The FSHD Workshop was held at the 48th annual meeting of the American Society of Human Genetics (ASHG) in Denver on Oct. 30, 1998. Researchers and clinicians from North America, Europe, Japan, Korea and South Africa met to discuss issues related to facioscapulohumeral muscular dystrophy.

Michael Altherr (Los Alamos National Laboratory) chaired the FSHD Workshop and suggested several topics to be covered during the meeting. These included mouse/animal studies, gene candidates, molecular models, diagnosis, muscle proteins and penetrance.

Kathy Mathews (University of Iowa) described her work in association with Pam Geyser on the development of a Drosophila (fruit fly) model for FSHD. Nine lines have been generated in which the D4Z4 (3.3 kb repeat) have been introduced in opposite orientations. One line has complete repression of normal eye color, four lines exhibit moderate repression of white eye gene expression, two lines are composed of flies which have normal eye color at birth but the color changes with age and two lines have no effect. Thus, preliminary results suggest that the D4Z4 repeat has the ability to suppress adjacent gene expression, which is in keeping with the current model of a position effect responsible for FSHD.

Silvere van der Maartel (Leiden University, The Netherlands) spoke briefly of the transgenic mouse experiments underway in Rune Frant's lab. He and a graduate student are using two YACs (a telomeric 4q YAC and a 400 kb YAC spanning the FSHD region) to introduce the FSHD region into mice.

Jane Hewitt (University of Nottingham, UK) presented the extensive work she and Pam Grewal have done on the evolution of the FSHD region. FRG1 is conserved between mouse and the pufferfish Fugu. Mouse chromosome 8 (a syntenic region with FSHD) contains two genes, PCM1 (pericentriole material gene 1) and ACER (acid ceramidase), which map between FRG1 and CPP32, disrupting the FSHD region. Michel Van Geel (Roswell Park Cancer Institute) described these two genes in detail. They are both very well conserved and all have the same map order in the FSHD region in dog, cow, mouse and Fugu, but not in monkey where the map order is similar to human.

Denise Figlewicz (University of Rochester) presented the results of direct selection in her lab. Using the 460 kb YAC 159F7, she has identified and sequenced over 100 cDNA fragments. Several known FSHD region sequences such as FRG1 and Tub4q were identified. In addition, at least two contigs not matching known sequences in BLAST map back to the FSHD region and will be pursued as candidate genes. Time did not permit Dr. Figlewicz to present her very eloquent analysis of quantitation of gene transcription in the FSHD region using competitive RT-PCR. Her poster at ASHG demonstrated that several genes throughout the region are upregulated in FSHD, including ANT-1, ALP and FRG1.

Alexandra Belayew (University of Leuven, Belgium) described her ongoing work examining the Dux-1 and Dux-2 sequences. Dux-1 (double homeobox-1) has 90 percent similarity to the D4Z4 repeat. In order to delineate the function and subcellular localization of Dux-1, she and Stephan Plaisance have generated antibodies as well as a GFP-Dux-1 fusion protein. After 30 hours, Dux-1 localizes to the nucleus, appearing to accumulate around the nucleolus. With an antibody to the peptide, they identify the protein as a dimer of the expected 42 kD monomer. They also have sequenced two repeats in patients, and find the identical promotor.

Min Dong Song (Kon-Kuk University, Korea) presented work done in collaboration with Kiichi Arahata (National Institute of Neuroscience, Tokyo) on the sequence and analysis of very small fragments (10 kb) from sporadic patients. Both have a long open-reading frame encompassing the double homeodomain on 4q. Their work also implies that there is a functional diversity of the homeodomain 2 on chromosome 10.

Rusella Tupler (HHMI, University of Massachusetts) presented her results utilizing PCR-based subtractive hybridization to examine gene expression FSHD. She generated two independent libraries, one representing genes that are over-expressed in the FSHD muscle, one representing genes that are under-expressed in the FSHD muscle. Sequencing of the clones, she has found that of these differentially expressed transcripts, 25 percent are known sequences, 17 percent are repetitive sequences and 25 percent aren't represented by any known sequence in the database. Interestingly, 40 percent of the transcripts are muscle specific.

