Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques, including an in-house–developed augmented whole-exome sequencing method (CoDE-seq) that enables simultaneous detection of whole-exome copy number variations (CNVs) and point mutations in coding regions. We identified 110 (49%) probands carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for nonsyndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of cases in the studied cohort are likely to have a discrete genetic cause, with 13% of these as a result of CNVs, demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible overlapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with a high prevalence of obesity provide unique, genetically enriched material in the quest of new genes/variants influencing energy balance.

The monogenic forms of obesity have defined the current concepts of the central regulation of energy balance and have opened new avenues for precision medicine (1,2). Monogenic nonsyndromic obesity is due to pathogenic mutations in genes involved in leptin-melanocortin signaling, resulting in extreme, early-onset obesity with an insatiable craving for food (2). In addition to excessive adiposity, syndromic obesity associates with other abnormalities such as dysmorphic features, intellectual disability, and organ-specific anomalies (3).

Pathogenic variations causing severe obesity include not only point mutations but also copy number variations (CNVs) (4). We have recently developed a new strategy that is based on an augmented whole-exome sequencing (WES) method, named CoDE-seq, that enables accurate and cost-effective detection of point mutations and CNVs in one step, thus expediting comprehensive genetic diagnosis (5).

Although only 5–10% of severe, early-onset obesity cases in outbred populations have been evidenced to have a monogenic condition, we previously reported a high prevalence of homozygous mutations in LEP, LEPR, MC4R, and ADCY3 in a Pakistani consanguineous population (69). Compared with other countries where consanguinity is practiced, the inbreeding coefficient reported for the Pakistani population is among the highest, with 60–65% of consanguineous marriages (10,11). Since Pakistan’s population comprises several endogamous subethnic groups that are more or less isolated, the country has a very high genetic diversity, which optimizes comprehensive genetic studies, particularly in obesity because Pakistan has the ninth highest prevalence of obesity worldwide (12).

Here, we describe 110 (49%) genetically elucidated cases in the world’s largest cohort of consanguineous subjects with severe, early-onset obesity (n = 225). Another 10% (n = 22) of obese case subjects were found to carry potentially causative CNVs associated with intellectual disability. Altogether, 59% of cases in the studied cohort are likely to have a genetic cause, with 13% of these potentially as a result of CNVs.

Participants

The investigation is based on 225 unrelated probands 0.2–22 years of age with severe, early-onset obesity and their family members from consanguineous families. Subjects were recruited on a voluntary basis from hospital pediatric units located in the province of Punjab, Pakistan. Patient/parent written informed consent was obtained for each subject. The study was approved by the institutional ethical committees. The selection criteria included a BMI SD score (SDS) for age ≥3 (using WHO Anthro version 3.2.2 and AnthroPlus), early-onset obesity and hyperphagia, and nonobese parents (with BMI of ≤30 kg/m2) of first- or second-degree relation. Family and medical history was obtained, and pedigrees spanning at least three generations were constructed. Anthropomorphic measurements were made, and a blood sample was obtained for subsequent genomic DNA extraction and hormone estimations.

Initial Screening of LEP and MC4R

DNA from all probands (n = 225) was screened for coding regions of LEP and MC4R by direct sequencing as previously described (7).

WES and CoDE-seq

The probands found negative for mutations in LEP and MC4R (n = 167) and their family members (n = 177) were screened using WES and DNA arrays or CoDE-seq. A first batch of 62 probands and 56 family members were screened by standard WES and DNA arrays (for CNV detection) as previously described by us (9). A second batch of 105 probands and 121 family members were screened with CoDE-seq (when the technology was available and validated). This technique combines standard capture targeting the whole exome (NimbleGen SeqCap EZ MedExome Target Enrichment) with an in-house–designed capture (NimbleGen SeqCap EZ Choice XL) (5). Sequencing was performed on an Illumina NovaSeq 6000 system. A mean sequencing depth of ∼100× was achieved for each individual using 150-bp paired-end reads. Computer analyses for variants detection and annotation have previously been described (5).

Variant Prioritization

The analysis, at first stage, was focused on homozygous variants. We excluded homozygous variants in obese probands that were also homozygous in family members with BMI ≤30 kg/m2 or BMI SDS for age <2. The variants with an allele frequency of >0.001% in the Genome Aggregation Database (gnomAD) were also ignored. The resulting list of rare mutations was searched in a list of known monogenic obesity genes (Supplementary Table 1). Subsequently, rare compound heterozygous variants (in genes with recessive inheritance) and heterozygous variants (in genes with known dominant inheritance) were also analyzed. All potentially causative variants were graded according to criteria of the American College of Medical Genetics and Genomics (13).

CNV Detection and Prioritization

For detection of CNVs from the genotyping data, integrated hidden Markov model algorithm (PennCNV) with its default method was used as described previously (8,14). For detection of CNVs from CoDE-seq data, we used eXome Hidden Markov Model (XHMM, version 1.0) as previously described (5,15). We excluded all CNVs with allele frequency >0.002 in the gnomAD structural variant data set. Furthermore, we looked for all well-known pathogenic CNVs on the basis of the literature, the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER), and the Database of Genomic Variants, as listed in our previous work (5).

Biochemical Analysis

Serum leptin, insulin, and cortisol concentrations were determined by commercially available ELISA kits. Assays were performed in duplicate with an automated analyzer (Bio-Rad Laboratories, Hercules, CA). The intra- and interassay variations were <11%.

Data and Resource Availability

The data sets generated during the current study are available from the corresponding author upon reasonable request. No applicable resources were generated or analyzed during the current study.

The genetic screening of 225 unrelated obese subjects, initially by Sanger sequencing (for LEP and MC4R only) followed by WES or CoDE-seq, revealed 55 pathogenic genetic variants (including point mutations or CNVs) in 110 probands (49% of the overall cohort) (Tables 1 and 2 and Fig. 1).

Table 1

Genetic and clinical data of probands with a pathogenic point mutation in genes associated with obesity

