Identification of two novel missense mutations in the human ... - Nature

International Journal of Obesity (1997) 21, 321±326 ß 1997 Stockton Press All rights reserved 0307±0565/97 $12.00

Identi®cation of two novel missense mutations in the human OB gene SM Echwald1, SB Rasmussen1, TIA Sùrensen2,3, T Andersen2,4, A Tybjñrg-Hansen2,5, JO Clausen6, L Hansen1, T Hansen1 and O Pedersen1 1

Steno Diabetes Center and Hagedorn Research Institute, Copenhagen, Denmark; 2Copenhagen City Heart Study, National University Hospital, Copenhagen, Denmark; 3Danish Epidemiology Science Centre at the Institute of Preventive Medicine, Copenhagen University Hospital, Denmark; 4Department of Internal Medicine, Elsinore Hospital, Denmark; 5Department of Clinical Biochemistry, Herlev University Hospital, Copenhagen, Denmark; and 6The Glostrup Population Studies, Medical Department C, Glostrup Hospital, University of Copenhagen, Denmark

OBJECTIVE: To clone the human OB gene and to investigate if mutations in the OB gene are related to juvenile onset obesity in Caucasians. DESIGN: Case-cohort study with mutational scanning of the OB gene in an obese cohort and in a population sample of young Caucasians. SUBJECTS: An obese cohort of 156 subjects with juvenile onset obesity and a population sample of 380 healthy young Caucasians. MEASUREMENTS: Various anthropometric and biochemical measures of obesity and insulin sensitivity and single strand conformation polymorphism scanning and nucleotide sequencing. RESULTS: Analysis of the coding region of the OB gene in the 536 participants revealed that one obese subject was heterozygous for a mutation at codon Phe17Leu and one normal weight subject was heterozygous for a mutation at codon Val110Met. The phenotypes of the carriers were not different from matched non-mutation carrying subjects. CONCLUSIONS: Mutations exist in the OB gene among obese as well as lean subjects although they are rare. However, it is unlikely that mutations in the coding region of the OB gene are a common cause of juvenile onset obesity among Caucasians. Keywords: juvenile onset obesity; human OB gene; mutations; weight change; insulin sensitivity

Introduction Obesity aggregates in families, and studies of twins,1,2 as well as adopted children3±5 show that most, if not all, familial aggregation is due to genetic in¯uence rather than to the shared family environment. On the other hand, it is obvious that there is a considerable in¯uence of the environment not speci®cally shared within the families, and it seems plausible to consider obesity as a condition originating from a variety of combinations of both genetic and environmental factors. The pattern of familial aggregation suggests that a major recessive gene may contribute to human obesity4 and this has been further substantiated by commingling and segregation analyses.6 However, only a few examples of genetically determined obesity in humans have been identi®ed so far, consisting of rare mutations and chromosomal rearrangements, often associated with mental retardaCorrespondence: Dr SM Echwald, Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. Received 11 November 1996; revised 2 January 1997; accepted 8 January 1997

tion, and these loci have been shown not to segregate with common human obesity.7 In rodents several obesity causing genes are known, one of which is the recently cloned OB gene. Mutations in the OB gene cause a recessively inherited, early-onset obesity in ob/ob mice. The OB protein (leptin) is a secreted protein. It contains a 22 amino acid signalling peptide which is cleaved off when the ligand is secreted.8 OB is almost exclusively expressed in white adipose tissue and injection of recombinant leptin in the OBde®cient ob/ob mouse decreases appetite and increases energy expenditure.9,10 This is probably mediated through the recently cloned putative leptin receptor expressed in the hypothalamus.11 Thus, leptin seems to be involved in a signalling pathway from the adipose tissue, regulating body weight. The human homologue, therefore, constitutes an interesting candidate gene for inherited obesity in humans.8 In the present study we have cloned the gene encoding the human OB protein and identi®ed the ¯anking intronic sequences of the gene. The coding region of the gene has been analysed for variations by PCR-single strand conformation polymorphism scanning in a cohort of 158 male Caucasians with juvenile onset and persistent obesity and in a population-based sample of 380 healthy, young Caucasians.

