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Molecular Scanning of ß-3-Adrenergic Receptor Gene in Total Congenital Lipoatrophic Diabetes Mellitus¹

Johns Hopkins University School of Medicine, Division of Endocrinology and Metabolism (K.S.), Division of Geriatric Medicine and Gerontology (J.W., A.R.S.), Division of Pediatric Endocrinology (L.P.), Baltimore, Maryland 21287; Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (S.I.T.), Bethesda, Maryland 20892; and the Joslin Diabetes Center (C.R.K.), Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Kristi Silver, 725 West Lombard Street, Room S-415, Baltimore, MD 21201. E-mail: ksilver{at}umppa1.ab.umd.edu

	Abstract

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Total congenital lipoatrophic diabetes is characterized by absence of subcutaneous adipose tissue, hypertriglyceridemia, and insulin resistance. We hypothesized that mutations in the ß-3-adrenergic receptor (ß3AR) gene might result in the lipoatrophic phenotype by preventing triglyceride storage in adipocytes; thereby, resulting in secondary insulin resistance. We screened the ß3AR gene in 7 subjects with total congenital lipoatropic diabetes. We found a heterozygous substitution of a guanine to cytosine at position -153 (G-153C) in the 5'-untranslated region of 3 African-American lipoatrophic siblings and 1 sibling without lipoatrophy but with insulin resistance. To determine whether the base change was related to the lipoatrophic phenotype, we genotyped 69 African-Americans without lipoatrophy and found the G-153C substitution in 2 control subjects (allele frequency = 0.01). No other single-stranded polymorphism variants were found in any of the 7 lipoatrophic subjects. Direct sequencing of both alleles of 1 lipoatrophic subject demonstrated a thymidine insertion at position -300 in both alleles. All lipoatrophic subjects along with 20 African-American control subjects were homozygous for the base insertion, suggesting an error in the published sequence. In conclusion, mutations in the ß3AR gene do not appear to be involved in the development of congenital total lipoatrophy.

	Introduction

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LIPOATROPHIC diabetes (Seip-Berardinelli syndrome) is a rare disorder characterized by absence or loss of subcutaneous adipose tissue, extreme insulin resistance, hypertriglyceridemia, and increased metabolic rate (1). There are four main types of lipoatrophic diabetes divided by the distribution of adipose loss (i.e. partial or total), and whether the loss is acquired or congenital (1). The acquired forms are often associated with a preceding viral infection, whereas congenital lipoatrophy is thought to have a genetic basis for both autosomal recessive and dominant forms. However, to date, mutations in specific genes that cause lipoatrophic diabetes have yet to be identified (2, 3).

A potential candidate gene for lipoatrophic diabetes is the ß-3-adrenergic receptor (ß3AR) gene. The ß3AR is a G protein-linked receptor found on adipocytes that is involved in lipolysis and thermogenesis (4). We hypothesize that activating mutations in the ß3AR might result in the lipoatrophic phenotype by preventing the storage of triglycerides in adipocytes (5). This defect might produce an increase in serum triglyceride and free fatty acid levels, both of which have been associated with insulin resistance (6, 7). Additionally, increased activity of the ß3AR might account for the observed increase in metabolic rate. To test our hypothesis, we screened seven subjects with total congenital lipoatrophy and one sibling with severe insulin resistance for mutations in the ß3AR gene.

	Materials and Methods

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Study subjects

All protocols were approved by the ethics committee at the Johns Hopkins University and were performed after informed written consent. Subjects 1–4 were siblings (8). Subjects 2, 3, and 4 had total congenital lipoatrophic diabetes. Subject 1 did not have lipoatrophy but did have severe insulin resistance. Subjects 5–8 were unrelated and had total congenital lipoatrophic diabetes (Table 1). Unrelated African-American subjects without lipoatrophy from the Baltimore/Washington, D.C. metropolitan area served as controls.

