Newcastle upon Tyne - Referral Centre for Limb Girdle Muscular Dystrophy
Funded through the National Specialist Commissioning Advisory Group (NSCAG), Department of Health; and the Specialised Health Services Commission for Wales
We now live in a time when molecular techniques can provide us with a more accurate diagnosis than was possible in the past from clinical assessment alone. Conditions that appeared very similar are now known to be caused by defects in different genes. Typical examples are the limb-girdle muscular dystrophies (LGMDs). As the name suggests, these are progressive diseases of muscle that produce weakness in a limb-girdle distribution. The severity can range from an onset in early childhood, reliance on a wheelchair by early teenage years and death in late teens or twenties; to an onset in adult years that is only slowly progressive with a normal life span. The LGMDs are now known to be caused by at least five genes with dominant inheritance (LGMD1A-1E) and ten genes that are inherited in a recessive manner (LGMD2A-J).
In 2001, the Government, in the form of the National Specialist Commissioning Advisory Group
(NSCAG) in the Department of Health, initiated central funding for a "Diagnostic and Advisory
Service for Rare Neuromuscular Diseases" for patients in England. The idea was to co-ordinate what was being undertaken on an academic 'research' basis in several parts of the country, bringing the expertise together under the umbrella of a unified NHS service.
Four groups of diseases are currently covered, all with involvement of multiple genes:
* Limb Girdle Muscular Dystrophies in Newcastle upon Tyne
* Congenital Muscular Dystrophies at Imperial College/Hammersmith Hosp. in London
* Ion Channel Disorders at the National Hospital/Institute of Neurology in London
* Congenital Myasthenic Syndromes in Oxford
Achieving an accurate diagnosis for a patient is important because useful drug therapies exist for some neuromuscular conditions (like the myasthenic syndromes or the muscle channelopathies). For all the inherited diseases the correct diagnosis is essential for genetic counselling within the family, and can provide an alert for potential clinical complications (some of the LGMDs are associated with heart problems or respiratory difficulties, for example). A definitive diagnosis may influence the prognosis and provide a family with a better idea of what to expect in the future and help to formulate their plans. Understanding that a wheelchair may be required in the future might influence a decision to move to a bungalow, for example.
Knowing what is 'wrong' often provides a psychological boost too - people appreciate being able to put a name to their disease. Finally, although forms of treatment are currently very limited for muscular dystrophies, trials towards gene therapy in some forms of LGMD are underway in the USA and Europe. Knowing which gene is at fault in any particular family is clearly the first step if and when gene therapy does become a serious possibility.
NSCAG Secretary, Muscle Immunoanalysis Unit
Lower Ground Floor, Dental Hospital, Richardson Road, Newcastle upon Tyne, NE2 4AZ
phone: 0191 282-0841 fax: 0191 222-5227 email n.scag@ncl.ac.uk

What is on offer for patients with Limb-Girdle Muscular Dystrophy (LGMD) in Newcastle ?
Different levels of service can be provided according to the needs of the patient and his referring clinician, but the basic levels of NSCAG referral are:
(1) In the first option, a patient may be admitted to a hospital in Newcastle for specialised clinical investigations, removal of a muscle biopsy and subsequent immunoanalysis, and gene mutation analysis.
(2) In the second option the patient attends as an outpatient only (the clinic is very close to the railway station), protein analysis is undertaken on a previously stored biopsy, and DNA analysis is performed.
(3) In the third option, the patient doesn't travel to the referral centre. Protein analysis is undertaken using a frozen muscle biopsy taken previously somewhere else and stored, and these investigations guide and focus subsequent genetic analysis on DNA from a blood sample.
Unfortunately, it is not possible to undertake DNA analysis alone for the LGMDs, without protein analysis, because the number of possible genes for study is so large.
Advice and appropriate information on physiotherapy will be also be available.
Although patients will be responsible for travelling to Newcastle, all the subsequent services are
FREE if they live in England, Scotland, or Wales. We do ask, however, that a Pre-referral Form is completed for each patient and returned with the muscle biopsy sample. If you need the biopsy results "Urgently" (for a genetic counselling appointment, for example), please tick the appropriate box on the front of the form and we will endeavour to provide a preliminary diagnostic opinion within 3 weeks. Please note that we are not able to accept any samples from patients that might be considered "High Risk".
Further information and copies of the Pre-referral Forms are available from the service administrator, Susan Robinson, whose contact details are below.
Telephone 0191-282 0841
Susan Robinson
NSCAG Administrator
Muscle Immunoanalysis Unit
Lower Ground Floor
Dental Hospital
Richardson Road
Newcastle upon Tyne NE2 4AZ
Fax 0191-222 5227 email n.scag@ncl.ac.uk

