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Polymorphisms in the human homologue of the drosophila Indy (I’m not dead yet) gene

Pauline Lee *, Ngoc Ho, Terri Gelbart, Ernest Beutler

Division of Hematology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

Received 10 April 2003; received in revised form 30 May 2003; accepted 11 June 2003

Abstract

Insertion of a P element in one copy of the Drosophila Indy gene results in a significant increase in Drosophila lifespan. In order to examine whether mutations or polymorphisms in the human Indy gene promoted human longevity, the Indy gene was sequenced in 13 subjects of age 90(cid:1)99, 12 subjects between age 38(cid:1)49 and 18 subjects Bage 23. Three coding region polymorphisms resulting / / / in an amino acid change, F62L, V80L and I550V, two silent coding region polymorphisms, A99A and I551I, and three intron polymorphisms, IVS2(cid:2)10 C deletion, IVS9(cid:2)40T/A and IVS10(cid:2)38C/T were identified. The I550V polymorphism occurred in / / / whites, Hispanics, Asians and African(cid:1)Americans at allele frequencies of 0.3514, 0.5104, 0.4274 and 0.5375, respectively. The I550V / polymorphism was genotyped in 2366 subjects and examined for its association with age and weight. # 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Aging; Indy; NADC; SLC13A2; Polymorphism; Sodium dependent dicarboxylate transporter

span by interference with normal metabolism resulting in ‘‘caloric restriction’’, because the expression of this gene in flies was limited to the fat body, midgut, and oenocytes, organs that are primary sites of intermediate metabolism, absorption, and metabolic storage.

1. Introduction

melanogaster and in Caenorhabditis elegans have been associated with increased life span. While aging in humans is clearly a complex phenomenon with a strong environmental component, there seems to be a genetic component as well (Cournil and Kirkwood, 2001). Accordingly, it seemed worthwhile to examine the gene that appears to have the most profound effect in D. melanogaster, viz. Indy (an acronym for I’m not dead yet) (Rogina et al., 2000), to determine whether it has an effect on human lifespan, or any other aspect of human health. In Drosophila interruption of Indy by a P element results in doubling of the life span in hetero- zygotes and a more modest increase in homozygotes. It was suggested (Rogina et al., 2000) that interruption of the Indy gene in D. melanogaster exerts its effect on life-

The Indy gene encodes a sodium dicarboxylate transporter protein (NADC). There are two Indy homologues in Drosophila, Indy and Indy 2. There are four Indy homologues in humans, NADC1, NADC2, NADC3 and NADC4 and two homologues in rat, SDCT1 and SDCT2. The closest human homologue of Indy is the sodium-dependent dicarboxylate transporter, member 2 (also called NADC2 or SLC13A2) (cDNA Genbank NM_003984, gene AC005726). It seemed possible that polymorphisms in the human analogue of the Indy gene might affect lifespan. We have, therefore, sequenced the Indy gene in 13 subjects age 90(cid:1)99 and 30 / subjects 5age 49 to identify potential ‘‘longevity / promoting’’ mutations and genotyped 2366 normal subjects for the common I550V polymorphism to determine whether it has any effect on aging or on body weight.

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0047-6374/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00149-0

Table 2

Oligomers used for allele specific oligomer hybridization of the F62L (186C0G), V80L (238 G0C) and I550V (1648 A0G) polymorph- / / / isms in the Indy gene

Anonymized DNA samples for this study were obtained from consenting subjects who had participated in a large epidemiological study (Beutler et al., 2000). No patient identifiers were associated with the samples; only age, height, weight and ethnic origin.

3. Results

3.1. Identification of single nucleotide polymorphisms (SNPs) by complete sequencing of the coding region

Amplification and sequencing was performed using the oligomers and conditions described in Table 1. Sequence analysis was done on a ABI Model 377 automatic sequencer.

Complete sequencing of the coding region of the Indy gene was performed on 13 subjects between the age of 90 and 99, 12 subjects between the ages of 38(cid:1)49 and 18 / subjects Bage 23. Three polymorphisms resulting in / amino acid changes in the human Indy gene (human NADC2) were identified by direct sequencing (Fig. 1).

For larger scale studies allele specific oligonucleotide hybridization (ASOH) was performed on the nt 186 C0 / G (F62L), nt 238 G0C (V80L) and nt 1648 A0G / / (I550V) polymorphisms with previously described meth- ods (Beutler and Gelbart, 2000) using oligomers de- scribed in Table 2.

The first polymorphism was identified at nt 1648 A0 / G (I550V). The frequency of the G allele was 0.4615 in the 13 subjects age 90(cid:1)99 and 0.3167 in the 30 subjects / 549. The isoleucine at this position is not conserved /

Primers used to amplify and sequence the human Indy gene