Figure 1.
Minigene analysis of c.840-2A>G mutation in patient 3A.
(A) The GNAS-IVS10 minigene and primer designs. The construct made by using primers a and b spans from introns 9 to 12 and contains exons 10, 11, and 12 with a total length of 815 bp. Primers c, d, and e were used for semi-nested PCR. (B) COS-7 cells were used for transient transfection study. An extra 362-bp band was found in the mutant (lane 3A) compared with a single 216-bp band in the wild type vectors (lane W). The 362-bp band in lane 3A was subcloned and sequenced. The electropherogram is shown in Panel C. The thick arrow indicates a faint 614-bp band amplified from DNA carried over from COS-7 cells or mini-gene plasmids. Lane H, healthy individual; M, 100 bp DNA ladder; E, empty pcDNA3.1 vector; W, IVS10 wild minigene; 3A, IVS10 mutant minigene. (C) TOPO TA subcloning and sequencing reveal the 362 bp band containing the complete intron 10 of 146 bp. The inclusion of intron 10 changes amino acid Trp to Phe at residue 281 and causes an earlier termination of translation at codon 284 (p.Trp281PhefsTer4 or p.W281Ffs*4). The vertical arrows in Panels A and C denote the site of the c.840-2A>G mutation. The vertical bars in Panel C indicate the exon-intron or intron-exon boundaries.
Figure 2.
RT-PCR analysis on the RNA from the peripheral blood leukocytes of a healthy control and patient 3A with primers c and e.
(A) The electropherogram shows a band of 268 bp in the healthy control (lane H) but two bands, 268 bp and 137 bp, in patient 3A (lane 3A). The 137-bp band is faint. M indicates 100 bp DNA ladder; H, healthy control; 3A, patient 3A. (B) Sequence analysis of the 137-bp band reveals no exon 11. The deletion of exon 11 results in a frameshift changing Arg to Ser at residue 280 and causing an earlier termination of translation at codon 300 (p.Arg280SerfsTer21 or p.R280Sfs*21). The vertical bar indicates the boundary of exons 10 and 12.
Figure 3.
Sonography of the left kidney.
A hyperechoic mass measuring 8(right panel, arrow).
Figure 4.
Radiography of the kidneys and urinary bladder.
A radio-opaque stone measuring 11×6 mm in size in the central portion of the left kidney (arrow).
Table 1.
Characteristics of patients with pseudohypoparathyroidism 1a (PHP1A) or pseudopseudohypoparathyroidism (PPHP).
Figure 5.
Pseudohypoparathyroidism 1a (PHPIA) caused by the c.840-2A>G mutation in Family 3.
The mutant c.840-2A>G allele of patient 3A (solid circle) was inherited from her mother (hatched circle) who had PPHP. Her father and younger brother were normal. The genotype of each member at this site is shown above the electropherogram.
Figure 6.
Pseudohypoparathyroidism 1a (PHPIA) caused by the c.1027_1028delGA mutation in Family 4.
The mutant c.1027_1028delGA allele of patient 4A (solid square) was inherited from his mother (hatched circle) who had pseudopseudohypoparathyroidism (PPHP). His father was normal. The deletion of two nucleotides in the mutant allele causes a frameshift resulting in a premature termination of translation at codon 343 (p.Asp343Ter or p.D343*). The genotype of each member at this site is shown above the electropherogram.