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ZusammenfassungGenetische Defekte werden vielfach noch als Schicksal empfunden, mit dem man sich Zeit seines Lebens abfinden muss. Es stimmt, dass vererbte Anlagen in vielen Fällen zu schweren Krankheiten führen, allerdings stimmt es auch, dass der Anteil von genetischen Defekten, bei denen eine Therapieoption besteht, stetig wächst und sich der Ausbruch von Krankheitssymptomen bei einigen davon bestenfalls gänzlich verhindern lässt. Die Kenntnis des genauen molekularen Krankheitsmechanismus liefert oft die Grundlage für einen Therapieansatz. Zum Auffinden des genetischen Defekts haben die Möglichkeiten der genomweiten Sequenzierung und ihr mittlerweile breiter Einsatz in der Diagnostik entscheidend beigetragen. Nach dem Nachweis einer genetischen Veränderung braucht es aber noch die Untersuchung der pathobiochemischen Konsequenzen auf zellulärer und systemischer Ebene. Dabei handelt es sich oft um einen längeren Prozess, da der volle Umfang von Funktionsausfällen nicht immer auf Anhieb erkennbar ist. Bei metabolischen Defekten kann die Therapie ein Auffüllen von fehlenden Produkten oder eine Reduktion von giftigen Substraten sein. Oft lässt sich auch die Restfunktion von betroffenen „pathways“ verbessern. Neuerdings haben Therapien mit direkter Korrektur des betroffenen Gendefekts Einzug in die therapeutische Anwendung gefunden. Da die ersten Krankheitssymptome in vielen Fällen früh im Leben auftreten, trifft die Kinderheilkunde eine Vorreiterrolle in der Entwicklung von Therapieansätzen.

Osteogenesis imperfecta (OI) is an inherited genetic disorder characterized by frequent bone fractures and reduced bone mass. Most cases of OI are caused by dominantly inherited heterozygous mutations in one of the two genes encoding type I collagen, COL1A1 and COL1A2. Here we describe a five-year-old boy with typical clinical, radiological and bone ultrastructural features of OI type I. Establishing the molecular genetic cause of his condition proved difficult since clinical exome and whole exome analysis was repeatedly reported negative. Finally, manual analysis of exome data revealed a silent COL1A2 variant c.3597 T > A (NM_000089.4), which we demonstrate activates a cryptic splice site. The newly generated splice acceptor in exon 50 is much more accessible than the wild-type splice-site between the junction of exon 49 and 50, and results in an in-frame deletion of 24 amino acids of the C-terminal propeptide. In vitro collagen expression studies confirmed cellular accumulation and decreased COL1A2 secretion to 45%. This is the first report of a cryptic splice site within the coding region of COL1A2. which results in abnormal splicing causing OI. The experience from this case demonstrates that routine diagnostic approaches may miss cryptic splicing mutations in causative genes due to the lack of universally applicable algorithms for splice-site prediction. In exome-negative cases, in-depth analysis of common causative genes should be conducted and trio-exome analysis is recommended., Highlights • Our study highlights a new category of mutations found in COL1A2. • Manual screening of WES data identified the cryptic splice site in COL1A2 • NGS may miss cryptic splicing mutations in causative genes • Universally applicable algorithms for splice-site prediction are needed

Purpose BiallelicCADvariants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. Methods Retrospective case series of 20 patients. Results Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. Conclusion We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Background: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. Methods: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. Results: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. Conclusions: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development., National Institutes of Health (U.S.) (Grant R01 HL124021), National Institutes of Health (U.S.) (Grant HL 122596), National Institutes of Health (U.S.) (Grant HL 138437), National Institutes of Health (U.S.) (Grant UH2 TR002073)

Zusammenfassung Die subkutan gestielte Plastik, auch VY-Plastik genannt, gehort zur Gruppe der Insellappenplastiken, deren Blutversorgung (im Gegensatz zu den horizontal perfundierten Verschiebe- und Transpositionslappenplastiken) durch senkrecht zur Hautoberflache fuhrende subkutane Blutgefase erfolgt. Aufgrund der hervorragenden Perfusion dieses Lappentyps entfernen wir bis zu einem Drittel des kranialen Lappenstiels und bilden dort einen 3–4 mm dicken Hautlappen, woraus ein wesentlicher Zugewinn im Hinblick auf die mit dem VY-Lappen deckbare Defektgrose resultiert. Daruber hinaus wird dieser Lappen in der Infraorbitalregion, soweit dies topographisch moglich ist, im kranialen Bereich durch eine schrag gesetzte Periostnaht weiter nach kranial in Richtung des zu verschliesenden Defektes gezogen. Dadurch wird ein eventuell vorhandener Zug des Lappens nach distal aufgehoben, was sich zur Verhinderung eines Ektropiums bewahrt hat. Summary The subcutaneously pedicled flap, also known as V-Y flap, belongs to the group of island flaps whose blood supply (unlike the horizontally perfused advancement and transposition flaps) comes from subcutaneous blood vessels perpendicular to the skin surface. Because of the outstanding perfusion of this type of flap, we remove up to one-third of the cranial flap pedicle to form a skin flap (3–4 mm thick), resulting in a significant increase in the defect size able to be covered by the V-Y flap. Moreover, in the infraorbital region – as far as topographically feasible – this flap is pulled further cranially (in the direction of the defect to be closed) by an obliquely placed periosteal suture. This offsets any potential distal tension on the flap and has thus proven beneficial in preventing an ectropion.

The subcutaneously pedicled flap, also known as V-Y flap, belongs to the group of island flaps whose blood supply (unlike the horizontally perfused advancement and transposition flaps) comes from subcutaneous blood vessels perpendicular to the skin surface. Because of the outstanding perfusion of this type of flap, we remove up to one-third of the cranial flap pedicle to form a skin flap (3-4 mm thick), resulting in a significant increase in the defect size able to be covered by the V-Y flap. Moreover, in the infraorbital region - as far as topographically feasible - this flap is pulled further cranially (in the direction of the defect to be closed) by an obliquely placed periosteal suture. This offsets any potential distal tension on the flap and has thus proven beneficial in preventing an ectropion.