SYNOPSIS ON

Phylogenetic Analysis of Facioscapulohumeral Muscular Dystrophy

(FSHD) using MATLAB

Under the supervision of:

Mr. Aashish Katyal

Submitted by:

(Assistant Professor, M.I.E.T)

Anmol Sharma (1306854011)

Arushi Garg (1306854017)

Purvi Bhasin (1306854046)

Department of Biotechnology
Meerut Institute of Engineering and Technology
2016-2017

CONTENT

S.No.
1

2
3
4
5
6

Contents
Introduction
Objective
Review of Literature
Materials and Methodology
Importance of project and expected outcome

Page No.
1-3
4
5
6
7

References

INTRODUCTION

FSHD
Facioscapulohumeral

muscular

dystrophy (FSHMD,

FSHD

or

FSH)originally

named Landouzy-Dejerine is a usually autosomal dominant inherited form of muscular

dystrophy (MD) that initially affects the skeletal muscles of the face (facio), scapula (scapulo)
and upper arms (humeral)Symptoms may develop in early childhood and are usually noticeable
in the teenage years with 95% of affected individuals manifesting disease by age 20 years.

Causes
In more than 95% of known cases, the disease is associated with contraction of the D4Z4 repeat
in the 4q35 subtelomeric region of Chromosome 4.
Seminal research published in August 2010 now shows the disease requires a second mechanism,
which for the first time provides a unifying theory for its underlying genetics. The second
mechanism is a "toxic gain of function" of the DUX4 gene.

Symptoms

Facial muscle weakness (eyelid drooping, inability to whistle, decreased facial

expression, depressed or angry facial expression, difficulty pronouncing the letters M, B,

and P)
Shoulder weakness (difficulty working with the arms raised, sloping shoulder)
Hearing loss
Abnormal heart rhythm
Unequal weakening of the biceps, triceps, deltoids, and lower arm muscles
Loss of strength in abdominal muscles (causing a protuberant abdomen and lumbar

lordosis) and eventual progression to the legs

Foot drop

A progressive skeletal muscle weakness usually develops in other areas of the body as well;
often the weakness is asymmetrical. Non-muscular symptoms frequently associated with FSHD
include subclinical sensorineural hearing loss and retinal telangiectasia.

Types
FSHD Type - 1

More than 95% of cases of FSHD are associated with the deletion of integral copies of a
tandemly repeated 3.2kb unit (D4Z4 repeat) at the region 4q35 on Chromosome 4 of
the human genome, of which a normal chromosome includes between 11-150 repetitions

of D4Z4.
There are both heterochromatin and euchromatin structures within D4Z4 and one putative
gene called DUX4. Inheritance is autosomal dominant, though up to one-third of the
cases appear to be from de novo (new) mutations. The heterochromatin is specifically lost

in the deletions of FSHD while the euchromatin structures remain

If the entire region is removed, there are birth defects, but no specific defects on skeletal
muscle. Individuals appear to require the existence of 11 or fewer repeat units to be at

risk for FSHD.

In addition, a few cases of FSHD are the result of rearrangements between subtelomeric
chromosome 4q and a subtelomeric region of 10q. This location contains a tandem repeat
structure highly homologous to 4q35 Disease occurs when the translocation results in a
critical loss of tandem repeats to the 4q site.

FSHD Type - 2

In 2012, a majority of FSHD2 cases were reported linked to mutations in the SMCHD1
gene on chromosome 18. This leads to substantially reduced levels of SMCHD1 protein,

and subsequently, hypomethylation of the 4q D4Z4 region.

The FSHD2 phenotype arises in individuals who inherited both the SMCHD1 mutations
plus a normal sized D4Z4 region on a permissive 4qA allele. This establishes a
genetic/mechanistic intersection of FSHD1 and FSHD2.

Diagnosis
Since the early 2000s, genetic testing that measures the size of the D4Z4 deletions on 4q35 has
become the preferred mechanism for confirming the presence of FSHD. However, because the
test is expensive, patients and doctors may still rely on one or more of the following tests, all of
which are far less accurate and specific than the genetic test:

Creatine kinase (CK) level

Electromyogram (EMG)

Nerve Conducting Velocity (NCV)

Muscle biopsy.

Treatment
1. Drug Treatment

ACVR2B is a compound identified in 2005/2006 by Johns Hopkins . It increased muscle

mass in a non-Muscular Dystrophy Mouse by up to 60% in two weeks.

MYO-029 is an experimental myostatin inhibiting drug developed
by WyethPharmaceuticals for the treatment of muscular dystrophy. Myostatin is a protein
that inhibits the growth of muscle tissue, MYO-029 is a recombinant human antibody
designed to bind and inhibit the activity of myostatin.

