Investigating

the Developmental
Requirements of Sex Chromosome Genes
Affected in Turner Syndrome
What is Turner Syndrome (TS)? RNA Sequencing Analysis Results
• Rare condition that affects 1/2000 women Zygote (3)
Morula (7) Figure 5: Stages of
8-cell (6) embryonic
• Associated with the missing second X chromosome 2-cell (4)
4-cell (5)
development. Image
• Common complications: short statue, infertility, and congenital cardiac from Xue et. al. 2013,
abnormalities Nature.
Oocyte (1) Pronuclei (2)
• Mosaicism is the term for when only some of the cells are affected by the
abnormality RNA sequencing analysis shows
Color Key

Gene Expression on X Chromosome

Short stature
Learning/behavioral
0 2 4 6
Value
8 RPS4X, TMSB4X, DDX3X, and
Hypothyroidism and disabilities
Hashimoto’s thyroiditis Figure 1: Typical TS RPS4X
TMSB4X
EIF1AX on X chromosome are
Shield-shaped thorax
Constriction of aorta phenotype. Image from DDX3X
EIF1AX
TBL1X highly expressed in the early
Ullrich, O. (1930) Zeitschrift
NLGN4X
Poor breast KDM6A

human embryo.
Widely spaced nipples SOX3
development AMELX
Shortened Für Kinderheilkunde Turner, H. PRKX
KDM5C
ZFX
metacarpal IV
Nail dysplasia
H. (1938) Endocrinology. USP9X
TXLNG
Figure 6: Hash maps showing gene expression
1 2 3 4 5 6 7

Oocyte_SRR893047
Oocyte_SRR893048
Pronuclei_SRR893049
Pronuclei_SRR893050
Pronuclei_SRR893051
Zygote_SRR893052
Zygote_SRR893053
2−cell_SRR893054
2−cell_SRR893055
2−cell_SRR893056
4−cell_SRR893057
4−cell_SRR893058
4−cell_SRR893059
4−cell_SRR893060
8−cell_SRR893061
8−cell_SRR893062
8−cell_SRR893063
8−cell_SRR893064
8−cell_SRR893065
8−cell_SRR893066
8−cell_SRR893067
8−cell_SRR893068
8−cell_SRR893069
8−cell_SRR893070
8−cell_SRR893071
Morula_SRR893072
Morula_SRR893073
Morula_SRR893074
Rudimentary
ovaries/gonadal
of TS candidate genes located on X
Brown spots (nevi)
streak Figure 2: TS karyotype. Image chromosome and PAR region. Darker areas
No menstruation
from NIH Genetics Home show more expression. Units in figure are
Reference. log2(TPM).

Hypothesis qPCR Analysis Results

A precise dosage of certain genes on the sex chromosomes is necessary in certain Relative Expression in Turner Syndrome 45,X
tissues for viable human development. Reduced expression of these genes in the Candidate Genes 46,XX
qPCR confirms
2.5
case of a missing second sex chromosome causes embryonic lethality and common
46,XY
that expression
TS phenotypes. of TS candidate
Relative Expression (log2(TPM))

2
genes KDM5C,
Methods RPS4X, and CD99
1.5
is lower in 45,X
Specific Aim 1 Specific Aim 2 samples.
1
To analyze embryonic tissue and search To find specifically which genes are
for chromosome material using implicated in TS. To do this, we must Figure 7: qPCR results
0.5
fluorescent in situ hybridization (FISH). analyze the expression of genes located normalized to GAPDH.
Then we can look at TS embryos for tissue on the sex chromosomes (TS candidate
that, despite the abnormality, still has genes) and assess deviations in TS. 0

two sex chromosomes. 1. Analyze expression of TS candidate KDM5C (X) RPS4X (X) CD99 (PAR)
1. Develop working FISH protocol (with genes in (non-TS) embryos using
appropriate digestion times that publically available datasets with RNA Conclusions and Future Directions
produces clear images) sequencing data using R programming • We made significant progress on FISH: both refining the List of Genes
2. Optimize X, Y, and chromosome 7 2. Analyze gene expression in TS samples protocol and developing the necessary tools. Potentially
probes on mouse tissue, then work on through quantitative polymerase • Future direction: Conduct FISH on TS tissue to survey Implicated in TS
human tissue chain reaction (qPCR) for mosaicism. RPS4X
3. FISH in TS embryos • This is first study ever to identify TS candidate genes TMSB4X
expressed in early embryonic development; qPCR results
showed loss of second sex chromosome decreases DDX3X
expression of these genes. EIF1AX
FISH Analysis Results • We found that DDX3X underexpression may RPS4Y1
Optimizing FISH protocol on mouse tissue successful: Y chromosome fish contribute to intellectual deficits associated with TS.
DDX3Y
• Future direction: Explore expression of these genes in
probe shows fluorescence in male mouse tissue. EIF1AY
actual TS (45,X) embryos.
A) B) C) D) SLC25A6
Figure 8: Genes that may be underexpressed in Turner Syndrome,
resulting in haploinsufficiency. CD99
88i20 Significance and Impact
(Cy3) • FISH provides a more detailed understanding of TS.
• We can determine which tissues require the second X chromosome.
Y • For example, imagine that through FISH we were to determine that even
in TS samples, liver tissue tends to have two sex chromosomes. We can
Figure 3: A) Diagram of Y chromosome probe. B) Y chromosome probe on male mouse shows one
then reasonably deduce that TS patients who are not mosaic in their liver
fluorescent spot in each nucleus. C) Y chromosome probe on female mouse shows no fluorescent
spots in nuclei. D) No probe on male mouse (control). tissue are not viable.
• Amniocentesis may be able to reveal the viability of a TS fetus prior to birth,
FISH on human embryo tissue successful for x chromosome probe. decreasing large abortion rates currently common in TS.
A) B) C) D) • By knowing which tissues are most affected in TS, recently diagnosed women
will know more about what to expect.
• By understanding TS better, we can make conclusions about the inactivation of the
42M20 second X chromosome in normal women as well.
(Cy3) • RNA sequencing results can be useful information in treatment for TS women.
• Targeted gene therapy and protein replacement can be used to compensate
X for deficiencies, improving quality of life for patients.
• Information about mosaicism can improve understanding of other chromosomal
Figure 4: A) Diagram of X chromosome probe. B) X chromosome probe on male human shows one conditions. For example, similar methods can be used to make discoveries about
fluorescent spot on each nucleus. C) X chromosome probe on female human shows two fluorescent
spots in each nucleus. D) No probe on male human (control).
Down Syndrome.

Additional Notes on Methodology Summary

• We developed experimental tools that help make it possible to test the hypothesis in the
• Embryos are good choice for models because we can sample all organs and tissues.
future by analyzing tissue of TS patients to search for mosaicism. To do this, we used a
• Probes chosen target DNA near centromere, which is advantageous since the centromere
method known as fluorescent in situ hybridization, which uses a probe made of DNA to
stays stable over time.
bind onto targeted genetic material, in this case the X and Y chromosomes.
• Embryos used for FISH were obtained from Human Developmental Biology Resource.
• We also used RNA-sequencing analysis to find genes located on the sex chromosomes
• Embryonic data analyzed for Specific Aim 2 was sequenced by Xue et. al.
that are highly expressed in normal (non-TS) embryo samples. TS patients lack a double
• qPCR results were analyzed using Delta Delta CT Method.
dosage of these genes by nature of only having one sex chromosome. Through
• Had to perform reverse transcription on RNA prior to qPCR.
quantitative polymerase chain reaction, we confirmed that TS samples do not express
these genes as highly as non-TS samples.
All figures made by student unless otherwise noted.