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Duchenne muscular dystrophy is a severe, X-linked disease characterized by decreased muscle mass and function in children. Genetic and biochemical research over the years has led to the characterization of the cause and the pathophysiology of the disease. Moreover, the elucidation of genetic mechanisms underlining Duchenne muscular dystrophy has allowed for the design of innovative personalized therapies. The identification of specific, accurate, and sensitive biomarkers is becoming crucial for evaluating muscle disease progression and response to therapies, disease monitoring, and the acceleration of drug development and related regulatory processes. This review illustrated the up-to-date progress in the development of candidate biomarkers in DMD at the level of proteins, metabolites, micro-RNAs (miRNAs) and genetic modifiers also highlighting the complexity of translating research results to clinical practice. We highlighted the challenges encountered in translating biomarkers into the clinical context and the existing bottlenecks hampering the adoption of biomarkers as surrogate endpoints. These challenges could be overcome by national and international collaborative efforts, multicenter data sharing, definition of public biobanks and patients’ registries, and creation of large cohorts of patients. Novel statistical tools/ models suitable to analyze small patient numbers are also required. Finally, collaborations with pharmaceutical companies would greatly benefit biomarker discovery and their translation in clinical trials.

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMDgene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.

Autozygosity is associated with an increased risk of genetic rare disease, thus being a relevant factor for clinical genetic studies. More than 2400 exome sequencing data sets were analyzed and screened for autozygosity on the basis of detection of >1 Mbp runs of homozygosity (ROHs). A model was built to predict if an individual is likely to be a consanguineous offspring (accuracy, 98%), and probability of consanguinity ranges were established according to the total ROH size. Application of the model resulted in the reclassification of the consanguinity status of 12% of the patients. The analysis of a subset of 79 consanguineous cases with the Rare Disease (RD)–Connect Genome-Phenome Analysis Platform, combining variant filtering and homozygosity mapping, enabled a 50% reduction in the number of candidate variants and the identification of homozygous pathogenic variants in 41 patients, with an overall diagnostic yield of 52%. The newly defined consanguinity ranges provide, for the first time, specific ROH thresholds to estimate inbreeding within a pedigree on disparate exome sequencing data, enabling confirmation or (re)classification of consanguineous status, hence increasing the efficiency of molecular diagnosis and reporting on secondary consanguinity findings, as recommended by American College of Medical Genetics and Genomics guidelines.

Eteplirsen, the first FDA-approved RNA-modifying therapy for DMD, is applicable to ∼13% of patients with DMD. Because multiple exonic deletions are amenable to exon 51 skipping, the isoforms resulting from the various exon 51-skipped transcripts may vary in stability, function, and phenotype. We conducted a detailed review of dystrophinopathy published literature and unpublished databases to compile phenotypic features of patients with exon 51 “skip-equivalent” deletions. Theoretically, 48 different in-frame transcripts may result from exon 51 skipping. We found sufficient clinical information on 135 patients carrying mutations that would result in production of 11 (23%) of these transcripts, suggesting the remainder have not been identified in vivo.The majority had mild phenotypes: BMD (n = 81) or isolated dilated cardiomyopathy (n = 3). Particularly interesting are the asymptomatic (n = 10) or isolated hyperCKemia (n = 20) patients with deletions of exons 45– 51, 48– 51, 49– 51 and 50– 51. Finally, 16 (12%) had more severe phenotypes described as intermediate (n = 2) or DMD (n = 14), and 6 reports had no definitive phenotype. This review shows that the majority of exon 51 “skip-equivalent” deletions result in milder (BMD) phenotypes and supports that exon 51 skipping therapy could provide clinical benefit, although we acknowledge that other factors, such as age at treatment initiation or ongoing standard of care, may influence the degree of benefit.

Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the DMDgene leading to the lack of dystrophin. Variability in the disease course suggests that other factors influence disease progression. With this study we aimed to identify genetic factors that may account for some of the variability in the clinical presentation. We compared whole-exome sequencing (WES) data in 27 DMD patients with extreme phenotypes to identify candidate variants that could affect disease progression. Validation of the candidate SNPs was performed in two independent cohorts including 301 (BIO-NMD cohort) and 109 (CINRG cohort of European ancestry) DMD patients, respectively. Variants in the Tctex1 domain containing 1 (TCTEX1D1)gene on chromosome 1 were associated with age of ambulation loss. The minor alleles of two independent variants, known to affect TCTEX1D1coding sequence and induce skipping of its exon 4, were associated with earlier loss of ambulation. Our data show that disease progression of DMD is affected by a new locus on chromosome 1 and demonstrate the possibility to identify genetic modifiers in rare diseases by studying WES data in patients with extreme phenotypes followed by multiple layers of validation.

Reported here is the case of a 1.8-year-old boy with a 9.6- Mb deletion in 6q13q14.1 and an 11.2-Mb deletion in 6q21q22.31, ascertained through array CGH, as the result of a complex de novo chromosome rearrangement. The clinical picture of this patient is characterized by severe psychomotor delay, dysmorphic features, and some congenital defects. Although, as reported in the literature, phenotypes associated with 6q deletions may vary, an attempt was made to associate the patient's symptoms to either deletion, comparing them to previously reported cases. Only a limited specific correlation was found, probably due to the prevalence of very common symptoms.

BackgroundVariants in the Structural Maintenance of Chromosomes flexible Hinge Domain-containing protein 1 (SMCHD1) can cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) and the unrelated Bosma arhinia microphthalmia syndrome (BAMS). In FSHD2, pathogenic variants are found anywhere in SMCHD1 while in BAMS, pathogenic variants are restricted to the extended ATPase domain. Irrespective of the phenotypic outcome, both FSHD2-associated and BAMS-associated SMCHD1variants result in quantifiable local DNA hypomethylation. We compared FSHD2, BAMS and non-pathogenic SMCHD1variants to derive genotype–phenotype relationships.MethodsExamination of SMCHD1variants and methylation of the SMCHD1-sensitive FSHD locus DUX4in 187 FSHD2 families, 41 patients with BAMS and in control individuals. Analysis of variants in a three-dimensional model of the ATPase domain of SMCHD1.ResultsDUX4methylation analysis is essential to establish pathogenicity of SMCHD1variants. Although the FSHD2 mutation spectrum includes all types of variants covering the entire SMCHD1locus, missense variants are significantly enriched in the extended ATPase domain. Identification of recurrent variants suggests disease-specific residues for FSHD2 and in BAMS, consistent with a largely disease-specific localisation of variants in SMCHD1.ConclusionsThe localisation of missense variants within the ATPase domain of SMCHD1 may contribute to the differences in phenotypic outcome.

Interstitial deletions of chromosome 3p are rare, and specific genotype-phenotype correlations cannot always be assessed. We report the case of a 3p14.2 proximal microdeletion in a 60-year-old female patient with mild intellectual disability, severe speech delay, and mild dysmorphism. An array-CGH analysis detected a 500-kb deletion in the 3p14.2 region, including FEZF2, CADPS, and PTPRG. FEZF2and CADPSare known to network within the neurodevelopmental pathways. It is possible that their rearrangements lead to the phenotypic features observed in the patient, and therefore, they can be considered candidate genes responsible for such abnormalities.

Background: Genetic alterations cause Hereditary Diseases (HDs) with a wide range of incidences. Some, like cystic fibrosis, occur frequently (1/1,000 newborns), whilst others, such as Pompe disease and other metabolic disorders are very rare (1/100,000 newborns). They are well under the threshold of 1/3,000, denoted by the European Community as Rare Diseases (RDs). Genetic alterations are also associated with multifactorial disorders like diabetes, and underline both somatic and germline mutations in cancer. Nowadays, thanks to the interventions of the European Union and the American National Health Institute as well as others, Hds are under an international lense, which has stimulated discussions and research targeting gene identification, prenatal diagnosis and care optimization leading to the development of new treatment options. Nanomedicine is paving the way toward some highly appealing clinical and research avenues in HDs. Nanotechnologies lend themselves to many aspects in human healthcare, such as in vitro diagnostics (nanobiosensors and nanoplatforms), drug delivery (nanovectors), drug monitoring (nanosensors) and artificial organs to study the genome variant meaning (nanostructures). Methods and Results: With a significant reduction in costs and simplified healthcare delivery, nanodiagnostics can potentially provide the tools to diagnose diseases at an early stage with precision. In vitro nanodiagnostics are already diagnosing RDs, with many nanodevices having been successfully introduced over the last few decades. Nanovectors represent an emerging approach in drug delivery and treatment for several diseases such as cancers, infectious diseases, cardiovascular disorders and neurological pathologies. Artificial tissues have valuable implications in replacing compromised organs, thus offering unique opportunities to explore pathogenic mechanisms as well as new drug targets in a personalized context. Conclusion: This article outlines and discusses the recent progress in nanotechnology and its potential applications in HDs. It is a pivotal field for research and innovation in healthcare, with emphasis on diagnostics, disease monitoring, biomarker assaying and drug delivery. We underlined the nanomethod's capacity to identify genetic alterations and the follow up of important aspects of the disease course, including therapies. We extensively described the new field of nanodelivery for experimental drugs, focusing on new genetic therapies and their implications in hereditary disorders. We also detailed innovative tools as artificial tissues based on nanomatrices and their use to identify or study genetic alterations.

Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population. To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients. In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age. Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions. This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.

BackgroundBohring-Opitz syndrome (BOS) is a rare genetic disorder characterised by a recognisable craniofacial appearance and a typical ‘BOS’ posture. BOS is caused by sporadic mutations ofASXL1. However, several typical patients with BOS have no molecular diagnosis, suggesting clinical and genetic heterogeneity.ObjectivesTo expand the phenotypical spectrum of autosomal recessive variants of KLHL7, reported as causing Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1)-like syndrome.MethodsWe performed whole-exome sequencing in two families with a suspected recessive mode of inheritance. We used the Matchmaker Exchange initiative to identify additional patients.ResultsHere, we report six patients with microcephaly, facial dysmorphism, including exophthalmos, nevus flammeus of the glabella and joint contractures with a suspected BOS posture in five out of six patients. We identified autosomal recessive truncating mutations in the KLHL7gene. KLHL7encodes a BTB–kelch protein implicated in the cell cycle and in protein degradation by the ubiquitin–proteasome pathway. Recently, biallelic mutations in the KLHL7gene were reported in four families and associated with CS/CISS1, characterised by clinical features overlapping with our patients.ConclusionWe have expanded the clinical spectrum of KLHL7autosomal recessive variants by describing a syndrome with features overlapping CS/CISS1 and BOS.

Duchenne muscular dystrophy (DMD) is a genetic, progressive neuromuscular condition that is marked by the long-term muscle deterioration with significant implications of pulmonary and cardiac dysfunction. As such, end-stage heart failure (HF) in DMD is increasingly becoming the main cause of death in this population. The early detection of cardiomyopathy is often challenging, due to a long subclinical phase of ventricular dysfunction and difficulties in assessment of cardiovascular symptomatology in these patients who usually loose ambulation during the early adolescence. However, an early diagnosis of cardiovascular disease in patients with DMD is decisive since it allows a timely initiation of cardioprotective therapies that can mitigate HF symptoms and delay detrimental heart muscle remodelling. Echocardiography and ECG are standardly used for screening and detection of cardiovascular abnormalities in these patients, although these tools are not always adequate to detect an early, clinically asymptomatic phases of disease progression. In this regard, cardiovascular magnetic resonance (CMR) with late gadolinium enhancement is emerging as a promising method for the detection of early cardiac involvement in patients with DMD. The early detection of cardiac dysfunction allows the therapeutic institution of various classes of drugs such as corticosteroids, beta-blockers, ACE inhibitors, antimineralocorticoid diuretics and novel pharmacological and surgical solutions in the multimodal and multidisciplinary care for this group of patients. This review will focus on these challenges and available options for HF in patients with DMD.

Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism, and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned.

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

IMPORTANCE In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)–mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. OBJECTIVE To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. MAIN OUTCOMES AND MEASURES Dystrophin mRNA and protein expression. RESULTS Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). CONCLUSIONS AND RELEVANCE Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

To test the effect of the single nucleotide polymorphism −66 T>G (rs28357094) in the osteopontin gene (SPP1) on functional measures over 12 months in Duchenne muscular dystrophy (DMD).

