to g.12627_g.12628ins113), an insertion mutation of retrotransposon L1 in exon 4 (c.634_c.635, corresponding to g.12293_g.12294), and a non-SNP deep intronic point mutation in intron 3 (c.547+395G>C, corresponding to g.11727G>C). It is difficult to detect these mutations with direct sequencing of only the exonic element. When exonic mutations are not found in the GLA gene from suspected FD patients, GLA cDNA and MLPA analyses should be performed to detect large deletion/insertion and intronic mutations including transcription abnormalities.

Keywords

Enzyme Replacement Therapy Inverted Repeat Sequence Female Family Member cDNA Sequence Analysis Large Deletion Mutation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgment

We thank our colleagues for their excellent technical assistance, Rie Ando (Oita Univ. Fac. of Med.), Miki Itohsa, Sayoko Iizuka, and Asako Morita (Div. of Gene Ther., The Jikei Univ.), and we thank Hidehito Kuroyanagi (Lab. of Gene Expres., Dep. of Func. Genomi., Med. Res. Inst., Tokyo Medical and Dental Univ.) for excellent discussions. We thank the primary medical doctors introducing the patients with FD to The Jikei Univ. hospital.

Supplementary material

432718_1_En_475_MOESM2_ESM.pptx (1.8 mb)
Fig. S1 Copy number of GLA Each Exons of Female Patient and locate in GLA mutation of Family A. (A) Graph showing the ratios between the relative peak areas (RPA) of the FD patient and female controls determined in all GLA exon elements. Intra-sample normalization: comparison of probe peak areas detecting each exons to other reference probe peak areas within each patient/control DNA sample. Inter-sample normalization: comparison of intra-sample normalized peak area of patient DNA sample to female control samples (n=3; female control RPA=1.0). (B) Sequence data of GLA gene between intron 1 and exon 5 (upper panel) and cDNA between exons 1 and 7 (lower panel) in II-3 Fig. S2 Locate in GLA mutation of Family B. Sequence data of GLA cDNA between exons 4 and 5 (upper panel) and intron 4 genomic DNA (lower panel) in III-1. Underlined: -gGAAT- repeat sequence Fig. S3 Locate in GLA mutation of Family D. Sequence data of GLA cDNA between exons 3 and 5 (upper panel) and exon 4 genomic DNA (lower panel) in II-2 Fig. S4 Locate in GLA mutation of Family E. Sequence data of GLA cDNA between exons 3 and 4 in II-1 (upper panel) and intron 3 genomic DNA in Family E (lower panel). Arrow, G>C heterozygous mutations; arrowhead, G>C hemizygous mutation; underlined, normal sequence
432718_1_En_475_MOESM1_ESM.xlsx (12 kb)
Table S1 Set of sequencing or RT-PCR primers

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Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Takashi Higuchi
    • 1
    Email author
  • Masahisa Kobayashi
    • 2
  • Jin Ogata
    • 2
  • Eiko Kaneshiro
    • 2
  • Yohta Shimada
    • 1
  • Hiroshi Kobayashi
    • 1
    • 2
  • Yoshikatsu Eto
    • 3
  • Shiro Maeda
    • 4
  • Akira Ohtake
    • 5
  • Hiroyuki Ida
    • 1
    • 2
  • Toya Ohashi
    • 1
    • 2
    Email author
  1. 1.Division of Gene TherapyResearch Center for Medical Sciences, The Jikei University School of MedicineTokyoJapan
  2. 2.Department of PediatricsThe Jikei University School of MedicineTokyoJapan
  3. 3.Front-Line Clinic CenterKanagawaJapan
  4. 4.Department of Advanced Genomic and Laboratory Medicine, Faculty of MedicineUniversity of the RyukyusOkinawaJapan
  5. 5.Department of PediatricsSaitama Medical UniversitySaitamaJapan

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