Mutations in the human OB gene SM Echwald et al

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Subjects and methods Subjects

The study comprises two independent study populations of Danish Caucasians. Before participation, the purpose and risks of the studies were explained to all of the volunteers and their informed consent was obtained. The protocols were approved by the local committees of ethics in Copenhagen and were in accordance with the Helsinki declaration (II). Study of men with juvenile onset obesity

This study population consists of young Danish men, who at the draft board examination around age 20 y, had a body mass index of at least 31 kg/m2, and who, in addition, had attended school in the Copenhagen municipality, where height and weight were measured as a part of the school health examination.12 The sample was further restricted to those who were examined at the Copenhagen City Heart Study Programme in 1981±8313 and again in 1992±94. This study sample consisted of 156 subjects, in whom whole blood samples, drawn at the last examination at the Copenhagen City Heart Study, were available. Phenotype study of a random sample of young healthy Caucasians

Three hundred and eighty subjects were randomly recruited from a population of young healthy subjects aged 18±32 y, who in 1979/80 and again in 1984/85 as children had participated in an epidemiological blood pressure survey in a representative speci®ed part of Copenhagen.14 Subjects were not selected for BMI. Physiological characteristics from this cohort have been presented previously.14 In short, anthropometric measures (height, weight, waist and hip circumference) as well as measures of insulin sensitivity were recorded.14 The examination of clinical physiology and anthropometric variables took place during 1992 and 1993 where also blood samples for DNA isolation were drawn. Cloning and sequencing of the human OB-gene

From the amino acid sequence of the human OBprotein published by Zhang et al.,8 degenerate primers were placed on the most conserved codon areas at the

start and at the end of the coding region of the gene (primers indicated in Table 1) to amplify the human cDNA sequence. RNA was isolated from human adipose tissue (kindly supplied by Dr Juleen Zierath, Karolinska Sjukhuset, Stockholm) and from this material cDNA was synthesised as described earlier.15 Nested PCR ampli®cation was performed on cDNA from approximately 10 ng of total RNA using primer set OBI-OBIV and OBII-OBIV (Table 1). This procedure ampli®ed an approximately 610 bp segment which was sequenced by the Sequenase kit (USB, Cleveland, Ohio) and was found to encode a part of the published human OB amino acid sequence.8 Primers were placed overlapping the putative intronposition at codon 49 in the gene (Primers OBIAOBIVA) and using the TaqPlus PCR kit (Stratagene, La Jolla, CA) and 100 ng of genomic DNA as a template, a 2100 bp segment was ampli®ed. Direct sequencing of the ends of this segment, showed that it contained the intron at codon 49 (data not shown). Subsequently, primers were placed to amplify a segment overlapping the intron/exon boundary in the 30 end of the intron (primer set 2AB1 from Table 1). This primer pair was used to screen a library of human genomic P1 clones (Genome Systems, St. Louis, MO), which identi®ed 2 genomic clones (DMPC-HFF#10398A5 and DMPC-HFF#1-0977D12 from Genome Systems). DNA was prepared from these two independent clones (The P1-manual, Genome Systems), and by using internal sequencing primers from the coding region the remaining sequences at the 30 and the 50 ends of the gene were sequenced directly by cycle sequencing (Fmol sequencing kit, Promega) using P1 DNA (DMPC-HFF#1-0977D12) as a template. The intron preceding the coding region was later PCR ampli®ed using primers OB-Sc1Ð Obprom1 (Table 1) from the previously published sequence of the OB promoter/gene structure (Genebank accession number D63708) and applying theTaqPlus kit as described in the protocol, with an extension time of 20 min per cycle. This revealed an intron size of 10 000 bp (data not shown). DNA isolation

Genomic DNA was isolated by two methods: (a) Human leukocyte nuclei from whole blood were

Table 1 Sequences of primers for cloning and PCR-SSCP analyses of the human OB gene

Primer set Cloning: OBI-OBIV OBII-OBIV OBIA-OBIVA OBprom1-OB-Sc 1 PCR-SSCP: OB1AB OB2AB1 OB2AB2

Upstream primer

Downstream primer

50 -GCG-GAG-GAA-ATG-CAT/C-TGG-30 50 -GGN-TTT/C-T/CTN-TGG-T/CTN-TGG-30 50 -GGA-TTC-TTG-TGG-CTT-TGG-30 50 -GCA-TGG-AGC-CCC-GTA-GGA-AT-30

50 -NAA/G-T/CTG-CCA-NAA/G-CAT-30 50 -NAA/G-T/CTG-CCA-NAA/G-CAT-30 5-GTC-CAG-CTG-CCA-CAG-CAT-3 50 -GTG-TGA-AAT-GTC-ATT-GAT-CC-30

