Genetic testing

Your doctor can order a genetic test for FSHD. Before seeking a test, consult a genetic counselor to make sure you fully understand the process and have considered how you and your family will respond to the information revealed by the test results.

Commercial genetic tests are now available for FSHD Type 1 and Type 2. Because approximately 95 percent of cases are Type 1, you will be tested for this first. If you test negative for Type 1, you may inquire about obtaining a test for Type 2.  About 2 percent of FSHD cases are still of unknown genetic origin. If your tests rule out FSHD1 and FSHD2, please contact us. Genetic researchers may be interested in collecting DNA samples from you to try to identify new genes that cause FSHD.

Where to Get a Genetic Test

Your doctor will need to requisition the tests at these labs. For a complete list, visit:

These labs do not offer direct patient consultation.

1) University of Iowa Diagnostic Laboratories Iowa City IA Client Services Phone: (866) 844-2522 Mary Sue Otis UI Diagnostic Laboratories Manager Email: marysue-otis@uiowa.edu Phone: (319) 356-3339 Fax: (319) 384-7213

Testing offered: FSHD1 4q35 deletion detection by PFGE and Southern blot analysis & 4qA/4qB allele determination; FSHD2 SMCHD1 sequencing; methylation testing (for FSHD1 and FSHD2). Details and requisition forms to order testing are available at these links:

Additional Testing Offered: Prenatal diagnosis and 4qA/4qB allele testing. Overseas clients can send blood specimens to UIDL via FedEx overnight using this International MD Requisition Form.  The lab requires payment prior to testing. Credit cards accepted.

2) Athena Diagnostics Inc. Worcester, MA Genetic Counselor: Carol Hoffman, PhD MS Email: carol.hoffman@athenadiagnostics.com Phone: (508) 756-2886, Ext. 3135 Fax: (508) 753-5601 Testing offered: FSHD1

3) Children’s Hospital of Eastern Ontario Molecular Genetics Diagnostic Laboratory Ottawa, Ontario, Canada Director: Nancy Carson, PhD, FCCMG Email: carson@cheo.on.ca Phone: (613) 737-7600, Ext. 3282 Fax: (613) 738-4822

4) University of Leiden, the Netherlands Testing for FSHD1 and FSHD2 can be ordered through Leiden University in the Netherlands. It takes about eight weeks for results to be reported. Download the University of Leiden genetic test request form (pdf).

If you have tested negative for FSHD1 The Jain Foundation and Genzyme are partnering to offer free genetic sequencing for limb-girdle muscular dystrophies (LGMD). Their genetic testing includes SMCHD1 mutations (associated with FSHD2).

FAQs about Genetic testing

This decision tree diagram depicts how FSHD genetic testing is carried out. (Reference: University of Iowa)

FSHDTestingWorkflow

How is FSHD genetic testing done?

The genetic test for FSHD1 consists of measuring the size of the DNA segment containing multiple repeats of identical DNA sequences called D4Z4. Molecular scissors (restriction enzymes called EcoRI and Blnl) are used to cut the DNA on either side of the repeats, and the fragments from chromosome 4 are sized. In healthy individuals, both copies of chromosome 4 will have more than 10 repeats of D4Z4, whereas in individuals with FSHD, one copy of chromosome 4 will have only between one and 9 repeats. This shortened, or “contracted”, chromosome must be combined with a “permissive” 4qA polyadenylation site in order for the person to have FSHD.

The genetic test report gives two numbers, one from each copy of chromosome 4, corresponding to the size of the D4Z4 region, measured in “kilobases.” If the D4Z4 region is more than 50 kilobases (>13 repeats), this is in the healthy range. Between 39 and 50 kilobases (10-13 repeats) is considered “borderline.” Less than or equal to 38 kilobases (1-9 repeats) is considered to be FSHD1. (Reference: University of Rochester Medical Center.)

The genetic test for FSHD2 consists of measuring the methylation levels in the D4Z4 region on chromosome 4. Recently, researchers found mutations in a gene called SMCHD1 in approximately 80 percent of patients with FSHD2. Over 52 mutations in SMCHD1 have been found in FSHD2 patients, and commercial testing for these mutations may become available in the future.

If you have tested negative for FSHD1

The Jain Foundation and Genzyme are partnering to offer free genetic sequencing for limb-girdle muscular dystrophies (LGMD). Their genetic testing includes SMCHD1 mutations (associated with FSHD2).

Interpreting FSHD1 Test Results

A member of the FSH Society made an inquiry as to what the following genetic test results report meant in terms of translating fragment sizes measured in kilobases (kb) of DNA to the numbers of repeats left (e.g. these are my test results and how does this translate into number of repeats?).

Enzymes EcoRI EcoRI/BlnI
Allele 1 >40 kb >40 kb
Allele 2 18 kb 15 kb

To make sense of the test results: Each one of the inquirer’s chromosome 4’s is being tested for FSHD. First they are cut with an enzyme called EcoRI and then further checked to see if it is an unusual arrangement of DNA with another enzyme which further cuts the DNA called BlnI.