Newcastle upon Tyne - Referral Centre for Limb Girdle Muscular Dystrophy
Funded through the National Specialist Commissioning Advisory Group (NSCAG), Department of Health; and the Specialised Health Services Commission for Wales
The Limb-Girdle Muscular Dystrophies (LGMDs) are a group of diseases that are clinically very heterogeneous. Although different sub-types of LGMD have characteristic patterns of muscle involvement, there are a number of other diseases that might be considered for differential diagnosis. All involve different genes, and analysis of their protein products may indicate which gene is at fault.
DOMINANT LGMDs
LGMD1A
LGMD1B / AD-EDMD
LGMD1C
LGMD1D
LGMD1E
RECESSIVE LGMDs
LGMD2A
LGMD2B / MM
5q TTID
1q11 LMNA
3p25
6q22
7q
CAV3
?
?
Myotilin
Lamin A/C
Caveolin 3
LGMD2C
LGMD2D
LGMD2E
LGMD2F
15q15
2p13
13q12
17q21
4q12
5q33
CAPN3
DYSF
SGCG
SGCA
SGCB
SGCD
Calpain 3
Dysferlin

-Sarcoglycan

-Sarcoglycan

-Sarcoglycan

-Sarcoglycan
LGMD2G
LGMD2H
LGMD2 I
LGMD2J
17q11
9q31
19q13
2q31
TCAP
TRIM32
FKRP
TTN
Telethonin
Trim 32
Fukutin-related Protein
Titin
OVERLAPPING CLINICAL CONDITIONS TO BE CONSIDERED
DMD / BMD / Carrier
EDMD
Xp21
Xq28
DMD
EDM
CMD 6q22 LAMA2
Dystrophin
Emerin
Laminin

2 chain (merosin)
Bethlem myopathy 21q22
FSHD 4q35
COL6A1-3
?
Collagen VI
Antibodies to the different proteins are used to help locate the primary genetic defect

Too many muscular dystrophy genes exist for genetic analysis to be undertaken on them all.
In order to limit the choice, and help focus the genetic analysis, we undertake immunolabelling of muscle biopsies using unfixed frozen sections and western blots. The muscle biopsies are usually taken as part of the routine diagnostic work-up, and most of the target proteins are not expressed in more accessible tissues (like blood). A panel of antibodies are used in each technique: some reveal primary genetic defects while others may show a pattern or profile of secondary alterations that contribute to a suggested diagnosis.
Protein
Dystrophin (N-term)
Dystrophin (rod)
Dystrophin (C-term)

-Sarcoglycan

-Sarcoglycan

-Sarcoglycan

-Sarcoglycan
Calpain 3 (exon 1)
Calpain 3 (exon 8
Dysferlin (exon 11-12 junction)
Dysferlin (exon 53)
Telethonin
Caveolin 3
Emerin
Lamin A/C
Collagen VI
Laminin

2 chain (80kd)
Laminin

2 chain (300kd)
Laminin

5 chain
Laminin

1 chain
Laminin

1 chain

-Spectrin

-Dystroglycan

-Dystroglycan
Desmin
Utrophin
Myosin heavy chain (neonatal)
Sections
Yes
Not routinely
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Not routinely
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Not routinely
Yes
Blots
Not routinely
Not routinely
Purpose
Not routinely 1 DMD / BMD / carriers
Yes
Yes
DMD / BMD / carriers
Yes
Not routinely
Yes
Not routinely
Yes
Yes
Yes
Not routinely
Not routinely
Not routinely
DMD / BMD / carriers

-Sarcoglycanopathy (LGMD2D)

