Molecular Genetics and Metabolism 106 (2012) 121–126
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism
j ourna l homepage: www.e lsev ie r .com/ locate /ymgme
High prevalence of leptin and melanocortin-4 receptor gene mutations in childrenwith severe obesity from Pakistani consanguineous families
Sadia Saeed a,⁎, Taeed A. Butt b, Mehwish Anwer c, Muhammad Arslan d, Philippe Froguel a,e
a Department of Genomics of Common Disease, Imperial College London, London W12 0NN, UKb Department of Paediatric Endocrinology, Children's Hospital, Lahore 54000, Pakistanc Centre for Research in Molecular Medicine, University of Lahore, Lahore 54000, Pakistand Department of Biological Sciences, Forman Christian College, Lahore 54600, Pakistane Centre National de la Recherche Scientifique, University Lille North of France, Pasteur Institute, 59000 Lille, France
⁎ Corresponding author at: Department of GenomicsCollege London, Burlington-Danes Building, HammersLondon W12 0NN, UK. Fax: +44 207 594 6537.
Recessive or co-dominant single-gene mutations disrupting leptin melanocortin pathway cause severe obe-sity and hyperphagia. Since Pakistan has a very high rate of consanguinity, therefore, a significantly higherincidence of monogenic obesity is expected in its population. We have assessed the incidence of LEP andMC4R mutations and associated hormonal profiles, in a cohort of randomly selected Pakistani children withearly onset of severe obesity. Sixty two unrelated children of consanguineous parents, with a weight-for-age percentile >97 were recruited in the study. Screening for mutations in the coding regions of LEP andMC4R was performed by direct sequencing. Serum hormone concentrations were determined by immunoas-say. LEPmutations were found in 16.1% of the probands. Of these, 9 probands carried the homozygous frame-shift mutation, G133_VfsX14, whereas one patient had a homozygous mutation involving deletion of 3 basepairs, (I35del). In these probands, leptin levels were very low or undetectable and insulin levels were in-creased in 33%. Homozygous MC4R mutations, M161T and I316S, identified separately in 2 subjects (3.2%),were associated with severe obesity, hyperphagia, hyperleptinemia and hyperinsulinemia. The heterozygousM161T sibling had normal body weight and hormone levels and the parents were only mildly overweight.Based on genetic analysis of LEP and MC4R genes only, we elucidated genetic causality of severe obesity in20% of our patients confirming high prevalence of monogenic form of obesity in this consanguineous popu-lation. Co-dominancy of MC4R is exacerbated in this group with non-penetrance of obesity in heterozygousloss-of-function MC4R mutation carriers. The sub-ethnic specificity of LEP mutation, G133_VfsX14, suggestsa founder effect.
The discovery of leptin in 1994 [1] has led to the elucidation of theleptin melanocortin pathway (LMCP) that has emerged as the criticalmechanism for regulation of food intake and body weight. Leptin, a167 amino acid protein encoded by leptin gene (LEP) (7q35.1) and se-creted by the adipocytes, is the initial peripheral stimulus to activatethis pathway. The other known key components of LMCP comprisethe leptin receptor, proopiomelanocortin, prohormone convertase 1and melanocortin-4 receptor (MC4R). During the last 15 years evi-dence has accumulated both in rodents and humans, indicating thata defect in any of the genes involved in the LMCP cause overt changes
of Common Disease, Imperialmith Hospital, Du Cane Road,
in food intake, body weight, energy expenditure and also some formsof neuroendocrine and immune dysfunctions [2]. The early signal em-anating from LMCP mainly in the form of α-MSH produced in the ar-cuate nucleus of the hypothalamus, is processed by different regionsof the brain where effected by efferent pathways into anorexigenicsignals, results in restricting food intake. Functional mutations involv-ing the LMCP result in hyperphagia and excessive body weight, bothin humans and mice [3].
Congenital leptin deficiency has so far been considered to be avery rare recessive genetic disorder resulting in severe hyperphagiaand early onset obesity that can be effectively reversed by leptintreatment. The first pathogenic mutation in human LEP was foundin two severely obese cousins of Pakistani origin, with non-detectable levels of leptin [4]. Since then, 6 mutations have beenreported in this gene [4–12].
