Structure of the GM2A Gene: Identification of an Exon 2 Nonsense Mutation and a Naturally Occurring Transcript with an In-Frame Deletion of Exon 2 Biao.

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Structure of the GM2A Gene: Identification of an Exon 2 Nonsense Mutation and a Naturally Occurring Transcript with an In-Frame Deletion of Exon 2 Biao Chen, Brigitte Rigat, Cynthia Curry, Don J. Mahuran The American Journal of Human Genetics Volume 65, Issue 1, Pages 77-87 (July 1999) DOI: 10.1086/302463 Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 1 Western blot analysis, done with a primary rabbit anti-Activator IgG, of fibroblast lysate (either 50 or 100 μg total protein) from N1, N2, and the patient. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 2 RT-PCR of total RNA from N1, N2, and the patient. Arrows indicate the normal-sized product (691 bp), the unexpected smaller product (529 bp) found only in the patient's cDNA, and, in lane M containing λDNA/ HindIII markers, the sizes of the surrounding markers. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 3 Partial nucleotide sequence of a cDNA fragment from a normal control and the patient. The arrows indicate the single G160→T transversion found. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 4 Partial-nucleotide sequence of the smaller cDNA RT-PCR product from patient samples, and cDNA fragments from normal controls. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 5 Determination of the length of intron 1 (∼6.45 kb) and intron 2 (∼6.60 kb) of GM2A. Intron-containing fragments were amplified by long PCR with use of exonic primers. Each fragment contained ∼150 bp exonic sequence at each end. Size markers (“M”) are also shown. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 6 A, Restriction map of GM2A; B = BamHI, E = EcoRI, S = SstI, X = XbaI, K = KpnI, and H = HindIII. No XhoI sites were present in intron 1 or 2. The numbers with each restriction site indicate the number of times it has appeared, reading from 5′ to 3′ in the gene structure. Intron lengths are given in brackets. The unblackened boxes refer to exons, and the blackened boxes refer to 5′ and 3′ UTRs of the cDNA. B, Sequence of the exons and exon/intron junctions of GM2A; intronic sequences are given in lowercase, the encoding exonic sequences in bold uppercase, and the untranslated exonic sequences in regular uppercase. The translation initiation (exon 1) and termination (exon 4) sites are wavy-underlined. The forward and reverse primers for amplification of the 4 exons and their flanking regions are single underlined and double underlined, respectively. The number followed by an arrow and either an “F” (forward) or “R” (reverse) in brackets above each primer indicates the exon being amplified and also the primer's direction (see table 1). The unique restriction site in each fragment, used to confirm its identity, is dotted-underlined. Three reported exonic polymorphisms are indicated with an asterisk (*). The two new polymorphisms identified during this study, G175A and IVS1 a-92t, and are indicated with a pound sign (#). We also report the correction of previously published sequences at IVS1–12 and IVS1–15 (Klima et al. 1991), which are indicated by a caret (^). The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 7 PCR fragments containing exons E1–E4 and their flanking intronic sequences, amplified with the primers in table 1. Each fragment is shown intact undigested (UD) or digested with its unique restriction site. X = XbaI, S = SstI, P = PstI, and E = EcoRI (table 1). Size markers (M; 100 bp) are also shown. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 8 Direct nucleotide sequence of a PCR fragment (fig. 6B and fig. 7 E2) of genomic DNA from the patient and a normal control. Arrows indicate the homozygous G160T mutation in the patient's DNA. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions

Figure 9 Nested PCR–amplified RT-PCR product from the patient and a normal control. “ΔE2” and “WT” refer to the cDNAs missing or containing exon 2, respectively. Undigested RT-PCR product (UD) or the RT-PCR products digested with HinfI prior to the second nested PCR procedure, HinfI, are indicated. Cloned WT and ΔE2 cDNA, as well as a H2O control sample (C) were also subjected to the nested PCR procedure. Size markers (M) are identified with arrows. The American Journal of Human Genetics 1999 65, 77-87DOI: (10.1086/302463) Copyright © 1999 The American Society of Human Genetics Terms and Conditions