Complex Genomic Rearrangement Within the GNAS ...

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Complex Genomic Rearrangement Within the GNAS Region Associated With Familial Pseudohypoparathyroidism Type 1b Akie Nakamura,* Erika Hamaguchi,* Reiko Horikawa, Yasuyuki Nishimura, Keiko Matsubara, Shinichiro Sano, Keisuke Nagasaki, Yoichi Matsubara, Akihiro Umezawa, Toshihiro Tajima, Tsutomu Ogata, Masayo Kagami,† Kohji Okamura,† and Maki Fukami† Departments of Molecular Endocrinology (A.N., K.M., S.S., M.K., M.F.) and Systems BioMedicine (K.O.) and Institute (Y.M.), National Research Institute for Child Health and Development, Tokyo 157-8535, Japan; Division of Endocrinology and Metabolism (R.H.), National Medical Center for Children and Mothers, Tokyo 157-8535, Japan; Department of Internal Medicine (E.H., Y.N.), Japanese Red Cross Kanazawa Hospital, Kanazawa 921– 8162, Japan; Division of Pediatrics, Department of Homeostatic Regulation and Development (K.N.), Niigata University Graduate School of Medical and Dental Sciences, Niigata 951– 8510, Japan; Department of Reproductive Biology (A.U.), Center for Regenerative Medicine, National Institute for Child Health and Development, Tokyo 157-8535, Japan; Department of Pediatrics (T.T.), Jichi Children’s Medical Center Tochigi, Shimotsuke 329 – 0498, Japan; and Department of Pediatrics (T.O.), Hamamatsu University School of Medicine, Hamamatsu 431–3192, Japan

Context: Pseudohypoparathyroidism type 1b (PHP-1b) results from methylation defects at the G protein stimulatory ␣ subunit (GNAS) exon A/B-differentially methylated region (DMR). Although microduplications in the GNAS region were recently identified in two PHP-1b patients, genetic information on these patients remained fragmentary. Case Description: A 20-year-old Japanese male and his mother presented with hypocalcemia and elevated blood levels of intact PTH. The proband had a maternal uncle who was previously diagnosed with PHP-1b. Methylation-specific multiplex ligation-dependent probe amplification, arraybased comparative genomic hybridization, pyrosequencing, fluorescence in situ hybridization, and whole-genome sequencing were performed for this family. The proband, mother, and uncle carried maternally derived approximately 133-kb duplication-triplication-duplication rearrangements at 20q13.32 involving NESP55, NESPAS, XL␣s, and exon A/B-DMR but not STX16 or the Gs␣ coding region. These individuals exhibited partial methylation defects of NESP55-, NESPAS-, and XL␣s-DMRs, which were ascribable to the increased copy numbers of these regions retaining the maternally derived methylation pattern and loss of methylation of exon A/B-DMR, which was inexplicable by the copy-number alterations. Fusion junctions of the rearrangement resided within non-repeat sequences and were accompanied by short-templated insertions. Conclusions: Our results indicate that maternally derived copy-number gains in the GNAS region mediated by nonhomologous end-joining and/or by break-induced replication can underlie autosomal dominant PHP-1b. These rearrangements likely affect methylation of exon A/B-DMR by disconnecting or disrupting its cis-acting regulator(s). This study provides a novel example of human disorders resulting from functional disturbance in the cis-regulatory machinery of DNA methylation. (J Clin Endocrinol Metab 101: 2623–2627, 2016) ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2016 by the Endocrine Society Received March 24, 2016. Accepted May 26, 2016. First Published Online June 2, 2016

doi: 10.1210/jc.2016-1725

* A.N. and E.H. contributed equally to this work as co-first authors. † M.K., K.O., and M.F. contributed equally to this work. Abbreviations: AHO, Albright’s hereditary osteodystrophy; BIR, break-induced replication; DMR, differentially methylated region; GOM, gain-of-methylation; GNAS, G protein stimulatory alpha subunit; LOM, loss of methylation; NHEJ, non-homologous end-joining; PHP, pseudohypoparathyroidism; PHP-1a, PHP type 1a; PHP-1b, PHP type 1b; STX16, syntaxin-16.

J Clin Endocrinol Metab, July 2016, 101(7):2623–2627

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seudohypoparathyroidism (PHP) is a rare endocrine disorder characterized by PTH resistance in the renal proximal tubules (1). Clinically, PHP is subdivided into PHP type 1a (PHP-1a) (OMIM no. 103580) with Albright’s hereditary osteodystrophy (AHO) and type 1b (PHP-1b) (OMIM no. 60323) without AHO. PHP-1b is caused by loss of methylation (LOM) at the G protein stimulatory ␣ subunit (GNAS) exon A/B-differentially methylated region (DMR) and includes sporadic and autosomal dominant forms. Sporadic PHP-1b is primarily associated with etiology-unknown broad methylation defects at NESP55-, NESPAS-, XL␣s-, and exon A/B-DMR (2). Autosomal dominant PHP-1b usually presents with isolated LOM of exon A/B-DMR due to microdeletions in syntaxin-16 (STX16) (3, 4); microdeletions involving