-Sarcoglycanopathy (LGMD2E)

-Sarcoglycanopathy (LGMD2C)

-Sarcoglycanopathy (LGMD2F)
LGMD2A
LGMD2A
LGMD2B / Miyoshi myopathy
LGMD2B / Miyoshi myopathy
LGMD2G
LGMD1C, Rippling Muscle Disease
Not routinely
Not routinely
Not routinely
X-linked EDMD
AD-EDMD, LGMD1B (not usually reduced)
Pos.

in Bethlem myopathy and Ullrich CMD
Yes Merosin-deficient CMD, some LGMDs eg LGMD2I
No
No
No
No
Not routinely
Yes
Not routinely
Not routinely
Merosin-deficient CMD
Often upregulated when

2 down

in some LGMDs (eg 2I), and dominant myopathies
Control for the other chains, pos.

in LGMD1A
Membrane integrity
Positive control (reduced in DMD/BMD)
Pos.

in some CMDs and LGMDs
Accumulates in desmin myopathies
Upregulated at membrane when dystrophin

Expressed in regenerating fibres
1 Not routinely: can be done, but not part of standard panel for efficiency/economy or other reasons
No: not possible because antibody doesn't work in this technique

Newcastle upon Tyne - Referral Centre for Limb Girdle Muscular Dystrophy
Funded through the National Specialist Commissioning Advisory Group (NSCAG), Department of Health; and the Specialised Health Services Commission for Wales
Note: We cannot accept samples unless this is completed.
The results and advice we are able to give you will be generated using a combined approach incorporating
 Clinical information
 Specialised analysis of a muscle biopsy
 Analysis of DNA from a blood sample
These are needed to offer an informed opinion because of the heterogeneity within this group of disorders.
Three levels of service are offered. Please indicate which you require below and then fill in
ALL of the form on the reverse side. Return form to: NSCAG Secretary
Muscle Immunoanalysis Unit, Lower Ground Floor
General Enquiries to Susan Robinson (NSCAG Sec) Dental Hospital, Richardson Road,
0191 282 0841 Newcastle upon Tyne NE2 4AZ
If you wish to discuss the case before deciding which level of service you prefer please contact
Dr Kate Bushby (email kate.bushby@ncl.ac.uk or telephone 0191 241-8737), or
Dr Richard Charlton (email richard.charlton@nuth.nhs.uk or tel: 0191 282 0849)
L
EVEL OF
NSCAG
REQUEST
Tick
□
1 Clinical assessment + rebiopsy (inpatient stay) plus biopsy and DNA analyses
□ 2 Clinical assessment (outpatient appointment) plus biopsy and DNA analyses
□ 3 Biopsy and DNA analyses
□
Please tick here if you need the preliminary biopsy results "Urgently". By what date?_______________
Please note that we cannot undertake DNA analysis alone due to the large number of possible genes involved
NSCAG Secretary, Neurobiology Department, University Medical School, Newcastle upon Tyne NE2 4HH phone:
0191 222-6973 fax: 0191 222-5227 email n.scag@ncl.ac.uk

Patient name __ ____________________________________________ Date of Birth _________________M / F ______
Address ______________________________________________ Hospital ref. __________________________
________________________________________________________ NHS number _________________________
_________________________________________________________
Patient Post Code (essential)
_______________________
Date recd __________________________
Biopsy ID H ________________________
Referring clinician ___________________________________________
Address for _____________________________________________ correspondence
NCL Hosp. ID ______________________
__________________________________________________________ please leave blank for admin
_________________________________________________________________________________________________
Specific reason for referral: _________________________________________________________________________
Pre-referral details (MUST BE PROVIDED IN ALL CASES)
Delete
Age at onset ___________________ Predominant pattern of muscle involvement: proximal / distal
Other comments _______________________________________ Limbs more affected: upper / lower
_____________________________________________________ Contractures? yes / no
______________________________________________ Which joints?___________________________________
Current functional level:
□
ambulant and able to climb stairs
□
unable to climb stairs
□
uses wheelchair full time
Other affected family members?
_________________________________________________________________________
CK level ________________ at age _______ yrs Results of investigations to date:
Upper normal limit in your labs = ________________ iu/l
EMG result:____________________________________________________________________________________
Muscle biopsy: Date taken____________________________ (or age at biopsy ____________________ ) if exact date unavailable
Has consent for processing and storage of the muscle sample been obtained? YES / NO
Results: Histology ________________________________________________________________________________
Please send a copy of report, if available
Immunohistochemistry ____________________________________________________________________________

