Pseudohypoparathyroidism type Ib (PHP-1b) is a rare imprinting disorder characterized by renal parathyroid hormone resistance, but without the physical features of Albright hereditary osteodystrophy. A common heterozygous 3-kb deletion of STX16 was previously reported in multiple unrelated probands. This deletion causes isolated loss of methylation at GNAS exons A/B. We aimed to identify STX16 deletions and the exact breakpoints to gain insights into the mechanism of STX16 deletion.
Methods
We investigated 10 patients who were diagnosed with pseudohypoparathyroidism type Ib, but did not have any pathogenic variants in GNAS. A methylation-specific multiplex ligation-dependent probe amplification assay of STX16 was performed to assess allelic dosage. Junction PCR and Sanger sequencing were performed around the presumptive breakpoint area to determine the exact breakpoint.
Results
STX16 gene deletion was detected in two of the 10 probands (20%). The deletion range was the same for both probands, ranging from g.57,243,566 to g.57,246,545 (2,979 bp), according to the February 2009 human reference sequence (hg19, build37), which is consistent with previous reports. The 5’ and 3’ break points were located in the mammalian-wide interspersed repeats (MIRs) sequences suggesting MIR-mediated non-allelic homologous recombination as the putative mechanism for the common STX16 3-kb deletion.
Conclusions
Non-allelic homologous recombination is a putative mechanism of STX16 deletion in our patients. To the best of our knowledge, this is the first study to show that MIRs are involved in genomic rearrangements.
배경
가성부갑상선기능저하증 1b형(PHP-1b)은 드문 각인 장애로, 신장에서 부갑상선 호르몬 저항성을 특징으로 하지만, Albright 유전성 골이영양증의 신체적 특징은 나타나지 않는다. 이전에 보고된 바에 따르면, 여러 비관련 환자들에서 공통적으로 나타나는 이형접합성 3kb STX16 결실이 있다. 이 결실은 GNAS 유전자 A/B 엑손에서 메틸화 문제를 유발한다. 저자들은 STX16 결실과 정확한 절단점을 확인하여 STX16 결실의 메커니즘을 규명하고자 하였다.
방법
저자들은 PHP-1b로 진단받았으나 GNAS 유전자에 병원성 서열 변이가 없는 10명의 환자를 대상으로 연구하였다. STX16의 복제수를 평가하기 위해 메틸화 특이적 복합결찰 의존 탐침 증폭법(methylation-specific multiplex ligation-dependent probe amplification, MS-MLPA) 분석을 수행하였다. 절단점 부근에서의 PCR과 생거 시퀀싱을 통해 정확한 절단점을 결정했다.
결과
10명의 환자 중 2명(20%)에서 STX16 유전자 결실이 확인되었다. 두 환자의 결실 범위는 g.57,243,566에서 g.57,246,545까지로 동일했으며, 이는 이전 보고와 일치하는 2,979 bp에 해당한다(2009년 2월 인간 참조 서열 hg19, build37 기준).
결론
5′ 및 3′ 절단점은 mammalian-wide interspersed repeats (MIRs) 내에 위치해 있으며, 이는 STX16의 일반적인 3-kb 결실에 대한 가설적 메커니즘으로서 MIRs 매개 비대립동종재조합(nonallelic homologous recombination, NAHR)을 시사한다. 저자들이 아는 한, 이 연구는 MIR가 유전체 재배열에 관여한다는 것을 처음으로 보여준 연구이다.
PseudohypoparathyroidismGene rearrangementDNA copy number variationsSTX16INTRODUCTION
Disorders of GNAS inactivation include pseudohypoparathyroidism types Ia, Ib, and Ic (PHP-Ia, -Ib, and -Ic), pseudopseudohypoparathyroidism (PPHP), progressive osseous heteroplasia (POH), and osteoma cutis (OC). PHP-Ia and PHP-Ic feature hormone resistance and characteristics of Albright hereditary osteodystrophy (AHO), such as short stature and subcutaneous ossification. PHP-Ib is primarily involved in parathyroid hormone resistance. PPHP manifests as AHO without hormone resistance. POH involves progressive bone formation in the muscles and fascia, whereas OC involves ossification of the dermis and subcutaneous tissues. Diagnosis involves identifying the clinical features and genetic or epigenetic alterations affecting the GNAS locus. GNAS inactivation can be caused by pathogenic variants, imprinting issues, epimutations, or paternal 20q disomy. PPHP and POH/OC are caused by pathogenic variants of the paternal GNAS allele [1].
PHP-1b is a rare imprinting disorder characterized by renal parathyroid hormone (PTH) resistance, but the absence of physical features of AHO [2]. This distinguishes PHP-Ib from PHP-Ia, which results from mutations in the GNAS gene encoding the G protein α subunit. The autosomal-dominant form of PHP-Ib (ADPHP-Ib) is linked to chromosome 20q13.3, where the GNAS locus is located. Notably, loss of methylation of exon the A/B differentially methylated region (DMR) of GNAS has been observed in both sporadic PHP-Ib cases and affected members of AD-PHP-Ib families. Recent findings indicate that affected individuals from 12 unrelated AD-PHP-Ib families and four patients with sporadic PHP-Ib, but not healthy controls, are heterozygous for an approximately 3 kb microdeletion approximately 220 kb upstream of GNAS exon A/B. This deleted region includes three exons of the STX16 gene, which does not show any imprinting. Microdeletion leads to loss of methylation at exons A/B, without affecting other GNAS DMRs, suggesting that it disrupts the cis-acting element necessary for exon A/B methylation, causing renal PTH resistance in AD-PHP-Ib [3].
However, there has been no research on the causes of recurrent 3-kb microdeletion. Therefore, we aimed to elucidate the putative mechanism through genomic characterization of the 3-kb microdeletion.