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NESP55 and/or NESPAS account for a small percentage of cases (1, 5). In 2015, copy-number gains at 20q13.32 were identified in two patients with PHP-1b (6). Case 1 carried a duplication involving NESP55, NESPAS, XL␣s, exon A/B, and GNAS exons 1–13, whereas case 2 had a duplication involving NESP55, NESPAS, and XL␣s. These results indicate that duplications in the GNAS region can underlie PHP-1b. However, these findings were based solely on the results of methylation-specific multiplex ligation-dependent probe amplification and array-based comparative genomic hybridization; therefore, the precise genomic structure and DNA methylation status have yet to be determined. Furthermore, the underlying mechanism of these rearrangements remains unknown.

Figure 1. Genomic rearrangements and methylation statuses of this family. A, Array-based comparative genomic hybridization. The black and red dots denote normal (log ratio from ⫹0.5 to ⫺1.0) and increased (log ratio ⭌ ⫹0.5) copy numbers, respectively. The orange and blue arrows indicate the genomic regions present in four and three copies, respectively. B, Pyrosequencing. The methylation index (the ratio of methylated clones) of each CpG site in the GNAS-DMRs is shown. The gray shaded areas represent the reference ranges obtained from 50 unaffected individuals. Methylation indexes of previously reported patients with maternally or paternally derived 850-kb deletions encompassing the entire GNAS region (8) are also indicated. C, Schematic representation of the genomic structure in the GNAS region. Genomic positions refer to the Human Genome (Build 37). Fragments A and C were duplicated, and fragment B was triplicated in our family. D, Genomic rearrangement in this family. Fragments A, B, and C were tandemly aligned in a head-to-tail position. Both of the two fusion junctions are accompanied by short nucleotide insertions. The fusion junction of fragments B and A shares no microhomology and is accompanied by an 11-bp insertion homologous to the nearby sequences (underlined). The junction of fragments C and B has taken place between sequences with 6-bp and 2-bp microhomologies and is associated with a 21-bp insertion homologous to a genomic region on the reverse strand approximately 470-bp centromeric to the breakpoint.


doi: 10.1210/jc.2016-1725

Subjects and Methods The proband was a 20-year-old Japanese male without AHO. Detailed data are provided in the Supplemental Data. At age 14, the proband experienced generalized convulsion and was diagnosed with epilepsy. He decided not to receive anticonvulsants. Electrolyte levels were not examined. At age 18, he had a second episode of convulsion. Blood examinations revealed hypocalcemia, hyperphosphatemia, and elevated intact PTH (Supplemental Table 1). The mother was asymptomatic and had no AHO features; however, laboratory examinations revealed mild hypocalcemia and elevated intact PTH (Supplemental Figure 1). A maternal uncle was previously diagnosed with PHP-1b based on laboratory findings. Thus, the proband was clinically diagnosed with autosomal dominant PHP-1b. The maternal grandmother was phenotypically normal and showed normal laboratory data. Detailed methods are available in the Supplemental Data. We performed direct sequencing of the Gs␣ coding region and copynumber analyses of the GNAS region. DNA methylation statuses of GNAS-DMRs were assessed by methylation-specific multiplex ligation-dependent probe amplification and pyrosequencing. The rearrangement of the proband was characterized by fluorescence in situ hybridization and whole-genome sequencing. The junction sequences of the mother, uncle, and grandmother were determined by Sanger sequencing.

Results No pathogenic mutations were detected in the Gs␣ coding region. Copy-number analyses identified two additional


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copies of an approximately 37-kb region involving NESP55, NESPAS exons 1– 4, and XL␣s and one additional copy each of an approximately 41-kb region involving NESPAS exon 5 and an approximately 18-kb region involving exon A/B in the proband, mother, uncle, and grandmother (Figure 1A and Supplemental Figure 1). Methylation analyses revealed that exon A/B-DMR was virtually unmethylated in the proband and uncle and mildly hypomethylated in the mother (Figure 1B and Supplemental Figure 2). In addition, partial LOM of NESP55DMR and gain of methylation (GOM) of NESPAS- and XL␣s-DMRs were similarly observed in these three individuals. In contrast, the grandmother showed partial GOM of NESP55-DMR, LOM of NESPAS- and XL␣sDMRs, and high-normal methylation of exon A/B-DMR. Fluorescence in situ hybridization suggested that the amplified DNA fragments were inserted into 20q13.32 (Supplemental Figure 3). Whole-genome sequencing successfully determined the structure of the rearrangement (Figure 1, C and D). Three DNA fragments, namely, an approximately 41-kb fragment containing NESPAS exon 5 (fragment A in Figure 1C); an approximately 37-kb fragment containing NESP55, NESPAS exons 1– 4, and XL␣s (fragment B); and an approximately 18-kb fragment containing exon A/B (fragment C), were amplified and in-

Figure 2. Genomic structures and methylation profiles of GNAS alleles in control subjects (top panel), the present patients (middle panel), and the previously described patients (bottom panel; Ref. 6). Filled, open, and gray shaded circles indicate methylated, unmethylated, and partially methylated DMRs, respectively. Dashed squares in the previously reported patients depict the region of copy-number gains of an unknown genomic position.


