Early-onset Obesity: Unrecognized First Evidence for GNAS Mutations and Methylation Changes

Annette Grüters-Kieslich; Monica Reyes; Amita Sharma; Cem Demirci; Terry J. DeClue; Erwin Lankes; Dov Tiosano; Dirk Schnabel; Harald Jüppner


J Clin Endocrinol Metab. 2017;102(8):2670-2677. 

In This Article


Height and Weight Data in Children With Later Diagnosis of PHP1A and PHP1B

At birth, patients with a subsequent diagnosis of PHP1A had mean z scores close to the 50th percentile for weight (mean, −0.5; range, −0.2 to −0.9) and for length (mean, −0.4; range, −0.2 to −0.6). In contrast, patients subsequently diagnosed with PHP1B had, at birth, higher z scores for length (mean, +1.4; range, +0.5 to +2.0) and weight (mean, +1.3; range, +0.2 to +2.4), consistent with our previous findings.[44] Subsequently, the mean body mass index remained well above the age-related normal ranges for both groups (z scores, +1.5 to +6.1) but appeared to improve by the end of the first decade of life (Figure 1). No appreciable differences were seen between PHP1A and PHP1B regarding the onset and severity of obesity; however, the underlying diagnosis was established >5 years earlier for PHP1A patients (Table 1). For some patients, the longitudinal measurements of body weight remained well above the reference range for the entire observation period, despite close medical monitoring, dietary efforts, and/or enrollment in childhood obesity programs. All the parents expressed concern that obesity in their children was caused by considerable hyperphagia.

Figure 1.

Early-onset obesity in PHP1A and PHP1B. Individual body mass index z scores and mean values (horizontal lines) at different ages of children affected by (left) PHP1A (n = 5) and (right) PHP1B (n = 7; Student t test with Welch correction: no significant differences between both groups). Note that most patients were monitored at their respective clinics several times within each age bracket; thus, several independent measurements were obtained.

TSH Resistance and Initiation of Therapy With L-thyroxine

With the exception of one patient (patient 5A), the PHP1A cohort revealed elevated serum TSH levels and low serum free thyroxine (fT4)/total thyroxine (TT4) concentrations when first evaluated for obesity (Table 1). Therapy with L-thyroxine was therefore initiated. Furthermore, in six of the seven patients with PHP1B, hypothyroidism was suspected because of increased TSH levels during the workup for obesity and despite normal fT4/TT4 concentrations; treatment with thyroid hormone was started in five patients aged 6 months to 3 years (i.e., well before the diagnosis of PHP1B was established). However, for both groups of patients, this therapeutic intervention failed to slow the weight gain.

Analysis of Epigenetic and Genetic Changes at the GNAS/STX16 Locus

In all patients, the diagnosis of PHP1A and PHP1B was confirmed by genetic testing. The calcium and phosphate levels were either normal or were not measured as a part of the initial obesity workup for most patients, in whom PHP1B was later diagnosed.

Because of obesity combined with elevated TSH levels, PHP1A was considered to be the underlying diagnosis in patients 1A, 2A, and 4A, although their mothers revealed no evidence of AHO. The PTH level was tested in only one patient with PHP1A (patient 3A) when first evaluated for obesity. This test was performed largely because of an equally affected older sibling (patient 2A), who had recently received the same diagnosis. Three different mutations in GNAS exons 7, 12, and 13 were identified in this cohort of patients, confirming their clinical diagnosis (Table 1).

For siblings 4B and 5B, both affected by PHP1B, sequence analysis of GNAS exons 1 to 13 was negative. Instead, complete loss-of-methylation at the differentially methylated region of GNAS exon A/B was noted. This epigenetic abnormality led to the identification of the 3-kb STX16 deletion in both children, which had been inherited from their unaffected mother and maternal grandfather (data not shown). Deletions in GNAS/STX16 could not be identified for patients 2B and 6B,[35] but both showed loss-of-methylation at exons A/B, XL, and AS, and gain-ofmethylation at exon NESP. Patient 3B was an affected member of a previously reported AD-PHP1B kindred and just as other affected familymembers, she had inherited the 4.4-kb STX16 deletion from an unaffected mother, thus explaining her elevated PTH level by 2 years of age.[19] However, at that time, early-onset obesity was thought to be unrelated to PHP1B, and she therefore underwent independent evaluations in an obesity clinic.

Screening for GNAS Methylation Variants in Unselected Children With Early-onset Obesity

Because of these findings, we hypothesized that hyperphagia leading to severe obesity in the first year of life might be an as-yet unrecognized symptom of PHP1B. We therefore investigated the cases of 24 patients, randomly selected from a cohort of 102 obese children, who had been evaluated at a single obesity clinic (Charité, Berlin, Germany). The exons encoding MC4R and POMC were sequenced in 10 individuals, including patient 7B; however, no genetic abnormality was detected (data not shown). Patients 1B and 7B showed a loss-of-methylation at GNAS exon A/B and MLPA revealed only one copy of STX16 exons 4 and 5, which led to the identification of the frequent 3-kb STX16 deletion (Table 1).

The mothers of both patients revealed the same deletion, as determined by polymerase chain reaction (PCR) of their genomic DNA, but they showed no GNAS methylation abnormalities indicating that their deletion resides on the paternal allele (data not shown). MLPA revealed only a barely detectable, very incomplete, loss of STX16 exons 4 and 5 for the mother of patient 1B (Supplemental Figure 1). Consistent with this observation, analysis of microsatellite 261P9-CA1 documented heterozygosity for this marker (mother, alleles 206 and 216; patient, only allele 210). The forward primer for amplification of this microsatellite is located within the 3-kb STX16 deletion, and the reverse primer resides outside this mutation. Although the 3-kb STX16 deletion was readily detectable by PCR, the identification of two maternal 261P9-CA1 alleles not shared with patient 1B indicated that the patient's mother has a de novo mosaic deletion.

After identification of the genetic defect for patient 1B, a review of his more recent medical records revealed that he had been admitted to a local hospital at the age of 11.5 years because of symptomatic hypocalcemia and an elevated PTH level, suggesting PTH resistance. Patient 7B had had no calcium or phosphate abnormalities found during routine follow-up examinations; however, after establishment of the genetic defect when he was 5.5 years old, the laboratory studies were repeated, and asymptomatic hypocalcemia and an elevated PTHlevel (86 ng/L) were noted. His leptin level was 20.0 ng/mL (normal, 3 to 21). MLPA and methylation-specific MLPA provided no evidence for abnormalities for the remaining 22 children.