X‐linked dilated cardiomyopathy (XLDC) represents a form of dystrophinopathy with exclusive heart involvement. Here a prenatal diagnosis of this condition performed in a family with XLDC is described. In this family, the causative mutation was a pure intronic deletion, which induces the splicing of a novel, aberrant, and out‐of‐frame exon into the dystrophin transcript. The genetic test was performed by defining both the DNA (villous) and the RNA (amniocyte) configuration. The prenatal diagnosis determined that the fetus was female, and a carrier of the genomic deletion. RNA analysis on cultured amniocytes revealed the presence of an easily detectable dystrophin transcript, as well as the co‐existence of both the wild‐type and the abnormal splicing profile. Our analysis represents the first report of a prenatal diagnosis in XLDC and also indicates the feasibility of dystrophin mutation detection on RNA from amniocytes. This finding suggests that the dystrophin splicing pattern in amniocytes and skeletal muscle is similar, and that, therefore, this approach could be used in other prenatal dystrophin mutation detection, where abnormal RNA splicing is thought to play a role, or for specific cases in which no mutations have been identified in the coding regions. © 2005 Wiley‐Liss, Inc.

Abstract: Complete or partial deficiency of the laminin α2 chain of merosin has been demonstrated in a proportion of children with classical congenital muscular dystrophy and linkage to the laminin α2 chain gene (LAMA2) on chromosome 6q2 has been established. As the laminin α2 chain is also expressed in the trophoblast, its detection and linkage analysis are useful tools for prenatal diagnosis. We report our experience of seven prenatal diagnoses in families with partial deficiency or total absence of the laminin α2 chain in the muscle of the propositi. In five instances, expression of the laminin α2 chain in the trophoblast was normal and linkage data suggested that the fetuses were unaffected. In one family, the immunocytochemical studies of the trophoblast showed the absence of laminin α2, suggesting that the fetus was affected. Linkage analysis confirmed that the fetus had inherited the two at-risk haplotypes. In one family with partial laminin α2 chain deficiency, the haplotype analysis was hampered by maternal DNA contamination. Immunocytochemical analysis of chorionic villus sampling showed a reduction in laminin α2 expression. The pregnancy was presumed to be at high-risk and terminated. However, subsequent analysis of fetal DNA indicated that the fetus was probably heterozygous. Our data suggest that immunocytochemical analysis of the trophoblast can detect abnormalities in affected fetuses and gives normal results in unaffected and carrier fetuses. Nevertheless, we recommend that linkage analysis to the LAMA2 locus is also studied in all cases.

We have characterised the 2371 bp 5′ end of intron 11 of the dystrophin gene. Comparative analysis of this intronic region revealed homologies with the following sequences: regions containing mobile elements; target sites for numerous transcription factors, two resolvases, and a histone-like DNA binding protein; three eukaryotic promoters. In addition, we identified three partially overlapping ORFs, and transcription analysis confirmed that one of these is expressed, representing the first gene reported to overlap the human dystrophin gene. We have also characterised a 136 bp sequence rearranged in intron 11 in a patient affected by X-linked dilated cardiomyopathy due to a dystrophinopathy. This is a multiple copy sequence with features of a repetitive element. Its comparative analysis showed a very high homology with human genomic and EST regions, adjacent and clustered withAlu, LINE1,andTHEelements. The pattern of homology suggests that it may represent a novelAlu-like, transcriptionally active sequence with a possible retrotransposable capacity. We hypothesise that the 5′ region of the dystrophin intron 11, containing common target areas for the insertion of mobile elements, may have a role in the rearrangement of this novelAlu-likesequence.

We previously reported that patients with familial amyloidotic polyneuropathy (FAP) (Met30) showed low plasma apolipoprotein AII (apoAII) levels and apolipoprotein AII/AI (apoAII/AI) ratio as the progression of the disease while plasma levels of apoAI, B, CII, CIII, and E were all within normal ranges. In the present study, we investigated these apolipoproteins contained in high density lipoprotein (HDL) extracted from plasma of FAP patients with various types of mutations (Gln89, Pro36, and Thr34) by ultracentrifugation in addition to FAP (Met30), and the levels of apoAII gene expression in the liver from patients with FAP (Met30). Plasma apoAII levels and the apoAII/AI ratio in extracted HDL in all the FAP types examined decreased compared with those in normal control subjects. Levels of apoAII mRNA expression in the liver of FAP (Met30) patients did not show significant changes compared with those in control subjects. These results suggest that decreased affinity of apoAII to HDL could not be compensated by overproduction of apoAII in the liver of the FAP patients and as a result of this phenomenon, plasma apoAII levels might decrease as the progression of FAP.

