PA-5 was a 14-year old girl, she was referred to our clinic for genetic evaluation, because of her abnormality of the dentition and premature loss of primary teeth. She has an elder sister with similar symptoms. Biochemical examination showed that the activity of ALP was significantly decreased to 9 u/L. Medical imaging examination showed carious teeth, alveolar bone loss around teeth, shortened dental roots, taurodontia and abnormality of the mandible (Figures 1E-E2).
Whole exome sequencing using the genomic DNA of the proband was performed in total and >18.1G clean data were generated covering 95.5% of exome target regions at least 20X. The detailed sequencing data of the variants are presented in Table 1. Based on the TGex Software (LifeMap Sciences, United States), we found variants existed in OMIM genes whose functions matched with known phenotypes. After PCR amplification, Sanger sequencing was used to identify variations. Genealogy and sequencing results are shown in Figure 2. We finally identified the cause of the disease in the five patients (PA-1: c.649_650insC and c.707A > G; PA2: c.98C > T and c.707A > G; PA3: c.407G > A and c.650delTinsCTAA; PA4: c.1247G > T (homozygous); PA5: c.406C > T and c.1178A > G; NM_000478.5), as shown in Table 2. The five patients had eight variants. Both c.1247G > T/p.Gly416Val, c.1178A > G/p.Asn393Ser and c.707A > G/p.Tyr236Cys were not presented in population and disease databases including gnomAD(https://gnomad.broadinstitute.org/), the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/), the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and LOVD (http://www.LOVD.nl/LTBP-4). All of these were novel variants.
CLUSTAL W was used for conservative analysis of three variations (Figure 3A). Using this method, it was found that c.1247G > T/p.Gly416Val; c.1178A > G/p.Asn393Ser and c.707A > G/p.Tyr236Cys were conservative substitutions. At the same time, we predicted the impact of c.1247G > T/p.Gly416Val, c.1178A > G/p.Asn393Ser and c.707A > G/p.Tyr236Cys with four in-silico tools: SIFT, Provean, Mutation Taster, and CADD (Figure 3F). Predictive software suggested that c.1247G > T/p.Gly416Val and c.707A > G/p.Tyr236Cys were harmful variations, while for c.1178A > G/p.Asn393Ser, only Mutation Taster and CADD supported the possibility that this variation was harmful. The c.1247G > T/p.Gly416Val was assessed to be likely pathogenic (PM1, PM2, PM3, PP3, and PP4), c.707A > G/p.Tyr236Cys was assessed to be likely pathogenic (PM1, PM2, PM3, PP3, and PP4) and c.1178A > G/p.Asn393Ser was assessed to be likely pathogenic (PM1, PM2, PM3, PM5, and PP4) by the ACMG/AMP guidelines. In combination with the clinical manifestations of the patients and the results of the pathogenicity assessment of the variants, we finally diagnosed that these five patients were hypophosphatasia.