A novel emerin gene mutation in Emery Dreifuss muscular dystrophy patient with spontaneous chordae tendinae rupture.

Emery Dreifuss muscular dystrophy (EDMD) is an inherited myopathy characterized by early contractures, slow progressive muscle weakness and cardiac involvement. To date at least seven genes have been associated to EDMD with different inheritance patterns, being emerin gene responsible for the X-linked form of the disease. We report a 40-year-old man who was referred for severe gait difficulty. At age 6 years the patient presented with a waddling gate, lumbar lordosis and heel contractures. Both electrophysiology and muscle biopsy were consistent with a neurogenic disorder and he received a diagnosis of spinal muscular atrophy type 3. At the age of 30 the patient developed heart involvement with junctional escape rhythm and, eight years later, had a spontaneous chordae tendinae rupture. A new clinical examination showed severe muscular weakness and atrophy in scapulohumeroperoneal pattern with significant involvement of the lower facial and intrinsic hand muscles and on a second muscle biopsy emerin was absent by immunohistochemistry and by immunoblot analysis. Sequence analysis of EMD gene revealed the presence of a novel mutation represented by an out-of-frame deletion spanning from the beginning of exon 1 to the half of intron 2 (p.Asp6Glyfs*27). Our study expands the clinical and molecular spectrum of X-linked EDMD.

A Family with γ-Thalassemia and High Hb A2 Levels.

We describe a family carrying a γ-globin gene deletion associated with an increase of Hb A2 level beyond the normal range. The family included the proband, his sister and their father, all with increased Hb A2 and normal Hb F levels. The proband and his sister showed borderline values of mean corpuscular volume (MCV) and reduced values of mean corpuscular hemoglobin (Hb) (MCH). The proband was referred to our Medical Genetics Service for preconception counseling together with his partner, a typical ß-thalassemia (ß-thal) carrier. The results were negative for the most frequent α-thalassemia (α-thal) mutations, and had no significant sequence variations of the coding sequences and promoter of the ß- and δ-globin genes. Quantitative analysis by multiplex ligation-dependent probe amplification (MPLA) of the ß-globin gene cluster detected a heterozygous deletion, ranging between 2.1 and 4.7 kb, in the proband, his sister and the father. The deletion involved the (G)γ gene and (G)γ-(A)γ intergenic region, whereas the 3' region of the (A)γ gene was preserved. A subsequent gap-polymerase chain reaction (gap-PCR) showed that a hybrid (GA)γ fusion gene was present. The deletion segregated with the elevation of Hb A2. The MLPA analysis of the ß-globin gene cluster in 150 control alleles excluded a common polymorphism. Despite stronger evidence being needed, the described family suggests a possible role of this γ-globin gene deletion in contributing to Hb A2 elevation, possibly by altering the transcription regulation of the cluster. We propose γ-globin gene dosage analysis to be performed in patients with unexplained elevated Hb A2 levels.

Paternal germline mosaicism in collagen VI related myopathies.

BACKGROUND: Collagen VI-related disorders are a group of muscular diseases characterized by muscle wasting and weakness, joint contractures, distal laxity, serious respiratory dysfunction and cutaneous alterations, due to mutations in the COL6A1, COL6A2 and COL6A3 genes, encoding for collagen VI, a critical component of the extracellular matrix. The severe Ullrich congenital muscular dystrophy (UCMD) can be due to autosomal recessive mutations in one of the three genes with a related 25% recurrence risk. In the majority of UCMD cases nevertheless, the underlying mutation is thought to arise de novo and the recurrence risk is considered as low. METHODS AND RESULTS: Here we report a family with recurrence of UCMD in two half-sibs. In both, the molecular analysis revealed heterozygosity for the c.896G > A missense mutation in COL6A1 exon 10 (Gly299Glu) and for the COL6A1 c.1823-8G > A variation within COL6A1 intron 29. The intronic variation was inherited from the father and RNA analysis in skin fibroblasts allowed to exclude its role in affecting COL6A1 transcript processing. The Gly299Glu mutation occurred apparently de novo in the two sibs. CONCLUSION: The described mutational segregation strongly suggests the occurrence of paternal germline mosaicism. This is the first report of UCMD recurrence due to a germline mosaic COL6 gene mutation. Mosaicism deserves to be considered as possible inheritance pattern in genetic counseling and recurrence risk estimation in collagen VI-related diseases.

A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.