The MC4R is a G protein-coupled receptor expressed mainly in thebrain. Mutations in the MC4R gene cause the most prevalent form ofmonogenic obesity known so far. Indeed, the prevalence ofMC4Rmu-tations associated with human obesity although variable among
122 S. Saeed et al. / Molecular Genetics and Metabolism 106 (2012) 121–126
studies is up to 6% of obese subjects [13].MC4R associated obesity hasa very high penetrance in young generations, causing early onset se-vere obesity in heterozygous carriers [14]
Pakistan has one of the highest rates of consanguinity in the worldand according to a report the frequency of consanguineous marriagesis 60 to 76% in this country [15,16]. Since consanguinity increases therisk of diseases caused by homozygosity of deleterious recessive mu-tations, it is fair to expect a higher prevalence of monogenic forms ofobesity such as associated with congenital leptin deficiency, in the Pa-kistani population. No systematic study has so far been carried out toassess the prevalence of LEP as well as ofMC4Rmutations in Pakistanipopulation and it is unknown whether MC4R is also the more com-mon cause of monogenic obesity in this part of world. The presentstudy was, therefore, conducted to assess the incidence of LEP andMC4R mutations in a cohort of randomly selected Pakistani childrenwith early onset of severe obesity. In addition, hormone profile ofsubjects carrying any of these mutations was also determined.
2. Materials and methods
2.1. Cohorts
A total of 62 probands with severe early onset obesity from centralPunjab, Pakistan, were recruited for this investigation. In addition 30normal-weight age-matched control subjects and in some cases par-ents and siblings of affected probands when available, were also in-cluded in the study. The study was approved by the ethicalcommittee of the Centre for Research in Molecular Medicine, The Uni-versity of Lahore. The patients and their parents were interviewed forfamily and medical history. Written informed consent was obtainedin all cases. Anthropomorphic measurements and physical examina-tion were made and a blood sample was obtained between1000–1100 h, in each case for subsequent DNA extraction and hor-mone estimations. The study was carried out in accordance with theprinciples of the Declaration of Helsinki.
2.2. Mutation screening
Screening for mutations in LEP andMC4Rwas performed in all the62 probands and some of their family members. In addition, LEP andMC4R sequencing in 60 alleles from randomly selected non-obesecontrol subjects was carried out to determine allele frequency.
Genomic DNA was extracted from peripheral blood leukocytesusing Gentra Puregene Blood Kit (Qiagen, Crawley, UK) andscreened for coding regions of LEP and MC4R. Coding exons (2and 3) of leptin gene were amplified using 2 sets of primers. Exon2 forward primer, 5′-GATGCATTTCATTAATACATATGTAG-3′ and re-verse primer, 5′-GTTTCTTGGACTATCTGGGTCCAGTGC-3′; exon 3forward primer, 5′-GCACTTGTTCTCCCTCTTCCT-3′ and reverseprimer, 5′-GTTCCTTCCCTTAACGTAGTCCT-3′. For exon 2, the PCRwas performed using AmpliTaq Gold (Applied Biosystems, FosterCity, CA, USA ) and the following conditions: 95 °C 8 min, followedby 39 cycles of 95 °C 30 s, 56 °C 40 s, 72 °C 1 min 10 s, followed by72 °C 10 min. Amplification of exon 3 of leptin gene was performedby using Clontech (Clontech Laboratories Inc, Mountain View, CA ,USA) under the following conditions: 95 °C 1 min, followed by 30cycles of 95 °C 30 s, 68 °C 1 min, followed by 68 °C 3 min. Codingregion of the MC4R was amplified using the forward primer 5′-GTGAGCATGTGCGCACAGATTC −3′ and the reverse primer 5′-GATATTCTCAACCAGTACCCTACA −3′. The PCR was performedusing BioTaq (Bioline, London, UK) and carried out under followingconditions: 95 °C 2 min, followed by 35 cycles of 95 °C 40 s, 56 °C40 s and 72 °C 1 m 20 s. The PCR products were sequenced usingBigDye Terminator kit (Applied Biosystems) and analysed on aABI 3730xl DNA sequencer. The sequences were assembled and an-alyzed with Mutation Surveyor software.
