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BackgroundUp to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing.MethodsRNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of the DMDgene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients.ResultsWe identified an alteration in the mRNA level in all the patients. We detected three pseudoexons in DMDcaused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity.ConclusionThese findings indicate that mRNA analysis of the DMDgene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing.

Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%).

Background: Desminopathies are a clinically heterogeneous group of myopathies, with common histological findings in muscle biopsy. Clinically, they usually present with distal and/or proximal muscle weakness often associated with cardiomyopathy. We present 8 patients from 3 unrelated families manifesting isolated respiratory insufficiency without skeletal muscle weakness or heart disease, as a result of a mutation in the DESgene.

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.

PurposePatients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients’ characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies.Methods68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCAmissense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies.ResultsWe identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCAvariants reported in mutations databases as ‘affecting functions’ are SNPs. Deep analysis of sequencing data revealed patients’ true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations)ConclusionWES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

Spinal muscular atrophy (SMA) is caused by deletions/mutations in SMN1. Most heterozygous SMA carriers have only one SMN1copy in one of the alleles (1/0 carriers). However, a few carriers lack SMN1in one of their chromosomes, but present two gene copies in the other. These “2/0 carriers” are undistinguishable from non-carrier individuals (1/1) with currently available methods. Previous association of SMN1variants c.*3 + 80 T > G and c.*211_*212del with two SMN1copies in cisin Ashkenazi population prompted us to analyze them in 270 Spanish individuals (SMA carriers, patients and general population). Both variants were much more frequently detected in chromosomes with 2 SMN1copies in cisin comparison with chromosomes carrying one copy (17.9 vs. 0.7%; p< 0.001). In particular, one-fifth of 2/0 SMA carriers harboured one or both variants compared to none of 99 non-carriers with two SMN1copies (p< 0.001). The c.*211_*212del variant was also much more frequent in exon 8 of SMN2–SMN1hybrids than in that of intact SMN1genes (20 vs. 0.83%, p< 0.001), suggesting its association with chromosomal rearrangements. Although absence of these variants does not exclude that a particular individual is a 2/0 SMA carrier, their presence is valuable to substantially increase residual risk in putative carriers, thus improving genetic counselling.

We report a family with a new phenotype of autosomal recessive muscle dystrophy caused by a dysferlin mutation. The onset of the illness is distal, in the muscles of the anterior compartment group. The disease is rapidly progressive, leading to severe proximal weakness. Muscle biopsy showed moderate dystrophic changes with no vacuoles. Dysferlin immunostaining was negative. Gene analysis revealed a frameshift mutation in the exon 50 (delG5966) of the DYSF gene. This phenotype further demonstrates the clinical heterogeneity of the dysferlinopathies. Ann Neurol 2001;49:130–134

Summary A recombinant probe specific for the proα2 chain of human Type V collagen has been used for the localization of the corresponding gene (COL5A2) to chromosome 2. Regional mapping by in situ hybridization and analysis of DNA from humanxrodent cell lines indicated that COL5A2 is confined within the segment 2q14→2q32, thus syntenic to the proα1 (III) collagen gene (COL3A1).

Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1 kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).

By in situ hybridization of normal human chromosomes with a cloned genomic probe specific for the constant region of the lambda immunoglobulin genes, band 22q11 was preferentially labelled. In two cell lines with t(8;22) derived from Burkitt's lymphoma a strong signal was noted on the 8q chromosome derivative, indicating that the constant region of the lambda Ig gene cluster was translocated from chromosome 22 to chromosome 8. In addition, the signal observed on the 22q- derivative chromosome was stronger than the background in one of the two cell lines tested, but not in the other. The implications are that the break point in chromosome 22 in some cases lies within the Ig gene itself or between clusters of such genes, and that different cases have different break points.