Facioscapulohumeral muscular dystrophy: epidemiological and molecular study in a north-east Italian population sample.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease associated with a partial deletion on chromosome 4q35. Few relevant investigations have been reported on its epidemiology and were essentially based on clinical diagnosis, having been performed before recognition of the molecular mutation. We report an epidemiological survey on FSHD patients, in which the diagnosis was obtained by combined clinical and molecular evaluation. The survey concerned the north-east Italian province of Padova, an area of 871,190 inhabitants (1 January 2004). We identified 40 patients affected by FSHD based on clinical diagnosis. In 33 of them, the EcoRI fragment size in the 4q35 region ranged from 14 to 35 kb. Four other patients belonging to the same family harbored a 38-kb fragment. In these four cases, the relationship between the borderline deletion with the mild FSHD phenotype was corroborated by additional haplotype reconstruction and segregation analysis. Interestingly, the same mild facial-sparing clinical pattern was apparent only in one other patient with an EcoRI fragment of 32 kb, suggesting that this unusual FSHD phenotype may be due to very small 4q35 deletions. On the whole, estimating a prevalence rate of 44 x 10(-6), our survey confirmed FSHD as one of the most frequent neuromuscular disorders in Western populations.

Phenotypic clustering of lamin A/C mutations in neuromuscular patients.

BACKGROUND: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. METHODS: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. RESULTS: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. CONCLUSIONS: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

Facioscapulohumeral muscular dystrophy presenting isolated monomelic lower limb atrophy. Report of two patients with and without 4q35 rearrangement.

Facioscapulohumeral muscular dystrophy has a distinctive regional distribution but variable clinical expression and may be markedly asymmetrical. We report two patients presenting weakness and wasting confined to a single lower limb. Creatine kinase was slightly increased, electromyogram and muscle biopsy were myopathic. Muscle computed tomography showed normal shoulder, mid-arm, pelvic and mid-thigh scans but involvement of calf muscles. In both cases, weakness of facial and periscapular muscles was found in other family members unaware of the disease. Molecular analysis showed 4q35 deletion in one family. These cases broaden the presentation of facioscapulohumeral muscular dystrophy to include isolated monomelic atrophy of lower limb with calf muscle involvement.

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.

Genetic localization of a new locus for recessive familial spastic paraparesis to 15q13-15.

OBJECTIVE: To characterize a new gene locus for familial spastic paraparesis (FSP). BACKGROUND: FSP is a genetically heterogeneous group of upper motor neuron syndromes. It can be inherited as an autosomal dominant, autosomal recessive, or X-linked disorder. Four loci for autosomal dominant FSP have been genetically mapped, and two genes have been shown responsible for the X-linked type. In addition, two loci for autosomal recessive type have been reported and mapped to chromosomes 8q and 16q. The gene for the 16q locus has been characterized as a mitochondrial protein. METHODS: Eight recessive FSP families from America and Europe were used for genetic linkage analysis. The known recessive loci (8q and 16q) and the X-linked loci (PLP and L1CAM genes) were screened through PCR amplification, followed by linkage analysis, single-strand conformational polymorphism, or both. RESULTS: All the families except one revealed lack of linkage to the known loci for recessive and X-linked types of FSP. One of the eight families showed data consistent with linkage to the previously characterized 8q locus. Analysis of all the families for possible linkage to other candidate loci revealed significant positive lod scores for markers in chromosome 15q. The maximum multipoint combined lod score for the non-8q families was Z = 3.14 for markers D15S1007, D15S971, D15S118, and D15S1012, at a distance of 6.41 cM from the marker D15S1007, in between D15S971 and D15S118. CONCLUSIONS: Our data suggest a new locus for recessive FSP linked to chromosome 15q, and that this may be the most common one.

Molecular analysis of 4q35 rearrangements in fascioscapulohumeral muscular dystrophy (FSHD): application to family studies for a correct genetic advice and a reliable prenatal diagnosis of the disease.

In the majority of facioscapulohumeral muscular dystrophy (FSHD) families (about 95%) the genetic defect has been identified as a deletion of a variable number of KpnI repeats in the 4q35 region, although no specific transcripts from this locus have been isolated so far. Molecular diagnosis is based on the detection by probe p13E-11 of EcoRI small fragments, in the range 10-28 kb, that are resistant to BlnI digestion. In family studies this probe is used with other 4q35 polymorphic markers to assign the haplotype associated with the disease. So far, we performed DNA analysis in 145 FSHD families and identified the 4q35 DNA rearrangement not only in affected individuals, but also in healthy subjects at risk of transmitting the disease, such as non-penetrant gene carriers and somatic mosaics. In addition we applied prenatal tests to 19 fetuses, using DNA extracted from chorionic villi samples (CVS) at 10-11 weeks of gestation. The FSHD status, as determined by the presence of BlnI-resistant small fragments associated with the at risk haplotype, was assessed in nine fetuses; in the remaining 10 cases the disease was excluded. Our results show that molecular analysis of 4q35 rearrangements is a reliable indirect method to perform diagnostic, predictive and prenatal tests in FSHD.

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.

Giant dystrophin deletion associated with congenital cataract and mild muscular dystrophy.