2.3. Hormone analysis
Serum hormone concentrations were determined in duplicateusing commercially available ELISA kits (leptin: DIAsource Immuno-Assays, Nivelles, Belgium; insulin and cortisol: Monobind Inc, LakeForest, CA, USA; TSH: BioCheck Inc, Foster City, CA, USA), with an au-tomated analyser (Bio-Rad Laboratories, Hercules, CA, USA). Assayswere carried out in accordance with manufacturers’ protocol.
3. Results
Among the 62 probands with early onset severe obesity, 10(16.1%) were identified with homozygous LEPmutations whereas ho-mozygous MC4R mutations were present in 2 (3.2%) subjects. Allthese children were unrelated to each other and the outcome offirst-cousin unions.
3.1. LEP mutations
G133_VfsX14 (398delG): This recessive frame shift mutation inexon 3 of the LEP was found in the homozygous state in 9 probands(Figs. 1A and 2A). This mutation disrupts the reading frame of the lep-tin gene resulting in 14 aberrant amino acids after Gly 132, followedby a premature stop codon.
The physical and hormonal characteristics of these subjects aregiven in Table 1. As expected serum leptin values were very low orundetectable. Insulin levels were increased in 3 (33%) probands.Serum TSH and cortisol concentration were within the normal range.
I35del (104_106delTCA): This homozygous mutation involvingdeletion of 3 base pairs was identified in an 18 month old girl(Fig. 2B). She weighed normal at birth but started gaining weightafter one month of age and at the age of 18 month weighed 15 kgwith a z-score of 5.85 (BMI for age). At this age she was unable tocrawl but suffered no other abnormalities suggestive of any other dis-ease. The parents and a 4 year old male sibling were found to be het-erozygous carriers. This mutation is the only one associated withextreme obesity that has so far been reported in exon 2 of LEP. Thephysical and endocrine data of the family are given in Table 2. Where-as hyperphagia was present in the proband, none of the family mem-bers who were heterozygous for this mutation had this condition andwere of normal body weight. Serum leptin levels in the proband werebelow the detectable range. Insulin and TSH values were normalwhereas cortisol levels were raised. Heterozygote carriers had normalhormone values except that leptin and insulin were raised in themother in spite of a normal BMI of 22.2. Serum leptin levels of the fa-ther and a 3.5 year old male sibling heterozygous for this mutation,were 1.5 and 1 ng/ml, respectively.
3.2. MC4R mutations
M161T (482 T>C): This mutation in the homozygous state wasidentified in a 7 month old girl with severe early onset obesity andhyperphagia (Figs. 1B1 and 2C). She weighed normal at birth butstarted gaining weight rapidly after two month and attained a bodyweight of 16 kg in the next 5 month. Homozygosity of this mutationassociated with extreme obesity is being reported here for the firsttime. The heterozygous parents were overweight (BMI>25) butone of the sibling which was found heterozygous for the mutationhad a normal body weight (Fig. 1B2) comparable to the wild type sib-ling (Fig. 1B3). Serum leptin and insulin levels were excessivelyraised in the proband and were also higher than normal in the twoparents (Table 3). The level of these two hormones was in the lowerrange, in both the siblings.
I316S (947 T>C): This homozygous mutation in MC4R was foundin a 15 year old male suffering from severe obesity (Figs. 1C and 2D).Hyperphagia and obesity in this boy were reported after 10 years of
Fig. 1. (A–C). Photographs of probands carrying LEP andMC4Rmutations associated with severe obesity in childhood: A: an eight month old girl with congenital deficiency due to aframeshift mutation (398delG) in LEP; B1: a six month old female proband with a homozygous M161T mutation in the MC4R, B2: an heterozygous five year old male sibling withnormal body weight and B3: a normal (wild type) four year male sibling, of proband B1; C: a fifteen year old boy carrying a homozygous MC4R mutation (I316S).