Sara Winokur (University of California, Irvine) announced that the Affymetrix GeneChip system has been installed in her laboratory and that analysis of FSHD muscle biopsies and myoblasts will be performed in the near future with the Human 6800 and 35,000 EST chips. This technology allows for the simultaneous monitoring of thousands of genes in a given tissue or cell line and is a powerful means of determining the primary defect in FSHD muscle. She also described a novel gene that maps to the FSHD region. This gene encodes a 7 kb skeletal muscle specific transcript that maps adjacent to ALP. Functional analysis of this gene is underway.

Silvere van der Maartel (Leiden University) in Rune Frant's lab announced the cloning of a gene, FRG2, which is a member of a multi-gene family with muscle specific expression. In patients with FSHD, this expression is dependent upon the D4Z4 repeat array. Expression in hybrid cell lines can be induced following MyoD adenovirus infection. Allele specific expression can be monitored as 4q specific PCR primers have been developed.

At this point, the focus of the workshop shifted to diagnostic and clinical aspects of FSHD. Kathy Mathews announced that the Molecular Pathology Department at the University of Iowa is offering diagnostic testing for FSHD with a two week turn-around time for a fee of $388.

Silvere van der Maartel (Rune Frant's lab) presented an additional diagnostic test they have developed for use when inconsistent results are obtained. This test can differentiate mono-, di-, tri- and tetrasomic alleles resulting from 4;10 translocations. A simple dosage technique without the use of PFGE relies on a BglII/BlnI digest since the BglII digest results in a 4kb p13E-11 fragment. Subsequent digestion with BlnI shortens only the chromosome 10 band to 1.7 kb. Therefore, a double digest will yield a 1.7 kb chr. 10 fragment and a 4.0 kb chromosome 4 fragment. The relative intensities of both fragments reflect the repeat composition on both chromosomes.

Peter Lunt (Bristol Children's Hospital, UK) discussed the potential for anticipation in FSHD families. He presented a family with a small fragment in which the disease is mild in the parent but in which two children have very severe symptoms. His poster at ASHG summarized the data from 250 molecular diagnostic tests for FSHD. In 8 percent of families, the Bln-resistant fragment occurs in apparently asymptomatic adult relatives of affected individuals. Dr. Lunt proposes a possible two-step mechanism for FSHD and suggests that three-generation families should be studied.

Meena Upadhyaya (University of Wales, Cardiff) presented a new mutation family with age of onset at 10 years. The mutation apparently arose from a de novo rearrangement that generated a 47 kb fragment. As presented in her poster at ASHG, the Medical Genetics Division has carried out prenatal diagnosis for FSHD in 10 pregnancies over the past five years, with assessment of risk information given to families.

Drs. Ricci (Catholic University, Rome) and Luciano Fellicetti (CNR, Rome) compiled data on 122 FSHD families. Eight families had no small fragment less than 30 kb (four families had a chromosome 10 Bln1 sensitive small fragment, while four families lacked a small fragment entirely). They also presented a strong correlation between the size of the fragment and clinical severity. 100 percent of patients with a fragment size between 10 and 13 kb (1 or 2 Kpn1 repeats left) fell into the most severely affected classification. The proportion of severe cases dropped to 21 percent and 17 percent in patients with fragment sizes of 21 to 24 kb and 24 to 27 kb, respectively.

Kiichi Arahata (National Institute of Neuroscience, Tokyo) summarized the data on 82 families in Japan. Of 39 isolated cases and 41 familial cases, all had a small fragment less than 35 kb. He also described an individual with a translocation t(4;9)(q33;q22.3) that didn't exhibit symptoms of FSHD.