50 -ATCGGCTCCTGCAGTGTGTG-30 50 -ACATGCTGAGCACTTGTTCTC-30 50 -GACCTGGAGAACCTCCGGGA-30

50 -GGCTGCAGTTCTACTTTGTCC-30 50 -AAGTGGCAGCTCTTAGAGA-30 50 -CCAGAGTTCCTTCCCTTAACG-30

Amplicon size (bp) 247 251 260


precipitated, washed and lysed in 0.5% SDS and Proteinase K. Proteins were precipitated by addition of a saturated NaCl solution, and genomic DNA was subsequently precipitated by ethanol precipitation.16 (b) Human leukocyte nuclei were isolated from whole blood by digestion with proteinase K and genomic DNA was obtained following phenol extraction on an Applied Biosystems 341 Nucleic Acid Puri®cation System (Applied Biosystems Inc., Foster City, CA, USA).

Measurement of insulin sensitivity index

Insulin sensitivity index was estimated from an intravenous glucose tolerance test in combination with intravenous injection of tolbutamide as previously described.14,17

Results Cloning and partial sequencing of the OB gene

PCR-SSCP and heteroduplex formation analysis

SSCP analysis was performed on the entire coding region of the OB gene in 156 subjects with juvenile onset obesity and in 380 healthy young Caucasians. From the genomic sequence, three primer pairs for PCR-SSCP speci®c to the genomic sequence were synthesised as shown in Table 1. Primers 1AB are intronic and amplify the entire exon 2 of the gene, including intron/exon splice sites, in a 247 bp segment. Primers 2AB1 and 2AB2 amplify exon 3 in two overlapping segments of 251 bp and 260 bp. PCR was carried out in 25 ml volumes, using 100 ng DNA with 5 pmol speci®c OB primers and 1.5 mmol/l MgCl2 and including 0.125 ml (a-32P(dCTP (3000 Ci/mmol; 1 Ci 37 Gbq; Amersham) and 0.5 U Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT). Samples were subjected to the following PCR conditions; initial denaturation at 95 C for 3 min, followed by 30 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s and extension at 72 C for 30 s, terminated by extension at 72 C for 5 min. Cycling was performed in thin-walled tubes on a GeneAmp 9600 Thermocycler (Perkin-Elmer/Cetus, Norwalk, CT). Samples were mixed with four volumes of loading buffer (90% formamide, 10% NaOH, xylencyanol and bromophenol blue). DNA was denatured and allowed to re-anneal prior to loading to generate heteroduplexes and ®nally analysed by non-denaturing gel electrophoresis through polyacrylamide gels at two different conditions (5% glycerol at 25 C and 1% glycerol at 6±8 C), as described previously.15 Using segments of approximately 250 bp this method has in our laboratory a sensitivity of more than 90%. Gels were exposed on X-ray ®lm overnight at 770 C and were analysed for variations in migration. Segments showing variations were re-ampli®ed by PCR and sequenced using the ThermoSequenase kit (Amersham, Buckingham PLC, UK).

Cloning and analysis of the OB gene identi®ed an intron at codon 49 and a putative intron in the 50 untranslated region. The deduced genomic structure of the gene is shown in Figure 1. The partial sequence of the gene identi®ed in this study (data not shown) was identical to sequences published after the termination of the present study (GeneBank accession number D63709 and D63710).18 The coding region is contained in exons 2 and 3 which in this study have been analysed by PCR-SSCP and heteroduplex analysis. PCR-SSCP and heteroduplex analysis of the human OB gene and leptin measurements

Analysis of the SSCP autoradiograms revealed that one of the 156 obese subjects showed an altered migration of the double-stranded DNA caused by heteroduplex formation (Figure 2a). The variant was found in the ®rst segment analysed covering until codon 49 (primer set 1AB, Table 1), and subsequent sequencing of DNA from this patient revealed a single base substitution changing a TTC to CTC (Figure 3). This nucleotide substitution predicts a change in amino acid from phenylalanine to leucine at codon 17. The carrier was heterozygous for this variant. The mutation creates a recognition site for the restriction enzyme Nsp 1, the presence of which can be used as a screening assay for the mutation. The BMI of the carrier at different ages (age 7, 13, 20 and 47 y) as shown in Table 2 showed no difference from the remaining group of obese subjects. The mean waist/hip ratio of the whole group was 1.01 0.10 whereas the same ratio was 0.96 in the carrier of the codon 17 variant. Circulating leptin concentration was measured in the Phe17Leu carrier and for comparison in two BMI matched wildtype subjects from the same cohort. The carrier had a serum leptin concentration of 36.3 ng/ml and the two matched subjects had values of 44.7 and 39.1 ng/ml, respectively. The samples were taken in the non-

Measurement of serum leptin

Serum leptin concentrations were measured using the human leptin RIA kit (Linco, St. Charles, MO). Two 6 100 ml of serum was analysed for each subject as described by the RIA kit protocol.

