Characteristics of the study subjects.

<p>Abbreviations: FD, osseous fibrous dysplasia; MAS, McCune-Albright syndrome; N.D., not detected; NGS, next generation sequencing; PNA, the peptide nucleic acid method; PNA-NGS, combinatory use of PNA and NGS; PP, precocious puberty</p>*<p>Hyperprolactinemia and GH-producing adenoma</p>**<p>GH-producing adenoma</p

Results of the serial dilution study.

<p>(<b>A</b>) Cloned mutant DNA (R201H mutation) was diluted into cloned wildtype DNA to 1/10 (10%), 1/100 (1%), 1/333 (0.3%), 1/1,000 (0.1%), 1/3,333 (0.03%) and 1/10,000 (0.01%). Serially diluted DNA samples were PCR-amplified with or without the peptide nucleic acid (PNA) probe (‘PNA’ and ‘Conventional’. Each PCR product was analyzed by Sanger sequencing and next generation sequencing (NGS). Partial chromatograms encompassing the GNAS codon 201 (indicated by CGT) are shown. Relative abundance of the c.602 nucleotide (G, A, T and C) measured by NGS is aligned with each chromatogram. The G allele is wildtype, while the A allele is the R201H mutant. Values with a positive test result (defined by z-score of measured mutant abundance; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060525#s2" target="_blank">Materials and Methods</a> for details) are colored red. Experiment-specific reference ranges are also shown. In the 12 chromatograms, the mutant signal could be detected in two PNA-treated samples and one non-treated sample (indicated by red arrows). Contrastingly, the mutation could be detected down to 0.03% by NGS alone, and down to 0.01% by combinatory use of PNA and NGS. (<b>B</b>) A serial dilution plot showing a linear correlation between true mutation abundance and measured mutation abundance. Note that both axes are logarithmic. Comparison of mutation abundance values of PNA-treated and untreated samples revealed that the fold enrichment by PNA is about 7, and it was independent of initial abundance.</p

Schematic diagramas showing an overview of mutation detection methods.

<p>(<b>A</b>) In patients with McCune-Albright syndrome, the proportion of mutation-carrying cells (colored red) is low in peripheral blood leukocytes (PBL). (<b>B</b>) In the present study, PCR amplification was conducted in the absence (<i>left panel</i>) or presence (<i>right panel</i>) of the peptide nucleic acid (PNA) probe. The PNA probe preferentially hybridizes to wildtype PCR fragments (colored black) and inhibits their amplification. This results in enrichment of mutant PCR fragments (colored red). We used chimeric PCR primers, containing both locus-specific and adapter sequences, to generate amplicons that are sequenced on the Illumina platform. (<b>C</b>) PCR without the PNA probe produces PCR amplicons, of which relative proportion between wildtype (colored black) and mutant (colored red) is similar to PBL (<i>left panel</i>). In contrast, PNA treatment enriches mutant amplicons (<i>right panel</i>). (<b>D</b>)<b>,</b> PCR amplicons were analyzed by both Sanger sequencing and next generation sequencing (‘NGS’). Due to low mutation abundance, mutations cannot be detected in amplicons generated without the PNA probe (<i>left panel</i>), while they can be detected in PNA-treated amplicons (<i>right panel,</i> an arrow indicates the mutation). In the MiSeq platform, clonal clusters, each derived from a single DNA molecule, are generated on a flow cell, and are sequenced base-by-base simultaneously and independently. The diagrams under the schematic flow cells show imaginative optically scanned data of the cycle corresponding to the mutated nucleotide. In a sample without PNA treatment, the mutant amplicons can be recognized on the flow cell (<i>left panel</i>). Mutant-enriched samples are also analyzable by NGS (<i>right panel</i>).</p

Endocrinological findings in Propositus of pedigree 1.