GeneMutation (zygosity)IDCarriers, nPatho-genicityMAF in gnomADSexAge (years)BMI SDS for age (children) BMI (adults)PhenotypeLeptin (ng/mL)Insulin (μIU/mL)Cortisol (μg/dL)
LEP c.-29+1G>C/p.? (hmz) #132, #155, #237, #255, #297, #342 M: 2
F: 4 
3.9 ± 0.9 9.1 ± 0.9 Excessive adiposity: #297, #342; hepatomegaly: #342; splenomegaly: #237; sleep apnea: #237, #342; delayed milestones ND 9.2 ± 2.5 13.0 ± 1.0 
LEP c.398delG/p.G133Vfs*15 (hmz) #130, #134, #138, #143, #153, #157, #162, #172, #194, #195, #199, #206, #228, #241, #261, #264, #262, #276, #278, #279, #281, #284, #289, #292, #295, #296, #305, #311, #322, #323, #324, #325, #326, #330, #344, #352, #341, #127, #154 39 0.00003 (7/245,914) M: 25
F: 14 
2.0 ± 0.421 (#127)§
25 (#154)§ 
8.4 ± 0.542 (#127)
50 (#154) 
Excessive adiposity: #206; undescended testes: #228; hepatomegaly: #264, #295; dyspnea: #264; hypersomnia: #305, #341; recurrent RTI: #228, #261, #262, #279, #281, #289, #295, #296, #330, #352; delayed milestones ND 21.8 ± 3.112 (#127)
36 (#154) 
17.6 ± 1.331 (#127)
9 (#154) 
LEP c.104_106del/p.I35del (hmz) #345, #336 0.00001 (2/246,258) M: 1
F: 1 
10 (#345)
1.3 (#336) 
3.7 (#345)
13.2 (#336) 
Excessive adiposity: #345; polyuria ND 26 (#345)
17 (#336) 
12 (#345)
15 (#336) 
LEP c.309C>A/p.N103K (hmz) #300, #293 0.00175 (430/24,625) 1.5 (#300)
0.7 (#293) 
7.2 (#300)
9.7 (#293) 
Excessive adiposity, delayed milestones 32 (#300)Π
83 (#293)Π 
17 (#300)
9 (#293) 
14 (#300)
16 (#293) 
LEP c.314G>A/p.R105Q (hmz) #309 LP 1.8 6.7 Excessive adiposity, recurrent RTI ND 
LEP c.298G>A/p.D100N (hmz) #249 0.00000 (1/246,244) 2.0 14.0 Excessive adiposity 12Π 11 12 
LEP c.417del/p.Y140Tfs*8 (hmz) #220 0.8 8.0 Excessive adiposity ND 36 12 
LEPR c.2396–2A>G/p.? (hmz) #183, #354 M: 1
F: 1 
18 (#183)§
0.7 (#354) 
64 (#183)
1.3 (#354) 
Excessive adiposity: #183; delayed milestones: #354; recurrent RTI 277 (#183)
136 (#354) 
25 (#183)
19 (#354) 
10 (#183)
15 (#354) 
LEPR c.2396–1G>T/p.? (hmz) #248, #321 0.00040 (1/250,546) M: 1
F: 1 
0.4 (#248)
0.6 (#321) 
8.4 (#248)
7.5 (#321) 
Excessive adiposity 38 (#248)
26 #(321) 
13 (#248)
18 (#321) 
15 (#248)
14 (#321) 
LEPR c.704–1G>A/p.? (hmz) #331, #173 0.5 (#331)
2.8 (#173) 
7.7 (#331)
11.4 (#173) 
Excessive adiposity 31 (#331)
61 (#173) 
23 (#331)
36 (#173) 
15 (#331)
14 (#173) 
LEPR c.40G>A/p.E14K (hmz) #14 2.3 10.6 Excessive adiposity 56 
LEPR c.2114G>A/p.W705* (hmz) #150 0.8 6.7 Excessive adiposity 30 12 
LEPR c.2899_2900insAT/p.A967Dfs*7 (hmz) #170 0.9 6.4 Excessive adiposity 10 16 
LEPR c.1738del/p.E580Kfs*37(hmz) #186 1.6 9.2 Excessive adiposity 19 13 16 
LEPR c.2T>C/p.? (hmz) #OB-1 18.5§ 38.6 Excessive adiposity 54 12 11 
LEPR c.2627C>T/p.P876L (hmz) #283 LP 0.00071 (2/282,696) 0.7 12.2 Excessive adiposity, recurrent RTI 31 23 
LEPR c.2153A>G/p.N718S (hmz) #142 LP† 0.00040 (1/251,374) 0.7 7.0 Excessive adiposity 45 19 
LEPR c.2213–3C>G/p.? (hmz) #328 LP† 3.0 7.8 Excessive adiposity, anemia, delayed milestones, aggressive behavior 28 17 17 
LEPR c.3268_3269del/p.S1090Wfs*6 (hmz) #312 14 6.6 Excessive adiposity, diabetes, delayed milestones 31 15 18 
MC4R c.493C>T/p.R165W (hmz) #269, #273, #286, #310 0.00002 (6/246,054) M: 1
F: 3 
5.1 ± 1.7 6.7 ± 0.5 Excessive adiposity 22 ± 9 67 ± 22 16 ± 6 
MC4R c.48G>A/p.W16* (hmz) #202 5.3 13.2 Excessive adiposity 55 16 
MC4R c.47G>A/p.W16* (hmz) #260, #337 4 (#260)
2 (#337) 
7.4 (#260)
9.6 (#337) 
Excessive adiposity 9 (#260)
11 (#337) 
7 (#260)
26 (#337) 
8 (#260)
13 (#337) 
MC4R c.482T>C/p.M161T (hmz) #232 LP 0.000004 (1/246,124) 6.9 11.0 Excessive adiposity, hepatomegaly, hepatosteatosis, aggressive behavior 27 13 14  
MC4R c.633_636del/p.Y212Sfs*5 (hmz) #233 0.000004 (1/246,020) 5.7 8.9 Excessive adiposity, bowlegs 10 8.6 
MC4R c.601_612del/p.F201_M204del (hmz) #239 LP 7.2 4.8 Excessive adiposity, recurrent tonsillitis 14 13 11 
MC4R c.63_64del/p.Y21* (hmz) #257 0.00001 (3/245,824) 4.6 8.0 Excessive adiposity 41 74 10 
MC4R c.206T>C/p.I69T (hmz) #308 0.000004 (1/246,114) 2.5 6.4 Excessive adiposity 10 40 12 
ADCY3 c.2173–10_2185del/p.? (hmz) #306 1.2 6.7 Excessive adiposity 14 11 
ADCY3 c.3315del/p.(I1106Sfs*3)‡ (hmz) #107 15 3.5 Excessive adiposity, anosmia, amenorrhea, moderate intellectual disability 30 48 18 
ADCY3 c.2578–1G>A/p.?‡ (hmz) #158 6.5 Excessive adiposity 22 11 11 
ADCY3 c.191A>T/p.N64I‡ (hmz) #174 LP† 0.00026 (66/250,536) 6.5 Excessive adiposity, anosmia, moderate intellectual disability 11 18 7.5 
BBS1 c.1570_1572del/p.N524del (htz); c.48–2A>C/p.? (htz) #252 LP 0 and 0.00001 (3/246,272) 1.4 Excessive adiposity, polydactyl 16 15 
BBS1 c.432+1G>A/p.? (hmz) #126 0.00001 (1/119,828) 3.2 6.5 Excessive adiposity, polydactyl 11 18 13 
BBS1 c.1339G>A/p.A447T (hmz) #184 LP 0.00004 (10/250,660) Excessive adiposity, polydactyl 10 22 18 
BBS2 c.406dup/p.A136Cfs*15 (hmz) #93 9.2 4.5 Excessive adiposity NA 47.5 NA 
BBS2 c.116A>G/p.K39R (hmz) #294 0.00001 (2/243,800) 11.0 5.0 Excessive adiposity, aggressive behavior, polydactyl 61 57 10 
BBS2 c.1759_1762del/p.P587Sfs*10 (hmz) #318 9.0 6.2 Excessive adiposity, intellectual disability, polydactyl 27 19 10 
BBS5 c.668_671del/p.E223Afs*14 (hmz) #316 10.3 5.6 Excessive adiposity, bowlegs, delayed milestones, poor vision, recurrent tonsillitis 14 86 11 
BBS5 c.2T>A/p.? (hmz) #198 0.000012 (3/249,784) 0.9 4.5 Excessive adiposity, polydactyl, recurrent RTI 16 11 
BBS5 c.206T>G/p.V69G (hmz) #335 LP† 14 4.1 Excessive adiposity, poor vision, intellectual disability, polydactyl 11 21 11 
BBS9 c.662A>G/p.E221G (htz), c.635T>C/p.F212S (htz) #128 LP 2.6 3.3 Excessive adiposity, polydactyl, recurrent RTI 14 25 
BBS9 c.400del/p.T134Qfs*5 (hmz) #131 1.5 9.4 Excessive adiposity, polydactyl 15 17 NA 
BBS10 c.271dup/p.C91Lfs*5 (hmz) #114, #73 1.1 (#114)
8.8 (#73) 
4.9 (#114)
4.2 (#73) 
Excessive adiposity, polydactyl 6 (#114)
42 (#73) 
3 (#114)
4 (#73) 
NA 
BBS10 c.257T>C/p.F86S(hmz) #214 LP 0.6 4.3 Excessive adiposity 10 14 
MKKS c.775del/p.T259Lfs*21(hmz) #275, #258, #339 0.00004 (11/245,790) 1.3 (#258)
13 (#339) 
10.8 (#258)
2.6 (#339) 
Delayed milestones; polydactyl: #258; sleep apnea: #339; poor vision in dark 25 ± 5 20 ± 5 12 ± 1 
ALMS1 c.4937C>A/p.S1646* (hmz) #139 0.000004 (1/245,290) 2.5 6.2 Excessive adiposity 10 48 12 
ALMS1 c.8008C>T/p.R2670* (hmz) #140 1.9 5.8 Excessive adiposity 39 17 
ALMS1 c.7436C>G/p.S2479* (hmz) #221 0.00082 (2/245,262) 1.1 5.7 Anemia, poor vision 36 14 
ALMS1 c.10975C>T/p.R3659* (hmz) #338 0.000004 (1/245,694) 2.0 8.6 Delayed milestones, poor vision 12 59 
