Below I want to present the significant (at least as I have
tried to understand them) genetic results for the case of Jackson Zuber, as
given to me by his mother Emily. While obviously not intended to be a whole
primer on the genetics, there should be enough detail so that we ourselves, and
any professional geneticist, protein experimentalist or modeler, neurologist,
neurobiologist, or radiologist clinician might extract the fuller picture and
hopefully generate a few additional lines of inquiry.
Jackson is the person the geneticists designate as the
'proband', meaning the one who initiated the study, in this case a one year old
boy. Exome sequencing revealed variants in four genes that were of significant
clinical interest:
NEB (nebulin) c.11450G>A; p.S3817N
PLP1 (proteolipid protein 1) c.194T>G; p.I65S
ERCC6 (excision repair cross-complementation group 1)
c.2924G>A; p.R975Q
PGAP1 (post-GPI attachment to proteins 1) c.2525+4C>T
The doctors logically focused on the PLP1 gene (and
initially diagnosed the associated Pelizaeus-Merzbacher disease or 'PMD')
because it is an X-linked homozygous gene. This means that Jackson only has the
one copy of the gene, and would be particularly suseptibilty to any deleterious
mutations in that gene . The other three genes are on 'autosomal chromosomes',
heterozygous, and would therefore not immediately be prime suspects by virtue
of the fact that another functioning copy of the gene is present…
That is not to say the other genes can be fully discounted,
particularly given the absence of a full genome sequence which would contain
any potential exome regions not analyzed in the exome sequence, including
regulatory regions generally at the beginning of genes. It is also possible
that one gene copy simply does not supply a required threshold level of the
protein, or that the defective protein itself causing some new pathology…
In order to look closer at the specific case of Jackson's
PLP1 variant we first need to decode and disambiguate the genetic notation for
the variant; 'c.194T>G; p.I65S'. I will need to verify what I write here
because errors are readily made.
The initial 'c.' indicates that we are looking at cDNA or
complementary DNA, as we are dealing with exome sequencing info. It refers to
an mRNA transcript's sequence expressed as DNA (GCAT) bases rather than as RNA
(GCAU) bases. Having a 'genomic sequencing' reference (g.) would be a little
more informative here for many reasons, namely, the presence of multiple
transcription initiation sites (promoters), alternative splicing, the use of
different poly-A addition signals, multiple translation initiation sites
(ATG-codons), and the occurrence of length variations. Potentially, if exome
sequencing draws on mRNAs after they are edited, (either in nucleus-specific or
cytosol-specific editing), this would be an issue too, although RNA editing
(post-transcriptional modification of bases, mostly A to G or A to I
substitutions in humans) is quite rare.
As I understand the notation 194T>G, the 194th base pair
position in Jackson's cDNA for PLP1 has a G while most normal cDNAs would have
a T. Because G (like A) is a purine and T (like C) is a pyrimidine, this
substitution is called a 'transversion' as opposed to a 'transition' (which
would occur in the case of a purine to purine or pyrimidine to pyrimidine
switch). Since there are natural mechanisms in the cell which more readily
convert one-ring purines to other purines, or convert two-ring pyrimidines to other
pyrimidines, transitions are significantly more common than transversions.
Because Jackson would have inherited his X chromosome with
the variant PLP1 from his mom, the grandparents were checked and it was found
that gramps had the same variant. Because gramps is asymptomatic the docs more
or less recanted the PMD diagnosis. One possible explanation of this situation
is that gramps could be a 'mosaic'. In other words the mutation was not present
at the level of the sperm but rather arose later in development (as a somatic
mutation), in which case it is possible that the cells that gave rise to
gramps' nervous system have a normal copy of PLP1, and he is therefore quite
normal. Another possibility is that gramps himself inherited the variant but
none the less was able to repair it in the cells of his nervous system.
Although it would be rare, it is also conceivable that
Jackson has the same mutation as gramps but it was independently gained, ie. it
arose again in the bloodline as de novo variant in Jackson. Perhaps not totally
inconceivable when you imagine that whatever genetic or metabolic background
the gramps mutation originated in, a similar background would be expected to be
present in Jackson. More typically the conventional thinking is that
'spontaneous' mutations arise more or less randomly during events like DNA
synthesis when there is some non-negligible error rate during copying that
escapes proofreading mechanisms.
It is also possible that the mutation does not have much
effect in gramps' genetic background, but does have significant effect when
occurring in the context of Jackson's genetic profile, ie. a 'facilitative'
mutation necessary but not sufficient for PMD. One curious feature of PMD is
that up to 70% of the patients have a duplicated PLP1 gene—an extra copy. It
looks like this was explicitly checked for with Jackson, as exome sequencing
wouldn't see it, but he did not have a duplication…
Isoleucine is a hydrophobic amino acid and serine is a polar
and uncharged amino acid. These are fairly different animals altogether and it
is normally assumed that this kind substitution should have some significant
effect on protein structure or function. The question is what effect? In
checking some of the common software tools and databases for this kind of thing
we find that 'PolyPhen2' says the substitution is probably damaging,
'MutationTaster' isn't happy with it either, and it is not recorded in either
ExAC or 1000G.
The canonical membrane structures of some of the various
splice variants of the normal PLP1 protein have been determined well over a
decade ago. It is a highly conserved protein that is virtually identical in
several species from mouse to man. More recently, a few 3-D protein
conformations, the actual crystal structures, have also been determined,
sometimes in combination with other bound proteins. The presumptive membrane
topology is four transmembrane helices, with the position 65 serine (or
thereabouts depending on where the amino acid start count is done) lying at the
extracellular apex of the first membrane helix. While serine can be
phosphorylated in various proteins this may not be likely in the observed
position.
As alternative splicing of the PLP gene yields four
products—the classic PLP and DM20 proteolipids, and the more recently described
proteolipids, srPLP and srDM20, it is important to try to understand how much
of these various products are getting made by various kinds of cells in the
nervous system, and their effects on those cells…
The main question I think, at each instance, is whether
there too much of this protein or not enough, and then also what is the effect
of a poorly functioning, nonfunctioning or otherwise obstructive protein in
each case? To this point, it is known that while transgenic mice that
overexpress the PLP gene exhibit neuronal degeneration and axonal
disintegration, perhaps paradoxically, the absence of PLP/DM20 in PLP null mice
also causes axonal swellings. Because this protein is normally so abundant,
around 50% of the total myelin protein, small changes can have large effects.
It is not known if the serine spot should affect splicing
(but note nearby splice site in picture below), or affect any of the protein's
cross-linked cysteines, or alternatively affect any critical cysteine
palmitoylations, but further study would be needed.
http://medicalxpress.com/news/2016-11-diagnosis-brave-world-genetics-based-medicine.html
[From the link in the post. I believe this to be erroneous as applied to Jackson. T>G means that T has been changed to a G. With this transposition, and the transpositions that follow from it, the paragraph works.] To get a better idea of how this T>G could have arisen I spoke with cell biologist Carl Smythe, a professor in the Department of Biomedical Science at University of Sheffield, and also geneticist Shane McKee, clinical director of the Belfast Health & Social Care Trust.The 'T>G' doesn't necessarily mean that a G has been directly changed into a T in the gene. For example, such a transversion could arise as a consequence of a mutation from a C to A on the non-coding strand. G-A bases can pair quite well (as do some others, although normal pairing is A to T and G to C) without causing major structural issues between the coding (sense) and noncoding (antisense) strands. As a consequence of this, the A would have a T inserted in opposite strand in the next round of synthesis…
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