PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available

Characterization of exRNA extracted from exosomes isolated using the four techniques.

<p>exRNA quality was evaluated using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using the different isolation techniques and serum volumes. The y-axis represents fluorescence, and the x-axis is the size of the RNA, measured in nucleotides (nt). (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.</p

ZetaView measurements of the exosomes extracted from three different serum starting volumes of six individual human donors samples using the 4 different isolation techniques.

<p>NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 1 ml, 500 μl, and 100 μl of human serum. The data in this graph are the mean values of the six individual human samples (±SEM), *p≤0.05.</p

Correlation between the volume of serum and the zeta potential of the isolated solutions.

<p>The data in this graph are the mean values of three experimental replicates (n = 3) ± SEM. (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.</p

Zeta potential measurements for exosomes isolated from the different techniques and serum volumes.

<p>Using the ZetaView instrument, the zeta potential (mV) was measured for the exosomes isolated with miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, and 100 μl of human serum. The data in this graph are the mean values of the zeta potential ± SEM (n = 3)</p

Size distribution of the exosomes isolated from pooled human serum using the different techniques and serum volumes.

<p>For each sample volume (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and technique, the number of particles per a specific particle size (nm) was measured using the ZetaView for NTA. Each graph represents quadratic interpolation of the mean number of particles isolated by each technique (n = 3). Data from the commercial kits miRCURY (blue), ExoQuick (green), and TEIR (red) are graphed on the left y-axis, while the UC (yellow) data, being at a significantly lower magnitude, are mapped on the right y-axis.</p

Correlation between the volume of serum and the total number of particles isolated.

<p>The relationship between the volume of serum and the total number of particles was linear for all four isolation techniques: (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC. The data in this graph represent the average number of isolated particles ± SEM (n = 3).</p

ZetaView measurements of the exosomes extracted from the six serum starting volumes of pooled human serum using the 4 different isolation techniques.

<p>NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl of human serum. The data in this graph are the mean values of three experimental replicates (±SEM), *p≤0.05.</p