The results show the inquirer’s unaffected chromosome 4 and affected chromosome 4. The unaffected chromosome (or allele 1) is the first when cut with enzymatic scissors. The length of this segment is greater than 40 and, therefore, according to the calculations below, the number of repeats is greater than the number of repeats needed to be affected, or positive, for FSHD.

The second number shows the affected chromosome (e.g., allele 2) is 18 kb in length when cut with only one scissors. It is checked for special cases and to see if there are 10q repeats within the shortened fragment. It is 3 kb less due to the test and is not a special case. The repeats are all from chromosome 4. D4Z4 repeat numbers were calculated from EcoRI-fragment sizes as follows: number of repeats = (fragment size in kb – 5 kb flanking sequence)/3.3 kb.

This would appear to be (18 – 5 )/3.3 = 3 or 4 repeats. Allele 2 is the disease-causing allele, as it contains a deletion within the FSHD diagnostic range.

Is There an Association Between the Size of the Deletion on Chromosome 4 and the Severity of FSHD in an Individual?

 There does appear to be a relationship between the size of the deletion and the severity and age of onset of FSHD. Large deletions (resulting in very small fragments) appear to be associated with earlier onset and more severe symptoms. Also, large deletions are more likely to be sporadic rather than inherited. Small deletions tend to be associated with later onset and milder symptoms.

How Accurate Is DNA Testing for FSHD?

 About 98 percent of individuals with FSHD can be accurately diagnosed by the DNA test due to the detection of a deletion. In comparison, 1-2 percent of individuals in the general population without FSHD show a deletion in the same region of chromosome 4. Within some FSHD families, some individuals have a deletion but do not show obvious symptoms of FSHD. This situation is more likely to occur when the deletion is small.

How Is DNA Obtained From an Individual Who Has FSHD?

 DNA is isolated from white blood cells. A single tube of blood (about a tablespoon) provides enough DNA to perform the test. DNA can also be obtained from saliva.

I Have Symptoms of FSHD. How Can I Arrange for a DNA Test to Confirm FSHD?

 You can ask your doctor to refer you to your local genetics clinic. There, a detailed family history will be taken. The geneticist will be able to identify those individuals in your family who could most benefit from DNA testing. The geneticist will also be able to arrange for the blood sampling and shipment of the blood to a testing center.

The DNA test results are reported to the referring physician (usually a geneticist). Since DNA test results can be difficult to interpret and understand, it is essential to have a skilled professional, such as a geneticist, explain the results of DNA testing.

What Is Meant by Sporadic FSHD and Inherited FSHD?

Sporadic FSHD means a single individual in a family has FSHD, but no one else in the family has symptoms. It also means that the FSHD deletion was identified in the affected individual but not in his/her parents. Once someone is diagnosed with sporadic FSHD, the risk of transmitting FSHD is the same as in the inherited form of FSHD. Inherited FSHD means that the disease is present in multiple members of the family over two or more generations.

I Have FSHD. What Is the Risk That I Could Have a Child With FSHD?

 Individuals with FSHD have a 50 percent chance of having a child with FSHD in each pregnancy.

We Have Been Told That Our Child Has Sporadic FSHD. Is There Any Risk FSHD Could Occur in Our Next Child?

 Up to 20 percent of apparently sporadic cases of FSHD arise due to mosaicism for the FSHD deletion in one parent. This means that one parent has a mixture of cells: some with the deletion and some without the deletion. This mixture of cells may or may not be detectable by the DNA test, depending upon the extent of mosaicism in the individual. Therefore, there is a risk of having another child with FSHD, even if there is no detectable deletion in either parent.

Is There a Prenatal Test for FSHD?

 Yes. Using the same technology as in the DNA test described above, prenatal testing is possible. Those who are interested in a prenatal test for FSHD should consult their physician and the genetic testing laboratories.

In prenatal diagnosis, fetal cells are obtained primarily by one of two procedures. The earliest procedure is called chorionic villus sampling (CVS). This procedure is performed at about the tenth to twelfth week of pregnancy. The alternative procedure is called amniocentesis. This procedure is performed at about the fifteenth to sixteenth week of pregnancy. Individuals at risk of having a child with FSHD should see a geneticist for counseling as early as possible in the pregnancy or even before becoming pregnant, since it is necessary for their DNA to be tested in order to obtain accurate results. Prenatal diagnosis must be arranged many weeks in advance, through a genetics clinic. Prenatal tests have risks associated with them, and therefore it is important to obtain genetic counseling and consider all the information about prenatal testing carefully before deciding to proceed. In general, molecular diagnostic laboratories make a special effort to process prenatal DNA samples as rapidly as possible.

Is Pre-implantation Genetic Diagnosis (PGD) Available for FSHD?