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serted into a region adjacent to the original locus. Fragments A, B, and C were tandemly aligned in a head-to-tail position. All breakpoints resided within nonrepetitive sequences. The junction between fragments B and A comprised an insertion of 11 nucleotides that were homologous to the nearby sequences. Sequences at the fusion junction shared no microhomology. The fusion junction between fragments C and B comprised a 21-bp insertion whose template was found on the reverse strand at a position approximately 470-bp centromeric to the breakpoint. The junction sequences shared microhomologies. The same breakpoint structures were shared by all family members.

Discussion This is the first report of complex copy-number gains within the GNAS region associated with autosomal dominant PHP-1b. The proband, his mother, and a maternal uncle carried identical duplication-triplication-duplication rearrangements on the maternally inherited chromosome 20 and exhibited aberrant methylation of GNAS-DMRs. Although mild LOM of NESP55-DMR and mild GOM of NESPAS- and XL␣s-DMRs are ascribable to the increased copy numbers of these regions retaining the maternally derived methylation pattern, the LOM of exon A/B-DMR is inexplicable by the copy-number alterations. Previous studies suggested that maternally inherited deletions containing STX16 or NESP55 induce isolated LOM of exon A/B-DMR (1, 3–5). The overlapping region of the deletions around STX16 exon 4 and NESP55 appear to be critical for the establishment or maintenance of the maternally derived methylation pattern of exon A/B-DMR (1). Although the rearrangement of our cases did not affect genomic structures around STX16, it disconnected STX16 from exon A/B-DMR (Figure 2). Furthermore, this chromosomal alteration may disrupt the putative cis-acting regulator of exon A/B-DMR in the upstream region of NESP55 (5). Thus, the rearrangement would impair the cis-regulatory machinery of the GNAS region. The contrastive methylation pattern of the grandmother indicates the paternal origin of the rearrangement, although the high-normal methylation of exon A/B-DMR is not consistent with paternal transmission (Figure 2). It is possible that the rearrangement on the paternally derived allele could also affect the methylation status of exon A/B-DMR to some extent. Notably, we observed a difference in the methylation status of exon A/B-DMR between the proband and his mother. The degree of LOM was correlated with the severity of hypocalcemia. Because we did not identify additional polymorphisms in the Gs␣ coding re-


gion, the underlying mechanism of this variation remains unknown. LOM at exon A/B-DMR was previously reported in a PHP-1b case with an approximately 180-kb duplication at 20q13.32 (case 2 in Ref. 6). Although the genomic structure of this case was not determined, the duplication likely dissociated STX16 from exon A/B-DMR as in our case (Figure 2). In contrast, a patient with an approximately 320-kb duplication involving the Gs␣ coding region (case 1 of Ref. 6) exhibited broad methylation defects similar to those in sporadic PHP-1b, which are inexplicable by the disconnection of STX16 from exon A/B-DMR (Figure 2). The rearrangement of our family consisted of insertion of three DNA fragments. Because all breakpoints resided within non-repeat sequences, nonallelic homologous recombination is unlikely to underlie this rearrangement. The duplication-triplication-duplication structure is indicative of break-induced replication (BIR), which originates from collapsed replication forks (7). Furthermore, we found microhomologies at the breakpoints, which are often seen in microhomology-mediated BIR (7). However, short-templated insertions at the fusion junctions are consistent with double-strand breakage leading to nonhomologous end-joining (NHEJ) (7). Collectively, the rearrangement seems to be mediated by BIR and/or NHEJ. In conclusion, our results indicate that copy-number gains within the GNAS region mediated by BIR and/or NHEJ can lead to autosomal dominant PHP-1b by disconnecting or disrupting a cis-acting regulator(s) of exon A/B-DMR. This study provides a novel example of human disorders resulting from functional disturbance in the cisregulatory machinery of DNA methylation.

Acknowledgments We thank Dr Masayuki Yamada for his assistance in the collection of clinical data. Address all correspondence and requests for reprints to: Maki Fukami, Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2–10-1 Okura, Setagaya, Tokyo 157-8535, Japan. E-mail: [email protected]. The work was supported by grants from the Japan Agency for Medical Research and Development (16ek0109049h0003, 16ek0109030h0003, and 16ek0109141h0002), the National Center for Child Health and Development (26 –11 and 25–10), and the Takeda Foundation and by Grants-in-Aid for Young Scientists (B) (26860715) and for Challenging Exploratory Research (15K15096) from the Japan Society for the Promotion of Science. Disclosure Summary: The authors have nothing to disclose.


doi: 10.1210/jc.2016-1725


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