PMC4880488_01
I Phenotypic and molecular studies has been performed on materials obtained from index patient, parents and control samples. A. Pedigree of the family. B. Clinical pictures of the patient (upper right panels) when she was 9 month-old (upper panels), 2 and half years old (left lower panel) and 4 and nine month-old (right lower panel). C and D. Representative sequence alignment figures cover the mutations in FTO and CETP, respectively. The top line in each panel represents the non-mutated reference sequence. The subsequent lines below the reference lines depict the results from exome sequence. Each line represents a distinct coverage read. Mean 20x coverage of all bases was above 81% for index patient. E and F. Chromatogram illustrations of FTO and CETP obtained via Sanger sequencing analysis of the index patient analyzed via whole-exome sequencing (NG1305-1) and her parents. Note that the respective mutations identified via whole-exome sequencing were confirmed as homozygous mutations in the index patient and as heterozygous in her parents. DNA from healthy individuals was also Sanger sequenced and these results are included as controls. G. CNV Segment Detection of NG1305-1: The log ratio comparing NG1305-1 and control sequence depths of coverage for each exon are depicted as gray dots. The black lines demonstrate regions of segmented copy neutral events, green lines are segmented deletion events and red lines are amplification events. H. mRNA levels of FTO in fibroblast cells were extracted from homozygous patient, heterozygous parents and control individual-same ethnicity and without a sequence variant- were analyzed using RT-PCR. I. The figure depicts FTO staining in patient fibroblasts. Significant nuclear accumulation is noted. J. Western blot of FTO and as an internal control GAPDH in patient, parental and control cells. No difference was detected.
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Table) (Fig. 1). Sanger sequencing of the entire coding region of FTO and CETP in the patient's parents revealed that both were heterozygous for the identified mutations (Fig. 1). The patient's exome data was analyzed for large scale CNV events, and no disease causing large duplications or deletions within coding regions were identified (Fig. 1). We have shown that FTO is located in nucleus of cells from the patient, her parents, and non-related controls (Fig. 1). Q-PCR analysis revealed slightly decreased levels of FTO expression in patient cells compared to controls (Fig. 1).
PMC4880488_02
I Phenotypic and molecular studies has been performed on materials obtained from index patient, parents and control samples. A. Pedigree of the family. B. Clinical pictures of the patient (upper right panels) when she was 9 month-old (upper panels), 2 and half years old (left lower panel) and 4 and nine month-old (right lower panel). C and D. Representative sequence alignment figures cover the mutations in FTO and CETP, respectively. The top line in each panel represents the non-mutated reference sequence. The subsequent lines below the reference lines depict the results from exome sequence. Each line represents a distinct coverage read. Mean 20x coverage of all bases was above 81% for index patient. E and F. Chromatogram illustrations of FTO and CETP obtained via Sanger sequencing analysis of the index patient analyzed via whole-exome sequencing (NG1305-1) and her parents. Note that the respective mutations identified via whole-exome sequencing were confirmed as homozygous mutations in the index patient and as heterozygous in her parents. DNA from healthy individuals was also Sanger sequenced and these results are included as controls. G. CNV Segment Detection of NG1305-1: The log ratio comparing NG1305-1 and control sequence depths of coverage for each exon are depicted as gray dots. The black lines demonstrate regions of segmented copy neutral events, green lines are segmented deletion events and red lines are amplification events. H. mRNA levels of FTO in fibroblast cells were extracted from homozygous patient, heterozygous parents and control individual-same ethnicity and without a sequence variant- were analyzed using RT-PCR. I. The figure depicts FTO staining in patient fibroblasts. Significant nuclear accumulation is noted. J. Western blot of FTO and as an internal control GAPDH in patient, parental and control cells. No difference was detected.
nihms742086f1.jpg
PMC4880488_02_nihms742086f1.jpg
F1
Table) (Fig. 1). Sanger sequencing of the entire coding region of FTO and CETP in the patient's parents revealed that both were heterozygous for the identified mutations (Fig. 1). The patient's exome data was analyzed for large scale CNV events, and no disease causing large duplications or deletions within coding regions were identified (Fig. 1). We have shown that FTO is located in nucleus of cells from the patient, her parents, and non-related controls (Fig. 1). Q-PCR analysis revealed slightly decreased levels of FTO expression in patient cells compared to controls (Fig. 1).
I Phenotypic and proliferation comparisons of fibroblasts extracted from index case, parents and control subjects. Upper panel shows comparison of fibroblast morphology of the patient, her parents and control fibroblasts. Images were taken after 2nd, 5th, 7th, 9th and 11thdays incubation under 20x objectives. Lower panel compares number of cells versus time in each study subject and there is no statistically difference between the patient and control samples.
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NG1305-3), and control cultures at 11 days incubation. No morphological differences between the patient and control fibroblasts were observed (Fig. 2). There were no differences in proliferation between patient and control fibroblasts (Fig. 2, S7 Table).
I The flow cytometric analysis of apoptosis in fibroblast cells after three days incubation using FITC-annexin V and 7AAD double staining. Quadrant analysis of the gated cells in FL-1 versus FL-3 channels was from 1,000 events. Annexin V+/7AAD- (lower left quadrant) areas stand for early apoptotic cells, and Annexin V+/7AAD+ (upper right quadrant) areas stand for late apoptotic or necrotic cells.
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The rates of apoptosis were assessed at five and nine days incubation; again, no differences were appreciated between patient and control fibroblasts (Fig. 3).