Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

Newbornscreening SMA - From Pilot Project to Nationwide Screening in Germany.

Now that targeted therapies for spinal muscular atrophy are available, attempts are being made worldwide to include screening for spinal muscular atrophy in general newborn screening. In Germany, after pilot projects from 2018-2021, it was included in the general newborn screening from October 2021. To ensure a smooth transition, criteria for follow-up were developed together with key stakeholders. At the beginning of the transition to nationwide screening, false positive findings were reported in 3 patients. After optimization of the screening method in the laboratories concerned, all findings have been subsequently confirmed. On average, the first presentation to a neuromuscular center occurred on day 12 of life, and in patients with 2 or 3 SMN2 copies, therapy started on day 26 of life. Compared with the pilot project, there was no significant delay in timing.

Increased transgenerational epigenetic variation, but not predictable epigenetic variants, after environmental exposure in two apomictic dandelion lineages.

DNA methylation is one of the mechanisms underlying epigenetic modifications. DNA methylations can be environmentally induced and such induced modifications can at times be transmitted to successive generations. However, it remains speculative how common such environmentally induced transgenerational DNA methylation changes are and if they persist for more than one offspring generation. We exposed multiple accessions of two different apomictic dandelion lineages of the Taraxacum officinale group (Taraxacum alatum and T. hemicyclum) to drought and salicylic acid (SA) treatment. Using methylation-sensitive amplified fragment length polymorphism markers (MS-AFLPs) we screened anonymous methylation changes at CCGG restriction sites throughout the genome after stress treatments and assessed the heritability of induced changes for two subsequent unexposed offspring generations. Irrespective of the initial stress treatment, a clear buildup of heritable DNA methylation variation was observed across three generations, indicating a considerable background rate of heritable epimutations. Less evidence was detected for environmental effects. Drought stress showed some evidence for accession-specific methylation changes, but only in the exposed generation and not in their offspring. By contrast, SA treatment caused an increased rate of methylation change in offspring of treated plants. These changes were seemingly undirected resulting in increased transgenerational epigenetic variation between offspring individuals, but not in predictable epigenetic variants. While the functional consequences of these MS-AFLP-detected DNA methylation changes remain to be demonstrated, our study shows that (1) stress-induced transgenerational DNA methylation modification in dandelions is genotype and context-specific; and (2) inherited environmental DNA methylation effects are mostly undirected and not targeted to specific loci.

Early and lethal neurodegeneration with myasthenic and myopathic features: A new ALG14-CDG.

OBJECTIVE: To describe the presentation and identify the cause of a new clinical phenotype, characterized by early severe neurodegeneration with myopathic and myasthenic features. METHODS: This case study of 5 patients from 3 families includes clinical phenotype, serial MRI, electrophysiologic testing, muscle biopsy, and full autopsy. Genetic workup included whole exome sequencing and segregation analysis of the likely causal mutation. RESULTS: All 5 patients showed severe muscular hypotonia, progressive cerebral atrophy, and therapy-refractory epilepsy. Three patients had congenital contractures. All patients died during their first year of life. In 2 of our patients, electrophysiologic testing showed abnormal decrement, but treatment with pyridostigmine led only to temporary improvement. Causative mutations in ALG14 were identified in all patients. The mutation c.220 G>A (p.Asp74Asn) was homozygous in 2 patients and heterozygous in the other 3 patients. Additional heterozygous mutations were c.422T>G (p.Val141Gly) and c.326G>A (p.Arg109Gln). In all cases, parents were found to be heterozygous carriers. None of the identified variants has been described previously. CONCLUSIONS: We report a genetic syndrome combining myasthenic features and severe neurodegeneration with therapy-refractory epilepsy. The underlying cause is a glycosylation defect due to mutations in ALG14. These cases broaden the phenotypic spectrum associated with ALG14 congenital disorders of glycosylation as previously only isolated myasthenia has been described.

Natural epigenetic variation contributes to heritable flowering divergence in a widespread asexual dandelion lineage.