<p>The conversion factors to the SI unit are as follows: GH 1.0 (µg/liter), LH 1.0 (IU/liter), FSH 1.0 (IU/liter), TSH 1.0 (mIU/liter), prolactin 1.0 (µg/liter), ACTH 0.22 (pmol/liter), cortisol 27.59 (nmol/liter), IGF-I 0.131 (nmol/liter), free T4 12.87 (pmol/liter), free T3, 1.54 (pmol/liter), and estradiol 3.671 (pmol/liter).</p>a<p>Reference data of pre-pubertal Japanese girls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>b<p>Reference data of pubertal (Tanner 2–3) Japanese girls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>c<p>Reference data of UK children (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>d<p>Reference data of UK children (older than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>e<p>Reference data of Japanese girls (5–7 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>f<p>Reference data of Japanese girls (15–17 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>g<p>Reference data of Japanese girls (15 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Japan1" target="_blank">[25]</a></p

Endocrine phenotype of 91 probands screened for 9 genes.

<p>Endocrine phenotype of 91 probands screened for 9 genes.</p

Results of MR scans of probands screened for 9 genes.

<p>Results of MR scans of probands screened for 9 genes.</p

Transactivation assays of R84X and V75I LHX4 using <i>POU1F1</i>(<i>PIT1</i>) and<i>αGSU</i> reporter.

<p><i>A and B</i>: COS7 cells were cotransfected with the pRL-CMV internal control vector, indicated amount (nanograms) of the effector plasmids, and the <i>POU1F1</i>(A) or<i>αGSU</i> (B) reporter. The data are the mean ± s.e.m. of at least three independent experiments performed in triplicate transfections. The white, black, red, and blue bars indicate the data of the empty expression vectors, expression vectors with wild type (WT) LHX4, expression vectors with R84X LHX4, and V75I LHX4, respectively. R84X LHX4 exhibited markedly reduced transactivation, whereas V75I LHX4 retained partial activity. The two mutants did not exhibit any dominant negative effect. The data are mean ± SEM of at least three independent experiments performed in triplicate transfections. <i>C and D</i>: GH3 cells were cotransfected with the pRL-CMV internal control vector, indicated amount (nanograms) of the effector plasmids, and the <i>POU1F1</i>(C) or<i>αGSU</i> (D) reporter.</p

Endocrinological findings in Propositus of pedigree 2.

<p>The conversion factors to the SI unit are as follows: GH 1.0 (μg/liter), TSH 1.0 (mIU/liter), LH 1.0 (IU/liter), FSH 1.0 (IU/liter), testosterone, 0.035 (nmol/liter), prolactin 1.0 (μg/liter), ACTH 0.22 (pmol/liter), cortisol 27.59 (nmol/liter), IGF-I 0.131 (nmol/liter), free T4 12.87 (pmol/liter), and free T3, 1.54 (pmol/liter).</p>a<p>Reference data of pre-pubertal Japanese boys (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>b<p>Reference data of UK children (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>c<p>Reference data of Japanese boys (younger than 1 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>d<p>Reference data of Japanese boys (7-9 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p

Functional characterization of two mutant LHX4.

<p><i>A</i>, Protein expression level of myc-tagged WT and two LHX4 mutants was assessed by western blot using a monoclonal anti-myc antibody. The expression of V75I LHX4 was comparable to that of WT, whereas R84X LHX4 was not detected. Tubulin was used as a control. <i>B</i>, Subcellular localization analysis. For subcellular localization analyses, we visualized and photographed COS7 cells transfected with GFP-tagged LHX4 using a Leica TCS-SP5 laser scanning confocal microscope, after mounting the cells in Vectashield-DAPI solution. The WT and V75I LHX4 are localized to the nucleus. <i>C</i>, EMSA experiments. WT LHX4 showed specific binding to the elements, which was competed by excess amount of (200 times) cold competitors. The V75I LHX4, which has an intact HD, bound with similar or slightly high efficiency to the WT LHX4.</p

The pedigrees of the affected families.

<p><i>A–C</i>, Pedigrees of families 1–3. Arrow indicates the propositus. ND: not determined.</p