Diagnosis Of Facioscapulohumeral Muscular Dystrophy Research Articles

Facioscapulohumeral muscular dystrophy with EcoRI/BlnI fragment size of more than 32 kb

Facioscapulohumeral muscular dystrophy (FSHD) is associated with the deletion of a variable number of 3.3-kb subunits of a tandemly arranged repeat (D4Z4) on chromosome 4q35. EcoRI/BlnI fragments in the range of 10-35 kb are currently defined as disease-associated. Diagnosis of FSHD is frequently complicated by interchromosomal exchange with a homologous locus on 10q26. We present clinical and laboratory data of six subjects from two unrelated families with a marked FSHD phenotype and EcoRI/BlnI fragments of 39 and 33 kb, respectively. Origin on chromosome 4q35 was confirmed by haplotype analysis in the first family and was supported by pulsed field gel electrophoresis data in the second family. Our data further confirm the existence of a region of overlap of normal and pathological fragments. Fragments from this region can obviously be associated with marked FSHD phenotypes. Furthermore, application of linked markers and resolution of all EcoRI/BlnI fragments by pulsed field gel electrophoresis in addition to routine laboratory tests considerably augments the information obtained from molecular tests, upon which genetic counselling can then be based.

Complete allele information in the diagnosis of facioscapulohumeral muscular dystrophy by triple DNA analysis.

Facioscapulohumeral muscular dystrophy is caused by partial deletion of the D4Z4 repeat array on chromosome 4q35. Genetic diagnosis is based on sizing of this repeat array, which is complicated by cross-hybridization of a homologous polymorphic repeat array on chromosome 10 and by the frequent exchanges between these chromosomal regions. The restriction enzyme XapI optimizes the diagnosis of facioscapulohumeral muscular dystrophy by uniquely digesting 4-derived repeat units and leaving 10-derived repeat units undigested, thus complementing BlnI, which uniquely digests 10-derived repeat units. A triple analysis with EcoRI, EcoRI/BlnI, and XapI unequivocally allows characterization of each of the four alleles, whether homogeneous or hybrid. This is particularly useful in the case of identical sized 4-derived and 10-derived arrays, in situations of suspected facioscapulohumeral muscular dystrophy with nonstandard allele configurations, and for assignment of hybrid fragments to their original alleles.

Improved characterization of FSHD mutations

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortest alleles of the 3.3kb-tandem repeat array D4Z4 at 4q35. Molecular diagnosis of FSHD depends upon the separation of unusually large alleles by pulse-field electrophoresis after EcoRI and EcoRI/BlnI digestion. The exact number of alleles could not however be directly inferred from the size of DNA fragments owing to polymorphisms in the telomeric region of the locus. Knowing the exact repeat number of disease causing alleles may benefit genetic counselling, help to understand the mechanism of this singular disease and the population dynamics of subtelomeric sequences variations. We present here a partial digestion mapping method giving the exact number of repeats for disease causing alleles, and we suggest that most inaccuracies induced by common polymorphisms could be reduced by using EcoRV in place of EcoRI. After studying more than 300 DNA samples with both the standard method and this new method, we show that alleles size can be evaluated with a precision of less than one half repeat, and that the variations in length of the truncated repeat in the telomeric region of the D4Z4 locus can be evaluated. The results suggest that at least one intact chromosome 4 type repeat at 4q35 is needed to cause FSHD.

A new dosage test for subtelomeric 4;10 translocations improves conventional diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the size reduction of a polymorphic repeat array on 4q35. Probe p13E-11 recognises this chromosomal rearrangement and is generally used for diagnosis. However,...

Progress in the molecular diagnosis of facioscapulohumeral muscular dystrophy and correlation between the number of KpnI repeats at the 4q35 locus and clinical phenotype.

Genotype analysis by using the p13E-11 probe and other 4q35 polymorphic markers was performed in 122 Italian facioscapulohumeral muscular dystrophy families and 230 normal controls. EcoRI-BlnI double digestion was routinely used to avoid the interference of small EcoRI fragments of 10qter origin that were found in 15% of the controls. An EcoRI fragment ranging between 10 and 28 kb that was resistant to BlnI digestion was detected in 114 of 122 families (93%) comprising 76 familial and 38 isolated cases. Among the unaffected individuals, 3 were somatic mosaics and 7, carrying an EcoRI fragment larger than 20 kb, could be rated as nonpenetrant gene carriers. In a cohort of 165 patients with facioscapulohumeral muscular dystrophy we found an inverse correlation between fragment size and clinical severity. A severe lower limb involvement was observed in 100% of patients with an EcoRI fragment size of 10 to 13 kb (1-2 KpnI repeats left), in 53% of patients with a fragment size of 16 to 20 kb (3-4 KpnI repeats left), and in 19% of patients with a fragment size larger than 21 kb (>4 KpnI repeats left). Our results confirm that the size of the fragment is a major factor in determining the facioscapulohumeral muscular dystrophy phenotype and that it has an impact on clinical prognosis and genetic counseling of the disease.

Definitive molecular diagnosis of facioscapulohumeral dystrophy.

To establish the usefulness of a molecular diagnostic protocol for the autosomal dominant disease facioscapulohumeral dystrophy (FSHD). The genetic defect underlying the majority of cases is a deletion on chromosome 4q35 that is not associated with the coding sequence of any known gene. Molecular diagnosis of FSHD involves the visualization of this deletion as a "small" EcoRI restriction fragment. However, molecular diagnostics are complicated because of the homology of the telomeric regions of chromosomes 4q and 10q; the homologous 10q26 EcoRI fragments are also detected, and can fall into the size range considered to be diagnostic for FSHD. It is therefore important to distinguish the 4q35 and 10q26 EcoRI fragments, taking advantage of the presence of additional restriction sites (BlnI) in the alleles of chromosome 10q origin. Paired digests of genomic DNA (EcoRI only and EcoRI/BlnI double digest), followed by pulsed field gel electrophoresis (PFGE), were used to establish the molecular diagnosis of FSHD in 82 unrelated index cases (46 familial, 24 proven sporadic with de novo mutations, and 12 with uncertain family history). In all cases fulfilling FSHD diagnostic criteria, a 4q35 EcoRI allele size of < or = 38 kb was present. The smallest 4q35 EcoRI allele in 205 normal control subjects was 41 kb. EcoRI alleles < or = 38 kb of chromosome 10q26 origin were present in 11.2% of this control group. In problematic cases, it was possible to resolve the diagnostic question. The combination of double digestion with EcoRI and BlnI followed by PFGE is the most reliable molecular protocol for distinguishing patients with FSHD.

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

A major advance in the molecular diagnosis of facioscapulohumeral muscular dystrophy is the recently reported elimination of confounding DNA fragments arising from homologous sequences located at 10q26. In order to...