Newcastle upon Tyne - Referral Centre for Limb Girdle Muscular Dystrophy
Funded through the National Specialist Commissioning Advisory Group (NSCAG), Department of Health; and the Specialised Health Services Commission for Wales
8.
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4.
5.
1.
NOTE: We are not able to accept samples designated, or suspected, as being "High Risk" (i.e. from patients with HIV, hepatitis, CJD, polio etc)
You must have permission from a member of our staff BEFORE you send a specimen. If you send email or leave a message on our answer phone, DO NOT send samples until a staff member contacts you and confirms that the sample can be sent. We accept no responsibility for the safety of samples sent without the prior consent of our unit.
2.
TEL: 0191 28 20847/20846 or 0191 282 0841
When you send the samples, please include a covering letter with the names and addresses of any additional people (e.g. GPs) who will require copies of letters or reports. More clinical information is always helpful. Please send a copy of the histopathology report, if available.
3. An unfixed frozen muscle sample, in transverse orientation, measuring approximately 3mm x 3mm x
3mm (25 mg) is the minimum required for immunoanalysis.
If a separate snap-frozen piece of the same size is not available for blotting, sections will be cut from the block and processed before the rest of the sample is passed on for western blotting.
6.
The muscle sample must be sent in dry ice, using a courier (TNT or FedEx – not Royal Mail special delivery ) for ‘door to door’ delivery.
Pack the muscle sample in a thick-walled (e.g. 2") polystyrene container, preferably enclosed in a cardboard box. Thinner walled boxes, without the outer cardboard support, are too often damaged during transit! Fill with sufficient dry ice to last 48 hours (e.g. several kg). Do not try to pack the dry ice with polystyrene chips - the chips mix with the dry ice during transit, effectively insulating the sample from the dry ice and causing it to thaw more rapidly.
Address the package to: Mr Keith Davison
NSCAG Biopsies
Muscle Immunoanalysis Unit
Lower Ground Floor (Room 2.026)
Dental Hospital,
Richardson Road
Newcastle upon Tyne NE2 4AZ
(N.B. not Neurology)
Put address labels on more than one side because the courier usually puts his label on top.
Please DO NOT include specimens for us in packages to other departments (e.g. The Mitochondrial
Service). Please do not forward specimens to us through other parties.
Use a courier service that will deliver the next morning. The Hospital delivery point is closed after
1pm.
10. Do not send samples to arrive on a Friday. Delays may well move the delivery of your package into the weekend. Our unit is not staffed at these times and your sample will thaw and be lost.
NSCAG Secretary, Muscle Immunoanalysis Unit
Lower Ground Floor, Dental Hospital, Richardson Road, Newcastle upon Tyne, NE2 4AZ
phone: 0191 282-0841 fax: 0191 222-5227 email n.scag@ncl.ac.uk

4.
5.
2.
3.
1. When you send the samples, please include a covering letter with the names and addresses of any additional people (e.g. GPs) who will require copies of letters or reports. Indicate when the muscle biopsy was sent.
Blood samples must be fresh (i.e. collected within the previous 24h).
Blood samples must be collected unclotted into EDTA tubes.
Ideally, 2 x 10ml samples should be sent.
Send at ambient temperature via First Class mail for next day delivery.
6. Do not send samples for delivery on a Saturday. The best days to send samples are
Monday to Wednesday.
7.
8.
Samples must be clearly labelled with patient name, date of birth, and post code.
Send to: NSCAG LGMD Samples
Diagnostic Molecular Genetics Unit
Institute for Human Genetics
International Centre for Life
Central Parkway
Newcastle upon Tyne
NE1 3BZ
We are able to undertake analyses for patients outside England, Scotland or Wales, but charges will have to be made (please enquire).