Do mutations in the TBL1X gene alter sensitivity to thyroid hormone?

Congenital central hypothyroidism is characterized by insufficient production
of thyroid hormone (TH) due to inadequate stimulation by thyroid stimulating
hormone (TSH) of an otherwise normal thyroid gland. Central hypothyroidism
occurs either isolated, or in combination with other pituitary hormone
deficiencies. Well-known genetic causes of isolated central hypothyroidism
include mutations in the TSHB, TRHR and IGSF1 genes.

Using X-exome sequencing in three related patients with isolated central
hypothyroidism, a novel mutation was identified in the Transducin *-like
Protein 1, X-linked (TBL1X) gene.

Plasma TH concentrations are continuously monitored and regulated by the
hypothalamic-pituitary-thyroid (HPT) axis, a multi-loop feedback system.
Production of TH is stimulated by TSH from the pituitary, which - in turn - is
stimulated by thyrotropin releasing hormone (TRH) from the hypothalamus. TRH
and TSH are both suppressed by the active form of TH, triiodothyronine (T3),
via so-called *negative regulation'. In this regulation, T3 suppresses hormone
production by inhibiting transcription of the TRH and TSH encoding genes.

Corepressors (CoRs) and coactivators (CoAs) serve as coregulatory factors in
T3-negative regulation for both gene transcription silencing and activation.
The TBL1X product, TBL1, is an essential core subunit of the main CoRs in this
pathway. TBL1 stabilises the binding of CoRs to the DNA for adequate
transcriptional modification. Additionally, TBL1 mediates the dismissal of CoRs
and recruitment of CoAs in the presence of T3. In genes negatively regulated by
T3, including TSHB and TRH, CoRs are critical in activation of gene
transcription in absence of T3. The mechanism of this pathway, and the role of
TBL1 herein, is still unknown.

In addition to the three above-mentioned patients, we found TBL1X mutations in
six other patients with isolated central hypothyroidism from five families. The
phenotype of these patients and several mutation carrying relatives with normal
thyroid function has been extensively evaluated. Besides isolated central
hypothyroidism, 11 of 16 mutation carriers were found to have mild high
frequency hearing loss.

Central hypothyroidism is characterised by a too low plasma free thyroxine
(FT4) concentration in combination with a normal TSH concentration. Although
most mutation carrying patients had FT4 concentrations in the *hypothyroid
range*, several patients had only mild, or no signs or symptoms of
hypothyroidism. Mice expressing a mutated NCoR - another component of the CoR *
were found to have low TH levels, but seemed clinically unaffected. Hereupon,
it was speculated that these mice might be more sensitive to TH, and do well
with lower plasma TH concentrations. Until now, this phenomenon has not been
encountered in humans. With respect to the low FT4 concentrations found in our
patients with mutations in TBL1X, we don*t know if they are harmful for
cellular function.

Therefore, we would like to study the functional consequences of mutations in
TBL1X in human liver cells, by generating such cells from induced pluripotent
stem cells (iPSCs), derived from peripheral mononuclear white blood cells
obtained from patients with a TBL1X gene mutation and their siblings (as
controls). Performing functional studies at the cellular level will make it
possible to test the hypothesis that patients with a (too) low plasma FT4
concentration due to a mutation in TBL1X are not hypothyroid at the target
tissue level because of increased sensitivity to TH. These studies will provide
the answer to the question patients with too low FT4 concentrations due to
TBL1X mutations need to be treated with TH yes or no.

To determine the effect of mutations in the TBL1X gene on TH dependent
signalling in liver cells generated from iPSCs.

Prospective descriptive study. To assess TH dependent signalling in liver
cells, induced pluripotent stem cells (iPSCs) will be generated from peripheral
mononuclear white blood cells and transformed into hepatocytes. This will be
done in collaboration with Prof. Hollenberg and Dr. Wilson, Harvard
University/Boston Medical Center, Boston.
Peripheral mononuclear white blood cells will be isolated from a venous blood
sample and shipped to Boston. To this end, blood will be taken from patients on
one occasion at a regular outpatient clinic visit. The remaining work -
generation of iPSC/hepatocytes and functional studies/gene expression - will be
done in Boston.

Confirmed carriers and non-mutation carrying first- or second-degree relatives
will be invited to the department of paediatrics or internal medicine of the
Academic Medical Center to undergo venous blood collection. As congenital
central hypothyroidism is a rare condition, and many known patients are
children, minors will be included in the study. Venous blood collection and
placement of an intravenous cannula may be uncomfortable and carry a small risk
of bruising and bleeding. The risks involved in participation are negligible
and the burden is considered minimal. There are no other risks or benefits
associated with participation.