GeneMutation (zygosity)IDCarriers, nPatho-genicityMAF in gnomADSexAge (years)BMI SDS for age (children) BMI (adults)PhenotypeLeptin (ng/mL)Insulin (μIU/mL)Cortisol (μg/dL)
LEP c.-29+1G>C/p.? (hmz) #132, #155, #237, #255, #297, #342 M: 2
F: 4 
3.9 ± 0.9 9.1 ± 0.9 Excessive adiposity: #297, #342; hepatomegaly: #342; splenomegaly: #237; sleep apnea: #237, #342; delayed milestones ND 9.2 ± 2.5 13.0 ± 1.0 
LEP c.398delG/p.G133Vfs*15 (hmz) #130, #134, #138, #143, #153, #157, #162, #172, #194, #195, #199, #206, #228, #241, #261, #264, #262, #276, #278, #279, #281, #284, #289, #292, #295, #296, #305, #311, #322, #323, #324, #325, #326, #330, #344, #352, #341, #127, #154 39 0.00003 (7/245,914) M: 25
F: 14 
2.0 ± 0.421 (#127)§
25 (#154)§ 
8.4 ± 0.542 (#127)
50 (#154) 
Excessive adiposity: #206; undescended testes: #228; hepatomegaly: #264, #295; dyspnea: #264; hypersomnia: #305, #341; recurrent RTI: #228, #261, #262, #279, #281, #289, #295, #296, #330, #352; delayed milestones ND 21.8 ± 3.112 (#127)
36 (#154) 
17.6 ± 1.331 (#127)
9 (#154) 
LEP c.104_106del/p.I35del (hmz) #345, #336 0.00001 (2/246,258) M: 1
F: 1 
10 (#345)
1.3 (#336) 
3.7 (#345)
13.2 (#336) 
Excessive adiposity: #345; polyuria ND 26 (#345)
17 (#336) 
12 (#345)
15 (#336) 
LEP c.309C>A/p.N103K (hmz) #300, #293 0.00175 (430/24,625) 1.5 (#300)
0.7 (#293) 
7.2 (#300)
9.7 (#293) 
Excessive adiposity, delayed milestones 32 (#300)Π
83 (#293)Π 
17 (#300)
9 (#293) 
14 (#300)
16 (#293) 
LEP c.314G>A/p.R105Q (hmz) #309 LP 1.8 6.7 Excessive adiposity, recurrent RTI ND 
LEP c.298G>A/p.D100N (hmz) #249 0.00000 (1/246,244) 2.0 14.0 Excessive adiposity 12Π 11 12 
LEP c.417del/p.Y140Tfs*8 (hmz) #220 0.8 8.0 Excessive adiposity ND 36 12 
LEPR c.2396–2A>G/p.? (hmz) #183, #354 M: 1
F: 1 
18 (#183)§
0.7 (#354) 
64 (#183)
1.3 (#354) 
Excessive adiposity: #183; delayed milestones: #354; recurrent RTI 277 (#183)
136 (#354) 
25 (#183)
19 (#354) 
10 (#183)
15 (#354) 
LEPR c.2396–1G>T/p.? (hmz) #248, #321 0.00040 (1/250,546) M: 1
F: 1 
0.4 (#248)
0.6 (#321) 
8.4 (#248)
7.5 (#321) 
Excessive adiposity 38 (#248)
26 #(321) 
13 (#248)
18 (#321) 
15 (#248)
14 (#321) 
LEPR c.704–1G>A/p.? (hmz) #331, #173 0.5 (#331)
2.8 (#173) 
7.7 (#331)
11.4 (#173) 
Excessive adiposity 31 (#331)
61 (#173) 
23 (#331)
36 (#173) 
15 (#331)
14 (#173) 
LEPR c.40G>A/p.E14K (hmz) #14 2.3 10.6 Excessive adiposity 56 
LEPR c.2114G>A/p.W705* (hmz) #150 0.8 6.7 Excessive adiposity 30 12 
LEPR c.2899_2900insAT/p.A967Dfs*7 (hmz) #170 0.9 6.4 Excessive adiposity 10 16 
LEPR c.1738del/p.E580Kfs*37(hmz) #186 1.6 9.2 Excessive adiposity 19 13 16 
LEPR c.2T>C/p.? (hmz) #OB-1 18.5§ 38.6 Excessive adiposity 54 12 11 
LEPR c.2627C>T/p.P876L (hmz) #283 LP 0.00071 (2/282,696) 0.7 12.2 Excessive adiposity, recurrent RTI 31 23 
LEPR c.2153A>G/p.N718S (hmz) #142 LP† 0.00040 (1/251,374) 0.7 7.0 Excessive adiposity 45 19 
LEPR c.2213–3C>G/p.? (hmz) #328 LP† 3.0 7.8 Excessive adiposity, anemia, delayed milestones, aggressive behavior 28 17 17 
LEPR c.3268_3269del/p.S1090Wfs*6 (hmz) #312 14 6.6 Excessive adiposity, diabetes, delayed milestones 31 15 18 
MC4R c.493C>T/p.R165W (hmz) #269, #273, #286, #310 0.00002 (6/246,054) M: 1
F: 3 
5.1 ± 1.7 6.7 ± 0.5 Excessive adiposity 22 ± 9 67 ± 22 16 ± 6 
MC4R c.48G>A/p.W16* (hmz) #202 5.3 13.2 Excessive adiposity 55 16 
MC4R c.47G>A/p.W16* (hmz) #260, #337 4 (#260)
2 (#337) 
7.4 (#260)
9.6 (#337) 
Excessive adiposity 9 (#260)
11 (#337) 
7 (#260)
26 (#337) 
8 (#260)
13 (#337) 
MC4R c.482T>C/p.M161T (hmz) #232 LP 0.000004 (1/246,124) 6.9 11.0 Excessive adiposity, hepatomegaly, hepatosteatosis, aggressive behavior 27 13 14  
MC4R c.633_636del/p.Y212Sfs*5 (hmz) #233 0.000004 (1/246,020) 5.7 8.9 Excessive adiposity, bowlegs 10 8.6 
MC4R c.601_612del/p.F201_M204del (hmz) #239 LP 7.2 4.8 Excessive adiposity, recurrent tonsillitis 14 13 11 
MC4R c.63_64del/p.Y21* (hmz) #257 0.00001 (3/245,824) 4.6 8.0 Excessive adiposity 41 74 10 
MC4R c.206T>C/p.I69T (hmz) #308 0.000004 (1/246,114) 2.5 6.4 Excessive adiposity 10 40 12 
ADCY3 c.2173–10_2185del/p.? (hmz) #306 1.2 6.7 Excessive adiposity 14 11 
ADCY3 c.3315del/p.(I1106Sfs*3)‡ (hmz) #107 15 3.5 Excessive adiposity, anosmia, amenorrhea, moderate intellectual disability 30 48 18 
ADCY3 c.2578–1G>A/p.?‡ (hmz) #158 6.5 Excessive adiposity 22 11 11 
ADCY3 c.191A>T/p.N64I‡ (hmz) #174 LP† 0.00026 (66/250,536) 6.5 Excessive adiposity, anosmia, moderate intellectual disability 11 18 7.5 
BBS1 c.1570_1572del/p.N524del (htz); c.48–2A>C/p.? (htz) #252 LP 0 and 0.00001 (3/246,272) 1.4 Excessive adiposity, polydactyl 16 15 
BBS1 c.432+1G>A/p.? (hmz) #126 0.00001 (1/119,828) 3.2 6.5 Excessive adiposity, polydactyl 11 18 13 
BBS1 c.1339G>A/p.A447T (hmz) #184 LP 0.00004 (10/250,660) Excessive adiposity, polydactyl 10 22 18 
BBS2 c.406dup/p.A136Cfs*15 (hmz) #93 9.2 4.5 Excessive adiposity NA 47.5 NA 
BBS2 c.116A>G/p.K39R (hmz) #294 0.00001 (2/243,800) 11.0 5.0 Excessive adiposity, aggressive behavior, polydactyl 61 57 10 
BBS2 c.1759_1762del/p.P587Sfs*10 (hmz) #318 9.0 6.2 Excessive adiposity, intellectual disability, polydactyl 27 19 10 
BBS5 c.668_671del/p.E223Afs*14 (hmz) #316 10.3 5.6 Excessive adiposity, bowlegs, delayed milestones, poor vision, recurrent tonsillitis 14 86 11 
BBS5 c.2T>A/p.? (hmz) #198 0.000012 (3/249,784) 0.9 4.5 Excessive adiposity, polydactyl, recurrent RTI 16 11 
BBS5 c.206T>G/p.V69G (hmz) #335 LP† 14 4.1 Excessive adiposity, poor vision, intellectual disability, polydactyl 11 21 11 
BBS9 c.662A>G/p.E221G (htz), c.635T>C/p.F212S (htz) #128 LP 2.6 3.3 Excessive adiposity, polydactyl, recurrent RTI 14 25 
BBS9 c.400del/p.T134Qfs*5 (hmz) #131 1.5 9.4 Excessive adiposity, polydactyl 15 17 NA 
BBS10 c.271dup/p.C91Lfs*5 (hmz) #114, #73 1.1 (#114)
8.8 (#73) 
4.9 (#114)
4.2 (#73) 
Excessive adiposity, polydactyl 6 (#114)
42 (#73) 
3 (#114)
4 (#73) 
NA 
BBS10 c.257T>C/p.F86S(hmz) #214 LP 0.6 4.3 Excessive adiposity 10 14 
MKKS c.775del/p.T259Lfs*21(hmz) #275, #258, #339 0.00004 (11/245,790) 1.3 (#258)
13 (#339) 
10.8 (#258)
2.6 (#339) 
Delayed milestones; polydactyl: #258; sleep apnea: #339; poor vision in dark 25 ± 5 20 ± 5 12 ± 1 
ALMS1 c.4937C>A/p.S1646* (hmz) #139 0.000004 (1/245,290) 2.5 6.2 Excessive adiposity 10 48 12 
ALMS1 c.8008C>T/p.R2670* (hmz) #140 1.9 5.8 Excessive adiposity 39 17 
ALMS1 c.7436C>G/p.S2479* (hmz) #221 0.00082 (2/245,262) 1.1 5.7 Anemia, poor vision 36 14 
ALMS1 c.10975C>T/p.R3659* (hmz) #338 0.000004 (1/245,694) 2.0 8.6 Delayed milestones, poor vision 12 59 

