Severe infantile obesity with a maternal GNAS deletion and multi-locus imprinting disturbance including hypomethylation of the KCNQ1OT1:TSS-DMR

Abstract.

Genetic defects in GNAS on the maternal allele cause pseudohypoparathyroidism type 1A (PHP1A) with PTH resistance. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome caused by aberrant methylation in the KCNQ1OT1:transcription start site (TSS)-differentially methylated region (DMR). PHP1A and BWS exhibit postnatal overgrowth. Recently, multi-locus imprinting disturbance (MLID) has been observed in cases with BWS. Deleterious variants in genes encoding proteins that maintain CpG methylation at DMRs have been found in MLID cases and/or their mothers, and ZAR1 is supposed to be one of the MLID causative genes. In this study, we identified a patient with a deletion involving Gsα-coding region and MLID including hypomethylation of the KCNQ1OT1:TSS-DMR by genome-wide copy number variation analysis, genome-wide methylation analysis, and whole-exome sequencing. The patient’s mother had a deletion of the same region on the paternal allele and carried a ZAR1 missense variant considered benign. The patient exhibited severe infantile obesity with a body mass index greater than 33 kg/m² and PTH resistance due to the comorbidity of PHP1A and MLID including hypomethylation of the KCNQ1OT1:TSS-DMR. This study highlights the importance of screening for PHP1A and imprinting disorders with overgrowth, in cases with severe infantile obesity, and for MLID causative genes in MLID cases.

Highlights

● We report a patient with a maternal GNAS deletion and MLID.

● The patient showed severe obesity due to the comorbidity of PHP1A and MLID.

● The patient’s mother had a ZAR1 missense variant considered benign.

Introduction

Imprinted genes functioning in a parental-origin-specific manner are regulated by differentially methylated regions (DMRs) exhibiting parentally distinct 5-methylcytosine modifications for CpGs (1). The abnormally expressed imprinted genes caused by structural variants involving DMRs, single nucleotide variants in imprinted genes, uniparental disomy, and epimutation lead to imprinting disorders (IDs) (2).

The GNAS locus on chromosome 20q13.32 harbors five DMRs (GNAS-DMRs) which comprise the maternally methylated GNAS-A/B:transcription start site (TSS)-DMR, GNAS-XL:exon 1 (Ex1)-DMR, GNAS-AS1:TSS-DMR, and GNAS-AS2:TSS-DMR and the paternally methylated GNAS-NESP:TSS-DMR (3) (Supplementary Fig. 1). Dysfunction of the α subunit of the stimulatory G protein (Gsα) encoded by the GNAS leads to GNAS-related diseases (4). Genetic defects in GNAS on the maternal allele cause pseudohypoparathyroidism type 1A (PHP1A), while similar defects on the paternal allele cause pseudopseudohypoparathyroidism (PPHP). Haploinsufficiency of Gsα leads to functional defects in most tissues with biallelic Gsα expression, such as bone and white adipose tissue, resulting in Albright hereditary osteodystrophy (AHO). AHO is characterized by short stature, ectopic ossification, brachydactyly, and a round face (4). Loss of Gsα function in tissues that predominantly express the maternal allele, such as the proximal tubules and thyroid, results in hormonal resistance (4, 5). Therefore, PHP1A and PPHP are associated with AHO features (6, 7), and PHP1A exhibit resistance to PTH and, in some cases, to TSH. In addition, infantile obesity, which is often observed in PHP1A, becomes less pronounced with age (4, 8,9,10).

Beckwith-Wiedemann syndrome (BWS) is one of the IDs caused by aberrant methylation at CpGs in the imprinting region of chromosome 11p15.5 (11). Cases with BWS exhibit various clinical features, including prenatal and postnatal overgrowth, macroglossia, and body asymmetry. The most frequent molecular etiology of BWS is hypomethylation of the KCNQ1OT1:TSS-DMR. Recently, multi-locus imprinting disturbance (MLID) with methylation defects in multiple DMRs other than the disease-responsible DMRs has been reported (12, 13). MLID was observed in approximately 12% of cases with BWS caused by hypomethylation of the KCNQ1OT1:TSS-DMR (13). The subcortical maternal complex (SCMC), composed of proteins, such as NLRP2 and NLRP5, is expressed in oocytes and preimplantation embryos and functions in maintaining CpG methylation at DMRs as a maternal factor (14). Loss-of-function variants in genes encoding SCMC proteins in a mother have been shown to cause MLID in the offspring, female infertility, biparental hydatidiform mole, and recurrent miscarriage (12, 13, 15). ZAR1 is also a maternal factor essential for maintaining oocyte quality (16). Several cases of female infertility with truncating variants of ZAR1 and a single case of MLID with a missense variant in the mother have been reported (17, 18).

