Genetic analysis of hypophosphatemic rickets in Malaysian patients through whole exome sequencing

Hypophosphatemic rickets (HR) is a rare subtype of rickets due to genetic defects in phosphate regulators. Low level of phosphate in the blood is due to low reabsorption and high excretion of phosphate by the kidneys. HR often manifests in childhood with skeletal deformities of rickets including bowing of legs, short stature and dental abnormalities. Several genes have been identified to cause HR. The most common gene is the phosphate-regulating endopeptidase homolog, Xlinked (PHEX) in which mutations in this gene cause X-linked dominant hypophosphatemic rickets (XLHR). Other less common genes include fibroblast growth factor-23 (FGF23), dentin matrix protein-1 (DMP1), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), chloride channel 5 (CLCN5), and solute carrier family 34 member 3 (sodium/phosphate cotransporter) (SLC34A3). HR is rare in Malaysia and a diagnosis is usually based on both clinical and biochemical results. In 2016, a study was conducted to look for mutations in the PHEX, DMP1 and FGF23 genes in four Malaysian children with HR. However, the results could not be concluded from the previous study because a causative mutation was not found in some patients. Therefore, alternative method was needed to identify the causative mutations in the patients. In the present project, a trio study was conducted with the aim to find the gene mutations responsible for HR in Malaysian patients using whole exome sequencing (WES). The candidate variants found from WES were validate by Sanger sequencing. Paternity investigation was performed by segregation of single nucleotide polymorphisms (SNPs) extracted from the WES data. High resolution melting curve profiles were established in healthy controls to identify the presence of the mutations based on the comparison of the patient melt profiles. Quantitative real time PCR was performed to compare the expression of the FGF23 between patients and controls. Plasma levels of FGF23 were measured using an ELISA assay. In this study, clinical data showed that patients had lower limb bowing, osteopenia, splaying and fraying of the metaphyses of the femur and distal tibia, and radius and ulna, which were consistent with hypophosphatemic rickets phenotypes. All patient’s parents did not show any phenotypic features of HR. Molecular genetic analysis revealed 37 variants in the six candidate HR genes. Since the parents of the patients were healthy, the variants were filtered based on two strategies; “de novo strategy” and “double-hit strategy”. After filtering, four candidate variants remained in which three were de novo PHEX gene mutations and one homozygous mutation in the DMP1 gene. Two of the variants found in PHEX have previously been reported as HR disease-causing mutation; Patient 1 (c.871C>T) had a stop-gain mutation (p.R291*) predicted to be damaging in all prediction software tools. Patient 2, had a PHEX (c.1970A>G) missense mutation, which led to the replacement of Tyr657 with cysteine. This mutation was also predicted to be damaging. The two remaining variants were novel and identified in the PHEX and DMP1 genes. The novel PHEX variant in patient 3 was an in-frame deletion (c.1946_1954delGCCTGCGGG, p.G649-651Rdel). Another novel variant was identified in patient 4, which was a homozygous splice donor variant of DMP1 (c.54+1G>A) and was inherited from the carrier parents. The presence of all four variants were confirmed by Sanger sequencing. The paternity analysis for mendelian transmission of SNPs confirmed the biological relationship between the probands and their parents. Therefore, the PHEX mutations were confirmed to be do novo. High resolution melting analysis revealed that the mutations found in the patients were absent in fifty DNA samples from healthy controls. From the FGF23 gene expression data, the fold change between the patients and controls demonstrated that overexpression of FGF23 was observed only in one patient and the expression difference between controls and patients was not significant (t (5)=0.39, p=0.71). This is likely due to bone tissue samples being unavailable and white blood cells not being the appropriate tissue for the qPCR experiment. Plasma FGF23 concentrations were higher than 30pg/mL in three of the patients, however, the differences between controls and patients were not significant (Mann-Whitney test, p=0.57). In conclusion, genetic studies for HR in Malaysia showed that sporadic XLHR cases occur frequently in these study cases, and that the PHEX gene is likely the most common cause of HR in Malaysia.

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