Figure 1 Intron/exon structure of the human OB gene, where introns are marked by the dotted line. The coding regions are designated by open boxes.

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324 Table 2 Phenotype of codon Phe17Leu carrier compared to 148 obese wildtype carriers of the human OB gene

Age at BMI measurement

Codon Phe17Leu BMI

7y 13 y 20 y 47 y

17.6 25.4 31.4 34.4

a

Figure 2 (a) Autoradiogram showing SSCP and heteroduplex gel analysis of a 247 bp DNA segment PCR ampli®ed from 4 wildtype (Wt) and one heterozygous (He) carrier (arrow) of the codon 17 variant in the human OB gene. Bands representing double-stranded (dsDNA) and single-stranded (ssDNA) DNA are indicated. The presence of the mutation is distinguished by a heteroduplex formation in the double stranded DNA. (b) Autoradiogram showing SSCP and heteroduplex gel analysis of a 251 bp DNA segment PCR ampli®ed from 5 wildtype (Wt) and one heterozygous (He) carrier (arrow) of the codon 110 variant in the human OB gene. Bands representing double-stranded (dsDNA) and single-stranded (ssDNA) DNA are indicated. The heterozygous carrier presents an extra single stranded band, representing the mutated allele.

Figure 3 Autoradiogram showing direct nucleotide sequencing of PCR-ampli®ed human OB DNA of a wildtype (Wt) carrier and in a subject heterozygous (He) for a codon Phe17Leu variant. The arrow indicates the position of the nucleotide variant.

kg/m2 kg/m2 kg/m2 kg/m2

Wildtype obese BMI (n 148)a 18.4 25.1 31.4 35.3

(2.3) (3.1) (2.6) (5.4)

kg/m2 kg/m2 kg/m2 kg/m2

Some values of BMI were missing in 8 subjects; Mean s.d.

Figure 4 Autoradiogram showing direct nucleotide sequencing of PCR-ampli®ed human OB DNA of a wildtype (Wt) carrier and in a subject heterozygous (He) for a codon Val110Met variant. The arrow indicates the position of the nucleotide variant.

fasted state. The mutation resides in the signal peptide of the protein and is therefore not contained in the circulating peptide ligand. Analysis of the SSCP autoradiograms from the random sample of 380 healthy, young Caucasians revealed two different SSCP conformers of which one is shown in Figure 2b. Sequencing revealed two variations. One single nucleotide substitution was identi®ed in the non-coding region of the gene, changing base number 9 downstream from the stop codon from A to G. The carrier was heterozygous for this silent mutation. Another single base substitution also in the heterozygous state was found at codon 110, changing GTG to ATG (Figure 4) and predicting an amino acid change from valine to methionine. The carrier of the Val110Met variant was a 23 y old male, with a BMI of 27.2 kg/m2. He had a waist/hip ratio of 0.84 as compared to 0.81 0.10 for the whole group. Compared to 18 BMI matched males he had a relatively high insulin sensitivity index as measured by an intravenous glucose tolerance test in combination with intravenous tolbutamide injection and analysed in accordance to Bergman's minimal model17 (23.7 vs 10.4 4.4 1075 min71 pmol71, mean s.d.). The fasting serum leptin concentration of this individual was 4.5 ng/ml. This value is within the normal


range (6.1 3.6 ng/ml, mean s.d.) for 18 males aged 23±30 y and with a BMI in the interval of 24±30 kg/ m2.