Bill Moore, Research Program Coordinator for the Muscular Dystrophy Association, spoke of the MDA gene therapy protocols, which are soon to enter clinical trials for Duchenne and limb-girdle muscular dystrophies. Although these initial trials will not involve FSHD, the information attained through these trials should, ultimately, benefit those with FSHD in terms of greater efficiency and efficacy of gene therapy if FSHD appears likely to benefit from gene therapy. Other protocols being developed through the MDA research program may also have benefit once the exact cause of FSHD is determined.

In summary, the workshop was a very productive one in which clinicians and researchers were able to communicate recent and compiled data on FSHD. Due to the high attendance and significant recent progress in FSHD, a suggestion was made to expand the workshop to a full-day conference in October 1999 in conjunction with the ASHG meeting in San Francisco. MDA has expressed support for such an all day conference in conjunction with ASHG.

MEETING ATTENDEES

Katherine D. Mathews, MD. University of Iowa, USA.
Alexandra Belayew, Ph.D. University of Leuven, Belgium
Chihiro Akazawa, M.D., Ph.D. Japan
David Picketts, Ottawa Research Institute, Ottawa, Ontario, Canada
Denise Figlewicz, Ph.D. University of Rochester, NY. USA
George W. Padberg. Nijmegen, The Netherlands.
Jane Hewitt, Ph.D. U. of Nottingham, Nottingham, UK
Kiichi Arahata, M.D. National Institute of Neuroscience, Tokyo, Japan.
Luciano Felicetti, MD, PhD Institute of Cell Biology. Rome, Italy.
Marc Jeanpierre. Laboratory of Genetics. Paris, France.
Meena Upadhyaya. Institute of Medical Genetics. Cardiff, Wales. UK.
Michael Altherr, Ph.D. Los Alamos National Laboratory, New Mexico. USA.
Michel Fardeau. Institute of Medical Research. Paris, France.
Michel Van Geel. Roswell Park Cancer Institute, Buffalo, NY. USA.
Min Dong Song. National Institute of Neuroscience. Tokyo, Japan.
Peter J. Bridge, Ph.D., FCCMG, FACMG. Alberta Children's Hospital. Calgary, Alberta, Canada.
Peter Lunt. Children's Hospital. Bristol, UK
Robert G Korneluk. Children's Hospital of Eastern Ontario. Ottawa, Ontario. Canada.
Rossella Tupler, M.D., Ph.D. University of Massachusetts, Worchester, MA. USA.
Rune Frants, Ph.D. Leiden University. Leiden, The Netherlands.
Sara T. Winokur, Ph.D. University of California at Irvine. USA.
Silvere Van Der Maarel. Leiden University. Leiden, The Netherlands.
Thomas H. Haaf. Institute of Medical Genetics. Berlin, Germany.
Jennifer Anderson, Duke University, Durham, NC
Egbert Bakker, Leiden University, Leiden, The Netherlands
Giancarlo Deidda, Leiden University, Leiden, The Netherlands
Giuliana Galluzzi, Institute for Cell Biology-CNR, Rome, Italy
Pam Grewal, University of Nottingham, Nottingham, UK
Kathleen Mills, University of Iowa, Iowa City, IA
Judith Van Deutekom, University of Pittsburg, PA
Kristi Viles, Duke University, Durham, NC
Stephan Plaisance, University of Leuven, Belgium
Elena Giulotto, University of Pavia, Pavia, Italy
Toshitumi Tsukahara, National Institute of Neuroscience, Tokyo, Japan
Daniel Perez, President FSH Society, Lexington, MA.
Carol Perez, Executive Director FSH Society, Lexington, MA.
Stephen J. Jacobsen, FSH Society, San Diego, CA.
Jack Shaw, Seattle, WA
Dr. Shaw, Seattle, WA.
Bill Moore, Research Program Coordinator, Muscular Dystrophy Association, Tucson, AZ.