MATERIALS AND METHODS1. Informed consent and study enrollment
Ten unrelated patients with PHP who visited Seoul National University Hospital between March 2009 and January 2019 were enrolled in the Korean Genetic Diagnosis Program for Rare Diseases Phase II [4]. This study was conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of the Seoul National University Hospital. Informed consent was obtained from all enrolled participants.
To investigate the genetic and epigenetic changes in the samples, the MS-MLPA technique was employed using the SALSA MSMLPA probemix ME031-B2 GNAS kit, following the manufacturer’ s instructions (MRC-Holland, Amsterdam, Netherlands). Genomic DNA (200 ng) was denatured at 98°C for 5 minutes and hybridized with the ME031-B2 probe mix for 16 hours at 60°C. After hybridization, the product was divided into two tubes for copy number and methylation analyses using a methylation-sensitive endonuclease. The PCR products were analyzed on an ABI 3130xl capillary sequencer (Applied Biosystems, Waltham, MA, USA), and the data were processed using GeneMarker v.1.51 software (SoftGenetics, State College, PA, USA). Peak intensities were normalized to internal control probes, and the intensity ratios of identical probes from the samples were compared with those of the controls.
3. Junction PCR and Sanger sequencing around the breakpoint areas
PCR primers were designed based on the consensus sequences from the split-read junctions. The amplified products were sequenced using an ABI PRISM 3730 xl DNA Analyzer (Applied Biosystems) with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems). Sequence analysis was conducted using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA).
4. Mapping of repetitive elements around the breakpoint areas
Repetitive elements within the breakpoint regions were identified using RepeatMasker in the UCSC Genome Browser (https://genome.ucsc.edu/). Sequence similarities were assessed using ClustalW (https://www.genome.jp/tools-bin/clustalw).
RESULTS1. Mutation analysis
Table 1 summarizes the 10 cases, their classifications, and the genetic testing results. Table 1 comprises data for individuals classified as having PHP-1a and PHP-1b. Genetic testing revealed various abnormalities, such as paternal 20q disomy, STX16 2,979bp deletion, and mutations in the GNAS gene. Some patients exhibited no detectable genetic abnormalities.
A 2,979 bp deletion of STX16 was detected in two of the 10 probands (20%). Heterozygous deletions in exons 5 and 6 of the STX16 gene were initially identified using MLPA. The deletion of exon 7, which was not detected using MLPA, was suspected based on the results of an in-house copy number variation screening method [5]. Subsequently, junction PCR was performed. Finally, we confirmed a 2,979 bp deletion (chr20:57,243,566–57,246,545) encompassing exons 5–7 (Fig. 1).
2. Junction sequence analyses
When analyzing similarity using ClustalW within 1-kb upstream and downstream of the breakpoints, a 319 bp homologous region was detected, which showed only 2 bp mismatches, as depicted in Fig. 2 (bottom). This region contains one of the mammalian-wide interspersed repeats (MIRs), MIRb, which spans 191 bp. A 1-bp difference was observed between the upstream and downstream MIRb sequences. The upstream homologous sequence was located within intron 4 of STX16, whereas the downstream homologous sequence was located within intron 7.
DISCUSSION
STX16 gene deletion was detected in two of the 10 probands (20%). This deletion range is consistent with previous reports [6-10]. Most genomic rearrangements occur through mechanisms such as non-allelic homologous recombination (NAHR), nonhomologous end joining, fork stalling, and template switching [11]. However, the presence of homologous sequences in proximity to breakpoints favors rearrangements mediated by NAHR [12]. While NAHR mediated by Alu elements is well documented [13, 14], to the best of our knowledge, this is the first cases that NAHR via MIRs is a putative mechanism of rearrangement.
Short interspersed elements are widespread in mammalian genomes. The significant diversity of these repeats across placental orders suggests independent amplification of each lineage following mammalian radiation. Here, we introduce an ancient family of repeats, termed MIRs, whose sequence variation and widespread presence among placental mammals, marsupials, and monotremes indicate amplification during the Mesozoic era. With approximately 120,000 copies still identifiable in the human genome (0.2–0.3% of DNA), MIRs serve as a “fossilized” record of a substantial genetic event preceding the radiation of placental orders [15].
However, the evolutionary role of repeated element sequences in genomes remains debatable. A recent study suggested that an expanded repertoire of repeat elements may enhance organismal fitness by promoting somatic diversity, akin to the beneficial genetic diversity observed in microbial populations. Specifically, the characterization of somatic recombination involving Alu and L1 in this study provides a foundation for future investigations into the dynamics of somatic NAHR events and their implications for genome structure and function [16]. We believe that our study will contribute to broadening our understanding of the mechanisms underlying genomic rearrangements.
In conclusion, the findings of this study suggest that the common 2,979 deletion of STX16 is mediated by MIR-mediated nonallelic homologous recombination.
Conflicts of Interest
None declared.
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Detection of the 2,979 bp deletion of STX16. Heterozygous deletion of exons 5 and 6 of the STX16 gene was initially identified using multiplex ligation-dependent probe amplification (MLPA). The deletion of exon 7, which was not detected using MLPA, was suspected base on the results of an in-house method. Junction PCR was subsequently performed. Finally, we confirmed the 2,979 bp deletion encompassing exon 5 to exon 7.
Junction analysis of the 2,979 bp deletion of STX16. The distribution of repetitive elements around the 2,979 deletion of STX16 is shown. Two MIRbs are indicated by green squares. Below, the 319 bp homology sequence is displayed. The green text corresponds to MIRb, and the blue text represents the remaining sequences. The upstream homologous sequences and the downstream homologous sequences were completely identical except for 2 bp.
Abbreviation: MIRb, mammalian-wide interspersed repeat b.