We report a patient with a large intragenic dystrophin deletion of exons 17-51 inclusive associated with congenital cataract and mild Becker muscular dystrophy. The cataract was similar to the congenital cataract described in the mdx mouse. The loss of 68% of the rod domain including hinge 2 and 3 regions did not adversely affect the correct localization of the dystrophin and the association with the dystrophin-associated glycoprotein complex. This observation may have implications for minigenes suitable for gene therapy.

Sequence homology between 4qter and 10qter loci facilitates the instability of subtelomeric KpnI repeat units implicated in facioscapulohumeral muscular dystrophy.

Physical mapping and in situ hybridization experiments have shown that a duplicated locus with a structural organization similar to that of the 4q35 locus implicated in facioscapulohumeral muscular dystrophy is present in the subtelomeric portion of 10q. We performed sequence analysis of the p13E-11 probe and of the adjacent KpnI tandem-repeat unit derived from a 10qter cosmid clone and compared our results with those published, by other laboratories, for the 4q35 region. We found that the sequence homology range is 98%-100% and confirmed that the only difference that can be exploited for differentiation of the 10qter from the 4q35 alleles is the presence of an additional BlnI site within the 10qter KpnI repeat unit. In addition, we observed that the high degree of sequence homology does facilitate interchromosomal exchanges resulting in displacement of the whole set of BlnI-resistant or BlnI-sensitive KpnI repeats from one chromosome to the other. However, partial translocations escape detection if the latter simply relies on the hybridization pattern from double digestion with EcoRI/BlnI and with p13E-11 as a probe. We discovered that the restriction enzyme Tru9I cuts at both ends of the array of KpnI repeats of different chromosomal origins and allows the use of cloned KpnI sequences as a probe by eliminating other spurious fragments. This approach coupled with BlnI digestion permitted us to investigate the structural organization of BlnI-resistant and BlnI-sensitive units within translocated chromosomes of 4q35 and 10q26 origin. A priori, the possibility that partial translocations could play a role in the molecular mechanism of the disease cannot be excluded.

Asymptomatic dystrophinopathy.

A 4-year-old girl was referred for evaluation for a mild but persistent serum aspartate aminotransferase (AST) elevation detected incidentally during routine blood screening for a skin infection. Serum creatine kinase activity was found to be increased. Immunohistochemical study for dystrophin in her muscle biopsy showed results consistent with a carrier state for muscular dystrophy. Molecular work-up showed the proposita to be a carrier of a deletion mutation of exon 48 of the dystrophin gene. Four male relatives also had the deletion mutation, yet showed no clinical symptoms of muscular dystrophy (age range 8-58 yrs). Linkage analysis of the dystrophin gene in the family showed a spontaneous change of an STR45 allele, which could be due to either an intragenic double recombination event, or CA repeat length mutation leading to identical size alleles. To our knowledge, this is the first documentation of an asymptomatic dystrophinopathy in multiple males of advanced age. Based on molecular findings, this family would be given a diagnosis of Becker muscular dystrophy. This diagnosis implies the development of clinical symptoms, even though this family is clearly asymptomatic. This report underscores the caution which must be exercized when giving presymptomatic diagnoses based on molecular studies.

Chromosome 4q35 haplotypes and DNA rearrangements segregating in affected subjects of 19 Italian families with facioscapulohumeral muscular dystrophy (FSHD).

Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810)1 detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new "small" fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.

Italian experience regarding the prevention of Duchenne and Becker muscular dystrophies.

The indirect approach to carrier detection and prenatal diagnosis of Duchenne and Becker muscular dystrophies based on the study of DNA polymorphisms closely linked to this gene has been followed by five Italian laboratories in the study of 106 pedigrees. Out of 354 women studied up to 1 May 1987, 147 were identified as carriers because of pedigree information and/or of increased creatine phosphokinase (CPK) values. Of the remaining 207, 184 could be assigned to three arbitrarily defined risk categories (low, intermediate and high) using linkage analysis. This disaggregation of women at risk is clearly more useful than that defined before DNA analysis, in which the same 184 women could be assigned only to the low or intermediate risk categories. Prenatal diagnosis was theoretically possible in 90% of carrier women, and was actually performed in 14 pregnancies, which led to the identification of four affected male foetuses, one also having Down syndrome.

Sporadic cases in Duchenne muscular dystrophy. A reappraisal through segregation analysis on 988 sibships.

A new estimation of the proportion of sporadic cases in Duchenne muscular dystrophy was attempted by means of segregation analysis in a sample of 988 sibships collected on a world-wide scale by different authors. Maximum likelihood estimates of ascertainment probability (pi), segregation frequency (p), and frequency of sporadic cases (x) were calculated by Morton's equations under different hypotheses. The best fit was found for p = 0.454 +/- 0.024 and x = 0.235 +/- 0.034. The possibility that the proportion of sporadic cases might be lower than the expected 1/3 is suggested.

Clinical study of proximal spinal muscular atrophy. Report on 89 cases.

A report on 89 cases of proximal Spinal Muscular Atrophy with observations on the clinical features, criteria of classification and modes of inheritance. The various forms into which SMA is divided probably represent a single disease that may begin at any age and may vary in severity, due, as a rule, to an autosomal recessive gene.