PMC7488884_01
Pedigree with genotypes and initial biochemical and clinical findings in the family.. After informed consent, genomic DNA of the index case (II-1) was subjected to WES analysis at the Yale Center of Genome Analysis. For validation, the identified SLC34A3 mutations were amplified by polymerase chain reaction (PCR) using primers 5'-CGTGGGTGCACACTCCCT-3' and 5'- GGGTGTCAGGCTGGCGGC-3' for SLC34A3.c.671delT, and primers 5'-CTCTGACCTCTGTCTGCC-3' and 5'-GGAAGGGGAAGTCTATGG-3' for SLC34A3.g.2259-2359del101bp and the Expand Long PCR kit (Roche) with annealing temperature 70 C for SLC34A3.c.671delT, and 62 C for SLC34A3.g.2250-2359,del101bp, respectively. PCR products were separated by 2% agarose TAE gel electrophoresis to visualize and verify the integrity of the product, purified using Qiagen (Valencia, CA, USA) reagents followed by Sanger sequencing at the Yale Keck Biotechnology Resource Laboratory. Sequence analysis was performed with Geneious R6 (Biomatters Ltd., San Diego, CA, USA).. Laboratory studies were performed in CLIA-certified laboratories (Mayo Labs, or Quest Labs as indicated). Phosphate was measured in fasting state. C-terminal FGF23 was measured by both laboratories using a commercially available ELISA [Immutopics, 60-6100, and Human FGF-23 (C-Term) ELISA Kit (Quidel, San Diego, CA, USA)]. IGF-1 was determined by liquid chromatography/mass spectroscopy. 25(OH)D, and 1,25(OH)2D were measured by liquid chromatography/tandem mass spectroscopy. Intact PTH was determined by electro-chemiluminescence immunoassay (Roche, South San Francisco, CA, USA). All biochemistry was performed by auto-analyzer in local laboratories.. CLIA, Clinical Laboratory Improvement Amendments; ELISA, enzyme-linked immunosorbent assay; FGF23, fibroblast growth factor 23; IGF-1, Insulin-like growth factor 1; MAF, minor allele frequency; PTH, parathyroid hormone; RU, relative units; TAE, Tris-acetate-EDTA; WES whole exome sequencing.
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Laboratory testing revealed normal serum calcium, but low serum phosphate, and elevated alkaline phosphatase (Figure 1). The middle brother (II-2) at age 8 years was also found to have hypophosphatemia along with elevated alkaline phosphatase, hypercalciuria, low serum PTH, and elevated 1,25(OH)2D (Figure 1) and was started on oral phosphate supplementation with sodium/potassium phosphate (neutral-Phos) 1000 mg/day 4 months later.
(a) Radiological bilateral full leg images of II-1 (Index) at age 12 years 3 months demonstrate the extent of corrective surgeries and the hardware left in place. (b) Radiological images of both knees of II-2 at age 13 years 6 months after bilateral distal medial hemiepiphysiodesis; notice diffuse osteopenia and metaphyseal irregularity.
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He required corrective orthopedic surgeries at 11 years 11 months, and at 13 years 6 months (Figure 2). Notwithstanding, he developed knee pain and bowing of both legs, and, at the age of 13 years 6 months, he underwent bilateral distal femoral medial hemiepiphysiodesis (Figure 2).
(a) Height chart of II-1 (Index) shows short stature and poor growth velocity. 1, epiphysiodesis and placement of supramalleolar pin, removed after 6 weeks; 2, epiphysiodesis and placement of permanent hardware in bilateral distal, and proximal tibia. (b) Height chart of II-2 shows short stature and poor linear growth with no significant improvement with phosphate treatment, and acceleration after the rHGH initiation (star). His bone age (triangles) was delayed compared to chronological age. 1, hemiepiphysiodesis and permanent hardware placed in bilateral distal femur. (c) Height chart of II-3, who is heterozygous.. rHGH, recombinant human growth hormone.
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By 16 years, he had reached his final adult height of 148.1 cm (0.08 percentile, or -3.14 SD; Figure 3). His bone age was 11 years (Figure 3).