ØFacioscapulohumeral muscular dystrophy
ØSpinal muscular atrophy


~Half of the human genome consists of repetitive DNA, significant proportion is organized in tandem
arrays.
•
•Repeat unit sizes 1- 4 nucleotides and spanning less than 100 bp are typically defined as
microsatellite repeats.
•Repeat unit sizes 10 - 40 nucleotides covering several hundreds of bp are referred to as
minisatellite repeats.
•The term midisatellite repeats has been proposed for loci containing repeat units of 40 - 100
nucleotides that can extend over distances of 250–500 kb.
•Macrosatellite repeats are the largest class of repeat arrays with unit sizes of >100 nucleotides
and can span hundreds of kb of DNA.
•Repeat sequences in the human genome

[USEMAP] An external file that holds a picture, illustration, etc., usually as some form of binary
object. The name of referred object is Figure_Seattle.jpg.
•Facioscapulohumeral muscular dystrophy, FSHD
•D4Z4 size: 3300 bp
• Normal DNA:
•11-100 D4Z4
•(36 000-330 000 bp)
• FSHD patients:
•1-10 D4Z4
•(<33 000 pb)
•The third most common muscular dystrophy after Duchenne muscular dystrophy and myotonic dystrophy
•The prevalence ∼1:8 000 – 22 000
•AD inheritance
•FSHD locus: 4q35, deletion of macrosatelite repetitive sequence D4Z4

• Disease onset - the second decade of life - initially weakness of shoulder and facial muscles.
• The spectrum of disease severity is wide, ranging from mildly affected to severely affected
wheelchair-bound individuals.
medwinging fshsm noc20407f2
http://upload.wikimedia.org/wikipedia/commons/thumb/d/da/Fshd.jpg/220px-Fshd.jpg
•FSHD, clinical manifestation

• Two allelic variants of 4q35 were identified - 4qA and 4qB.
• FSHD is associated with deletion of D4Z4 on 4qA (FSHD1).
• 5% of FSHD patients do not have D4Z4 deletion on 4q35 but 4qA allele is present (FSHD2).
F1
•FSHD, 4qA and 4qB alleles

•D4Z4, 290 CpG - candidates for DNA methylation (modification of DNA associated with chromatin
condensation and gene silencing)
•DNA methylation of D4Z4 is significantly reduced in
• FSHD1 chromosome (D4Z4 deletion)
• FSHD2 chromosome (without D4Z4 deletion, both 4q35 chromosomes)
•FSHD, DNA methylation

fig1
•Histone modifications
•Chromatin - DNA, histones and other chromosomal proteins; a major function of chromatin is
packaging of DNA in the nucleus.
•Histones may undergo posttranslational modifications (acetylation, methylation, phosphorylation
and ubiquitination).
•Specific histone modifications are associated with either transcriptional activation or
transcriptional repression. Methylation at lysine residues 4, 36 and 79 of histone H3 has been
correlated with transcriptional activation. In contrast, methylation at lysine residues 9 and 27 of
histone H3 has been linked to heterochromatin and gene repression.

nrg2270-f5
•FSHD is associated with changes of DNA methylation and histone modification (H3K9me3).
•
• Normal allele 4q35:
•DNA methylation, H3K9me3
•
• FSHD1 chromosome (D4Z4 deletion):
•DNA hypomethylation,
•H3K9me3 ↓
•
• FSHD2 chromosome:
•DNA hypomethylation,
•H3K9me3 ↓ (both chromosomes)
•FSHD, epigenetic modifications

•The DUX4 gene located within D4Z4 unit. FSHD chromosomes, the last copy of DUX4 splices to a third
exon located in the region immediately flanking the repeat and stabilizing the transcript owing to
the presence of a polyadenylation signal (association of FSHD and 4qA).

