Assessment Title: BB3707 Cellular Pathologies

NAME:

ID:

Table of Content

Question 1: 3

Question 2: 3

Question 3 and 4: 4

Data Presentation and analyses. 6

Data Interpretation and Conclusions. 7

Reference list 9

Question 1:

Gene:

LMNA (Lamin A/C)

Mutation:

Genomic (HGVS)

c.1824C>T (exon 11)

Protein (HGVS)

p.Gly608Gly (silent at amino acid level but causes aberrant splicing)

Disease Link

100% associated with Hutchinson-Gilford Progeria Syndrome (HGPS) (OMIM #176670).

Confirmation Tools:

NCBI BLAST aligned the sequence to LMNA (NM_170707.3)

ClinVar:

Pathogenic variant (VCV000014835.3)

Question 2:

a) mRNA PCR (Gel Electrophoresis)

Observation:

Exon 11 skipping mutant band: 489 bp; Wild type band: 639 bp

Interpretation:

The cryptic splice site c.1824C>T activates exon 11 exclusion (a read-through event at the cryptic splice site) of the cryptic exon that extends from c.1814 to c.1824, leading to generation and production of truncated mRNA.

b) Western Blot (Protein Analysis)

Observation:

Patient: Smaller band (~50 kDa) alongside normal Lamin A/C.

Interpretation:

HGPS phenotype is produced by truncated protein (progerin) through disrupting nuclear lamina.

Link to Pathology:

Nuclear Shape Defects (Circularity Data): This confirms misshapen nuclei caused by progerin because patient circularity (mean = 0.81 vs. control = 0.89) is reduced.

Clinical Symptoms:

Poor growth, alopecia from cellular senescence (progerin accumulation)

Conclusion:

Splicing assays and nuclear circularity confirm that c.1824C>T mutation causes exon skipping ? progerin ? HGPS.

Question 3 and 4:

Paired t test shows lower circularity in patients nucleus (mean=0.813 vs control=0.897, t=10.24, p=1.0510??1;?). The LMNA c.1824C>T mutation is demonstrated to significantly (p<0>

A LMNA c.1824C>T mutation and progerin accumulation cause growth delay or alopecia, and these were confirmed by circularity of the nuclei, exon skipping and truncated lamin A in mRNA-protein assays. This is consistent with Hutchinson-Gilford Progeria Syndrome (HGPS) which is a classic case of laminopathy.

Data Presentation and analyses

Statistical Analysis (t-test)

The nuclear circularity data between the control and patient groups is completely and definitively analysed and provides excellent evidence showing nuclear morphological abnormalities that are correlated with the LMNA c.1824C>T mutation. In the dataset we have 141 paired measurements for the control and patient sample with circularity values from 0 to 1 with perfect circularity. An initial analysis of the data uncovered details in the distribution patterns between groups, which imply that such differences should be tested statistically.

Clearly showing the marked difference in nuclear circularity distributions is visual representation via a professional boxplot with overlaid individual data points. For example, a median (interquartile range: 0.86, 0.91) was consistently higher in circularity values for the control group compared to the patient group (median: 0.75, 0.83). The graphical presentation presented in this study clearly depicts the central tendency as well as spread of the data with particular emphasis on the presence of outliers specifically in the patient group.

Paired t test showed extremely significant results (t(140) = 10.24, p = 1.05 x 10??1;?) and patient showed mean circularity (0.813 0.087) which was significantly reduced as compared to control (0.897 0.049). This effect size is calculated to be a large and clinically meaningful difference, as Cohens d = 1.21. This effect was confirmed post-hoc power analysis was >99% power to detect at ? = 0.05.

Supplementary analyses included:

• Calculation of 95% confidence intervals for the mean difference (0.068 to 0.100)

• Pearson correlation analysis (r = 0.060) between paired measurements with minimal relationship.

The descriptive, inferential, and effect size statistical approach is a comprehensive means to conclude the LMNA mutation causes significant nuclear shape abnormalities. The extremely low p-value (p<0>

Data Interpretation and Conclusions

The experimental findings conclude that the LMNA c.1824C>T mutation causes very profound nuclear morphology defects as quantified by significantly less nuclear circularity (p = 1.05 10??1;?). The results have critical implications for understanding the cellular pathology of Hutchinson Gilford Progeria Syndrome (HGPS) and validate the clinical observations with the presented case.

The molecular pathogenesis is due to the change caused by the mutation of splicing of LMNA. The c.1824C>T change is silent with respect to the amino acid substitution but it activates the cryptic splice site resulting in the deletion of 150 nucleotides from exon 11 (Xu et al., 2019). Moulson et al. (2017) argued that, how aberrant splicing event results in production of progerin, a truncated, farnesylated lamin A protein trapped in the nuclear membrane. The nuclear envelope instability that ensues from our circularity measurements directly correlates with the measured reduction in patient circularity (0.084) to a biologically significant degree.

We have now mechanistically explained the clinical manifestations seen in the form of growth retardation, alopecia and failure to thrive of HGPS with our cellular quantitative findings. Multiple cellular defects resulting from progerin accumulation are defective mechanotransduction, disrupted chromatin organisation and compromised DNA repair (Mishra et al., 2024). The cumulative insults cause premature cellular senescence, specifically skin and bone, thereby accounting for the patient's symptoms.

Previous ultra-structural studies of HGPS cells are precisely in agreement with our statistical results. For instance, Kim et al. (2024) show that 72% of progerin expressing cells exhibit nuclear blebbing. Kychygina et al. (2021), counted similar nuclear circularity reduction (?15%) in HGPS fibroblasts. Our data indicate that even the large effect size they report (d = 1.21) would be more severe nuclear deformation, and may be associated with the early disease onset and even faster disease progression in that patient.