Here, we report a severe infantile obesity patient with PHP1A caused by a maternal deletion involving Gsα-coding region and MLID, including hypomethylation of the KCNQ1OT1:TSS-DMR. This study highlights the utility of combined genetic and methylation analyses in severe infantile obesity.

Patients and Methods

Case presentation

The girl was born as the first child to healthy and unrelated parents at 42 wk of gestation by vaginal delivery. Her mother was 30 yr old and had no history of miscarriage and infertility treatment. Fetal abnormality was not observed during the pregnancy. At birth, her length was 52.0 cm (+1.0 standard deviation, SD), her weight was 3168 g (–0.1 SD), and her newborn screening test was negative. She exhibited rapid postnatal weight gain and developed marked obesity by 10 mo of age, with a body mass index (BMI) of 33.6 kg/m² (BMI-SDS: +10.1 SD) (Figs. 1, A–C). In infancy, the patient showed an extreme appetite despite being breastfed. After the introduction of solid foods, hyperphagia subsided, and her body weight gradually normalized without medical intervention. At 2 yr and 10 mo, she had an elevated serum TSH level (11.8 μIU/mL, reference range: 0.4–4.0 μIU/mL) and a low FT4 level (0.7 ng/dL, reference range: 0.99–1.90 ng/dL) and started receiving treatment with levothyroxine. At 6 yr of age, she was referred to a hospital due to tetany. Physical and imaging examinations revealed a round face and intracranial calcifications, without evidence of brachydactyly (Fig. 1A). She had an intellectual disability with a Wechsler Intelligence Scale for Children score of 68 at six years of age. Laboratory tests demonstrated hypocalcemia with a serum calcium level of 5.6 mg/dL (reference range: 8.8–10.4 mg/dL) and a markedly elevated intact PTH level of 1330 pg/mL (reference range: 10–65 pg/mL). Based on these findings, she was clinically diagnosed with PHP1A and began treatment with alfacalcidol. She did not exhibit the typical characteristics of BWS, such as macroglossia and body asymmetry. She corresponds to patient 25 in reference 7 and was not included in reference 13. She has one unaffected sibling who shows no clinical features of PHP1A, BWS, or infantile obesity.

Fig. 1.

Clinical information of the patient. A: The top left image is a photograph of the patient aged 5 mo, and the top right image is a photograph of the patient aged 10 yr. The bottom left image is a computed tomography scan of the head, with red arrows indicating ectopic calcifications. The bottom right image is an X-ray of the patient’s left hand. y, year; m, month. B: The patient’s growth chart plotted on the growth curves of Japanese girls. Black circles indicate the patient’s height, and blue circles indicate the patient’s weight. C: The patient’s BMI plotted on the BMI curves of Japanese girls. Black circles indicate the patient’s BMI. D: The BMI chart of the patient plotted on the BMI curves of cases with BWS (pink background, reproduced from reference 22) and PHP1A (modified from reference 21). Black circles indicate the patient’s BMI, and red circles and whiskers indicate the mean and range of BMI standard deviation scores in cases with PHP1A. The green background represents the World Health Organization female reference data. SD, standard deviation; BMI, body mass index; BWS, Beckwith-Wiedemann syndrome; PHP1A, pseudohypoparathyroidism type 1A.

Her mother had a height of 159 cm, a weight of 45 kg, and normal serum calcium levels. The mother exhibited no infantile obesity or AHO features, such as clinically apparent brachydactyly, and no ectopic ossification was identified on chest-abdominal computed tomography performed for follow-up of gastric cancer, although whole-body skeletal radiography was not performed.

Molecular studies

This study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development (518) and conducted after obtaining written informed consent to publish clinical and molecular findings, including photographs.