•Schematic of the FSHD locus.
•(a) The D4Z4 repeat (triangles) is located in the subtelomere of chromosome 4q and can vary
between 11 and 100 copies in the unaffected population. This repeat structure has a closed
chromatin structure characterized by heterochromatic histone modifications (dense springs), high
DNA methylation levels (closed circles) and complex bidirectional transcriptional activity (gray
arrows). Candidate genes DUX4, FRG2, FRG1 and ANT1 are indicated.
•(b) In patients with FSHD, the chromatin structure of D4Z4 adopts a more open configuration (open
springs and open circles) leading to inefficient transcriptional repression (black arrows) of the
D4Z4 repeat.
•(c) The DUX4 gene is located within each D4Z4 unit. On permissive chromosomes, the last copy of
the DUX4 genes splices to a third exon located in the region immediately flanking the repeat and
stabilizing the transcript owing to the presence of a polyadenylation (polyA) signal.
•
•Silvère M van der Maarel, Trends in Molecular Medicine May 2011

•FSHD1, FSHD2: the D4Z4 repeat array adopts a more open chromatin configuration leading to the
expression of DUX4 mRNA. This mRNA is stabilized owing to the presence of a polyadenylation signal
immediately distal to the D4Z4 repeat array. The DUX4 mRNA encodes for a nuclear double-homeobox
protein that when expressed in muscle induces cell death.

•A unifying mechanism for FSHD.
•Upon (a) contraction of the D4Z4 repeat (FSHD1) or by (b) a yet unknown mechanism (FSHD2,
phenotypic FSHD), the D4Z4 repeat array (triangles) adopts a more open chromatin configuration
(orange > green dots) leading to the leaky expression of DUX4 mRNA. On permissive chromosomes, this
mRNA is stabilized owing to the presence of a canonical polyadenylation signal immediately distal
to the D4Z4 repeat array. (c) Nonpermissive chromosomes do not have this polyadenylation signal and
therefore DUX4 mRNA becomes rapidly degraded. The DUX4 mRNA encodes for a nuclear double-homeobox
protein that when expressed in muscle induces apoptosis.
•
•Silvère M van der Maarel, Trends in Molecular Medicine 2011

• D4Z4 contains the open reading frame of a double-homeobox transcription factor DUX4.
• 4qA, 4qB; the difference between 4qA and 4qB - the presence of a DUX4 polyadenylation signal;
full-length DUX4 is produced from the last D4Z4 unit in early development and suppressed during
cellular differentiation; in differentiated tissues, the D4Z4 array is associated with DNA
methylation and H3K9me3.
• In FSHD, the expression of the full-length DUX4 transcript is not completely suppressed in
skeletal muscle and results in expression of  the full-length DUX4 mRNA and protein inducing death
of muscle cells.
•FSHD, pathogenesis

rflp
•FSHD, DNA diagnostics


•Genomic map of the facioscapulohumeral muscular dystrophy locus region containing 4qA-defined and
4qB-defined 4qter subtelomeres.
•J Med Genet 2007;44:215-218
• Pacienti s FSHD: detekce EcoR1 fragmentu < 43 000 b
•(< 10 repetic)
•
• Kontrola: detekce EcoR1 fragmentu 43 000 - 340 000 (11-100 repetic)

•The SMCHD1 gene - Structural Maintenance of Chromosomes flexible Hinge Domain containing 1
•The SMCHD1 protein is associated with the D4Z4 array in skeletal muscle cells, this association is
required to maintain array silencing.
•FSHD2 occurs in individuals who inherited  normal-sized D4Z4 array on 4qA and a SMCHD1 mutation.
•FSHD2
•J.R. Lupski, Nature Genetics 2012

•The disease mechanisms of FSHD1 and FSHD2 - D4Z4 chromatin relaxation and DUX4 expression
•FSHD1 and FSHD2 require inheritance of two independent genetic variations: the DUX4 gene with a
polyadenylation signal (4qA) and a second genetic variant.
•FSHD1, FSHD2
•For FSHD1, a second genetic variant is contraction of the D4Z4 array.
•For FSHD2, a second genetic variant is
•L.H.Wang, Neurol Neurosci Rep (2016) 16: 66

•In 1990, the gene locus responsible for FSHD was mapped to 4q35.
•In 1992, contraction in the number of macrosatellite repeats at 4q35 was identified as the genetic
defect.
•In 2010, the DUX4 gene, a copy of which is located in each macrosatellite repeat D4Z4, was found
to be aberrantly expressed.
•FSHD, history
•In 2012 and 2016, mutations in two other genes (SMCHD1, DNMT3B) on different chromosomes resulting
in decreased heterochromatin condensation and subsequent DUX4 de-repression were described in small
percentage of FSHD families (5%) with a normal D4Z4 repeat size.
•The molecular mechanism leading to FSHD is complex and not yet adequately understood.
•L.H.Wang, Neurol Neurosci Rep (2016) 16: 66