Epigenetic variation has been proposed to contribute to the success of asexual plants, either as a contributor to phenotypic plasticity or by enabling transient adaptation via selection on transgenerationally stable, but reversible, epialleles. While recent studies in experimental plant populations have shown the potential for epigenetic mechanisms to contribute to adaptive phenotypes, it remains unknown whether heritable variation in ecologically relevant traits is at least partially epigenetically determined in natural populations. Here, we tested the hypothesis that DNA methylation variation contributes to heritable differences in flowering time within a single widespread apomictic clonal lineage of the common dandelion (Taraxacum officinale s. lat.). Apomictic clone members of the same apomictic lineage collected from different field sites showed heritable differences in flowering time, which was correlated with inherited differences in methylation-sensitive AFLP marker profiles. Differences in flowering between apomictic clone members were significantly reduced after in vivo demethylation using the DNA methyltransferase inhibitor zebularine. This synchronization of flowering times suggests that flowering time divergence within an apomictic lineage was mediated by differences in DNA methylation. While the underlying basis of the methylation polymorphism at functional flowering time-affecting loci remains to be demonstrated, our study shows that epigenetic variation contributes to heritable phenotypic divergence in ecologically relevant traits in natural plant populations. This result also suggests that epigenetic mechanisms can facilitate adaptive divergence within genetically uniform asexual lineages.

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Phylogenetic relationships within Luzula DC. and Juncus L. (Juncaceae): A comparison of phylogenetic signals of trnL-trnF intergenic spacer, trnL intron and rbcL plastome sequence data.

Juncus and Luzula are the largest, almost cosmopolitan, genera in the Juncaceae. Relationships within Juncus and Luzula and among other genera of Juncaceae (Distichia, Marsippospermum, Oxychloë, Patosia and Rostkovia) remain incompletely resolved. RbcL sequence data resolved a part of the supraspecific phylogeny, but many clades remain polytomic. For this reason, the non-coding cpDNA regions, trnL intron and trnL-trnF intergenic spacer, were sequenced. We intended to create hypotheses of relationships within Juncaceae and to test the classification of the sections, but a primary goal to this study was to assess the relationships within Juncus and Luzula and to test for monophyly of groups recognized from rbcL data (especially the monophyly of genus Luzula and the Southern Hemisphere Clade (SHC)). Furthermore, we tested the influence of different rooting and ingroup composition on the tree topology. The parsimony analyses revealed several well-supported lineages. The traditionally distinguished genus Luzula is monophyletic and Juncus is non-monophyletic. Two subgenera of Luzula (Pterodes and Luzula) are non-monophyletic, while subg. Marlenia forms a sister group to the whole Luzula clade (trnL-F data set). Within Juncus, both subgenus Juncus and subgenus Agathryon are non-monophyletic. SHC is clustered not only with the South African J. lomatophylus and J. capensis, but also together with members of the section Juncus, Caespitosi and Graminifolii. These sections form a well-separated sister group to the SHC. Within the genera Juncus and Luzula, monophyly is demonstrated for a number of groups (e.g., Juncus section Stygiopsis, Luzula section Luzula) but questioned for others (e.g., Juncus section Graminifolii). The unusual, separate positioning of Juncus trifidus and J. monanthos were clarified by trnL-trnF sequence data, but vary within the tree topology depending on outgroup selection and also due to LBA phenomenon.

TrnL- trnF intergenic spacer and trnL intron define major clades within Luzula and Juncus (Juncaceae): importance of structural mutations.

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL- trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion "o" within a pseudogene) within the chloroplast trnL- trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The "Southern Hemisphere clade" was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5' acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL- trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.

Clinical impact of antibody formation to botulinum toxin A in children.

We studied the clinical impact of neutralizing antibodies to botulinum toxin A that occurred during long-term treatment of children between 1993 and 2001. Antibodies were found in high titers in 35 of 110 (31.8%) samples from individual patients. Antibody formation correlated with secondary nonresponse (p < 0.001). The most significant risk factors for antibody formation were the frequency of treatments (p = 0.0001) and the injection of a higher weight-adapted maximum dose per treatment (p = 0.001).