Array-based methylation analysis using EPIC

We conducted genome-wide methylation analysis using Infinium MethylationEPIC Kit (EPIC) (Illumina) with genomic DNA from leukocytes of patient and from saliva of the mother. A sample from the patient’s sibling was not available. We obtained β values indicating the methylation levels (MLs) for 842 CpGs in 78 DMRs and defined aberrantly methylated DMRs based on a previous report (Supplementary Tables 1 and 2) (13). In brief, the median β value for each CpG within a DMR was determined as ML of the DMR. For the patient, an aberrantly hypomethylated DMR was defined as ML < –3 SD from the mean ML of leukocyte DNA from 16 healthy child controls, and an aberrantly hypermethylated DMR was defined as ML > +3 SD. For the mother, the thresholds were calculated using the mean ML of saliva DNA from three healthy controls.

Whole exome sequencing

We performed trio exome sequencing using SureSelect Human All Exon V6 (Agilent Technologies). We screened for variants of reported MLID-related genes, candidate MLID-causative genes, and associated with BWS-like phenotypes, as previously reported (19). We filtered rare variants with minor allele frequencies of ≤ 0.01 in public databases and in-house database. We also searched for other potential causative genes for genetic disorders. The pathogenicity of the identified rare variants was evaluated using combined annotation dependent depletion phred (http://cadd.gs.washington.edu/), Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), and sorting intolerant from tolerant (http://sift.jcvi.org/). We assessed pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) (20).

Results

At 10 yr, she underwent genetic analysis for PHP. No pathogenic variants were identified by targeted next-generation sequencing of the GNAS locus. We then conducted methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) analysis targeting multiple ID-related DMRs (SALSA MS-MLPA Probe-mix ME034) (MRC-Holland), which obtained the results of methylation analysis and copy number variation analysis at the same time, using genomic DNA from leukocytes of the patient and her mother (Figs. 2A and 2B). The patient showed hypermethylation of the GNAS-NESP:TSS-DMR and hypomethylation of the GNAS-A/B:TSS-DMR, GNAS-AS1:TSS-DMR, GNAS-XL:Ex1-DMR, and KCNQ1OT1:TSS-DMR, together with copy number loss of the GNAS locus. Her mother showed hypomethylation of the NESP-DMR and hypermethylation of the GNAS-A/B:TSS-DMR, GNAS-AS1:TSS-DMR, GNAS-XL:Ex1-DMR, together with copy number loss of the GNAS locus. These results were consistent with a diagnosis of PHP1A in the patient and PPHP in the mother.

Fig. 2.

Results of molecular analyses. A, B: Result of MS-MLPA analysis in the patient (A) and the mother (B). MS-MLPA shows copy number changes (upper panel) and methylation levels (lower panel). Probe ratios below 0.7 (red line) indicate a deletion, while those above 1.3 (blue line) indicate a duplication (upper panel). Probe ratios below 0.3 (red line) or above 0.3 (blue line) for the corresponding reference ranges indicate loss or gain of DNA methylation, respectively (lower panel). C: The result of array-based methylation analysis using the Infinium MethylationEPIC Kit (Illumina). Red boxes show hypermethylation in the DMRs, and blue boxes show hypomethylation in the DMRs. Yellow backgrounds are clinically associated DMRs. D: Results of Sanger sequencing. E: Results of array-based comparative genomic hybridization analysis in the patient. Black dots indicate normal copy numbers and green dots indicate low copy numbers. MS-MLPA, methylation-specific multiple ligation-dependent probe amplification. DMRs, differentially methylated region. Chr, chromosome; Pt, patient.

To evaluate comprehensive MLs of CpGs in DMRs, we conducted EPIC analysis. In addition to the KCNQ1OT1:TSS-DMR and GNAS-DMRs, the DIRAS3:Ex2-DMR, FAM50B:TSS-DMR, PLAGL1:altanation-TSS-DMR, IGF2R:intron (Int) 2-DMR, HTR5A:TSS-DMR, and FANCC:Int1-DMR showed aberrant methylation in the patient (Fig. 2C). In addition to the GNAS-DMRs, PPIEL:Ex1-DMR, DMR related to LOC151121, and DMR related to LIPI exhibited aberrant methylation in the mother’s saliva sample (Supplementary Fig. 2).

Trio exome sequencing identified a heterozygous missense variant in ZAR1 in the patient and her mother. Sanger sequencing further confirmed the variant in the patient, her mother, and her maternal grandmother. (Fig. 2D). This variant was rare and was classified as a variant of uncertain significance according to the ACMG guidelines (Table 1).

Table 1. Summary of ZAR1 variants.