Discussion The OB gene product, leptin, plays an important role in regulating energy metabolism, especially in the control of body fat turnover in rodents.9,10 The amino acid sequence of the mouse OB protein has an 83% homology to the human protein8 suggesting a similar function of leptin in humans. In this study we report the ®nding of two novel missense mutations in the OB gene. Mutations in the OB as reported here seem to be very rare (2 out of 536). Thus, homozygous carriers or compound heterozygotes will be extremely uncommon (0.0014% of the population). One of the examined study groups consisted of subjects with juvenile onset obesity. By selecting juvenile obese subjects we expected to select for a highly penetrant genotype. A high degree of inheritance of obesity is expressed already at age seven19 and onset of obesity prior to adulthood increases the relative risk for ®rst degree relatives of obese parents. 20 A missense mutation in the heterozygous form was identi®ed at codon Phe17Leu in the signal peptide of the OB protein in one of 156 male subjects with juvenile onset obesity. Phenylalanine is, however, not a conserved amino acid21 and judging from BMI, waist/hip ratio and the circulating leptin level this subject is comparable to the rest of the cohort indicating that this variant in the heterozygous form may not have any major impact on the pathogenesis of obesity. However, as the mutation resides in the signal peptide of the protein, the mutation may lead to erroneous processing or degradation of leptin. Circulating leptin concentrations vary considerably in the high range of BMI 22 and we cannot exclude the possibility that this subject would have produced more leptin and consequently perhaps would have been leaner if he did not carry this variant. Thus, further studies are necessary to exclude a functional importance of this mutation. The potential pathogenic implications of this OB variant may be tested in family studies and a possible alternative processing of the mutated proteins from the subject may be examined by Western blotting or high performance liquid chromatography. Also analysing the mutated protein in vitro by transfection studies may evaluate the posttranslational processing. A second mutation, also in the heterozygous form, was identi®ed at codon 110 in a normal weight subject. This subject was part of the population sample of young Danish Caucasians, which represents the general population and thus functions as a nonselected control material. This mutation is located in the C-terminal part of leptin, and is therefore contained in the secreted ligand. The conformational

aspects of how leptin binds to its receptor are unknown and prediction of the functional effect of this mutation on binding or activation of the OB receptor is not possible. The valine at codon 110 is not conserved in mice or rat, both of which have a leucine at this residue,21 and the change from Val to Met constitutes a conservative change. Further, in vitro studies are necessary to examine receptor binding and other characteristics of this mutated ligand. The carrier of the codon Val110Met mutation had a fasting serum leptin level which was within the normal range when considering BMI. It is obvious that the interpretation is dependent on whether the leptin RIA kit we have used is able to recognise the circulating variant protein with a similar af®nity as the circulating wildtype protein. SSCP scanning of the OB gene in obese subjects and NIDDM patients from several ethnic groups23±26 has revealed only one mutation (Val94Met) in the OB gene with no apparent association to obesity.26 However, recent studies have shown a possible linkage of extreme obesity (BMI > 35±40 kg/m2) with the OB gene region27,28 indicating a possible pathogenic role of the OB gene in these very obese subjects. This region has been linked to obesity in a study of a Quebec family,29 showing linkage between the KELL locus and BMI. The distance between the OB locus and KELL is, however, approximated to be 25 cM28 which is a large distance with respect to linkage. The indication of linkage to the OB gene region in patients with severe obesity27,28 may be an example of a subtype of obese subjects where speci®c loci may in¯uence body weight. Mutational analysis of the OB gene in the present and previous studies23±25 has been con®ned to include subjects in the BMI range from 35±40 kg/m2. Thus, if the linkage between the OB locus and extreme obesity is valid, the promoter region of the OB gene is an obvious candidate for mutations in these subjects.

Conclusions Two novel missense mutations have been identi®ed in the OB gene showing that variations in the gene exist in both lean and obese subjects. However, it seems unlikely that genetic variation in the coding region of the OB gene is a major contributor to the pathogenesis of obesity in Caucasians. Acknowledgements

The study was supported by grants from University of Copenhagen, the Velux Foundation, the Danish Diabetes Association, EEC Grant (BMH4-CT 95-0662), Danish Medical Research Council, the Danish Heart Foundation, the Danish Hospital Foundation for Medical Research, Region of Copenhagen, the Faeroe Islands and Greenland, the Else and Mogens

325


326

Wedell-Wedellsborg Foundation, Bristol-Myers Squibb, Frimodt-Heineke Foundation, House of Prince, Novo's Foundation, Ingeniùr August Frederik Wedell Erichsens Foundation, Grosserer A.V. Lykfeldts og Hustrus Foundation Legat, Foundation of 1870 and Leo Chemicals. The authors thank Helle Fjordvang, Sandra Urioste, Lene Aabo, Bente Mottlau, Annemette Forman, Susanne Kjellberg, Jane Brùnnum, Quang Truong, Hanne Damm Kia Olsen, Mette Sadolin, Lone Westh, Miguel Lee, Birgitte Stumann and Karen Grunnet for dedicated and careful technical assistance and Grete Lademan for secretarial support.

15

16 17

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References

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