Genomic DNA was prepared from leukocytes or immortalized cell lines by established methods (9). Genomic DNA was amplified by PCR (10). Ten pairs of primers were used to generate 10 overlapping PCR products encompassing the entire coding region, the 5'-untranslated region, 521 bp of the 5'-regulatory region, and the exon-intron junctions of the ß3AR gene as described previously (11, 12). SSCP analysis was performed as previously described using four gel conditions: with and without 10% glycerol at 4 C and at 25 C (11). Variants detected by SSCP were subjected to direct sequence analysis using asymmetric PCR followed by dideoxy sequence analysis with Sequenase Version 2.0 (United States Biochemical, Cleveland, OH) (10).

For complete sequencing of both alleles of the ß3AR gene, a 1893-bp product was generated from genomic DNA of subject 2 by PCR with upstream primer 5'-GAAAGGGGACAGATCTCACCAA-3' and downstream primer 5'-CCACGGACACATCGCATGCTT-3' using standard conditions (10). The PCR product encompassed the entire first exon, splice junction, and beginning of the intron of the ß3AR gene. This product was subcloned into pCRII (Invitrogen, San Diego, CA). Clones containing each allele were selected based on the PCR-restriction fragment length polymorphism (RFLP) for the G-153C polymorphism as described below. The remainder of the coding region in the second exon and its adjacent exon-intron junction were directly sequenced from a 170-bp PCR product amplified with upstream primer 5'-CCAGGGTCTCTGATCTCTGTCATT-3' and downstream primer 5'-CAGAGGCCAGAGGTTTTCCACAGGT-3'. Automated sequence analysis was performed using fluorescent dideoxy nucleotides with an ABI 373 Sequencer (Applied Biosystems, Inc., Foster City, CA).

Detection of G^-153->C^-153 (G-153C) by PCR-RFLP analysis

A 110-bp fragment of the ß3AR gene encompassing the mutation site was generated by PCR from genomic DNA using upstream primer 5'-GCCCCTCCAGACTATAGGCAGCT-'3 and a mutagenic downstream primer (5'-GTCCCAGCCAGAGCGCTCAGCCTCCGC-3'), which introduced a HhaI restriction site only if the G-153C substitution was present (13). The PCR product was digested with HhaI, electrophoresed on a 4% agarose gel, and visualized by staining with ethidium bromide. The expected products after digestion with HhaI were 110 bp for normal homozygotes; 110, 82, and 28 bp for heterozygotes and 82 and 28 bp for G-153C homozygotes.

Allele-specific oligonucleotide (ASO) hybridization detection of thymidine insertion at position -300

A 229-bp fragment of the ß3AR gene encompassing the insertion site was amplified by PCR from genomic DNA using upstream primer 5'-CCAGGGAGTGCTATGCTGAG-3' and downstream primer 5'-AACTCCCTCGGTGCCACCGCTCTT-3'. The PCR products were blotted in duplicate onto nylon membranes. Hybridization was accomplished with ³²P-radiolabeled oligonucleotides corresponding to either the published sequence of the ß3AR gene (5'-TCCTCCAGATTAGCTAAAG-3') or the sequence with the insertion (5'-TCCTCCAGATTTAGCTAAAG-3'). The membranes were washed twice for 20 min in 2 x sodium chloride-sodium-phosphate-EDTA (SSPE) and 0.05% SDS at 53 C for the probe specific for the normal sequence and 55 C for the probe specific for the thymidine insertion, and autoradiography was performed.

	Results

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Four siblings (3 with total congenital lipoatrophy and 1 without lipoatrophy but with extreme insulin resistance) and 4 unrelated subjects with total congenital lipoatrophy were screened by SSCP for variant patterns in the ß3AR gene. In all 4 siblings, a variant pattern was found in the segment encoding the 5'-untranslated region (Fig. 1). Dideoxy sequence analysis revealed a guanine to cytosine substitution at position -153 (G-153C) (Fig. 2). This site is located three bases from one of four transcription initiation sites (14). All 4 siblings were heterozygous for the G-153C substitution. No other SSCP variants were found in the 8 subjects. Because subjects 1–4 were African American, to determine whether the base change was related to the lipoatrophic or insulin-resistant phenotype, we genotyped 69 African Americans without lipoatrophy from the Baltimore metropolitan area for the G-153C substitution using PCR-RFLP. In this group, the base change was found in 2 subjects (allele frequency = 0.01).

