Functional characterization of all missense variants in LEPR, PCSK1, and POMC genes arising from single-nucleotide variants

Figures & data

Figure 1. The MC4R pathway [1–3]. LEPR, leptin receptor; MC4R, melanocortin-4 receptor; MSH, melanocyte-stimulating hormone; PCSK1, proprotein convertase subtilisin/kexin type 1; POMC, proopiomelanocortin.

Figure 2. In vitro functional assays for (A) LEPR, (B) PCSK1, and (C, D) POMC variants. AMC, 7-amino-4-methylcoumarin; cAMP, cyclic adenosine monophosphate; HEK, human embryonic kidney; LEPR, leptin receptor; MC4R, melanocortin-4 receptor; MSH, melanocyte-stimulating hormone; PCSK1, proprotein convertase subtilisin/kexin type 1; POMC, proopiomelanocortin; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SRE, serum response element.

Table 1. Validation of assays against published variants of known functional impact.

Gene and variant	Published functional classification^a	Rhythm data (% of WT activity)^b	Validation^c
LEPR
GFP_Control	NA	1.84	–
W664R[11]	Significant LOF	53.21	No
A409E[11]	Significant LOF	0	Yes
H684P[11]	Significant LOF	13.58	Yes
P878S[32]	Significant LOF	0	Yes
P876S[32]	Significant LOF	0	Yes
Q270P[13]	Significant LOF	6.75	Yes
Y1141F[27]	Significant LOF	0	Yes
R612H[11]	Moderate LOF	81.28	No
Q223R[16]	WT-like	89.19	Yes
PCSK1^c
GFP_Control	NA	1.29	–
G226R[7]	Significant LOF	0.91	Yes
G209R[15]	Significant LOF	1.84	Yes
N423K[15]	Significant LOF	1.28	Yes
F548S[15]	Significant LOF	1.57	Yes
G593R[15]	Significant LOF	1.19	Yes
S307L[10]	Significant LOF	2.09	Yes
N309K[17]	Significant LOF	0.89	Yes
T175M[7]	Significant LOF	6.66	Yes
M125I[7]	Significant LOF	17.73	Yes
S24C[38]	WT-like	78.34	Yes
S690T[34]	WT-like	93.84	Yes
K26E[7]	WT-like	84.97	Yes
H72L[35]	WT-like	118.21	Yes
T558A[7]	WT-like	42.89	No
POMC
GFP_Control	NA	1.30	–
E105*[38]^d	Significant LOF	0	Yes
F144L[9]	Significant LOF	23.56, >1 µM	Yes
H143Q[14]	Significant LOF	44.79, >1 µM	Yes
R236G[6]^e	Moderate LOF	88.56	No
Y221C[14]^e	Moderate LOF	98%, 173 nm	Yes
D53G[39]	WT-like	95.86	Yes

^aDetermined qualitatively on the basis of data in publication. Functional classification is based on that reported by the references cited within the first column for each respective gene. ^bGiven that negative values do not have biological relevance, negative results were reported as 0. ^cDetermined by comparing percent of WT activity category with published LOF category. ^cWild-type activity measured at 1 hour. ^dStop codon (nonsense mutation). ^eMutation in β–melanocyte-stimulating hormone. GFP, green fluorescent protein; LOF, loss of function; NA, not applicable; WT, wild-type.

Table 2. Criteria for variant classification.

	WT-like	LOF
LEPR, PCSK1, and POMC^a variants
% of CI overlap with WT	Overlap	No overlap
α-MSH variants^b
EC50	<119 nM	>120 nM
Activity at 1 µM	≥60%	<60%
β-MSH variants^b
EC50	<53 nM	>54 nM
Activity at 1 µM	≥60%	<60%

^aPOMC variants classified according to CI overlap with WT assay were performed for variants outside of the α-/β-MSH peptide region. ^bClassification was based on POMC variants within specified peptide region. CI, confidence interval; EC₅₀, half-maximal effective concentration; LOF, loss of function; MSH, melanocyte-stimulating hormone; WT, wild-type.

Figure 3. Distributions of the mean percent of WT of all observed variants characterized in the functional assays. Each data point represents the mean (95% confidence interval in blue) of replicate assays and is compared with mean (95% confidence interval) results from replicate WT assays, shown in gray. Variants in red demonstrate a functional deficit, characterized by lack of overlap with confidence interval with results from WT assays. (A) All observed LEPR variants (n = 910). (B) All observed PCSK1 variants (n = 619). (C) All observed variants outside α-/β-MSH peptide region (n = 314). (D) Representation of variant by functional categorization for variants within the α-MSH peptide region (138–150; n = 17) and β-MSH region (213–234; n = 27). cAMP, cyclic adenosine monophosphate; MSH, melanocyte-stimulating hormone; pERTKR-AMC, Pyr-Glu-Arg-Thr-Lys-Arg-7-amino-4-methylcoumarin; STAT3, signal transducer and activator of transcription 3; WT, wild-type.