•The central component of FSHD molecular pathogenesis is the de-repression of the DUX4
transcription factor, which is present in each D4Z4 repeat (orange triangle). At least one D4Z4
repeat must be present. FSHD is only manifested in chromosomes carrying a haplotype that contains a
functional polyadenylation site (green rectangle) just distal to the last D4Z4 repeat. Decreased
DNA methylation (red circles) is associated with a more open chromatin structure (open orange
triangle). These factors allow for transcription of the DUX4 gene and stabilization of DUX4 mRNA
and ultimately expression of the transcription factor that is normally repressed.
•L.H.Wang, Neurol Neurosci Rep (2016) 16: 66

•Marked telomere shortening occurs in myoblasts between the neonatal and adult growth stages (in
patients with muscular dystrophy more extensive from the reason of pathological degeneration and
regeneration of skeletal muscles).
•Telomere position effects - reversible silencing of genes near telomeres.
•DUX4 expression is upregulated by telomere shortening in cells from patients, in some cases by
100-fold between cells with long and short telomeres. The effect is progressive with decreasing
telomere length.
•Stadler G., Nature structural & molecular biology 2013
•FSHD, Telomere position effect

•Autosomal recesive disease
•The prevalence: 1: 6 000 – 10 000
•The second most frequent fatal disease with autosomal recesive inheritance (after cystic fibrosis)
•Characterised by degeneration of alpha-motor neurons
•
3501716680_17c4df3759 gwendolyn motor-unit-somatic-motor-neuron
•Spinal muscular atrophy (SMA)

•4 clinical groups on the basis of age of onset and clinical course:
•Type I (Werdnig-Hoffmann), characterized by severe, generalized muscle weakness and hypotonia at
birth or within the first three months. Death from respiratory failure usually occurs within the
first two years.
•Type II (intermediate form), with clinical manifestation starting 6–18 months after birth and life
expectancy of 2–30 years; children with type II SMA are able to sit, although they cannot stand or
walk unaided.
•Type III (Kugelberg-Welander disease), first impacts are typically observed after the second year
of life; patients often get wheelchair-bound within or after adolescence.
•Type IV, symptoms emerging during adulthood.
5865074 Dominick and Oliver
•SMA, clinical manifestations

•More than 95% of all SMA cases are due to a homozygous deletion of the SMN1 gene (Survival of
Motor Neuron 1) located on chromosome region 5q12-5q13.38. This region contains a 500-kb inverted
duplication.
•The SMN1 and SMN2 genes differ in only a few nucleotides (none of which affect the encoded protein
sequence).
•The number of SMN1 and SMN2 genes may be different.
Schematic representation with localization of genes on chromosome 5q13
•SMA, the SMN1 gene

•Human Molecular Genetics, 2010, Vol. 19
•SMN1: TTC (Phe)
•SMN2: TTT (Phe)
•SMA, the SMN1 and SMN2 genes

•Schematic of the human SMN locus. The human SMN genes, SMN1 and SMN2, are located in close
proximity on chromosome 5. The SMN2 locus is likely derived from a recent duplication event of a
genomic region spanning 500 kb which contains additional genes and microsatellite markers. The SMN
genes comprise nine exons and eight introns and encode an identical protein product. A silent C–T
transition in exon 7 of SMN2 alters a critical exonic splice enhancer and results in a strong
reduction of exon 7 inclusion during splicing. Consequently, 85% of the mature mRNA lacks exon 7
(D7), highlighted by the RT–PCR in the bottom panel. The truncated protein is defective in SMN
self-association and is degraded rapidly.
•Human Molecular Genetics, 2010, Vol. 19, Review Issue 1, R111–R118

•No phenotype-genotype correlations were initially observed -  the homozygous deletion of SMN1 is
present in 95% of SMA patients, independent of the SMA type (I-IV).
•The SMN2 copy number modifies the severity of disease.
•The majority of patients with SMA I have one or two copies of SMN2; most patients with SMA II have
three SMN2 copies; and most patients with SMA III have three or four SMN2 copies.
smadel3
•SMA, phenotype-genotype correlations