View larger version (74K): [in this window] [in a new window]   Figure 1. SSCP of a segment in the 5'-untranslated region of the ß3AR gene in one insulin-resistant subject (lane 1) and six lipoatrophic subjects (lanes 2–7). Arrow, Shows a variant pattern in lanes 1–4, which corresponds to four African-American siblings.

View larger version (21K): [in this window] [in a new window]   Figure 2. Dideoxy sequence analysis of region giving variant SSCP pattern in subject 2. Sequence shows both a guanine and cytosine at nucleotide -153, indicating that the subject is heterozygous at this position.

View larger version (14K): [in this window] [in a new window]   Figure 3. DNA sequence from promotor of subject 2 demonstrating thymidine insertion at position -300.

View larger version (27K): [in this window] [in a new window]   Figure 4. Detection of thymidine insertion at position -300 by ASO hybridization. TT and TTT control DNA indicate that hybridization was specific for each sequence. DNA from four unrelated African-American subjects hybridized only with the TTT probe, indicating that they were homozygous for the thymidine base insertion.

	Discussion

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In this group of seven subjects, lipoatrophic diabetes does not appear to be the result of mutations in the ß3AR gene. Although we found a base change in the promotor region in an area close to a transcription initiation site that could potentially have an effect on expression of the receptor, the base change occurred in all three siblings with total congenital lipoatrophic diabetes as well as the sibling with only severe insulin resistance. Additionally, the base change was present in two unrelated nonlipoatrophic African-American control subjects, indicating that the G-153C substitution in the ß3AR is not responsible for lipoatrophy.

Although studies employing SSCP indicate that this method has a sensitivity of approximately 70% (16), in our hands, with the use of four gel SSCP conditions, the sensitivity to detect mutations in the ß3AR gene approaches 100% (12). Thus, although it is unlikely that mutations were missed, we cannot rule out this possibility. Further, we cannot rule out the possibility that relevant mutations are present in regions that lie outside those that were scanned.

Sequencing of the complete nucleotide sequence of the ß3AR gene in one patient with total congenital lipoatrophy revealed an insertion of a thymidine at position -300. Studies of other subjects, both lipoatrophic and nonlipoatrophic and nondiabetic, indicate that all subjects were homozygous for this base change. In addition, screening by SSCP in this group and other previously studied populations (11, 12, and the unpublished observations of K. Silver) have not revealed any abnormal patterns in this region. These studies suggest that the thymidine insertion is normally present, and the originally published sequence lacking this thymidine is inaccurate.

In conclusion, the ß3AR gene does not appear to play a role in the development of total congenital lipoatrophic diabetes mellitus. Studies of other candidate genes or positional cloning approaches will be necessary to determine its genetic etiology.

	Acknowledgments

 
We thank Keith Tanner for his technical assistance.

	Footnotes

 
¹ This work was supported by grants from Paul Beeson Faculty Scholarship Program from the American Federation of Aging Research (to A.R.S.), the American Diabetes Association (to A.R.S.), the Chesapeake Educational and Research Trust (to K.S., J.W., A.R.S.), the National Institutes of Health, National Research Service Award (1F32DK09340–01, to K.S.), and the Brookdale Foundation (to J.W.)

Received April 3, 1997.

Revised May 15, 1997.

Accepted July 3, 1997.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Silver, K. Articles by Shuldiner, A. R. Search for Related Content PubMed PubMed Citation Articles by Silver, K. Articles by Shuldiner, A. R.