123S. Saeed et al. / Molecular Genetics and Metabolism 106 (2012) 121–126
age and in the next 5 years he had attained a body weight of 120 kgand a BMI of more than 49. The proband had a family history of dia-betes and obesity. Leptin levels were markedly increased whereas in-sulin levels were only slightly higher than the upper limit of thenormal range (Table 4). Serum TSH and cortisol levels were normal.
3.3. Non-obese control subjects
LEP and MC4R mutations were not found in 60 alleles from a co-hort of non-obese children, selected randomly from the same popula-tion. The physical and endocrine characteristics of 20 of thesechildren are shown in Table 5.
4. Discussion
In this study we have screened 62 unrelated children with severeobesity, for mutations in LEP and MC4R and have found that amongthese 16.1 percent (n=10) had mutations in LEP whereas MC4R mu-tations were present in 3.2 percent (n=2), all at homozygous state.By the end of 2010 only 14 children had been identified worldwidewith congenital leptin deficiency [4–11]. Eight of these affected chil-dren belonged to Pakistani ethnicity and were homozygous for anidentical frameshift mutation (G133_VfsX14) in exon 3 of LEP. Inthe present study we report another 9 unrelated children with thehomozygous G133_VfsX14 mutation and one subject with I35del inexon 2 of LEP [12]. In a recent study, 9 of 25 (36%) severely obese
Fig. 2. (A–D). Sequencing scans of LEP and MC4R at the site of mutations demonstrat-ing: a homozygous 1 base deletion (398delG) in exon 3 of LEP disrupting the readingframe (A); a homozygous 3 base deletion (I35del) involving codons 35 and 36 inexon 2 of LEP resulting in deletion of Ile at position 35 (B); a homozygous missense mu-tation (M161T) of MC4R resulting in a change from Met to Thr (C); and, a homozygousmissense mutation (I316S) of MC4R resulting in a change from Ile to Ser (D).
Table 1Anthropometric and endocrinological characteristics of probands with G133_VfsX14(c.398delG) homozygous mutation in exon 3 of LEP.
Proband no.a
Variable 4 13 16 22 30 53 56 60 62
Sex F M M M M F F M FAge (yr) 0.7 1.4 1 7 3.3 0.7 0.7 0.7 2Weight (kg) 12 14 31 41.5 44 16.2 18.5 14 25.5Height (m) 0.65 0.72 0.89 1.2 1.03 0.72 0.78 0.68 0.78BMI 28 33 39 29 41 31 30 30 42z-scoreb 3.3 5.56 14.18 5.8 16.16 7.49 7.1 8.12 14.22Leptin (ng/ml) 0.8 0.7 0.45 0.9 0.68 1.0 b0.2 b0.2 b0.2Insulin (μIU/ml) 1.6 16.7 3.67 14 28.1 7.5 2.64 1.62 3.15TSH (μIU/ml) 0.1 0.1 4.26 0.15 2.83 1.34 1.98 N.A. N.A.Cortisol AM (μg/dl) N.A. 5.3 23.2 1.0 10.8 8.0 17.9 13.6 11.5
a All probands were offspring of consanguineous parents. With the exception ofno.22, all subjects belonged to the Arain caste. Hyperphagia was reported in all pro-bands.
b BMI z -score for age: calculated using WHO Anthro, version 3.2.2 and AnthroPlussoftwares.
124 S. Saeed et al. / Molecular Genetics and Metabolism 106 (2012) 121–126
children have been reported to carry LEP mutations [12]. However,this group of severely obese children was apparently selected mostlyon the basis of clinically low leptin levels and, therefore, the reportmay grossly overestimate the incidence of congenital leptin deficien-cy among obese children of this region.
