Precise mapping of 17 deletion breakpoints within the central hotspot deletion region (introns 50 and 51) of the DMD gene.

Exon deletions in the human DMD gene, which encodes the dystrophin protein, are the molecular defect in 50-70% of cases of Duchenne/Becker muscular dystrophies. Deletions are preferentially clustered in the 5' (exons 2-20) and the central (exons 45-53) region of DMD, likely because local DNA structure predisposes to specific breakage or recombination events. Notably, innovative therapeutic strategies may rescue dystrophin function by homology-based specific targeting of sequences within the central DMD hot spot deletion region. To further study molecular mechanisms that generate such frequent genome variations and to identify residual intronic sequences, we sequenced 17 deletion breakpoints within introns 50 and 51 of DMD and analyzed the surrounding genomic architecture. There was no breakpoint clustering within the introns nor extensive homology between sequences adjacent to each junction. However, at or near the breakpoint, we found microhomology, short tandem repeats, interspersed repeat elements and short sequence stretches that predispose to DNA deletion or bending. Identification of such structural elements contributes to elucidate general mechanisms generating deletion within the DMD gene. Moreover, precise mapping of deletion breakpoints and localization of repeated elements are of interest, because residual intronic sequences may be targeted by therapeutic strategies based on genome editing correction.

Prenatal molecular diagnosis of inherited neuromuscular diseases: Duchenne/Becker muscular dystrophy, myotonic dystrophy type 1 and spinal muscular atrophy.

BACKGROUND: Neuromuscular disease is a broad term that encompasses many diseases that either directly, via an intrinsic muscle disorder, or indirectly, via a nerve disorder, impairs muscle function. Here we report the experience of our group in the counselling and molecular prenatal diagnosis of three inherited neuromuscular diseases, i.e., Duchenne/Becker muscular dystrophy (DMD/BMD), myotonic dystrophy type 1 (DM1), spinal muscular atrophy (SMA). METHODS: We performed a total of 83 DMD/BMD, 15 DM1 and 54 SMA prenatal diagnoses using a combination of technologies for either direct or linkage diagnosis. RESULTS: We identified 16, 5 and 10 affected foetuses, respectively. The improvement of analytical procedures in recent years has increased the mutation detection rate and reduced the analytical time. CONCLUSIONS: Due to the complexity of the experimental procedures and the high, specific professional expertise required for both laboratory activities and the related counselling, these types of analyses should be preferentially performed in reference molecular diagnostic centres.

Unraveling unusual X-chromosome patterns during fragile-X syndrome genetic testing.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID). Together with fragile X-associated tremor and ataxia (FXTAS) and fragile X-associated premature ovarian failure (POF)/primary ovarian insufficiency (POI), FXS depends on dysfunctional expression of the FMR1 gene on Xq27.3. In most cases, FXS is caused by a >200 CGG repeats in FMR1 5'-untranslated region (UTR) and by promoter hypermethylation that results in gene silencing. Males and females with unmethylated premutated alleles (repeats between 55 and 200) are at risk for FXTAS and POF/POI. METHODS: FXS molecular testing relied on PCR and methylation-specific Southern blot analysis of the FMR1 5'UTR. Atypical Southern blot patterns were studied by X-chromosome microsatellite analysis, copy number dosage at DMD locus, amelogenin gender-marker analysis and array-comparative genomic hybridization (array-CGH). RESULTS: Six men affected by ID and three women affected by ID and POF/POI underwent FXS molecular testing. They had normal FMR1 CGG repeats, but atypical X chromosome patterns. Further investigations revealed that the six males had Klinefelter syndrome (XXY), one female was a Turner mosaic (X0/XX) and two women had novel rearrangements involving X chromosome. CONCLUSIONS: Diagnostic investigation of atypical patterns at FMR1 locus can address patients and/or their relatives to further verify the condition by performing karyotyping and/or array-CGH.

A larger spectrum of intragenic short tandem repeats improves linkage analysis and localization of intragenic recombination detection in the dystrophin gene: an analysis of 93 families from southern Italy.

Duchenne/Becker muscular dystrophies (D/BMD) are X-linked recessive disorders resulting from dystrophin gene mutations. Intragenic recombination in the dystrophin gene occurs with a high frequency. Therefore, determination of the location and frequency of recombination improves D/BMD carrier detection and prenatal diagnosis in families in which the disease-causing mutation cannot be detected by most conventional methods. We describe herein a linkage analysis performed using a fast method based on capillary gel electrophoresis of fluorescent-labeled amplified alleles of 15 intragenic short tandem repeats spanning the entire dystrophin gene. On characterization of recombination events in 93 unrelated D/BMD families from southern Italy, we mapped 25 intragenic recombinations out of 273 informative meioses analyzed. The terminal regions of a gene are notoriously challenging for linkage analysis because some recombination events could be missed in case of lack of informativeness of the outermost markers. Many recombination events (10/25) identified in this study were located at the terminal regions of the dystrophin gene, and some were found by typing of several informative short tandem repeats located in these regions. Moreover, about 24% of the recombination events found in this study mapped to the 3' region of the gene, in contrast with the low frequency (4 to 15%) reported by others.

A 15-year case-mix experience for fragile X syndrome molecular diagnosis and comparison between conventional and alternative techniques leading to a novel diagnostic procedure.

Fragile X syndrome (FXS) is the main cause of heritable mental retardation. In most patients, it is associated with an increased number of CGG repeats (>200) within the 5'-untranslated region of the FMR1 gene, and with methylation of the expanded repeats and of the promoter. FXS female carriers and transmitting males have expansions of between 55 and 200 repeats (premutated alleles). Alleles with premutations are unstable in female meioses. Normal and premutated repeats are unmethylated in males and subject to lyonization in females. Here, we report the postnatal and prenatal molecular diagnoses of FXS made with conventional PCR and Southern blotting in a cohort of Italian patients and their families over a period of 15years. Moreover, we tested two novel high-performance PCR procedures (PCR with a chimeric primer, and the AmplideX™ FMR1 kit) in our patients and compared the results with our previous observations. We concluded that the high-performance PCR assays complement the results obtained by conventional methods, but they cannot replace the Southern blot procedure. Consequently, also based on cost-benefit considerations, our FXS diagnostic flowchart now consists of conventional PCR and Southern blotting plus the chimeric primer PCR procedure, whereas the AmplideX™ procedure is reserved for doubtful cases.

A 15-year molecular analysis of DMD/BMD: genetic features in a large cohort

Duchenne (DMD) and Becker muscular dystrophies (BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene. In most cohorts, DMD/BMD are due to deletions (60-80%) and duplications (6-10%) involving one or more exons. The remaining cases are caused by different type of point mutations. We analyzed 179 unrelated male patients, 296 women belonging to 137 DMD/BMD families, and 93 independent patients referred for hyperCKemia. We identified 121 deletions and 11 duplications involving one or more exons and one complex rearrangement in the DMD/BMD patients, and 9 deletions in males referred for high levels of serum CK. Carrier status was investigated in 219 female relatives of deleted or duplicated DMD/BMD males, and by linkage analysis in 77 women belonging to families in which the causative mutation was not identified. Four carrier women with clinical manifestations of the disease had unbalanced X inactivation with a degree of X skewing between 70% and 93%. Large cohort studies from different geographic areas may be important for mutation typology comparisons and their appropriate analytical approach.