A novel nonsense mutation c.424G>T (p. G142X) in the first exon of XLas leading to osteopetrosis

GNAS is one of the most complex gene loci in the human genome and encodes multiple gene products. XLas, the extra-large isoform of alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gas), is paternally inherited. Although XLas can mimic the action of Gas, its significance remains largely unknown in humans. Here we report a patient presented with increased bone mass, hypophosphatemia, and elevated parathyroid hormone levels. His serum calcium was in the lower limit of normal range. DEXA scan revealed progressive increase in the bone density of this patient. Whole exome sequencing of this subject found a novel nonsense mutation c.424G>T (p. G142X) in the first exon of XLas, which was inherited from his father and transmitted to his daughter. This mutation was predicted to exclusively influence the expression of XLas, while may have no significant effects on other gene products of this locus. SaOS2 cells transfected with mutant XLas failed to generate cAMP under parathyroid hormone stimulation, indicating skeletal resistance to this hormone. This subject showed higher circulating SOST, DKK1 and OPG levels, while lower RANKL levels and RANKL/OPG ratio, leading to reduced bone resorption. It is speculated that this patient may belong to a very rare type of pseudohypoparathyroidism with selective skeletal resistance but normal renal tubular response to parathyroid hormone. Our findings indicate that XLas plays a critical role in bone metabolism and GNAS locus should be considered as a candidate gene for high bone mass. Author summary GNAS has been regarded as one of the most complex gene loci and encodes multiple transcripts, including Gsα, XLαs, NESP55 and A/B transcripts. These isoforms share the same 2-13 exons with alternative first exons. Previously reported mutations often disrupt multiple protein-coding transcripts in addition to that encoding Gsα, making it difficult to distinguish the contributions of each transcript to disease phenotypes. Here we first report a novel nonsense mutation c.424G>T (p. G142X) in the first exon of XLas in a subject presenting with high bone mass, unclosed cranial suture, and persistent hypophosphatemia, and elevated parathyroid hormone (PTH) levels. This is the first report of a mutation located in the first exon of XLas in humans, which was predicted to exclusively influence the expression of XLas, while may have no significant effects on other gene products of this locus. SaOS2 cells transfected with mutant XLas failed to generate cAMP under PTH stimulation, indicating skeletal resistance to this hormone. Our study suggests that XLas has an important physiological role in humans, and is involved in skeletal PTH/cAMP pathway. Our findings also indicate GNAS locus should be considered as a candidate gene for high bone mass. Funding The National Natural Science Foundation of China. Declaration of Interests The authors declare no competing interests.


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GNAS has been regarded as one of the most complex gene loci in the human genome 71 and undergoes tissue-specific imprinting [1]. Epigenetic event contributes to tissue 72 specific imprinting of Gsα, which leads to phenotypic variability in GNAS mutations.  The GNAS complex locus encodes multiple transcripts, including Gsα, XLαs, 79 NESP55 and A/B transcripts [3]. These isoforms share the common exons 2-13 with increased. He had normal thyroid stimulating hormone (TSH) and free T4 (FT4) levels.

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His mean bone mineral density, T and Z scores of lumbar vertebrae, L1-L4, were 1.936 105 (gm/cm 2 ), 7.1 and 7.6, respectively. His mean bone mineral density, T and Z scores of 106 femoral neck were 1.406 (gm/cm 2 ), 3.3 and 3.8, respectively (table 1). This patient had 107 no history of fluorosis and hepatitis C infection.

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Circulating SOST, DKK1, receptor activator of RANKL and OPG levels 109 Plasma levels of SOST, RANKL and OPG were detected using kits from abcam 110 (Cambridge, UK). Serum DKK1 levels were also measured using ELISA methods 111 (R&D Systems, Inc., Minneapolis, USA). Twenty gender-and age-matched healthy 112 adult males were selected as controls.  Osteoclast culture 118 Osteoclasts from human peripheral blood were cultured as previously described 10 .

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PBMCs were isolated using Ficoll-Hypaque Solution. Cells were washed in PBS twice, 120 and plated on 24-well plates at a density of 1×106/well at 37°C in α-MEM,       transfected SaOS2 cells (Fig. 4). The mRNA expression of WT and mutant XLas was 210 significantly higher in transfected cells than in cells transfected with empty vector 211 (Fig.3H). lower cAMP production as that in SaOS2 cells transfected with empty vector (Fig.3I).

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This is the first report of a mutation located in the first exon of XLas in humans, which PHP1b [35]. Gsa in skeleton is biallelically expressed. Therefore, no matter the origin 285 of mutation is, GNAS mutation may lead to decreased skeletal response to PTH [36, formation, leading to increased bone mass ( Figure 5). Therefore, we believe this patient 305 belongs to a type of HBM with low bone turnover, primarily resulting from impaired 306 bone resorption. Our study also suggests that exogenous RANKL may alleviate the 20 307 progressive increase in bone mass in this patient.

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In the classic form of PHP, the PTH resistance is confined to the renal proximal 309 tubule, leading to hypocalcemia, hyperphosphatemia, and elevated levels of PTH levels 310 [39]. In fact, PTH resistance can occur either in the renal tubular level or the skeleton 311 [39]. Given that the proband showed elevated serum PTH concurrently with increased 312 phosphate excretion, it appears that PTH resistance is not present in the proximal tubule.

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In addition, urinary calcium excretion was extremely low, consistent with elevated PTH 314 and a lack of resistance in distal nephron. In renal proximal tubules, Gsα is expressed 315 only from the maternal allele, which may also apply to XLas based on patient-specific 316 clinical data. Therefore, in this proband, the renal tubular response to PTH may be 317 normal. We speculated the paternally inherited mutation located in XLas impaired 318 skeletal response to PTH, leading to reduced serum calcium and elevated PTH level.

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The persistently elevated PTH caused hypophosphatemia. As a compensation, his 320 serum calcium levels were maintained at the lower limit of the normal range.

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It is notable that the patient showed unclosed coronal suture and sagittal suture.

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Unclosed cranial suture has also been reported in patients carrying mutations in 323 runt-related transcription factor 2 (Runx2), which is known to affect metopic suture Runx2 expression ( Figure 5).

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His father also carried this mutation and the origin of mutant allele was unknown.

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It seemed that his father manifested a similar but milder phenotype. His daughter also 336 carried the same mutation and presently showed no similar synptoms, possibly due to 337 her young age or incomplete penetrance. It is suggested that XLas may be essential for kg and was well after birth. This family will be regularly followed up.

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In conclusion, we first report a novel mutation located in the first exon of XLas in