Data are mean ± SEM. hmz, homozygous; htz, heterozygous; LP, likely pathogenic; MAF, minor allele frequency; NA, not available; ND, not detectable (or <0.5 ng/mL); P, pathogenic; RTI, respiratory tract infection.

§Young adults (≥18 years of age).

ΠImmunoreactive but bioinactive leptin protein.

†Upgraded from VUS to LP on the basis of strong phenotypic relevancy with regard to the genetic mutation.

‡Previously published by us (9).

Table 2

Genetic and clinical data of probands with CNVs causing obesity and/or intellectual disability

Proband IDCNVGenomic interval (hg38)Gene(s)Size (kb)gnomAD region MAF SizePatho-genicityGene or CNV- associated disorderAge (years)SexBMI SDS for agePhenotype (other than obesity)Additional pathogenic obesity-associated variantsLeptin (ng/mL)Insulin (μIU/mL)Cortisol (μg/dL)
#234 1q42.12 loss hmz chr1:224923245–225051803 DNAH14 128.56 VUS Panventriculomegaly (CNV del), intellectual disability, hydrops fetalis, nonimmune 2.4 9.4 Slow learner, intellectual disability  10 109 
#238 1p31.3 loss hmz chr1:65532214–65592881 LEPR 60.67 Morbid obesity 0.8 9.1   38 26 12 
#263 1p31.3 loss hmz chr1:65592634–65637041 LEPR 44.41 Morbid obesity 1.8 8.4 Delayed milestones, mental retardation  27 46 13 
 7q31.1 loss htz chr7:111337755–111407620 IMMP2L 69.87 chr7:111209525–111532640 0.00004656 323 kb VUS Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#343 1q43 loss htz chr1:240207914–240208747 FMN2 0.83 Intellectual disability, mental retardation, short stature, premature ovarian failure (CNV), intellectual disability 2.1 6.6 Delayed milestones  117 106 11 
 7q31.1 loss htz chr7:111180340–111569415 IMMP2L 389.08 Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#304 10q21.3 loss htz chr10:66402469–66676594 CTNNA3 274.13 VUS Autism spectrum disorder (CNV), cardiomyopathy NA  29 25 10 
#334 10q25.3 loss htz chr10:115799104–116272282 ATRNL1. GFRA1 473.18 VUS ATRNL1: cognitive impairment, autism, dysmorphic features (CNV). GFRA1: Hirschsprung disease (CNV), central hypoventilation syndrome 2.7 Delayed milestones  22 19 
#299 12p13.31 loss htz chr12:9192171–9208382 PZP 16.21 chr12:9172788–9215201 0.00004656 42.4 kb VUS Autism spectrum disorder, congenital heart disease, neurodevelopmental disorder, breast cancer early onset 15 Metrorrhagia, severe body ache  40 23 12 
#354 12q24.33 loss htz chr12:133210742–133234403 ZNF268. ANHX 23.66 VUS ZNF268: autism spectrum disorder, congenital heart disease 0.7 1.6 Frequent diarrhea LEPR (c.2396–2A>G) 19 15 
 7q22.2 loss htz chr7:101039237–101041145 MUC17 1.91 chr7:101034793–101041958 0.0005203 7.17 kb VUS Autism spectrum disorder         
#271 14q11.2 loss htz chr14:20350503–20357181 PARP2 6.68 VUS Schizophrenia, prostate cancer, breast cancer 3.1 Slow learner  11 19 
#339 16p13.3 loss htz chr16:4959295–4992063 SEC14L5 32.77 chr16:4958364–5031681 0.00004656 73.3 kb VUS Autism spectrum disorder 13 5.5 Intellectual disability, delayed puberty, polydactyly MKKS (c.775del. p.T259Lfs*21) 14 32 13 
#347 16p12.2 loss htz chr16:21510262–21728466 OTOA. METTL9. IGSF6 218.21 OTOA: autism spectrum disorder, hearing loss (CNV). METTL9: cognitive impairment 4.0 Delayed milestones, memory loss  25 14 
#294 18p11.31 loss htz chr18:4233040–4421599 DLGAP1. DLGAP1-AS5 188.56 VUS DLGAP1: obsessive-compulsive disorder (CNV), schizophrenia, autism spectrum disorder, developmental disorder 11 Poor vision, aggressive behavior BBS2 (c.116A>G. p.K39R) 61 57 10 
#303 19p13.2 loss htz chr19:8904672–8906876 MUC16 2.21 chr19:8688936–9141019 0.00004656 453 kb VUS Autism spectrum disorder 0.4 NA  21 10 
#247 19p13.2 loss htz chr19:12398272–12431993 ZNF443. ZNF799 33.72 chr19:12386649–12433971 0.0002328 47.3 kb VUS ZNF799: autism spectrum disorder, Tourette syndrome 6.5 4.5 NA  10 10 17 
#244 2q11.2 loss htz chr2:97077521–97244014 FAHD2B. ANKRD36 166.49 VUS ANKRD36: autism spectrum disorder, psychosis 22  PWS-like features, no facial hair  31 16 16 
#270 2q32.1 loss htz chr2:183023784–184276709 NCKAP1. DUSP19. NUP35 1,252.93 VUS NCKAP1: autism spectrum disorder 4.1 8.4 Sleep apnea, aggressive behavior, recurrent RTI  22 61 
#215 2q37.3 loss htz chr2:239281474–239282694 HDAC4 1.22 chr2:239281331–239282793 0.001498 1.46 kb VUS Brachydactyly, mental retardation 1.1 6.5 Sleep apnea, gastric problems  23 83 14 
#219 20p12.1 loss htz chr20:14867224–15009042 MACROD2 141.82 chr20:14677820–15136935 0.00004656 459 kb VUS Early-onset obesity (CNV dup); Kabuki syndrome, ADHD, schizophrenia (CNV loss) 0.8 8.7 Slow learner  18 18 
   17q24.2 loss htz chr17:68145129–68515603 ARSG. AMZ2. PRKAR1A. WIPI1. SLC16A6 370.48 VUS ARSG: Usher syndrome, neurodevelopmental disorder. PRKAR1A: Carney complex (CNV)         
#224 22q12.3 loss htz chr22:32247382–32255353 SLC5A4 7.97 VUS ADHD 9.5 4.1 Asthma, mild intellectual disability  12 12 
 7q31.1 loss htz chr7:111407385–111596185 IMMP2L 188.8 chr7:111380309–111597224 0.00004656 217 kb Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#277 6p22.3 loss htz chr6:17754387–17781333 KIF13A 26.95 VUS Autism spectrum disorder 0.6 Wakes up at night demanding food  28 28 
 4q35.1-q35.2gain chr4:186017165–186210660 TLR3. FAM149A. CYP4V2 193.5 VUS TLR3: herpes simplex encephalitis/encephalopathy; immunodeficiency. FAM149A: pulmonary arterial hypertension. CYP4V2: Bietti crystalline corneoretinal dystrophy (CNV loss); retinitis pigmentosa         
#298 6p22.3 loss htz chr6:17893182–18021242 KIF13A 128.06 VUS Autism spectrum disorder 11 3.8 Intellectual disability, hypothyroidism  25 31 11 
  15q21.1 gain chr15:45106503–45178335 DUOX2. DUOXA1. DUOX1. SHF 71.83 VUS DUOX2: hypothyroidism.
DUOXA1: schizophrenia, hydrops fetalis.
DUOX1: intellectual disability, hypothyroidism 
        
#250 8p22 loss htz chr8:16098745–16164282 MSR1 65.54 chr8:16087944–16166408 0.001723 78.5 kb VUS Schizophrenia (CNV loss), Barrett esophagus/esophageal adenocarcinoma, prostate cancer 0.9 3.7 Mild intellectual disability, slow movements  10 
#316 10q26.13 gain chr10:121503759–121870059 FGFR2. ATE1 366.3 VUS FGFR2: Apert syndrome (CNV dup), craniosynostosis.
ATE1: hearing impairment, tinnitus, atrioventricular septum defect 
10.3 5.5 Slow learner, bowlegs, poor vision BBS5 (c.668_671del. p.E223Afs*14) 14 86 11 
#333 4p16.3 loss htz chr4:3444014–3448310 HGFAC 4.3 VUS Autism spectrum disorder 19 BMI: 39 Delayed milestones  45 102 14 
#39 15q11.2-q13.1 loss htz chr15:23235221–26108349 (hg19) MKRN3. MAGEL2. NDN. NPAP1. SNRPN. SNURF. UBE3A. ATP10A 2,873.1 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 6.1 5.2 Atrophied uterus and ovaries  130 36 
#87 15q11.2-q13.1 loss htz chr15:23996462–28544359 (hg19) NPAP1. SNRPN. SNURF. UBE3A. ATP10A. GABRB3. GABRA5. GABRG3. OCA2. HERC2 4,547.9 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 22 BMI: 43 No menarche, moderate intellectual disability  28 19 
#320 15q11.2-q13.1 loss htz chr15:22781870–26561186 NIPA1. NIPA2. CYFIP1. TUBGCP5. GOLGA6L1. LOC102723534. GOLGA8S. MKRN3. MAGEL2. NDN. LOC105370733. NPAP1. SNRPN. SNURF. UBE3A. ATP10A. GABRB3 3,779.320 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 4.8 6.0 PWS-like features, intellectual disability  12 28 
  5q23.2 gain chr5:127447531–127539157 MEGF10. PRRC1 91.63 chr5:127045431–127965806 0.00004902 920 kb VUS MEGF10: minicore myopathy, congenital myopathy, muscle weakness         
#282 16p11.2 loss htz chr16:29581926–30231884 SPN. QPRT. C16orf54. ZG16. KIF22. MAZ. PRRT2. PAGR1. MVP. CDIPT. SEZ6L2. ASPHD1. KCTD13. TMEM219. TAOK2. HIRIP3. INO80E. DOC2A. C16orf92. FAM57B. ALDOA. PPP4C. TBX6. YPEL3. GDPD3. MAPK3. CORO1A. BOLA2B. SLX1A. SULT1A3. NPIPB13 649.96 16p11.2 loss CNV, severe obesity, autism 3.2 Slow learner, delayed milestones  18 16 
#338 11p12 gain chr11:36592557–36957106 RAG2. C11orf74 364.55 VUS RAG2: immunodeficiency (CNV), Omenn syndrome 8.6 Poor vision, delayed milestones ALMS1 (c.10972C>T. p.R3658*) 12 60 
#315 11q23.3 gain chr11:118077018–118152698 TMPRSS4. SCN4B 75.68 chr11:118076058–118161781 0.00004902 85.7 kb VUS TMPRSS4: cerebral atrophy, autosomal recessive. SCN4B: atrial fibrillation 2.6 7.2 NA  15 25 10 
#313 16p13.3 gain chr16:43545–176835 POLR3K. SNRNP25. MPG 133.29 VUS POLR3K: Hypomyelinating leukodystrophy.
MPG: schizophrenia, colorectal cancer 
5.2 5.5 Intellectual disability, recurrent RTI  11 50 13 
#290 18q21.33 gain chr18:63641736–63659552 SERPINB3. SERPINB4 17.82 VUS SERPINB3: liver cirrhosis.
SERPINB4: diabetes MODY, autism spectrum disorder 
17  Secondary amenorrhea  31 96 11 
#321 19q13.43 gain chr19:57477251–57491801 ZNF772. ZNF419 14.55 chr19:57461161–57532262 0.00004902 71 kb VUS ZNF419: autism spectrum disorder 0.6 7.4 NA LEPR (c.2396–1G>T) 26 17 13 
#307 2p11.2 gain chr2:85302449–85322813 TCF7L1. TGOLN2 20.36 VUS TCF7L1: glaucoma, primary congenital (CNV), autism 6.0 5.2 Intellectual disability  17 34 11 
  4q35.1-q35.2 gain chr4:186017165–186210660 TLR3. FAM149A. CYP4V2 193.5 chr4:185990671–186121496 0.00009804 131 kb VUS TLR3: herpes simplex encephalitis/encephalopathy, influenza-associated.
CYP4V2: Bietti crystalline corneoretinal dystrophy, retinitis pigmentosa.
TLR3: inflammatory bowel disease, schizophrenia 
        
#319 2q14.2 gain chr2:119195166–119810001 STEAP3. DBI. TMEM37. CFAP221. TMEM177. PTPN4 614.84 VUS STEAP3: hypochromic anemia.
PTPN4: Rett-like syndrome (CNV loss), autism spectrum disorder, neurodevelopmental disorder 
4.4 Delay in mental age, aggressive behavior  122 11 
#285 21q11.2 gain chr21:13251123–14109107 POTED. LIPI 857.99 VUS LIPI: hypertriglyceridemia 11 3.7 NA  30 41 23 
#275 21q22.3 gain chr21:42259283–42296415 ABCG1 37.13 VUS Abnormal HDL cholesterol, autism spectrum disorder, heart disease   NA MKKS (c.775del.p.T259Lfs*21) 29 14 
#342 22q11.22 gain chr22:21955538–22110518 TOP3B 154.98 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 1.8 8.8 Delayed milestones LEP (c.-29+1G>C) 19 15 
 3q29 gain chr3:197791254–197875785 LRCH3 84.53 chr3:197791715–197915689 0.00009804124 kb VUS Neurodevelopmental disorder         
#266 22q11.22 gain chr22:21955538–22110518 TOP3B 154.98 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 4.4 6.1 NA  23 26 
#329 22q11.22 gain chr22:21957088–22215308 TOP3B 258.22 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 2.8 8.3 NA  19 26 
#205 4p13 gain chr4:42806238–43454858 GRXCR1 648.62 VUS Deafness, dizziness 1.6 5.3 NA  
#252 5q31.3 gain chr5:141332929–141341211 PCDHGA1. PCDHGA2 8.28 VUS PCDHGA2: Cardiovascular malformation        
#332 5q32 gain chr5:149860860–149886410 PDE6A 25.55 chr5:149629797–150001191 0.00009804 371 kb VUS Retinitis pigmentosa, Leber congenital amaurosis 16 3.2 Rudimentary external genitalia, autistic  35 27 10 
Proband IDCNVGenomic interval (hg38)Gene(s)Size (kb)gnomAD region MAF SizePatho-genicityGene or CNV- associated disorderAge (years)SexBMI SDS for agePhenotype (other than obesity)Additional pathogenic obesity-associated variantsLeptin (ng/mL)Insulin (μIU/mL)Cortisol (μg/dL)
#234 1q42.12 loss hmz chr1:224923245–225051803 DNAH14 128.56 VUS Panventriculomegaly (CNV del), intellectual disability, hydrops fetalis, nonimmune 2.4 9.4 Slow learner, intellectual disability  10 109 
#238 1p31.3 loss hmz chr1:65532214–65592881 LEPR 60.67 Morbid obesity 0.8 9.1   38 26 12 
#263 1p31.3 loss hmz chr1:65592634–65637041 LEPR 44.41 Morbid obesity 1.8 8.4 Delayed milestones, mental retardation  27 46 13 
 7q31.1 loss htz chr7:111337755–111407620 IMMP2L 69.87 chr7:111209525–111532640 0.00004656 323 kb VUS Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#343 1q43 loss htz chr1:240207914–240208747 FMN2 0.83 Intellectual disability, mental retardation, short stature, premature ovarian failure (CNV), intellectual disability 2.1 6.6 Delayed milestones  117 106 11 
 7q31.1 loss htz chr7:111180340–111569415 IMMP2L 389.08 Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#304 10q21.3 loss htz chr10:66402469–66676594 CTNNA3 274.13 VUS Autism spectrum disorder (CNV), cardiomyopathy NA  29 25 10 
#334 10q25.3 loss htz chr10:115799104–116272282 ATRNL1. GFRA1 473.18 VUS ATRNL1: cognitive impairment, autism, dysmorphic features (CNV). GFRA1: Hirschsprung disease (CNV), central hypoventilation syndrome 2.7 Delayed milestones  22 19 
#299 12p13.31 loss htz chr12:9192171–9208382 PZP 16.21 chr12:9172788–9215201 0.00004656 42.4 kb VUS Autism spectrum disorder, congenital heart disease, neurodevelopmental disorder, breast cancer early onset 15 Metrorrhagia, severe body ache  40 23 12 
#354 12q24.33 loss htz chr12:133210742–133234403 ZNF268. ANHX 23.66 VUS ZNF268: autism spectrum disorder, congenital heart disease 0.7 1.6 Frequent diarrhea LEPR (c.2396–2A>G) 19 15 
 7q22.2 loss htz chr7:101039237–101041145 MUC17 1.91 chr7:101034793–101041958 0.0005203 7.17 kb VUS Autism spectrum disorder         
#271 14q11.2 loss htz chr14:20350503–20357181 PARP2 6.68 VUS Schizophrenia, prostate cancer, breast cancer 3.1 Slow learner  11 19 
#339 16p13.3 loss htz chr16:4959295–4992063 SEC14L5 32.77 chr16:4958364–5031681 0.00004656 73.3 kb VUS Autism spectrum disorder 13 5.5 Intellectual disability, delayed puberty, polydactyly MKKS (c.775del. p.T259Lfs*21) 14 32 13 
#347 16p12.2 loss htz chr16:21510262–21728466 OTOA. METTL9. IGSF6 218.21 OTOA: autism spectrum disorder, hearing loss (CNV). METTL9: cognitive impairment 4.0 Delayed milestones, memory loss  25 14 
#294 18p11.31 loss htz chr18:4233040–4421599 DLGAP1. DLGAP1-AS5 188.56 VUS DLGAP1: obsessive-compulsive disorder (CNV), schizophrenia, autism spectrum disorder, developmental disorder 11 Poor vision, aggressive behavior BBS2 (c.116A>G. p.K39R) 61 57 10 
#303 19p13.2 loss htz chr19:8904672–8906876 MUC16 2.21 chr19:8688936–9141019 0.00004656 453 kb VUS Autism spectrum disorder 0.4 NA  21 10 
#247 19p13.2 loss htz chr19:12398272–12431993 ZNF443. ZNF799 33.72 chr19:12386649–12433971 0.0002328 47.3 kb VUS ZNF799: autism spectrum disorder, Tourette syndrome 6.5 4.5 NA  10 10 17 
#244 2q11.2 loss htz chr2:97077521–97244014 FAHD2B. ANKRD36 166.49 VUS ANKRD36: autism spectrum disorder, psychosis 22  PWS-like features, no facial hair  31 16 16 
#270 2q32.1 loss htz chr2:183023784–184276709 NCKAP1. DUSP19. NUP35 1,252.93 VUS NCKAP1: autism spectrum disorder 4.1 8.4 Sleep apnea, aggressive behavior, recurrent RTI  22 61 
#215 2q37.3 loss htz chr2:239281474–239282694 HDAC4 1.22 chr2:239281331–239282793 0.001498 1.46 kb VUS Brachydactyly, mental retardation 1.1 6.5 Sleep apnea, gastric problems  23 83 14 
#219 20p12.1 loss htz chr20:14867224–15009042 MACROD2 141.82 chr20:14677820–15136935 0.00004656 459 kb VUS Early-onset obesity (CNV dup); Kabuki syndrome, ADHD, schizophrenia (CNV loss) 0.8 8.7 Slow learner  18 18 
   17q24.2 loss htz chr17:68145129–68515603 ARSG. AMZ2. PRKAR1A. WIPI1. SLC16A6 370.48 VUS ARSG: Usher syndrome, neurodevelopmental disorder. PRKAR1A: Carney complex (CNV)         
#224 22q12.3 loss htz chr22:32247382–32255353 SLC5A4 7.97 VUS ADHD 9.5 4.1 Asthma, mild intellectual disability  12 12 
 7q31.1 loss htz chr7:111407385–111596185 IMMP2L 188.8 chr7:111380309–111597224 0.00004656 217 kb Autism, epilepsy, intellectual disability, multiple congenital abnormalities (CNV loss), neurodevelopmental disorder (CNV dup)         
#277 6p22.3 loss htz chr6:17754387–17781333 KIF13A 26.95 VUS Autism spectrum disorder 0.6 Wakes up at night demanding food  28 28 
 4q35.1-q35.2gain chr4:186017165–186210660 TLR3. FAM149A. CYP4V2 193.5 VUS TLR3: herpes simplex encephalitis/encephalopathy; immunodeficiency. FAM149A: pulmonary arterial hypertension. CYP4V2: Bietti crystalline corneoretinal dystrophy (CNV loss); retinitis pigmentosa         
#298 6p22.3 loss htz chr6:17893182–18021242 KIF13A 128.06 VUS Autism spectrum disorder 11 3.8 Intellectual disability, hypothyroidism  25 31 11 
  15q21.1 gain chr15:45106503–45178335 DUOX2. DUOXA1. DUOX1. SHF 71.83 VUS DUOX2: hypothyroidism.
DUOXA1: schizophrenia, hydrops fetalis.
DUOX1: intellectual disability, hypothyroidism 
        
#250 8p22 loss htz chr8:16098745–16164282 MSR1 65.54 chr8:16087944–16166408 0.001723 78.5 kb VUS Schizophrenia (CNV loss), Barrett esophagus/esophageal adenocarcinoma, prostate cancer 0.9 3.7 Mild intellectual disability, slow movements  10 
#316 10q26.13 gain chr10:121503759–121870059 FGFR2. ATE1 366.3 VUS FGFR2: Apert syndrome (CNV dup), craniosynostosis.
ATE1: hearing impairment, tinnitus, atrioventricular septum defect 
10.3 5.5 Slow learner, bowlegs, poor vision BBS5 (c.668_671del. p.E223Afs*14) 14 86 11 
#333 4p16.3 loss htz chr4:3444014–3448310 HGFAC 4.3 VUS Autism spectrum disorder 19 BMI: 39 Delayed milestones  45 102 14 
#39 15q11.2-q13.1 loss htz chr15:23235221–26108349 (hg19) MKRN3. MAGEL2. NDN. NPAP1. SNRPN. SNURF. UBE3A. ATP10A 2,873.1 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 6.1 5.2 Atrophied uterus and ovaries  130 36 
#87 15q11.2-q13.1 loss htz chr15:23996462–28544359 (hg19) NPAP1. SNRPN. SNURF. UBE3A. ATP10A. GABRB3. GABRA5. GABRG3. OCA2. HERC2 4,547.9 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 22 BMI: 43 No menarche, moderate intellectual disability  28 19 
#320 15q11.2-q13.1 loss htz chr15:22781870–26561186 NIPA1. NIPA2. CYFIP1. TUBGCP5. GOLGA6L1. LOC102723534. GOLGA8S. MKRN3. MAGEL2. NDN. LOC105370733. NPAP1. SNRPN. SNURF. UBE3A. ATP10A. GABRB3 3,779.320 15q11.2-q13.1 loss CNV, feeding difficulties in infancy, hypogonadism, intellectual disability, muscular hypotonia, truncal obesity, schizophrenia 4.8 6.0 PWS-like features, intellectual disability  12 28 
  5q23.2 gain chr5:127447531–127539157 MEGF10. PRRC1 91.63 chr5:127045431–127965806 0.00004902 920 kb VUS MEGF10: minicore myopathy, congenital myopathy, muscle weakness         
#282 16p11.2 loss htz chr16:29581926–30231884 SPN. QPRT. C16orf54. ZG16. KIF22. MAZ. PRRT2. PAGR1. MVP. CDIPT. SEZ6L2. ASPHD1. KCTD13. TMEM219. TAOK2. HIRIP3. INO80E. DOC2A. C16orf92. FAM57B. ALDOA. PPP4C. TBX6. YPEL3. GDPD3. MAPK3. CORO1A. BOLA2B. SLX1A. SULT1A3. NPIPB13 649.96 16p11.2 loss CNV, severe obesity, autism 3.2 Slow learner, delayed milestones  18 16 
#338 11p12 gain chr11:36592557–36957106 RAG2. C11orf74 364.55 VUS RAG2: immunodeficiency (CNV), Omenn syndrome 8.6 Poor vision, delayed milestones ALMS1 (c.10972C>T. p.R3658*) 12 60 
#315 11q23.3 gain chr11:118077018–118152698 TMPRSS4. SCN4B 75.68 chr11:118076058–118161781 0.00004902 85.7 kb VUS TMPRSS4: cerebral atrophy, autosomal recessive. SCN4B: atrial fibrillation 2.6 7.2 NA  15 25 10 
#313 16p13.3 gain chr16:43545–176835 POLR3K. SNRNP25. MPG 133.29 VUS POLR3K: Hypomyelinating leukodystrophy.
MPG: schizophrenia, colorectal cancer 
5.2 5.5 Intellectual disability, recurrent RTI  11 50 13 
#290 18q21.33 gain chr18:63641736–63659552 SERPINB3. SERPINB4 17.82 VUS SERPINB3: liver cirrhosis.
SERPINB4: diabetes MODY, autism spectrum disorder 
17  Secondary amenorrhea  31 96 11 
#321 19q13.43 gain chr19:57477251–57491801 ZNF772. ZNF419 14.55 chr19:57461161–57532262 0.00004902 71 kb VUS ZNF419: autism spectrum disorder 0.6 7.4 NA LEPR (c.2396–1G>T) 26 17 13 
#307 2p11.2 gain chr2:85302449–85322813 TCF7L1. TGOLN2 20.36 VUS TCF7L1: glaucoma, primary congenital (CNV), autism 6.0 5.2 Intellectual disability  17 34 11 
  4q35.1-q35.2 gain chr4:186017165–186210660 TLR3. FAM149A. CYP4V2 193.5 chr4:185990671–186121496 0.00009804 131 kb VUS TLR3: herpes simplex encephalitis/encephalopathy, influenza-associated.
CYP4V2: Bietti crystalline corneoretinal dystrophy, retinitis pigmentosa.
TLR3: inflammatory bowel disease, schizophrenia 
        
#319 2q14.2 gain chr2:119195166–119810001 STEAP3. DBI. TMEM37. CFAP221. TMEM177. PTPN4 614.84 VUS STEAP3: hypochromic anemia.
PTPN4: Rett-like syndrome (CNV loss), autism spectrum disorder, neurodevelopmental disorder 
4.4 Delay in mental age, aggressive behavior  122 11 
#285 21q11.2 gain chr21:13251123–14109107 POTED. LIPI 857.99 VUS LIPI: hypertriglyceridemia 11 3.7 NA  30 41 23 
#275 21q22.3 gain chr21:42259283–42296415 ABCG1 37.13 VUS Abnormal HDL cholesterol, autism spectrum disorder, heart disease   NA MKKS (c.775del.p.T259Lfs*21) 29 14 
#342 22q11.22 gain chr22:21955538–22110518 TOP3B 154.98 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 1.8 8.8 Delayed milestones LEP (c.-29+1G>C) 19 15 
 3q29 gain chr3:197791254–197875785 LRCH3 84.53 chr3:197791715–197915689 0.00009804124 kb VUS Neurodevelopmental disorder         
#266 22q11.22 gain chr22:21955538–22110518 TOP3B 154.98 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 4.4 6.1 NA  23 26 
#329 22q11.22 gain chr22:21957088–22215308 TOP3B 258.22 Mild mental retardation, generalized overgrowth (CNV dup), intellectual disability, schizophrenia 2.8 8.3 NA  19 26 
#205 4p13 gain chr4:42806238–43454858 GRXCR1 648.62 VUS Deafness, dizziness 1.6 5.3 NA  
#252 5q31.3 gain chr5:141332929–141341211 PCDHGA1. PCDHGA2 8.28 VUS PCDHGA2: Cardiovascular malformation        
#332 5q32 gain chr5:149860860–149886410 PDE6A 25.55 chr5:149629797–150001191 0.00009804 371 kb VUS Retinitis pigmentosa, Leber congenital amaurosis 16 3.2 Rudimentary external genitalia, autistic  35 27 10 

ADHD, attention deficit hyperactivity disorder; chr, chromosome; del, deletion; dup, duplicate; hmz, homozygous; htz, heterozygous; MAF, minor allele frequency; MODY, maturity-onset diabetes of the young; NA, not available; P, pathogenic; RTI, respiratory tract infection.

Figure 1

Genetic etiologies of obesity in a cohort of 225 patients from Pakistani consanguineous families.

Figure 1

Genetic etiologies of obesity in a cohort of 225 patients from Pakistani consanguineous families.

LEP: 47% of Elucidated Cases

We identified seven homozygous pathogenic mutations in LEP carried by 52 probands (Table 1 and Fig. 1). These included two frameshift (p.G133Vfs*15 and p.Y140Tfs*8), one in-frame (p.I35del), one splice site (c.-29+1G>C), and three missense variations (p.D100N, p.N103K, and p.R105Q). Of these mutations, c.-29+1G>C (identified in six unrelated probands) and p.Y140Tfs*8 (identified in one proband) were novel (i.e., not reported in the literature or listed in gnomAD) (Supplementary Fig. 1). The remaining five LEP mutations have previously been reported (2). Of note, p.G133Vfs*15 was identified in 39 unrelated children and, by itself, accounted for obesity in 38% of the elucidated cases.

Besides excessive obesity and hyperphagia, the leptin-deficient probands often presented with respiratory infections, hepatomegaly, undescended testes, and delayed milestones. As anticipated, in leptin-deficient children, leptin levels were <0.2 ng or nondetectable (Supplementary Tables 2 and 3), with the exception of three probands with p.D100N and p.N103K variants with mean levels of 46 ± 12 ng/mL (Table 1 and Supplementary Table 4). Serum insulin and cortisol levels were variable but mostly in the higher range (Supplementary Tables 24).