•Splicing architecture of exon 7 of the human SMN1 and SMN2 genes.
•The diagram represents exon 7 (yellow box) and its flanking intronic regions (lines). Elements
inhibiting exon 7 inclusion are shown in red, whereas the positive elements are represented in dark
blue. The branch point (BP) and polypyrimidine tract (PP tract) are indicated in light blue.
SF2/ASF and Tra2/β1 bind to the exonic splicing enhancers SE1 and SE2, respectively.
•The recognition of SE1 by SF2/ASF is prevented in SMN2, due to the C→U transition. This sequence
alteration also creates a heterogeneous nuclear RNPA1-dependent splicing silencer. Exon 7 is
extremely short (only 54 bp).
•RNA Biology 7:4, 430-440; 2010

•SMN2 - the loss of amino acids that are encoded by exon 7 results in the production of SMN protein
with severely decreased oligomerization efficiency and stability. The SMN monomers are rapidly
degraded. Thus, loss of SMN1 results in reduction of SMN levels in most tissues.
•Nat Rev Neurosci. 2009 August ; 10(8): 597–609.

•SMN1 and SMN2 genes: structure and splicing
•The SMN1 and SMN2 have identical gene structure and are 99.9% identical at the sequence level. The
essential difference between the two genes is a single nucleotide change in exon 7 (C or T as
indicated). This single nucleotide change affects the splicing of the gene. Thus the majority of
SMN transcripts from SMN2 lack exon 7 whereas those from SMN1 contain exon 7. However, because SMN2
does produce some full-length SMN it can be viewed as a gene with reduced function but not loss of
function. The loss of amino acids that
•are encoded by exon 7 results in the production of SMN protein with severely decreased
oligomerization efficiency and stability. The SMN monomers are rapidly degraded. Thus, loss of SMN1
results in reduction of SMN levels in most tissues. The SMN oligomer is represented as an octomer
based on gel filtration of SMN complexes formed in vitro.
•
•Nat Rev Neurosci. 2009 August ; 10(8): 597–609.

•
• The SMN protein has an essential function involving production of small nuclear
ribonucleoproteins (snRNPs).
•
•  Each snRNP particle is composed of small nuclear RNA (snRNA) of approximately 150 nucleotides,
several Sm proteins and a number of specific proteins that are unique for each snRNP.
•
•snRNPs are active in recognizing and removing introns from pre-mRNA in the nucleus.
17_11snRNPsSpliceosomes_L
•SMA, splicing

•SMN function in snRNP assembly: In the cytoplasm: Sm proteins bind to the SMN complex. The SMN
complex is composed of SMN, Gemins 2-8 and Unrip. The snRNA is transcribed in the nucleus and then
transported to the cytoplasm. The SMN complex places the Sm proteins onto the snRNA. The 5´cap of
the snRNA is hypermethylated, allowing the SMN complex with the snRNA to bind snurportin and
importin, which mediates transport of the SMN complex with an assembled snRNP into the nucleus. In
the nucleus, the SMN complex and snRNPs localize to the Cajal body and snRNPs undergo further
maturation.