The most frequent transthyretin (TTR) variant associated with hereditary amyloidosis is TTR Met 30, which has its major focus in Portugal, although it also occurs in many other countries. The distribution of the mutation and its occurrence in a CpG dinucleotide lead us to question the origin of the mutation and the possibility of its having originated in Portugal. In order to investigate these questions, we studied the distribution of haplotypes associated with the Met 30 mutation in families from different European countries. All the analysed Portuguese families presented the same haplotype associated with the Met 30 mutation (haplotype I). The same was found for the Swedish and Spanish families studied. However, a distinct haplotype (haplotype III) was found in three families, one Italian, one English and one Turkish. These results suggest that, although the Portuguese Met 30 carriers might have one founder, the mutation probably recurred in populations in Europe in a similar manner to that reported in Japan. In this study, we have also analysed the haplotypes associated with other TTR variants frequent in the Portuguese population.

Knowledge about the parental origin of new mutations and the occurrence of germline mosaicism is important for estimating recurrence risks in Duchenne (DMD) and Becker muscular dystrophy (BMD). However, there are problems in resolving these issues partly because not all mutations can as yet be directly detected, and additionally because genetic ratios are very sensitive to ascertainment bias. In the present study, therefore, analysis was restricted to currently detectable mutations (deletions and duplications) in particular types of families which tend to be rare. In order to obtain sufficient data we pooled results from 25 European centers. In mothers of affected patients who were the first in their family with a dystrophin gene deletion or duplication, the ratio between the paternal and the maternal origin of this new mutation was 32:49 (binomial test P = 0.075) for DMD. In five BMD families the ratio between paternal and maternal origin of new mutations was 3:2. Recurrence risk because of maternal germline mosaicism was studied in sisters or subsequent sibs of isolated cases with an apparently new detectable mutation. In 12 out of 59 (0.20; 95% CI 0.10–0.31) transmissions of the risk haplotype the DMD mutation was transmitted as well. No recurrences were found in nine BMD families.

Nine unrelated pedigrees in which Duchenne muscular dystrophy (DMD) was not present in more than one sibship were studied, using 6 DNA polymorphisms closely linked to the DMD gene. The reconstruction of grandparental haplotypes indicates the occurrence of at least three new mutations, two in grandpaternal chromosomes and one in a grandmaternal chromosome. Two additional (but less well documented) new mutations might have occurred respectively in a grandfather's and in a grandmother's chromosome, the latter being represented by a deletion mutation. The new mutations detected in this study therefore add to a total of either three or five out of nine apparently independent mutations present in pedigrees without recurrence of the disorder.

Transthyretin‐related hereditary (TTR) amyloidoses represent a clinically heterogeneous group of diseases associated with various point mutations of the TTR gene. We propose a molecular strategy for the diagnosis of this group of disorders.— Ferlini, A.; Fini, S.; Salvi, F.; Patrosso, M. C.; Vezzoni, P.; Forabosco, A. Molecular strategies in genetic diagnosis of transthyretinrelated hereditary amyloidosis. FASEB J.6: 2864‐2866; 1992.

As part of an epidemiological study that aims to characterize chemically the mutation(s) in transthyretin (TTR) related to familial amyloidotic polyneuropathy (FAP) of different ethnic origins, studies were carried out on TTR from two FAP kindreds of Italian origin. Two different criteria were employed in the characterization of TTR from these kindreds: (1) immunoblotting of cyanogen bromide fragments for screening of TTR(Met30) and (2) isoelectric focusing. TTR(Met30) was not detected but other substitutions were demonstrated using isoelectric focusing techniques. One of the variants found is a basic TTR variant. The substitutions occurring in the variant TTRs of these two kindreds are not known and are presently under study.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca2+-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene.