Fig 1.
Appearance of two brothers with hypophosphatemic rickets of an Indian family (E0023).
Clinical examination of both individuals yielded decreased tubular phosphate reabsorption, growth retardation, and bone malformations typical of hypophosphatemic rickets. Proband II-1 (left) shows shows genu valgum “knock-kneed” features while his brother II-2 shows the characteristic signs of genu varum “bowlegs”.
Table 1.
Clinical data and biochemical results of four patients with hypophosphatemic rickets from two Indian families.
Fig 2.
Pedigrees and PHEX gene mutation chromatograms from two Indian families with X-linked hypophosphatemic rickets.
(A,B) Pedigrees of two XLH families. Filled symbols represent affected individuals. Circles and squares indicate females and males, respectively. (C) Genomic sequence chromatograms showing a novel acceptor splice site mutation c.1080-3C>A in intron 9 of the PHEX gene (arrows). The mutation in family E0023 co-segregates with the affected status and is present in the mother (I-2) and her two affected sons (II-1 and II-2). The father (I-1), who is healthy, exhibits a hemizygous wild-type allele. The mutation was identified after whole exome sequence analysis in E0023-II-2 and confirmed by direct Sanger sequencing. (D) A novel heterozygous insertion/deletion mutation (c.1211_1215delACAAAinsTTTACAT) in PHEX exon 11 leading to a frameshift (p.Asp404Valfs*5) was identified by direct Sanger sequencing in a female patient with hypohosphatemic rickets (E0024-II1). Note, that both her father (I-1) and her mother (I-2) show only the wild-type sequence. Therefore, we conclude that this mutation is most likely a de novo change, although the presence of germ line mosaicism in one of the parents cannot entirely be ruled out by analyzing RNA from blood only. The two bottom chromatograms represent the c.1211_1215delACAAAinsTTTACAT frameshift mutation (bottom) and the wild-type allele (above) after the respective patients’ PHEX exon 11 PCR products have been cloned into TA-cloning plasmid vector pCR2.1 (Invitrogen) and subsequently sequenced. Black boxes highlight deleted (ACAAA) and inserted (TTTACAT) bases accordingly.
Fig 3.
Consensus sequences and frequencies of human splice site regions.
Pictograms representing the comprehensive in silico analysis of all human splice sites concerning 327,293 exons across 81,814 different transcripts among 20,345 human genes. A) Frequencies and consensus sequences of 15 human splice donor nucleobases. B) Frequencies and consensus sequences of 26 human splice acceptor nucleobases. Note, that the GT (+1, +2) and AG (-1, -2) positions adjacent to donor and acceptor splice junctions are highly conserved and nearly invariant. Consensus flanking bases are found at frequencies higher than expected compared to a random distribution. Frequencies of each nucleobase across various splice site positions are given in a table aligned with the respective bases in the pictograms above.
Fig 4.
RT-PCR analysis of the mutated PHEX 1080-3C>A acceptor splice site.
(A) Schematic representation of the exon/intron structure of the PHEX gene up and downstream of the mutated 1080-3C>A splice site in intron 9 (arrow). Primers used for amplification are indicated (PHEX_ex6_F, PHEX_ex22_R). (B) Schematic representation of the aberrant splice product found in male patient (E0023-II-2) who carries a hemizygous PHEX (c.1080-3C>A) splice acceptor site mutation (arrow). RT-PCR revealed an aberrant splice product which generates an in-frame mRNA transcript with exon 9 directly joined to exon 15, thereby skipping five consecutive exons. (C) Agarose gel electrophoresis of RT-PCR fragments produced after RNA was extracted from EBV-transformed peripheral lymphocytes of patient E0023-II-2 and of a healthy control individual. Using primers PHEX_exon6_forward and PHEX_exon22_reverse resulted in a 1,636 bp wild-type RT-PCR product in both samples. Note, that an additional fainter aberrant 1,129 bp splice product, which corresponds to an in-frame transcript and skipping exon 10–14, is only present in the patient’s sample. 100 bp Marker (New England Biolabs) (D) Sequence chromatograms of the aberrant splice product of 1,129 bp next to the sequence of the wild-type canonical splice product. Sequence traces of the aberrant fragment demonstrate that exon 9 is spliced directly to exon 15 indicating that the PHEX 1080-3C>A mutation may alter the strength of the intron 9 splice acceptor site.