•Function of SMN in snRNP assembly
•Small nuclear ribonucleoproteins (snRNPs) are active in recognizing and removing introns from
pre-mRNA in the nucleus. Each snRNP particle is composed of small nuclear RNA (snRNA) of
approximately 150 nucleotides, several Sm proteins and a number of specific proteins that are
unique for each snRNP. Survival motor neuron (SMN) functions in the cytoplasm to assemble Sm
proteins onto the snRNAs to produce an active snRNP.
•A) In the cytoplasm the 7 Sm proteins bind to the chloride conductance regulatory protein (pICln).
In vitro studies reveal that pICIn first binds the Sm proteins as two separate complexes: SmB,
SmD3, and SmD1, SmD2. The latter subsequently binds SmE, SmF and SmG44 The protein arginine
methyltrasferase (PRMT5 complex) and PRMT7 methylate the Sm proteins SmB, SmD1 and SmD3. Sm
proteins are released from pICln-PRMT5 complex and bind the SMN complex.
•B1) The SMN complex is composed of SMN, Gemins2-8 and unrip. SMN is shown in the figure as an
oligomer as it has been shown to self-associate and it has been suggested that oligomerization is
critical for SMN function. The exact numbers of SMN monomers in a SMN complex is unknown (it has
been suggested to be an octomer). The Gemins are shown as single units for simplicity as the exact
stoichiometry of the SMN complex has not been determined.
•B2) snRNA is transcribed in the nucleus and then binds the export proteins phosphorylated adaptor
for RNA export (PHAX), Cap-binding complex (CBC), exportin (Xpo1) and rasrelated nuclear protein
GTP (Ran), which transport it to the cytoplasm. In vertebrates, the snRNA is brought into the Sm
protein-bound SMN complex by binding to Gemin5.
•C) The SMN complex places the Sm proteins onto the snRNA. The m7G cap of the snRNA is
hypermethylated by trimethylguanosine sythetase 1 (TGS), allowing the SMN complex with the snRNA to
bind snurportin and importin, which mediates transport of the SMN complex with an assembled snRNP
into the nucleus.
•D) In the nucleus the SMN complex and snRNPs localize to the Cajal body and snRNPs undergo further
maturation. Depending on the cell type and developmental stage, SMN can localize as a separate body
adjacent to the Cajal body.
•
•Nat Rev Neurosci. 2009 August ; 10(8): 597–609.

•SMA is characterised by the degeneration of alpha motor neurons of the anterior horns of the
spinal cord - not all cell types are equally affected in SMA patients.
•The vast majority of functional SMN protein is produced by SMN1 and low level by SMN2 (poor
inclusion of exon 7).
•SMA (homozygous deletion of SMN1): SMN2 is the only source of functional SMN protein, the degree
of exon 7 inclusion becomes critical.
•SMA, splicing

Mouse models:
•Motor neurons express 4-fold less full-length SMN2 mRNA than dorsal horn cells from the same
spinal segment. This difference is due to changes not in SMN2 gene transcription but in the
efficiency of exon 7 inclusion in the SMN2 mRNA.
•SMA (the loss of SMN1): 4-fold difference in the levels of full length SMN2 mRNA is critical for
motor neuron function. Studies showed that two copies of the SMN2 gene produce enough protein for
normal function of most cells except motor neurons.
•SMA, splicing

•Model for the selective vulnerability of motor neurons in SMA.
•Under normal conditions, the SMN2 gene produces smaller amounts of the full-length SMN mRNA and
protein in motor neurons (MN) than in non-motor neurons (non-MN).
•Upon loss of the SMN1 gene (SMA), reduced snRNP levels trigger a negative feedback loop affecting
exon 7 splicing that might contribute to further decreasing SMN expression and enhancing downstream
defects specifically in SMA MNs.
•Ruggiu M., Molecular and Cellular Biology 2012
•SMA, splicing

•MLPA (Multiple ligation dependent probe amplification)
• stanovení počtu kopií SMN1 genu na genom,
• stanovení přenašečství SMA v rodinách s výskytem SMA (1 kopie - přenašeč, 2 kopie - zdravý),
• vytipování pacientů u kterých se bude provádět sekvenční analýza SMN1 genu (1 kopie SMN1).
•95% způsobeno homozygotní delecí SMN1 genu
•5% způsobeno delecí SMN1 genu na jednom chromozomu a bodovou mutací na druhém.
•SMA, DNA diagnostika

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•Denatured genomic DNA is hybridised with a mixture of probes. Each MLPA probe consists of two
oligonucleotides. The two parts of each probe hybridise to adjacent target sequences and are
ligated by a thermostable ligase. All probe ligation products are amplified simultaneously by PCR
using a single primer pair labeled with a fluorescence mark. The amplification product of each
probe has a unique length. Amplification products are separated by capillary electrophoresis.
Relative amounts of probe amplification products reflect the relative copy number of target
sequences.
•The SMA probe mix contains 18 different control probes as well as exon 7 and 8 probes specific for
SMN1 and SMN2.
•Multiplex Ligation-dependent Probe Amplification (MLPA)