LEPR: 15% of Elucidated Cases

We identified 14 homozygous pathogenic variants of LEPR in 17 probands (Tables 1 and 2 and Fig. 1). These included four splice-site (c.704–1G>A, c.2213–3C>G, c.2396–1G>T, and c.2396–2A>G), three frameshift (p.E580Kfs*37, p.A967Dfs*7, and p.S1090Wfs*6), one nonsense (p.W705*), and three missense mutations (p.E14K, p.N718S, and p.P876L); two copy-loss CNVs of 44.4 and 61 kb each; and a unique change at translation initiation site of LEPR (c.2T>C/p.?). Twelve of these LEPR variants were novel (Supplementary Fig. 2).

In contrast to previous findings (16), LEPR-deficient probands were phenotypically indistinguishable from leptin-deficient subjects. Apart from excessive obesity and hyperleptinemia (mean 41 ± 7.6 ng/mL, n = 15) (Supplementary Table 2), no other noticeable clinical problems except respiratory infection and/or delayed milestones in three subjects were reported.

MC4R: 11% of Elucidated Cases

We identified eight pathogenic homozygous mutations in MC4R in 12 probands that included three nonsense (c.47G>A/p.W16*, c.48G>A/p.W16*, and p.Y21*), one frameshift (p.Y212Sfs*5), one in-frame (p.F201_M204del), and three missense mutations (p.I69T, p.M161T, and p.R165W) (Table 1). Three of these variants were novel (Table 1 and Supplementary Fig. 3). In addition to excessive adiposity, hyperinsulinemia (44 ± 11 μIU/mL; n = 12) was recorded in most MC4R-deficient probands (Supplementary Table 2). No other significant clinical abnormalities were observed (Table 1).

Monogenic Syndromic Obesity (Bardet-Biedel Syndrome, ALMS1, and Prader-Willi Syndrome): 23% of Elucidated Cases

Twelve homozygous and two compound heterozygous mutations were identified in six genes linked to Bardet-Biedel syndrome (BBS) (BBS1, BBS2, BBS5, MKKS [BBS6], BBS9, and BBS10) in 17 probands (Table 1). The 12 homozygous mutations identified here included 6 frameshift, 2 splice-site, and 3 missense variants, and 1 was a substitution affecting the translation initiation codon (Table 1). Carriers of BBS gene mutations presented with central obesity and hyperphagia accompanied with other dysmorphic features (Table 1).

Four novel homozygous pathogenic, nonsense mutations in ALMS1 were identified in four male subjects with severe obesity (Table 1). The majority of probands with these mutations presented hyperinsulinemia (Supplementary Table 2).

Furthermore, we identified three probands carrying heterozygous deletions in the 15q11–13 Prader-Willi syndrome (PWS)–associated region (Table 2). Whereas one subject carried the typical 4.5-Mb deletion in the PWS-associated region, the other two subjects carried unique deletions of ∼3.3 and ∼3.8 Mb each (Supplementary Figs. 46). All three probands presented typical features of PWS (Table 1).

ADCY3: 4% of Cases

A girl with severe obesity was found with a unique 23-bp homozygous deletion (c.2173–10_2185del) in ADCY3 (Table 1). In the same cohort, we have previously reported three other homozygous loss-of-function mutations in this gene (9).

Variants of Uncertain Significance: Point Mutations in Obesity Genes

We identified a homozygous novel mutation in VPS13B (p.P2207T), CEP19 (p.M38T), and BBS9 (p.Q748L). These mutations were variants of uncertain significance (VUS) (Supplementary Table 5).

Pathogenic Copy-Loss CNV in Chromosome 16p11.2

A proband was identified with an ∼650-kb heterozygous deletion of chromosome 16p11.2 (Supplementary Fig. 7). Besides severe obesity, delayed milestones and intellectual disability were reported in the carrier.

CNVs Associated With Intellectual Disability and Potentially Causing Obesity (n = 22)

In addition to CNVs in known obesity-causing genes/regions, we found 47 CNVs, including 10 rare CNVs (allele frequency 0.001 in gnomAD) and 37 novel CNVs, in coding regions. These comprised 25 copy-loss and 11 copy-gain CNVs (Table 2). Of these, 36 CNVs have previously been associated with intellectual disability. Importantly, 28 of these 36 CNVs (21 copy loss and 7 copy gain) were found in 22 subjects who were negative for any other mutation (Table 2 and Fig. 1).

Novel CNVs Overlapping Genes Potentially Associated With Obesity (n = 4)

Four novel CNVs affecting candidate genes for energy balance impairment were also identified. This includes a small 8-kb heterozygous copy-loss CNV in SLC5A4 (Table 2). SLC5A4 is involved in the neuronal glucose sensing mechanism and control of food intake (17). Furthermore, we found a novel heterozygous 473-kb copy-loss CNV involving ATRNL1. This gene, mainly expressed in the brain, modulates the melanocortin signaling pathway (18). A 366.3-kb copy-gain CNV encompassing FGFR2 and ATE1 was identified in a proband also deficient for BBS5 (Table 1). ATE1 is reported to affect adipogenesis and adipocyte function (19). Another 858-kb copy-gain CNV affecting the LIPI gene, which is involved in regulation of fat metabolism (20), was identified in a severely obese proband (Table 2).

The main result of this largest genetic study of patients with childhood-onset, severe obesity is genetic elucidation of obesity in 110 (49%) probands from a consanguineous population. This unexpectedly high percentage includes loss-of-function point mutations or CNVs in loci classically known to cause obesity. We confirm our earlier findings that leptin deficiency alone explains obesity in one-fifth of patients, making leptin deficiency the predominant etiology of monogenic obesity in this Pakistani population (7,8,21). The proportion of elucidated variants may even be much higher because we also identified an additional 22 cases with rare or novel CNVs linked to intellectual disabilities often associated with a severely obese phenotype. These cases provide credence to the notion that loci causing intellectual disability may also be involved in obesity (4). Thus, altogether, we elucidate up to 132 (59%) obesity cases, excluding severely obese patients in which VUS were identified in obesity genes (Supplementary Table 5).

This investigation highlights the importance of CNVs in the diagnosis of obesity. Although suggested by some of the pioneering studies (4,22), this hypothesis has so far received little attention from the clinical viewpoint (23) possibly because of the high cost of microarray technology, thus restricting its use to syndromic phenotypes only. In the current study, CoDE-seq has allowed us to directly detect CNVs in addition to point mutations (5). This comprehensive genetic screening in obesity is powerful because our retrospective clinical investigations unraveled the yet-undiagnosed intellectual disability in the probands carrying CNVs.

In conclusion, we show that next-generation sequencing approaches make it possible to uncover the genetic causality of severe obesity in a large proportion of subjects from the consanguineous population of Pakistan. An unrelenting quest for the discovery of new genes and variants, and associated pathways predisposing to obesity, is crucial in the development of specific and effective pharmacotherapies for the treatment of obesity.

This article contains supplementary material online at https://doi.org/10.2337/figshare.12200843.

Acknowledgments. The authors thank the patients and their families for participation in the study. The authors are grateful to H. Crouch (Imperial College London) for help in single nucleotide polymorphism array experiments and I. Qureshi (University of Lahore) for technical assistance. The authors also thank F. Allegaert and N. Larcher (CNRS-UMR 8199–European Genomic Institute for Diabetes) for DNA extraction and storage.

Funding. This study was supported by the Fédération de Recherche 3508 LabEx European Genomics Institute for Diabetes (ANR-10LABX-46), the ANR EquipEx 2010 session (ANR-10-EQPX-07-01, “LIGAN-PM”), the European Community (FEDER), and the Region Hauts-de-France. The research leading to this study was also supported by funding from the Pakistan Academy of Sciences (PAS003 to M.A.) and the European Research Council (GEPIDIAB 294785 to P.F.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.S., M.A., S.M.D., Q.M.J., Q.A., and L.I. collected samples and performed biochemical analysis. S.S., M.A., A.Bo., and P.F. designed the study and wrote the first draft of the manuscript. S.S., E.V., E.D., M.D., S.A., A.Ba., L.B., and A.Bo. performed whole-exome sequencing and analyzed the genetic data. J.M., H.A., W.I.K., and T.A.B. identified and recruited obese families. S.L. carried out microarray experiments. S.G. managed the database. All authors contributed to the final version of the manuscript. S.S. and P.F. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

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