2. Therapies
No approved therapies exist specifically for FSHD.
Based on the consensus model of pathophysiology, researchers propose four approaches
for therapeutic intervention :
o enhance the epigenetic repression of the D4Z4
o target the DUX4 mRNA, including altering splicing or polyadenylation;
o block the activity of the DUX4 protein
o inhibit the DUX4-induced process, or processes, that leads to pathology.

Physiotherapy could improve patients' functional status by providing therapeutic

exercises.

OBJECTIVE

Phylogenetic Analysis of Facioscapulohumeral Muscular Dystrophy (FSHD) using MATLAB.

REVIEW OF LITERATURE

E. Bakker et al. (1996) observed that in the majority of FSHD families the gene defect (FSHD1)
has been localised to chromosome 4q35.Although a number of classical FSHD families have
been reported that exclude this region, indicating locus heterogeneity in at least 5% of FSHD
families.
E. Bakker et al. (1996) stated that the use of indirect methods for diagnostic purposes in FSHD1
has been hampered since its localisation in 1990 because (1) no flanking markers are available,
(2) genetic heterogeneity exists in about 5 to 10% of the families, and (3)homologous
chromosome 10q alleles identified by p 1 3E- 11 occur, whose size interferes with the FSHD
associated deletion fragments, making interpretation of data very difficult.
M. Upadhyay et al. (1997) reported about the detection of sequence divergence between the
KpnI tandem repeat units located at 4q and 10q. They identified a different distribution of
restriction enzyme sites, and that the enzyme BlnI specifically cleaves the 3.3kb repeats derived
from 10q, leaving intact the tandem repeat units at 4q. Thus they inferred a double restriction
enzyme digest (DD) with the enzymes EcoRI and BlnI allows the specific detection of 4q
fragments and should greatly facilitate the molecular diagnosis of FSHD.

M. Upadhyay et al. (1997) described that the presence of a deletion of an integral number of
D4Z4 repeats which do not appear to contain any expressed sequences and are in close proximity
to 4q telomeric sequences has led to the hypothesis that the mechanism underlying the disease
could be position effect variegation (PEV)
M. Upadhyay et al. (1997) observed that the occurrence of a normal size fragment in the FSHD
patients indicates toward another mechanism for the causal of disease

Michael R. Green et al (2002) showed that the 4q35 genes located upstream to the D4Z4 are
inappropriately overly expressed.
Michael R. Green et al (2002) showed that an element within D4Z4 specifically binds a
multiprotein complex consisting ofYY1, a known transcriptional repressor, HMGB2, an
architectural protein, and nucleolin and mediates the transcriptional repression of 4q35 genes.

MATERIALS AND METHODOLOGY

Material Requirement

Kyoto Encylopedia of Genes and Genomes (KEGG) - (http://www.genome.ip/kegg/)

MATLAB- (www.mathworks.com)
o ORF
o PHYLIP

Methodology

Sequence Retrival
Selection of
species for amino
acid selection
ORF Prediction

Multiple Sequence
Alignment
Phylogenetic
Analysis

IMPORTANCE OF PROJECT AND EXPECTED OUTCOMES

The resultant outcomes of the project will be phylogenetic analysis of the genes and proteins
involved in FSHD in humans and other species represented in the form of phylogenetic tree.
The gene (D4Z4 macrosatellite and DUX4 in human beings) sequence that are retrieved from
KEGG database followed by the expression of genes using ORF finder tool in order to identify
the frame consisting of amino acid sequence (that causes the disease). This is followed by the
alignment of the expressed sequence with 10 species. The results would be represented in the
form of phylogenetic tree that will tell about the species in which the disease first occurred.

REFRENCES

BakkerE.,Van der WielenM.J.R.,VoorhoveE.,IppelP.F., PadbergG.W.,FrantsR.R.,

WijmengaC. (1996)Diagnostic, predictive, and prenatal testing forfacioscapulohumeral
muscular dystrophy:diagnostic approach for sporadic and familial cases.

UpadhyayaM.,Maynard J.,RogersM.T.,LuntP.W.,JardineP., RavineD., Harper, P.S.

(1997)Improved molecular diagnosis of facioscapulohumeral muscular
dystrophy(FSHD): validation of the differential double digestion for FSHD.

GabelliniD., Green M.R., TuplerR., Howard Hughes Medical Institute Program in

Gene Funtion and Expression., Program in Molecular Medicine, University of
Massachusetts, Medical School Worcester, Massachusetts Biologia Generale e
Genetica Medica Universita degli Studi di Pavia, Italy (2002) Inappropriate Gene
Activation in FSHD: A Repressor Complex Binda a Chromosoal repeat Deleted in
Dystrophic Muscle