Globally, monogenic forms of obesity have so far been demonstratedin only about 5% of the most severely obese patients [3]. This study,therefore, indicates a potentially much higher incidence of monogenicobesity in Pakistani population. Quantification of homozygosity in indi-viduals with a long history of consanguinity reveals that an average of11% of their genomemay be homozygous resulting in an increased inci-dence of autosomal recessive disease [16]. Pakistan's population com-prises many ethnic groups as a result of successive waves of migrationfrom its neighbouring countries, mainly Afghanistan, Iran, and India,during its history. The major ethnic groups include Punjabi, Sindhi,Pashtun, Baluchi and Mohajirs [17]. This ethnic composition also corre-sponds to the linguistic and to a large extent regional distribution. Thesemajor ethnic groups are composed of castes or sub-ethnic groups[18,19]. Our study subjects are representatives of the general popula-tion of the province of Punjab and mostly of Punjabi descent. It is inter-esting to note that eight of the nine families with the G133_VfsX14mutation belonged to the Arain caste that constitutes one of the largersub-ethnic populations of Punjab and that has preferred consanguine-ous marriages for many generations [20]. The foregoing leads us tohypothesise that G133_VfsX14 is a founder mutation in this caste. Thisis also supported by the observation that other LEP mutations,
Table 2Anthropometric and endocrinological characteristics of a proband and family mem-bers, with I35del (c.104-106delTCA) mutation in exon 2 of LEP.
Variable Proband Sibling Mothera Fathera
(41) (41B) (41A) (41 C)
m/mb n/mb n/m n/m
Sex F M F MAge (yr) 1.5 3.5 20 27Weight (kg) 15 13 48 53Height (m) 0.75 0.89 1.47 1.62BMI 27 16 22.2 20.2z-scorec 5.85 0.35 - -Leptin (ng/ml) b0.2 1.5 24 1.0Insulin (μIU/ml) 3.4 1.4 30 3.2TSH (μIU/ml) 3.1 1.1 2.2 1.9Cortisol AM (μg/dl) 31 12 7 8
a Parents were first cousins.b m/m: homozygous; n/m: heterozygous.c BMI z -score for age
125S. Saeed et al. / Molecular Genetics and Metabolism 106 (2012) 121–126
Arg105Trp (Turkish), Asn103Lys (Egyptian), Leu72Ser (Australian),that have so far been reported are population specific and none hasbeen replicated so far in individuals of another ethnicity.
In contrast to the LEP mutations, a large number of MC4R mutantsassociated with obesity have previously been reported with morethan 150 distinct genetic variants [21]. Thus MC4R dysfunction hasbeen regarded as the most common type of monogenic obesity[14,22,23]. In a previous study 5.8% of 500 probands with earlyonset obesity were shown to have MC4R mutations [13]. The greatmajority of probands thus far described are heterozygous carriersfor this mutation. Most of these studies, both in vivo and in vitro, indi-cate a co-dominant mode of inheritance with heterozygous subjectshaving a less severe phenotype than homozygous carriers [14,23].We report for the first time a proband with a homozygous M161Tmutation in MC4R. This mutation has previously been reported onlyin the heterozygous state in a 12 years old obese Turkish boy [23]. In-terestingly, in our study a 5 year old heterozygous sibling of the pro-band with homozygous M161T mutation, had no obesity (z-score forBMI: −0.38) and was not seen as hyperphagic. Also, levels of leptinand insulin of this heterozygote were indistinguishable from a fouryear old unaffected sibling although the homozygous proband, hadsevere hyperleptinemia and hyperinsulinemia consistent with previ-ous observations [4,13,24]. The heterozygous parents were over-weight but not obese though their serum leptin and insulin wereraised over normal values. Another homozygous MC4R mutation,I316S, previously reported in both homozygous and heterozygouscarriers [25], was found by us in a 15 year old boy. It is of note thatthe onset of obesity and hyperphagia, in this proband were reportedto occur at a much advanced age (10–11 years) as compared to thatof the child with the homozygous M161T mutation. The penetranceof obesity in individuals with heterozygous MC4R mutations hasbeen previously considered almost complete [13]. However, in a
Table 4Anthropometric and endocrinological characteristics of a proband with a homozygousI316S (c. 947 T>G) mutation in MC4R.
