SLIDE ONE

The zebrafish is a well-elucidated vertebrate model organism. The

gene of interest, KLHL40, has two zebrafish orthologs: KLHL40A
and KLHL40B.

A: The location of KLHL40A/B expression was ascertained through

RT-PCR.

Both at 24 hours and 48 hours post-fertilisation, KLHL40A and B

were expressed in zebrafish. Furthermore, in adult zebrafish,
strong expression of KLHL40A is seen in heart and skeletal muscle
tissue. Similarly, KLHL40B is seen expressed primarily in skeletal
muscle tissue. EF1-alpha, or elongation factor 1 alpha, is used as
the control.

B: Testing the efficacy of the morpholino antisense

oligonucleotides in knocking down KLHL40A/B levels.

Genetic knockdowns are an invaluable tool in reverse genetics.

Here, too, the authors sought to knockdown KLHL40A and B in
hopes of understanding its function in skeletal muscle. This figure
shows an immunoblot in which KDs of 40A, 40B, and both are
shown in comparison to wild type levels. As one can see, protein
levels are greatly diminished in the knockdowns.

SLIDE TWO
Piggybacking off the understanding gained through RT-PCR, the
authors then proceeding to use in situ hybridisation to identify
how the two isoforms KLHL40A and B are expressed in the
developing organism. As the figure A shows, at 16 and 24 hours
post fertilisation, KLHL40A and B expression is restricted to the
skeletal muscle.

To better understand the effects of decreased KLHL40 expression,

the authors decided to use inject the previously-referenced
morpholinos into the zebrafish embryos. The results as seen in B
and C were strikingembryos injected with klhl40a-MO, klhl40b-
MO, and klhl40a-MO/40b-MO (double morpholinos) showed a
curved trunk and small head at 48 hours post fertilisation. C: they
categorised the phenotypes at 48 hpf into normal (normal
appearance), mild (curved trunk), and severe (tail defect and
severe development delay) (n 14 111130).

However, this alone would not provide the full picture as to

KLHL40s role in the myofibril. To better understand this role, the
authors immunostained slow myosin heavy chains, found in slow
myofibrils. The image labelled D shows this in more detail. [The
upper panels show labelling with a myosin antibody (F59), and
the lower panels with phalloidin, which labels F-actin.] In
comparison to the wild-type embryos, the embryos treated with
the morpholinos showed disruption of muscle
patterning with an irregular, wavy appearance of the myofibres,
and significant gaps between the fibres. E: they categorised the
phenotypes for these, in a manner similar to C.

SLIDE THREE

The presence of nemaline bodies, which are essentially lateral

polymers of Z-disc units, is the defining feature of the Nemaline
Myopathy. To test the potential link between aberrant KLHL40 and
NEM, the authors took isolated myofibres from the double
morpholinos, and coimmunostained them with both phalloidin
(which labels F-actin) and an antibody against alpha-actinin, a Z-
disc protein. As seen in F, the morpholinos had irregular
patterning, and fibre organisation seemed to be almost entirely
absent. The white arrows in the second morpholino panel point to
small aggregates of alpha-actinin. These aggregates suggest the
presence of nemaline bodies in the morpholino-treated zebrafish.

Further electron microscopic analysis, seen in G, showed that,

indeed, the Z-discs are widened and misshapen in the myofibrils.

These results suggest that Klhl40a and Klhl40b are required for
muscle development and function and that loss of either isoform
in the early embryo is sufficient to impair normal mobility.

SLIDE FOUR
Of course, studies regarding genes involved in disease come full
circle when the phenomenon seen in model organisms can be
related to symptoms in the disease itself.

The table on this slide summarises the clinical features of NEM-

afflicted patients with KLHL40 mutations. These individuals were
from various ethnicities, such as European, Middle and Near
Eastern, or Asian. Eighty-three percent of affected individuals
showed prenatal symptoms, and 76% displayed fetal akinesia or
hypokinesia. Most persons had severe respiratory compromise
(97%), and approximately a third required ventilatory support
(38%). Almost all affected individuals (96%) also had swallowing
problems, and half required tube feeding or gastrostomy.

The authors wanted to see if there was a correlation between

specific KLHL40 mutations and intensity of symptoms. They
looked at individuals with one of the most frequent mutation,
c.1582G>A, and compared their symptomatology with that of
individuals without said mutation. They found that individuals with
this mutation had relatively less severe symptoms, specifically
with respect to ventilator necessity, muscle weakness, and death
rate.

CONCLUSION
The elucidation of disease mechanisms requires a multi-
disciplinary approach. These scientists, in trying to identify
potential genes leading to pathology, found a potential link
between KLHL40 and certain forms of nemaline myopathy. Thanks
to techniques culled from biochemistry, immunochemistry,
bioinformatics, and proteomics, they were able to ascertain the
following:

Mutations in conserved regions of KLHL40 impair protein

stability
KLHL40 necessary for muscle patterning and function
Implication of KLHL40 as contributing factor in autosomal
recessive NEM
KLHL40c.1582G>A mutation leads to relatively milder
phenotype