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Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia

Glazov et al. (2011)

PLoS Genetics 7(3):e1002027

When two siblings presented with a novel skeletal disorder closely resembling inherited anauxetic dysplasia but lacking the expected causative mutations in the RNase mitochondrial RNA processing (RMRP) gene, exome sequencing of the immediate family was able to illuminate disease etiology. The affected siblings bore compound heterozygous mutations in POP1 (processing of precursor 1 RNA), encoding a core component of the RNase MRP complex, which resulted in impaired complex activity and reduced cell proliferation. POP1 interacts with the structural RNA subunit of RNase MRP complex, which is mutated in anauxetic dysplasia, thus explaining the resemblance of these two skeletal disorders.

Skeletal dysplasias (SDs) are monogenic disorders that affect skeletal development and result in limb and spine deformities and dwarfism. In this article, Glazov et al. report on two siblings (from unaffected parents) with prenatal growth retardation and abnormalities in the long bones of the lower limb and in the spine, which is reminiscent of the SD, anauxetic dysplasia. Sequencing of the disease-causing gene in anauxetic dysplasia, RMRP, excluded this diagnosis in the siblings. Given the lack of family history, authors undertook whole-exome sequencing to elucidate the probable genetic basis of this novel SD. Exome sequencing is currently a much more affordable and rapid approach for solving the basis of rare Mendelian diseases than traditional linkage studies because of major advances in high-throughput sequence capture and next-generation sequencing technologies.

Whole-exome sequencing was performed on the two unaffected, unrelated parents and two affected offspring. Like the piecing together of a puzzle, the millions of sequence reads (approximately 100 base pairs) were aligned to a reference human sequence with specialized software (Burrows–Wheeler alignment tool and sam tool). Polymorphic sites within the aligned exomes were identified using the Genome Analysis toolkit (1). A number of filtering steps narrowed down the possible disease-causing alleles (Fig. 2). From around 17,000 single-nucleotide polymorphisms (SNPs) initially detected, 97% were eliminated because of their presence in a database of known SNPs (NCBI dbSNP), which was assumed to preclude them from causing such a rare disease. SNPs in genes for which there was no functional information were also discarded, thus reducing the candidate pool to 483 novel coding non-synonymous SNPs. Given the inheritance pattern of the observed disease, autosomal recessive homozygous and autosomal compound heterozygous inheritance paradigms were considered most likely. No SNPs satisfied an autosomal recessive homozygous inheritance model but SNPs in four candidate genes satisfied criteria for an autosomal compound heterozygous inheritance model: that is two novel coding non-synonymous SNPs were present in the same gene in both affected siblings whereas only one of the SNPs was present in each parent, in a mutually exclusive manner. When the SNPs of these four genes were analyzed with SIFT (2), an algorithm that predicts effects of amino acid substitutions on protein function, only one gene appeared to bear mutations that would disrupt protein function. This was found to be a human homolog of the yeast POP1, encoding a core component of the RNase P and RNase MRP complexes. These complexes are eukaryotic ribonucleoproteins with multiple essential cell functions, including mitochondrial DNA replication.


Figure 2. Steps in exome-sequence data reduction process to elucidate novel disease-causing variant in skeletal dysplasia.

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The two POP1 variants identified [a nonsense mutation (c.1573C>T; pArg513Ter) in exon 10 and a missense mutation (c.1748G>A; p.Gly583-Glu) in exon 12] were validated in the four individuals by classical Sanger sequencing. The variants were found to be relatively rare in the normal population, given that they were not detected in 186 controls. The amino acids altered by both mutations are highly conserved among species indicating their critical role in POP1 and/or RNase P and RNase MRP complexes. Consistent with this, similar POP1 mutations in yeast affect stability and activity of RNase P and RNase MRP complexes and POP1 ablation in yeast and drosophila is lethal.

Further studies were undertaken to understand the physiological impact of these POP1 mutations. One cellular function of the RNase MRP complex is to process precursor 5.8S rRNA to the mature form. To assess the activity of this complex, the relative abundance of unprocessed 5.8S rRNA and RMRP transcripts was measured by quantitative real-time PCR in the family members and unrelated controls. The relative abundance of mature RMRP RNA was significantly reduced and the abundance of unprocessed pre-5.8S rRNA was increased in the affected individuals compared with the unaffected parents and unrelated controls indicating POP1 mutations reduced the stability and activity of the RMRP complex, as previously shown in yeast. Cell division rates of stimulated peripheral blood mononuclear cells (PBMC) were markedly reduced in the affected individuals compared with healthy controls as measured by progressive dilution of fluorescent marker intensity by fluorescence activated cell sorting. Interestingly, the father who carried the nonsense mutation (c.1573C>T; pArg513Ter) also exhibited a somewhat reduced PBMC proliferation rate, indicating the more severe nature of this mutation. Thus, reduced activity of RNase MRP complex impairs cell proliferation suggesting a mechanism for poor bone development observed in the affected individuals.

In summary, this study identifies loss-of-function mutations in POP1 underlying a novel skeletal dysplasia, which reduce RNase MRP complex activity and cell proliferation. The fact that POP1 directly interacts with the same RMRP domain that is altered in anauxetic dysplasia suggests a shared molecular mechanism underlies these SDs. The authors speculate that the novel skeletal phenotype reported here may differ from anauxetic dysplasia because of altered processing of additional, as yet unidentified, regulatory RNAs by RNase P and RNase MRP complex during bone development. Moreover they suggest the existence of additional RMRP or POP1 variants or variants in other components of RNase P and RNase MRP complexes in currently unmapped short-stature syndromes. Additionally, this study highlights the tremendous power of exome sequencing in identifying rare Mendelian disorders with limited pedigrees.


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