Variable Proband (58)
Sex MAge (yr) 15Weight (kg) 120Height (m) 1.56BMI 49.3z-scorea 5.17Leptin (ng/ml) 66.6Insulin (μIU/ml) 11.8TSH (μIU/ml) 2.1Cortisol AM (μg/dl) 6.4
a BMI z -score for age
French multigenerational familial study on MC4R mutation carrierswe demonstrated an age and generation related effect on the pene-trance of obesity [14]. We showed that in older French generations(>52 years of age), the penetrance of heterozygous loss-of-functionMC4R mutations was not more than 40%, and only a small minorityof these individuals were self- reported to be obese (BMI>30) atthe age of 20 years. It was suggested that the challenging environ-ment in Europe after World War II was less permissive for develop-ment of obesity than the current obesogenic environment [14].These results together with our current data suggest that the pene-trance of MC4R mutations is variable and to a certain extent depen-dent on environmental and other factors. Most of the patientsincluded in this study belonged to lower classes and had a ruralbackground.
The majority of the previous studies on clinical phenotype of LEPand MC4R mutants indicate that apart from leptin and insulin levelsthere is little change in thyroid and adrenal functions from the nor-mal [7,13]. Our results are consistent with these findings and lendsupport to the notion that metabolic hormones may use alternatepathways to modulate endocrine and reproductive functions [26].
The recent trend of characterization of individual disease disposi-tion based on genomic, transcriptomic and proteomic analyses, hasled to an emerging era of personalized medicine. Although use of per-sonalized medicine for obesity has so far been shown to be exception-ally successful only in relation to congenital leptin deficiency, acondition that can be completely reversed by treatment with recom-binant leptin [6,27,28], yet new strategies to counteract obesity dueto specific pathogenic mutations in MC4R are already underway.Novel small molecule compounds acting as pharmacological chaper-ones have been identified that can facilitate in vitro trafficking of re-ceptor protein to plasma membrane in cases of human MC4Rmutants that result in intracytoplasmic retention of receptor proteinand poor surface expression [29]. Furthermore, novel peptide ago-nists of α-MSH have been shown to activate mutant MC4R fromobese patients with impaired endogenous agonist response [30].There is also the possibility that these pharmacological chaperonesand highly potent MC4R agonists may find their use in the treatmentof some types of common obesity for inducing a decrement in food in-take and normalization of energy homeostasis [29,30]. It is to beexpected that personalized medicine based on molecular diagnosisshall in future play a key role in management of severe obesity withan unfolding of the genetic cause of different forms of this disease.The foregoing emphasizes the importance of elucidating monogenicforms of obesity in consanguineous populations, in this perspective.
5. Conclusions
These findings indicate a high prevalence of monogenic obesity inthis consanguineous population as 20% of cases of severe obesity inchildren could be ascribed to the presence of pathogenic LEP andMC4R mutations. As other obesity causing genes are identified, it islikely that the true proportion of monogenic obesity may even behigher. The results of this study underscore the interest of a moleculardiagnosis in children with severe obesity coming from consanguineous
126 S. Saeed et al. / Molecular Genetics and Metabolism 106 (2012) 121–126
families, for evidence-based counselling, management and where pos-sible, individualized treatment.
Acknowledgments
We thank the participating children and their family members, fortheir cooperation and help. This study was supported by the Depart-ment of Genomics of Common Disease, Imperial College London.
References
[1] Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, J.M. Friedman, Positionalcloning of the mouse obese gene and its human homologue, Nature 372 (1994)425–432.
[2] A.J. Walley, J.E. Asher, P. Froguel, The genetic contribution to non-syndromichuman obesity, Nat. Rev. Genet. 10 (2009) 431–442.
[3] A.I. Blakemore, P. Froguel, Investigation of Mendelian forms of obesity holds outthe prospect of personalized medicine, Ann. N. Y. Acad. Sci. 1214 (2010) 180–189.
