Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets
Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets
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DOI：
10.1016/j.bone.2019.05.017
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发表时间：
2019-08-01
期刊：
影响因子：
4.1
通讯作者：
Shi, Yufei
中科院分区：
文献类型：
--
作者：
BinEssa, Huda A.;Zou, Minjing;Shi, Yufei
x连锁低磷血症佝偻病（XLH）是由PHEX基因失活突变引起的，是遗传性佝偻病最常见的形式。剪接位点突变占所有报道的PHEX突变的17%。这些剪接位点突变的功能后果尚未得到系统的研究。目的：目前的研究是对先前报道的PHEX基因的22个剪接位点突变进行功能注释。方法：采用位点定向诱变方法制备不同剪接位点突变的PHEX小基因，并在HEK293细胞中表达。通过RT-PCR、克隆和测序分析mRNA转录本。结果：这些剪接突变导致多种后果，包括外显子跳跃、内含子保留和隐剪接位点的激活。在22个剪接位点突变中，外显子跳变是最常见的事件，占73%（16/22）。非规范剪接位点突变可导致剪接错误，其程度与规范剪接位点突变相同，如C .436+3G > C、C .436+ 4A > C、C .436+ 6T > C、C .437- 3c > G、C .850- 3c > G、C .1080- 3c > A、C .1482 +5G > C、C .1586 + 6T > C、C .1645 +5G > A、C .1645 + 6T > C、C .1701- 16t > A、C .1768 +5G > A和C .1899 +5G > A。非典型（C .436 + 6T > C和C .1586 + 6T > C）和典型剪接位点突变（C .1769- 1g > C）可产生部分剪接错误（检测到野生型和突变型转录本），导致PHEX基因不完全失活，这可能解释了先前报道的轻度疾病表型，为基因型-表型相关提供了证据。c.1645C > T （p.R549*）虽然位于典型剪接供体位点gt附近，但对pre-mRNA剪接没有影响。结论：剪接位点突变导致外显子跳变是最常见的结果。典型和非典型剪接位点突变都可能导致严重或轻微的RNA剪接缺陷，从而导致表型异质性。非典型剪接位点突变在遗传筛选中不可忽视，尤其是那些位于典型剪接位点50 bp以内的突变。
Context X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene and is the most common form of hereditary rickets. The splice-site mutations account for 17% of all reported PHEX mutations. The functional consequence of these splice-site mutations has not been systemically investigated. Objective: The current study was undertaken to functionally annotate previously reported 22 splice-site mutations in the PHEX gene.Methods: PHEX mini-genes with different splice-site mutations were created by site-directed mutagenesis and expressed in HEK293 cells. The mRNA transcripts were analyzed by RT-PCR, cloning, and sequencing.Results: These splicing mutations led to a variety of consequences, including exon skipping, intron retention, and activation of cryptic splice sites. Among 22 splice-site mutations, exon skipping was the most common event accounting for 73% (16/22). Non-canonical splice-site mutations could result in splicing errors to the same extent as canonical splice-site mutations such as c.436+3G > C, c.436 + 4A > C, c.436 + 6T > C, c.437-3C > G, c.850-3C > G, c.1080-3C > A, c.1482 + 5G > C, c.1586 + 6T > C, c.1645 +5G > A, c.1645 + 6T > C, c.1701-16T > A, c.1768 + 5G > A, and c.1899 + 5G > A. Interestingly, non-canonical (c.436 + 6T > C and c.1586 + 6T > C) and canonical splice-site mutations (c.1769-1G > C) could generate partial splicing errors (both wild-type and mutant transcripts were detected), resulting in incomplete inactivation of PHEX gene, which may explain the mild disease phenotype reported previously, providing evidence of genotype-phenotype correlation. c.1645C > T (p.R549*) had no impact on pre-mRNA splicing although it is located next to canonical splice donor site GT.Conclusions: Exon skipping is the most common outcome due to splice-site mutations. Both canonical and non canonical splice-site mutations can result in either severe or mild RNA splicing defects, contributing to phenotype heterogeneity. Non-canonical splice-site mutations should not be overlooked in genetic screening especially those located within 50 bp from canonical splice site.