Diffuse Optical Characterization of the Healthy Human Thyroid Tissue and Two Pathological Case Studies

<div><p>The <i>in vivo</i> optical and hemodynamic properties of the healthy (n = 22) and pathological (n = 2) human thyroid tissue were measured non-invasively using a custom time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) system. Medical ultrasound was used to guide the placement of the hand-held hybrid optical probe. TRS measured the absorption and reduced scattering coefficients (<i>μ</i>_a, <i>μ</i>_s′) at three wavelengths (690, 785 and 830 nm) to derive total hemoglobin concentration (THC) and oxygen saturation (StO₂). DCS measured the microvascular blood flow index (BFI). Their dependencies on physiological and clinical parameters and positions along the thyroid were investigated and compared to the surrounding sternocleidomastoid muscle. The THC in the thyroid ranged from 131.9 <i>μ</i>M to 144.8 <i>μ</i>M, showing a 25–44% increase compared to the surrounding sternocleidomastoid muscle tissue. The blood flow was significantly higher in the thyroid (BFI_thyroid = 16.0 × 10^-9 cm²/s) compared to the muscle (BFI_muscle = 7.8 × 10^-9 cm²/s), while StO₂ showed a small (StO_{2, muscle} = 63.8% to StO_{2, thyroid} = 68.4%), yet significant difference. Two case studies with thyroid nodules underwent the same measurement protocol prior to thyroidectomy. Their THC and BFI reached values around 226.5 <i>μ</i>M and 62.8 × 10^-9 cm²/s respectively showing a clear contrast to the nodule-free thyroid tissue as well as the general population. The initial characterization of the healthy and pathologic human thyroid tissue lays the ground work for the future investigation on the use of diffuse optics in thyroid cancer screening.</p></div

New Splice Site Acceptor Mutation in <i>AIRE</i> Gene in Autoimmune Polyendocrine Syndrome Type 1

<div><p>Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this <i>AIRE</i> mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the <i>AIRE</i> gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.</p></div

Thyroid schematics and results for the nodule cases.

<p>(A) <i>CASE 1</i>: Two locations were added to the standard protocol. The nodule was located within the left thyroid gland with a maximum diameter of 65 mm, as indicated by the shaded region. (B) <i>CASE 1</i>: One location was added on the right lobe to the standard protocol. This case had a maximum diameter 40 mm nodule (shaded region) together with a second nodule of 3 mm diameter, both located in the right thyroid gland. The results ((C) and (D)) are shown for the total hemoglobin concentration (THC), oxygen saturation (StO₂), blood flow index (BFI) and the reduced scattering coefficient (<i>μ</i>_s′) for 785 nm. Note, that these color plots do not include the muscle locations. For the detailed representation please refer to the Tables in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147851#pone.0147851.s004" target="_blank">S4 Table</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147851#pone.0147851.s005" target="_blank">S5 Table</a>.</p

Vital records.

<p>Vital records.</p

AIRE overview family pedigree and detection of the c.653-1G>A AIRE mutation.

<p>(A) Schematic AIRE protein representation showing the different protein domains: HSR domain (HSR), conserved bipartite nuclear localization signal (NLS), PHD zinc finger motif (PHD), proline-rich region (PRR), LXXLL motif (L), and SAND domain (SAND). (B) Schematic <i>AIRE</i> gene representation, where rectangles indicate exons and the dashed line the introns. Finally, (C) schematic representation of the consensus sequences for the 5′ splice site donor (SSD), branch site and 3′ splice site acceptor (SSA). The star indicates the mutation. (D) Pedigree of the Spanish consanguineous family. Genotypes were shown as wild-type (G/G), heterozygous (G/A) and homozygous (A/A) of the c.653-1G>A <i>AIRE</i> mutation. The arrow indicates the index case. (E) Above, the schematic representation of the junction between intron 5 and exon 6, and below, the direct sequence analysis of the <i>AIRE</i> gene identified homozygous carriers of the c.653-1G>A mutation in APS-1 patients, while heterozygous carriers were found in unaffected relatives (I-1, I-2, III-2) of the family.</p

Reduced scattering coefficients (<i>μ</i>_s′) from TRS data.

<p>Reduced scattering coefficients (<i>μ</i>_s′) from TRS data.</p

Protocol schematics with measurement locations, tissue dimensions and probe geometry.

<p>Panel (A) Shows the location of the thyroid, its dimensions and the defined measurement points for this study together with a head turn used to expose the thyroid. The ultrasound images illustrate the locations of the jugular vein (JV), carotid artery (CA), esophagus (EPG), the (left) thyroid gland, its position and superficial structures (sternocleidomastoid muscle, skin and adipose layers) in normal neck (right) and rotated neck (left) position; Panel (B) shows an excerpt of an ultrasound image from a thyroid gland. The total tissue depth (TTD) is defined by the sum of the superficial tissues (STT) and the tissue thickness (TT). Typical values vary between different tissue types; Panel (C) illustrates the three different measurement points per side. The probe size and its placement are shown in relation to the thyroid gland size; Panel (D) shows the hybrid diffuse optics probe which consisted of one source per modality (DCS and TRS) and two different detector locations per source. For a good overlap for the probed regions by both modalities, we have used a cross-geometry with the DCS source on the left side, its detectors on the right side of the probe and for TRS vice-versa. The shorter source-detector separation (SD) was used in a subset of eleven subjects, which is half of the study population, as well as the two pathology cases.</p

Tissue dimensions recorded by ultrasound.

<p>Tissue dimensions recorded by ultrasound.</p

Absorption coefficients (<i>μ</i>_a) from TRS data.

<p>Absorption coefficients (<i>μ</i>_a) from TRS data.</p

Clinical features and analytical data.

<p>Abbreviations: IDDM (Insulin-dependent Diabetes Mellitus), T2D (Type 2 Diabetes), PTH (Parathyroid hormone), ACTH (Adrenocorticotropic hormone), GAD (Glutamic acid decarboxylase), IA-2 (Islet Antigen 2) and Ab (antibodies).</p