[4] C.T. Montague, I.S. Farooqi, J.P. Whitehead, M.A. Soos, H. Rau, N.J. Wareham, C.P.Sewter, J.E. Digby, S.N. Mohammed, J.A. Hurst, C.H. Cheetham, A.R. Earley, A.H.Barnett, J.B. Prins, S. O'Rahilly, Congenital leptin deficiency is associated with se-vere early-onset obesity in humans, Nature 387 (1997) 903–908.
[5] A. Strobel, T. Issad, L. Camoin, M. Ozata, A.D. Strosberg, A leptin missense muta-tion associated with hypogonadism and morbid obesity, Nat. Genet. 18 (1998)213–215.
[6] W.T. Gibson, I.S. Farooqi, M. Moreau, A.M. DePaoli, E. Lawrence, S. O'Rahilly, R.A.Trussell, Congenital leptin deficiency due to homozygosity for the Delta133G mu-tation: report of another case and evaluation of response to four years of leptintherapy, J. Clin. Endocrinol. Metab. 89 (2004) 4821–4826.
[7] I. Mazen, M. El-Gammal, M. Abdel-Hamid, K. Amr, A novel homozygous missensemutation of the leptin gene (N103K) in an obese Egyptian patient, Mol. Genet.Metab. 97 (2009) 305–308.
[8] P. Fischer-Posovszky, J. von Schnurbein, B. Moepps, G. Lahr, G. Strauss, T.F. Barth,J. Kassubek, H. Mühleder, P. Möller, K.M. Debatin, P. Gierschik, M. Wabitsch, Anewmissense mutation in the leptin gene causes mild obesity and hypogonadismwithout affecting T cell responsiveness, J. Clin. Endocrinol. Metab. 95 (2010)2836–2840.
[9] S. O'Rahilly, Human obesity and insulin resistance: lessons from experiments ofnature, Biochem. Soc. Trans. 35 (Pt 1) (2007) 33–36.
[10] M. Ozata, I.C. Ozdemir, J. Licinio, Human leptin deficiency caused by a missensemutation: multiple endocrine defects, decreased sympathetic tone, and immunesystem dysfunction indicate new targets for leptin action, greater central than pe-ripheral resistance to the effects of leptin, and spontaneous correction of leptin-mediated defects, J. Clin. Endocrinol. Metab. 84 (1999) 3686–3695.
[11] G.J. Paz-Filho, T. Babikian, R. Asarnow, T. Delibasi, K. Esposito, H.K. Erol, M.L.Wong, J. Licinio, Leptin replacement improves cognitive development, PLoS One3 (2008) e3098.
[12] W. Fatima, A. Shahid, M. Imran, J. Manzoor, S. Hasnain, S. Rana, S. Mahmood, Lep-tin deficiency and leptin gene mutations in obese children from Pakistan, Int. J.Pediatr. Obes. 6 (2011) 419–4127.
[13] I.S. Farooqi, J.M. Keogh, G.S. Yeo, E.J. Lank, T. Cheetham, S. O'Rahilly, Clinical spec-trum of obesity and mutations in the melanocortin 4 receptor gene, N. Engl. J.Med. 348 (2003) 1085–1095.
[14] F. Stutzmann, K. Tan, V. Vatin, C. Dina, B. Jouret, J. Tichet, B. Balkau, N. Potoczna, F.Horber, S. O'Rahilly, I.S. Farooqi, P. Froguel, D. Meyre, Prevalence of melanocortin-4 receptor deficiency in Europeans and their age-dependent penetrance in multi-generational pedigrees, Diabetes 57 (2008) 2511–2518.
[15] M.A. Hashmi, Frequency of consanguinity and its effect on congenitalmalformation-a hospital based study, J. Pak. Med. Assoc. 47 (1997) 75–78.
[16] C.G. Woods, J. Cox, K. Springell, D.J. Hampshire, M.D. Mohamed, M. McKibbin, R.Stern, F.L. Raymond, R. Sandford, S. Malik Sharif, G. Karbani, M. Ahmed, J. Bond,D. Clayton, C.F. Inglehearn, Quantification of homozygosity in consanguineous in-dividuals with autosomal recessive disease, Am. J. Hum. Genet. 78 (2006)889–896.
