Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets.

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.

Molecular Analysis of Congenital Hypothyroidism in Saudi Arabia: SLC26A7 Mutation Is a Novel Defect in Thyroid Dyshormonogenesis.

Context: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder, affecting one in 3000 to 4000 newborns. Since the introduction of a newborn screening program in 1988, more than 300 cases have been identified. The underlying genetic defects have not been systematically studied. Objective: To identify the mutation spectrum of CH-causing genes. Methods: Fifty-five patients from 47 families were studied by next-generation exome sequencing. Results: Mutations were identified in 52.7% of patients (29 of 55) in the following 11 genes: TG, TPO, DUOX2, SLC26A4, SLC26A7, TSHB, TSHR, NKX2-1, PAX8, CDCA8, and HOXB3. Among 30 patients with thyroid dyshormonogenesis, biallelic TG mutations were found in 12 patients (40%), followed by biallelic mutations in TPO (6.7%), SLC26A7 (6.7%), and DUOX2 (3.3%). Monoallelic SLC26A4 mutations were found in two patients, one of them coexisting with two tandem biallelic deletions in SLC26A7. In 25 patients with thyroid dysgenesis, biallelic mutations in TSHR were found in six patients (24%). Biallelic mutations in TSHB, PAX 8, NKX2-1, or HOXB3 were found once in four different patients. A monoallelic CDCA8 mutation was found in one patient. Most mutations were novel, including three TG, two TSHR, and one each in DUOX2, TPO, SLC26A7, TSHB, NKX2-1, PAX8, CDCA8, and HOXB3. SLC26A7 and HOXB3 were novel genes associated with thyroid dyshormonogenesis and dysgenesis, respectively. Conclusions: TG and TSHR mutations are the most common genetic defects in Saudi patients with CH. The prevalence of other disease-causing mutations is low, reflecting the consanguineous nature of the population. SLC26A7 mutations appear to be associated with thyroid dyshormonogenesis.

Clinical and genetic characteristics of 15 families with hereditary hypophosphatemia: Novel Mutations in PHEX and SLC34A3.

BACKGROUND: Hereditary hypophosphatemia is a group of rare renal phosphate wasting disorders. The diagnosis is based on clinical, radiological, and biochemical features, and may require genetic testing to be confirmed. METHODOLOGY: Clinical features and mutation spectrum were investigated in patients with hereditary hypophosphatemia. Genomic DNA of 23 patients from 15 unrelated families were screened sequentially by PCR-sequencing analysis for mutations in the following genes: PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. CytoScan HD Array was used to identify large deletions. RESULTS: Genetic evaluation resulted in the identification of an additional asymptomatic but intermittent hypophosphatemic subject. Mutations were detected in 21 patients and an asymptomatic sibling from 13 families (86.6%, 13/15). PHEX mutations were identified in 20 patients from 12 families. Six of them were novel mutations present in 9 patients: c.983_987dupCTACC, c.1586+2T>G, c.1206delA, c.436+1G>T, c.1217G>T, and g.22,215,887-22,395,767del (179880 bp deletion including exon 16-22 and ZNF645). Six previously reported mutations were found in 11 patients. Among 12 different PHEX mutations, 6 were de novo mutations. Patients with de novo PHEX mutations often had delayed diagnosis and significantly shorter in height than those who had inherited PHEX mutations. Novel compound heterozygous mutations in SLC34A3 were found in one patient and his asymptomatic sister: c.1335+2T>A and c.1639_1652del14. No mutation was detected in two families. CONCLUSIONS: This is the largest familial study on Turkish patients with hereditary hypophosphatemia. PHEX mutations, including various novel and de novo variants, are the most common genetic defect. More attention should be paid to hypophosphatemia by clinicians since some cases remain undiagnosed both during childhood and adulthood.