Yes. An important aspect to know about PGD is that the statistic mapping techniques are used to infer if the actual deletion that causes FSHD itself is inherited. A study is done on the inheritance pattern of map markers on each allele to ascertain whether the disease-carrying allele has been inherited. There are documented cases in which the disease allele is inherited but the deletion of D4Z4 is not inherited due to rearrangement (e.g., the D4Z4 region comes from the other chromosome). With FSHD, a prenatal diagnosis usually follows the PGD to be sure the deletion was not passed on.

Adapted from Marsha Speevak, PhD, Molecular Diagnostic Laboratory, Genetics Department
Children’s Hospital of Eastern Ontario, Ontario, Canada
FSH Watch, Vol. 5 No. 1, Spring 1998 [amended January 2014]

FSHD Molecular Diagnosis Flowchart

An excellent paper published in the journal Chromosoma and co-authored by FSH Society grantees Melanie Ehrlich, Ph.D., Tulane University, and Richard Lemmers, Ph.D., Leiden University Medical Center, Leiden, the Netherlands introduces improvements to genetic testing and education in genetic testing for FSHD. Their article contains one of the finest depictions of a graphic showing a flowchart of recommended procedures for molecular diagnosis for FSHD. The paper illustrates “the inherent complexity of FSHD molecular diagnosis due to the 4q and 10q homology between D4Z4 arrays and adjacent sequences, translocations between 4q and 10q D4Z4 arrays, mitotic D4Z4contractions, deletions encompassing p13E-11, and the wide range of sizes of D4Z4 arrays combined with the need for high resolution of bands in the 30- to 45-kb range.” They also make an improvement to the genetic testing FSHD. “An important advantage of using a D4Z4 probe for molecular diagnosis of FSHD in conjunction with the p13E-11 probe is that it permits the identification of about 3% of FSHD patients who have a deletion of the p13E-11genomic sequence next to a short 4q D4Z4 array (Lemmers et al. 2003).”

The article is an excellent overview of the state of the art complex genetics of FSHD. In particular, figure 6 on page 114 has a flowchart for the molecular diagnosis of FSHD. The diagram depicts testing scenarios for both confirmation of clinical FSHD as well as exclusion to prove clinically no FSHD. Many thanks go to Drs. Ehrlich and Lemmers and co-authors for this significant improvement in genetic tests for FSHD as well as providing materials and documentation of how FSHD genetic testing works.

Chromosoma. 2007 Apr;116(2):107-16. Hybridization analysis of D4Z4 repeat arrays linked to FSHD. Ehrlich M, Jackson K, Tsumagari K, Camaño P, Lemmers RJ. Abstract.

To download the diagnostic flowchart reprinted with the authors’ permission, please click here

Determining the Number of D4Z4 Repeats Based on Deletion Size

The FSH Society receives numerous inquiries about understanding genetic test results.

The following excerpts are from Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management. Clin Neurosci. Basel, Karger, 2000. vol 18. pp 44-60. The chapter title “Facioscapulohumeral Muscular Dystrophy: Diagnostic and Molecular Aspects” is by Peter Lunt, Ph.D., Clinical Genetics Unit, Bristol Royal Hospital for Sick Children, Bristol, UK.

Pages 48-49 have a section headed “Molecular Testing: Confirmation of Diagnosis” that states: “In 90-95% of cases of FSHD, as defined by meeting the diagnostic criteria, the diagnosis can effectively be confirmed by showing the presence of a shortened (<35 kb) DNA fragment at 4q35 (recognized by probe pl3E-11), which arises from deletion of an integral number of copies of the 3.3-kb repeats from that region. The DNA probe used (pl3E-11) also detects the closely homologous 3.3-kb repeat array from 10q26. However, each chromosome 10-type repeat has an additional BlnI restriction site. For the specific diagnostic test, a double digest with EcoRi/BlnI is employed on genomic DNA (obtained from peripheral blood), which removes chromosome 10-type repeats, but leaves chromosome 4-type repeats intact (albeit reduced by 3 kb in size compared to EcoR1 single digest) {Source: Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.Chapter p 48-49 “Facioscapulohumeral Muscular Dystrophy: Diagnostic and Molecular Aspects,” by Peter Lunt, Ph.D.]

Page 45 of the chapter defines the generally accepted correlation between clinical severity and D4Z4 repeat number calculation. “It is found that the age at onset and severity of clinical presentation correlates broadly and inversely with the size of the residual DNA fragment at 4q35, and, by inference, therefore correlates directly with the number of repeat units deleted. Thus, the smallest residual fragment lengths at 10-17 kb (1-3 repeat copies) are usually associated with a severe infantile or childhood presentation, medium lengths (18 30 kb, or 4-7 repeat copies) are often found in the largest recognised dominant families, while the largest lengths (31-38 kb, or 8-10 repeat copies) have been associated with a milder predominantly scapulohumeral presentation and may well have reduced penetrance, particularly in females. New mutation cases are seen predominantly with the smallest residual fragment lengths, giving matching clinical severity, and may originate predominantly on the maternal copy of chromosome 4. Study of parental DNA suggests that around 20-30% of new mutations occur as somatic and germline events in one of the parents, this usually also being the mother.”