SNORD116 and growth hormone therapy impact IGFBP7 in Prader-Willi syndrome

Abstract

Purpose: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with hypothalamic dysfunction due to deficiency of imprinted genes located on the 15q11-q13 chromosome. Among them, the SNORD116 gene appears critical for the expression of the PWS phenotype. We aimed to clarify the role of SNORD116 in cellular and animal models with regard to growth hormone therapy (GHT), the main approved treatment for PWS.

Methods: We collected serum and induced pluripotent stem cells (iPSCs) from GH-treated PWS patients to differentiate into dopaminergic neurons, and in parallel used a Snord116 knockout mouse model. We analyzed the expression of factors potentially linked to GH responsiveness.

Results: We found elevated levels of circulating IGFBP7 in naive PWS patients, with IGFBP7 levels normalizing under GHT. We found elevated IGFBP7 levels in the brains of Snord116 knockout mice and in iPSC-derived neurons from a SNORD116-deleted PWS patient. High circulating levels of IGFBP7 in PWS patients may result from both increased IGFBP7 expression and decreased IGFBP7 cleavage, by downregulation of the proconvertase PC1.

Conclusion: SNORD116 deletion affects IGFBP7 levels, while IGFBP7 decreases under GHT in PWS patients. Modulation of the IGFBP7 level, which interacts with IGF1, has implications in the pathophysiology and management of PWS under GHT.

Trial registration: ClinicalTrials.gov NCT01298180.

Fig. 1. Elevated IGFBP7 plasma levels decrease under growth hormone therapy (GHT) in Prader–Willi syndrome (PWS) patients.

(a) IGF1 (n = 21) and (b) IGFBP3 (n = 20) values in plasma of PWS patients at day 0 (D0) and after one year (M12) of GHT in 21 PWS patients. (c) IGFBP7 values in the plasma of PWS (n = 13) at D0 and M12 of GHT, and in age-matched controls (n = 18). The immunogen of antibodies used for enzyme-linked immunosorbent assay (ELISA) corresponded to the IGFBP7 sequence (ser28-thr264). Data are represented as mean ± SEM. (d) Pearson correlation between IGFBP7 (ng/ml) decreased and IGF1 (SDS) increased within one year of GHT (M12-D0 values) (n = 13). Pearson product-moment correlation coefficients (r) were used to assess the relationship between variables for PWS patients. ***P < 0.001, ns nonsignificant.

Fig. 2. Structure of the Prader–Willi syndrome (PWS) locus and consequence of large deletion (LD) or microdeletion (MD) on IGFBP7 and proconvertases 1 and 2 (PCSK1/PCSK2) and NHLH2 expression in iPSC-derived neurons at day 34, and responses to IGF1.

(a) Diagram of the human PWS locus 15q11-q13. Maternally expressed genes are indicated in pink, paternally expressed genes in blue. Protein-coding genes are shown as ovals; small nucleolar RNAs (snoRNAs) as bars (SNORD116: 29 copies, SNORD115: 48 copies) the imprinting center (IC) is shown by ovals. LD large deletion, MD microdeletion including SNORD116. Drawing is not to scale. (b) Values of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of IGFBP7. (c) PCSK1, NHLH2, and PCSK2 messenger RNA (mRNA) expression (left, middle, and right, respectively) in untreated induced pluripotent stem cell (iPSC)-derived neurons and treated with IGF1 in phosphate buffered saline (PBS) 10 ng/ml for 2 hours. Data are from a male control subject (CON) (056LB), MD (2 clones: MD-A and MD-C) female, and LD (031MP) female. TBP (TATA-binding protein) was used as a housekeeping gene as reference. Results were from three independent experiments. The comparison was assessed by a two-tailed Student’s t-test. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns nonsignificant.

Fig. 3. Intact IGFBP7 expression is increased in large deletion (LD) and microdeletion (MD) cells, but there is no difference in IGFBP7 editing between controls and Prader–Willi syndrome (PWS) patients in induced pluripotent stem cell (iPSC)-derived neurons.

(a) Western blot of IGFBP7 secreted in a medium of immortalized fibroblasts from controls and PWS subjects cultured for 96 hours. 50 µg of total protein were deposited per well. Detection was performed with antibodies directed against the (Ser28-Thr264) IGFBP7 sequence, which detect both intact (32 kDa) and cleaved (22 KDa) forms. The cleaved form was less detected in LD and MD cell extracts than in controls. PWS-LD large deletion, PWS-MD PWS microdeletion. (b) Nucleotide frequencies from RNA sequencing of raw data from Burnett et al. performed at the position hg19: chr4:57,976,229 (hg38: chr4:57,110,063), which includes the A to I editing site of IGFBP7 at codon 95, hypothetical target for proteases and proconvertase 1. Comparison between iPSC-derived neurons from 6 unaffected subjects (CON) (056LB, 1111, 1034, 1043, 1058-6, and 1058-B7), 1 PWS patient with large deletion LD (031MP), and 1 PWS patient with microdeletion (MD) (2 clones were used: MD A and MD C). Readings were mapped to a reference genome (human: NCBI/build37.2).

Fig. 4. IGFBP7 is elevated in the plasma and brain of PWScr^p−/m+ mice and proposed schema of IGFBP7 regulation by SNORD116 deletion.

(a) Schematic representation of the orthologous PWS region on mouse chromosome 7qC. Small nucleolar RNAs (snoRNA) genes are represented by bars (Snord116: 71 copies and Snord115: 136 copies), other genes locations are depicted with rectangles and ovals. Paternally expressed genes are indicated in blue, maternally expressed genes in pink. The imprinting center (IC) is shown by a gray oval. The orange line indicates the deleted area in the studied model of PWScr^p−/m+ mice. Drawing is not to scale. (b) Circulating Igfbp7 was 2.2-fold higher (P = 0.00034) in P7 PWScr^p−/m+ mice (n = 7) compared to wild-type (WT) littermates (n = 7). Normality was assessed by Shapiro–Wilk normality tests, and statistical differences were analyzed using the paired Student’s t-test. Data are expressed as mean ± SEM, ***P < 0.001. (c) Transcript levels of Igfbp7 in different mouse tissues PWScr^p−/m+ (n = 7) compared to WT littermates (n = 7). Igfbp7 were highly expressed in PWScr^p−/m+ brains. Data are shown as mean delta Ct values compared to pgk1 as the reference gene giving the more stable expression besides other housekeeping genes. A comparison was drawn by a 2-tailed Student’s t-test. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001. (d) Proposed schema of IGFBP7 regulation by SNORD116 deletion and GHT in PWS. Both increased expression of IGFBP7 and proconvertase PCSK1/ PC1 deficiency could explain the raised IGFBP7 level in PWS patients, IGFBP7 being a potential target of PC1, which is downregulated in SNORD116-deleted neurons. Loss of SNORD116 results in an increase in IGFBP7 mRNA expression and a decrease in potential IGFBP7 cleavage by PC1, with high levels of secreted IGFBP7 in naive PWS patients. Growth hormone therapy (GHT) stimulates IGF1 production, which downregulates IGFBP7 mRNA expression and lowers its circulating level.