We further performed array comparative genomic hybridization analysis using a catalog human array (1×1M format, catalog no. G4447A; AgilentTechnologies) on the patient, mother, and father. We identified a heterozygous deletion of approximately 1.8-Mb at 20q13.3 in both the patient and her mother (Fig. 2E and Supplementary Fig. 3). This deletion included VAPB, APCDD1L, STX16, NPEPL1, GNAS, TUBB1, ATP5F1E, NELFCD, CTSZ, PREILD3B, ZNF831, EDN3, PHACTR3, SYCP2, FAM217B, PPP1R3D, and CDH26 (Fig. 2E).

Discussion

In this study, we first reported a severe infantile obesity patient caused by the coexistence of PHP1A due to a maternal deletion involving Gsα-coding region and MLID, including hypomethylation of the KCNQ1OT1:TSS-DMR, with a ZAR1 variant detected in the mother. The BMI of our patient far exceeded the typical BMI trajectories observed in either PHP1A or BWS alone (Fig. 1D) (21, 22). This suggests a potential additive effect of the two phenotypes, contributing to the development of severe obesity. We emphasize the importance of performing GNAS abnormality screening and methylation analysis for IDs, such as BWS, which are characterized by overgrowth, in cases with severe infantile obesity.

We identified a heterozygous missense variant on ZAR1 in the patient, her mother, and her maternal grandmother. ZAR1 is a maternal-effect gene involved in early mitotic divisions of the embryo and the zygote-to-embryo transition (16). Truncating variants of ZAR1 have been reported in cases of female infertility due to premature ovarian failure (17). Additionally, a ZAR1 missense variant was reported in a mother with a history of miscarriage, and her child showed prenatal and postnatal overgrowth and mild macroglossia with MLID, including hypomethylation of the KCNQ1OT1:TSS-DMR (18). Our patient’s mother had no history of miscarriage or infertility treatment; however, in silico analysis of the ZAR1 variant revealed a rare variant with pathogenicity comparable to the previously reported case. Nevertheless, because the mother showed no methylation defects in clinically associated DMRs other than the GNAS-DMRs despite her mother (the patient’s maternal grandmother) carrying the same ZAR1 variant, the variant may be considered putatively benign. However, MLID causative genes acting as maternal-effect genes are challenging to interpret under ACMG criteria and remain incompletely understood. Putative pathogenic variants in MLID causative genes are identified in only approximately 30% of MLID cases, and the underlying cause remains unknown (13). Therefore, further accumulation of cases and additional research are necessary.

We considered that the maternal deletion involving Gsα-coding region and hypomethylation of the KCNQ1OT1:TSS-DMR were highly associated with the patient’s phenotype. Among the aberrant DMRs detected by the EPIC in our patient, only the PLAGL1:TSS-DMR, other than GNAS-DMRs and KCNQ1OT1:TSS-DMR, was a clinically associated DMR (12), although MS-MLPA analysis showed normal methylation at the DMR. Given that EPIC analysis can overestimate hypermethylation depending on the criteria (13), the PLAGL1:TSS-DMR may not be truly abnormal in our patient. The impact of MLID on phenotypes has not been fully elucidated (12, 13), so the effects of abnormal DMRs in the patient other than the KCNQ1OT1:TSS-DMR on her phenotype are unknown.

The patient carried a maternally inherited 1.8-Mb deletion of chromosome 20. The methylation defect patterns in the GNAS-DMRs suggest that the patient’s mother had same deletion on the paternal allele. Despite having the deletion, the mother showed no AHO features. Previous reports have indicated that 70–80% of cases with PPHP have brachydactyly, and 18–100% have ectopic ossification (10). Because whole-body skeletal radiography was unavailable for the mother, it remains unclear whether AHO features are truly absent. Of the genes included in the deleted region, VAPB has been suggested to be associated with amyotrophic lateral sclerosis (23), and EDN3 with Hirschsprung’s disease (24). Given that the patient and mother had no shared phenotype, it is unlikely that the haploinsufficiency of genes containing the deleted region other than the GNAS locus may have influence on their phenotypes.

Conclusion

We identified the patient with PHP1A caused by the maternal deletion involving Gsα-coding region, accompanied by MLID, including hypomethylation of the KCNQ1OT1:TSS-DMR. We concluded that the comorbidity of PHP1A and MLID led to severe infantile obesity in the patient. This study highlights the importance of screening for genomic abnormalities in GNAS and performing methylation analysis for IDs in cases with severe infantile obesity.

Conflict of interests

The authors have no conflicts of interest to declare.