A deletion of the HBII-85 class of small nucleolar RNAs (snoRNAs) is associated with hyperphagia, obesity and hypogonadism

Abstract

Genetic studies in patients with severe early-onset obesity have provided insights into the molecular and physiological pathways that regulate body weight in humans. We report a 19-year-old male with hyperphagia and severe obesity, mild learning difficulties and hypogonadism, in whom diagnostic tests for Prader-Willi syndrome (PWS) had been negative. We carried out detailed clinical and metabolic phenotyping of this patient and investigated the genetic basis of this obesity syndrome using Agilent 185 k array comparative genomic hybridization (aCGH) and Affymetrix 6.0 genotyping arrays. The identified deletion was validated using multiplex ligation-dependent probe amplification and long-range PCR, followed by breakpoint sequencing which enabled precise localization of the deletion. We identified a approximately 187 kb microdeletion at chromosome 15q11-13 that encompasses non-coding small nucleolar RNAs (including HBII-85 snoRNAs) which were not expressed in peripheral lymphocytes from the patient. Characterization of the clinical phenotype revealed increased ad libitum food intake, normal basal metabolic rate when adjusted for fat-free mass, partial hypogonadotropic hypogonadism and growth failure. We have identified a novel deletion on chromosome 15q11-13 in an individual with hyperphagia, obesity, hypogonadism and other features associated with PWS, which is normally caused by deficiency of several paternally expressed imprinted transcripts within chromosome 15q11-13, a region that includes multiple protein-coding genes as well as several non-coding snoRNAs. These findings provide direct evidence for the role of a particular family of non-coding RNAs, the HBII-85 snoRNA cluster, in human energy homeostasis, growth and reproduction.

Figure 1.

Mapping of a chromosome 15 deletion in a 19-year-old male with suspected PWS. (A) Ideogram of chromosome 15, showing the approximate chromosomal position of the microdeletion as highlighted by the red block. (B) Detection of the PWS-region deletion by array CGH on the Agilent 185 k microarray, as viewed in CGH Analytics 3.4. The red box highlights the 15 probes that appear to be deleted, with an average log₂ ratio of −1.143, compared with an expected ratio of 0 for a normal 2n copy number state. The blue bar below the red box shows the position of the MLPA probe (probe 1318-L07970 from the SALSA MLPA P245-A1 Microdeletion Syndromes probe mix) that was found to be deleted when compared with control probes in the patient only. (C) The red bar represents the novel deletion we have detected (chr15: 22 757 219–22 944 158), which is shown to overlap the deletion detected by Sahoo et al. (13) by ∼108 kb.

Figure 2.

MLPA of patient and patient's father using the SALSA MLPA P245-A1 Microdeletion Syndromes probemix. Peak height data for each probe has been normalized against five control probes (2n). The probemix contains a total of 49 different MLPA probes, but only four are shown here: NDN probe 6282 (ligation site at chr15:21482517), SNRPN probe 2026 (chr15:22652723), SNRPN probe 1318 (chr15:22764291) and UBE3A probe 4620 (chr15:23167784). SNRPN probe 1318 is shown to be deleted in the patient but not in the father, whereas the other probes are of equal copy number in both individuals.

Figure 3.

Positions of the up- and downstream breakpoints in the patient's DNA relative to the human reference genome sequence (hg17_DNA). In the upstream breakpoint, the red box shows the position of a LINE1 element, and in the downstream breakpoint the blue box represents the position of an Alu element, according to RepeatMasker (30). In both cases, the deletion breakpoint occurs within the poly-A tail of the repeat element.

Figure 4.

Expression analysis using RT–PCR of lymphocyte-derived RNA obtained from the proband and his father who does not carry the deletion. The 186 940 bp deletion results in loss of expression of several members of the HBII-85 snoRNA cluster in the patient, but these snoRNAs are expressed in the father. However, UBE3A, MAGEL2 and NECDIN, which lie outside the deleted region, are expressed in both individuals. GAPDH was used as an internal control. The experiments were conducted with and without reverse transcriptase.

Figure 5.

Obesity and endocrine phenotype. (A) Food intake at an 18 MJ ad libitum test meal expressed per kg lean body mass, for the proband with the deletion affecting the HBII-85 snoRNA cluster compared with subjects with mutations in other genes (LEP and MC4R) responsible for obesity syndromes and controls. Food intake is expressed per kg lean body mass measured by dual energy X-ray absorptiometry to allow comparison between subjects of different ages (*=P < 0.05). (B and C) Gonadotropin hormone pulsatile secretion. Samples were obtained every 10 min overnight during sleep.

Figure 6.

Patient aged 4 years showing obesity, upslanting palpebral fissures and tented mouth.