Table 3. List of observed variants for potential reclassification based on in vitro functional assays.

Gene and variant	Interpretation	AF gnomAD v2.1.1	AF URO	Activity^a
LEPR
LEPR_p.Tyr422Phe	VUS	4.00E-06	2.21E-05	0 (0–0)
LEPR_p.Ala458Val	VUS	5.66E-05	8.85E-05	43.23 (18.71–67.74)
LEPR_p.Pro540Thr	VUS	1.42E-05	2.21E-05	53.5 (23.22–83.77)
LEPR_p.Leu568Arg	VUS	NR	2.21E-05	23.39 (10.12–36.66)
LEPR_p.Pro639Ala	VUS	NR	2.21E-05	0 (0–0)
LEPR_p.Trp664Arg	Likely pathogenic	2.80E-05	8.85E-05	53.21 (29.01–77.41)
LEPR_p.His684Pro	Pathogenic	1.77E-05	2.21E-05	13.58 (4.19–22.97)
LEPR_p.Asn718Ser	VUS	3.98E-06	2.21E-05	0 (0–0)
LEPR_p.Leu729Val	VUS	3.18E-05	0	0(0–0)
LEPR_p.Leu786Pro	Suspected pathogenic	4.00E-06	4.43E-05	29.85 (12.96–46.74)
LEPR_p.Gly858Arg	VUS	NR	2.21E-05	19.02 (10.41–27.62)
LEPR_p.Pro878Ser	VUS	3.98E-06	0	0(0–0)
LEPR_p.Cys881Phe	VUS	3.98E-06	2.21E-05	0 (0–0)
LEPR_p.Pro1044Leu	VUS	3.19E-05	2.21E-05	59.77 (25.95–93.59)
LEPR_p.Arg1114Ser	VUS	4.02E-06	2.21E-05	56.02 (23.95–88.08)
LEPR_p.Gln1146Leu	VUS	1.08E-05	0	46.1 (19.99–72.2)
LEPR_p.Glu1157Gln	VUS	NR	2.21E-05	52.7 (28.97–76.43)
PCSK1
PCSK1_p.Ala27Val	VUS	4.24E-05	4.43E-05	34.16 (32.77–35.56)
PCSK1_p.Gly42Asp	VUS	7.08E-06	4.43E-05	6.9 (5.91–7.89)
PCSK1_p.Lys71Gln	VUS	3.99E-06	4.43E-05	76.35 (70.83–81.87)
PCSK1_p.Met125Ile	VUS	1.80E-04	3.32E-04	17.4 (16.03–18.77)
PCSK1_p.Met125Lys	VUS	1.99E-05	6.64E-05	29.85 (27.36–32.34)
PCSK1_p.Ile156Thr	VUS	3.89E-05	4.43E-05	24.52 (23.83–25.21)
PCSK1_p.Val165Ile	VUS	2.00E-05	4.43E-05	59.15 (56.68–61.61)
PCSK1_p.Thr175Met	Likely pathogenic	1.47E-04	1.99E-04	6.67 (5.11–8.23)
PCSK1_p.Tyr181His	VUS	1.06E-04	1.33E-04	51.71(47.33–56.1)
PCSK1_p.Asp191Glu	VUS	3.98E-06	2.21E-05	76.03 (70.28–81.77)
PCSK1_p.Gly209Arg	Suspected pathogenic	1.99E-05	6.64E-05	1.84 (1.62–2.06)
PCSK1_p.Ala217Val	VUS	5.30E-05	4.43E-05	71.1 (65.19–77.01)
PCSK1_p.Gly226Arg	Suspected pathogenic	1.06E-05	2.21E-05	0.92 (0.32–1.52)
PCSK1_p.Tyr290Cys	VUS	5.66E-05	6.64E-05	54.86 (53.26–56.46)
PCSK1_p.Ser307Leu	Pathogenic	1.19E-05	2.21E-05	2.1 (1.94–2.26)
PCSK1_p.Ala344Thr	VUS	1.94E-04	4.43E-05	21.86 (17.91–25.81)
PCSK1_p.Thr366Met	VUS	3.59E-05	4.43E-05	86.85 (82.97–90.73)
PCSK1_p.Ser382Leu	VUS	NR	4.43E-05	0.97 (0.92–1.01)
PCSK1_p.Asn423Lys	VUS	2.39E-05	0	1.29 (0.85–1.72)
PCSK1_p.Asn429Lys	VUS	NR	4.43E-05	3.74 (3.35–4.13)
PCSK1_p.Ala446Val	VUS	7.95E-06	4.43E-05	22.39 (21.5–23.27)
PCSK1_p.Val461Met	VUS	2.78E-05	8.85E-05	72.29 (71.15–73.43)
PCSK1_p.Pro462Thr	VUS	3.98E-06	4.43E-05	82.32 (79.38–85.26)
PCSK1_p.Arg517Gln	VUS	8.13E-05	1.99E-04	4.01 (3.79–4.22)
PCSK1_p.Arg541Trp	VUS	3.89E-05	4.43E-05	58.58 (51.93–65.24)
PCSK1_p.Val556Ile	VUS	2.48E-05	1.11E-04	49.49 (47.43–51.55)
PCSK1_p.Asp573Asn	VUS	NR	4.43E-05	4.13 (3.83–4.42)
PCSK1_p.Arg654Gln	VUS	2.83E-05	8.85E-05	71.44 (65.81–77.06)
PCSK1_p.Arg669Gln	VUS	2.39E-05	6.64E-05	76.77 (73.21–80.32)
POMC
α-MSH peptide (138–150)
POMC_p.His143Tyr	Suspected benign	1.24E-04	4.43E-05	35.5 (>1 µM)
POMC_p.His143Gln	VUS	1.79E-04	2.21E-04	44.8 (>1 µM)
POMC_p.Phe144Leu	VUS	1.68E-04	2.21E-04	23.6 (>1 µM)
POMC_p.Arg145Leu	VUS	2.96E-05	6.64E-05	12.7 (>1 µM)
POMC_p.Arg145Cys	Likely pathogenic	2.11E-05	2.21E-05	6.7 (>1 µM)
POMC_p.Arg145His	VUS	4.48E-05	2.21E-05	24.9 (>1 µM)
POMC_p.Arg145Gly	Suspected pathogenic	4.22E-06	0	3.1 (>1 µM)
POMC_p.Trp146Leu	VUS	1.00E-05	0	5.4 (>1 µM)
POMC_p.Gly147Asp	VUS	8.42E-06	0	71.3 (255.7 nM)
POMC_p.Lys148Glu	VUS	8.39E-06	0	180.6 (127.4 nM)
POMC_p.Pro149Thr	VUS	2.94E-05	0	57.8 (22.5 nM)
β-MSH peptide (213–234)
POMC_p.Tyr221His	VUS	NR	2.21E-05	56.9 (111.1 nM)
POMC_p.Tyr221Cys	VUS	8.27E-04	8.85E-04	98.4 (173.6 nM)
POMC_p.Arg222Trp	VUS	3.99E-06	0	53.8 (>1 µM)
POMC_p.Met223Thr	VUS	4.97E-05	4.43E-05	30.7 (>1 µM)
POMC_p.Met223Val	VUS	1.60E-05	0	101.4 (83 nM)
POMC_p.His225Gln	VUS	7.98E-06	0	29.2 (>1 µM)
POMC_p.His225Pro	VUS	1.60E-05	8.85E-05	26.9 (>1 µM)
POMC_p.Phe226Leu	VUS	3.99E-06	0	10.4 (>1 µM)
POMC_p.Arg227Cys	VUS	6.57E-06	2.21E-05	3.2 (>1 µM)
POMC_p.Trp228Gly	VUS	6.58E-06	0	1.5 (>1 µM)
POMC_p.Trp228Ser	VUS	1.60E-05	2.21E-05	2.9 (>1 µM)
Other region (1–137, 151–212, 235–267)
POMC_p.Leu12Pro	VUS	6.57E-06	0	75.6 (73.1–78.1)
POMC_p.Pro52Thr	VUS	3.19E-05	0	75.9 (66.4–85.4)
POMC_p.Arg90Gly	VUS	1.11E-05	0	70.8 (63.6–78)
POMC_p.Ala109Val	VUS	NR	2.21E-05	75.4 (64.8–85.9)
POMC_p.Pro161Ala	VUS	NR	2.21E-05	69.4 (67–71.8)
POMC_p.Gln202Arg	VUS	9.58E-06	0	82 (68.2–95.8)
POMC_p.Ser208Arg	VUS	1.13E-05	0	74.3 (59.2–89.3)

Frequencies of select observed variants were determined using gnomAD database and sequencing results from the URO program. The URO program is a comprehensive genetic testing program for rare genetic diseases of obesity. ^aActivity reported as percent of WT activity (95% CI) for LEPR and PCSK1 variants and POMC other region variants and percent activity at 1 µM (EC₅₀) for POMC α-/β-MSH peptide region variants. CI, confidence interval; EC₅₀, half-maximal effective concentration; MSH, melanocyte-stimulating hormone; NR, not reported; URO, Uncovering Rare Obesity; VUS, variant of uncertain significance; WT, wild-type.

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