[17] R. Hussain, A.H. Bittles, The prevalence and demographic characteristics of con-sanguineous marriages in Pakistan, J. Biosoc. Sci. 30 (1998) 261–275.
[18] D. Ibbetson, Panjab Castes (being a reprint of the chapter on the races, castes andtribes of the people in the report on the Census of the Panjab published in 1883by the late Sir Denzil Ibbetson), Government Printing, Lahore, 1916.
[19] A. Shaw, Kinship, cultural preference and immigration: consanguineous marriageamong British Pakistanis, J. R. Anthropol. Inst. 7 (2001) 315–334.
[20] S.A. Shami, J.C. Grant, A.H. Bittles, Consanguineous marriage within social/occupational class boundaries in Pakistan, J. Biosoc. Sci. 26 (1994) 91–96.
[21] Y.X. Tao, The melanocortin-4 receptor: physiology, pharmacology, and patho-physiology, Endocr. Rev. 31 (2010) 506–543.
[22] L.H. Larsen, S.M. Echwald, T.I. Sorensen, T. Andersen, B.S. Wulff, O. Pedersen, Prev-alence of mutations and functional analyses of melanocortin 4 receptor variantsidentified among 750 men with juvenile-onset obesity, J. Clin. Endocrinol.Metab. 90 (2005) 219–224.
[23] K. Tan, I.D. Pogozheva, G.S. Yeo, D. Hadaschik, J.M. Keogh, C. Haskell-Leuvano, S.O'Rahilly, H.I. Mosberg, I.S. Farooqi, Functional characterization and structuralmodeling of obesity associated mutations in the melanocortin 4 receptor, Endo-crinology 150 (2009) 114–125.
[24] C.E. Martinelli, J.M. Keogh, J.R. Greenfield, E. Henning, A.A. van der Klaauw, A.Blackwood, S. O'Rahilly, F. Roelfsema, C. Camacho-Hübner, H. Pijl, I.S. Farooqi,Obesity due to melanocortin 4 receptor (MC4R) deficiency is associated with in-creased linear growth and final height, fasting hyperinsulinemia, and incomplete-ly suppressed growth hormone secretion, J. Clin. Endocrinol. Metab. 96 (2011)E181–E188.
[25] G.S. Yeo, E.J. Lank, I.S. Farooqi, J. Keogh, B.G. Challis, S. O'Rahilly, 2003Mutations inthe human melanocortin-4 receptor gene associated with severe familial obesitydisrupts receptor function through multiple molecular mechanisms, Hum. Mol.Genet. 12 (2003) 561–574.
[26] T.A. Dardeno, S.H. Chou, H.S. Moon, J.P. Chamberland, C.G. Fiorenza, C.S.Mantzoros, Leptin in human physiology and therapeutics, Front. Neuroendocri-nol. 31 (2010) 377–393.
[27] I.S. Farooqi, G. Matarese, G.M. Lord, J.M. Keogh, E. Lawrence, C. Agwu, V. Sanna,S.A. Jebb, F. Perna, S. Fontana, R.I. Lechler, A.M. DePaoli, S. O'Rahilly, Beneficial ef-fects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency, J. Clin. Invest. 110(2002) 1093–1103.
[28] G. Paz-Filho, M.L. Wong, J. Licinio, Ten years of leptin replacement therapy, Obes.Rev. 12 (2011) 315–323.
[29] Z.C. Fan, Y.X. Tao, Functional characterization and pharmacological rescue ofmelanocortin-4 receptor mutations identified from obese patients, J. Cell. Mol.Med. 13 (2009) 3268–3282.
[30] P. Roubert, B. Dubern, P. Plas, C. Lubrano-Berthelier, R. Alihi, F. Auger, D.B.Deoliveira, J.Z. Dong, A. Basdevant, C. Thurieau, K. Clément, Novel pharmacologi-cal MC4R agonists can efficiently activate mutated MC4R from obese patient withimpaired endogenous agonist response, J. Endocrinol. 207 (2010) 177–183.
