Abstract Background As clinical genetics evolves towards the broader field of clinical genomics, the diagnostic approach to rare diseases is undergoing a paradigm shift. This transformation has significantly impacted rare disease diagnostics, increasingly done through gene panels, whole exome and whole genome sequencing. To advance beyond genomics into precision medicine and encompass the breadth of relevant clinical scenarios, a true systems shift is required that challenges conventional barriers and enables the formation of cross-disciplinary, integrated environments. Methods The Genomic Medicine Center Karolinska Rare Diseases (GMCK-RD) has, for the past 10 years, brought together healthcare and academia to enable large-scale genome sequencing in a clinical diagnostics context. Within GMCK-RD, experts from various medical disciplines collaborate closely with clinical geneticists, bioinformaticians, and researchers to integrate genome sequencing into healthcare. Results In total, 15 644 individuals with suspected rare diseases were analyzed using clinical genome sequencing, including pediatric (48%), adult (48%) and fetal (4%) samples. The overall diagnostic yield was 22.6%, providing a diagnosis for 3 538 individuals with variants in 1 570 genes. Moreover, a rare disease analysis tool suite developed and validated in house includes a bioinformatic pipeline allowing for comprehensive data analysis covering a wide range of genetic variants including SNVs, INDELs, repeat expansions, uniparental disomies, balanced and unbalanced structural variants as well as insertions of mobile elements. Results are visualized and interpreted in custom-developed decision support systems functioning as an interpretation portal as well as a knowledge-base to capture the interpretation efforts made in a structured format allowing future secondary use. Conclusions Altogether, GMCK-RD has shifted healthcare in our region towards precision diagnostics. We emphasize the need to transition from traditional clinical genetic diagnostics to a broader clinical genomics approach. Beyond this shift, we advocate integrating genomics with specialized clinical and laboratory medicine, a concept pioneered for inborn errors of metabolism (IEM) with stepwise spread to additional disease groups. In this model, a multidisciplinary unit combines screening, targeted diagnostics, individualized treatment, and long-term patient follow-up. Here we provide a road map and guide for inspiration for centers aiming to implement genome sequencing in rare disease diagnostics.

Objective:. VASCERN (https://vascern.eu/) is the European Reference Network for Rare Multisystemic Vascular Diseases. VASCERN-VASCA is the working group within VASCERN that focuses on the study of vascular anomalies. One of the objectives of this group is to establish patient pathways to guide physicians toward efficient diagnostic and management measures. The patient pathway presented here is focused on capillary malformations (CMs). Methods:. The Nominal Group Technique, a structured variation of small group discussion was used. Two facilitators were identified: one to propose initial discussion points and draw the pathway and another to chair the discussion. A dermatologist (E. Baselga) was chosen as the first facilitator due to her specific clinical and research expertise. The draft was subsequently discussed within VASCERN-VASCA monthly virtual meetings and biannual face-to-face meetings. Results:. The pathway starts from the clinical recognition of a vascular red stain, describing clinical characteristics and location. Depending on the clinical features, a subsequent workup for associated manifestations or complications is suggested. These steps should enable the establishment of 6 subtypes of CMs: (1) nevus simplex; (2) isolated CM, syndromic or nonsyndromic; (3) CM of microcephaly CM syndrome; (4) CM of CM–arteriovenous malformation syndromes; (5) “pseudo” CM of arteriovenous malformation; (6) cutis marmorata telangiectatica congenita. Management according to the recognized phenotype is detailed in subsequent pages of the pathway. A color code is used to differentiate (1) clinical evaluations, (2) investigations, (3) associated genes, and (4) treatments. Actions relevant to all types are marked in separate boxes, for example, when to perform specific imaging. Conclusion:. The collaborative efforts of VASCERN-VASCA, a European network of the 14 Expert Centers for Vascular Anomalies, have led to a consensus pathway for CMs. This pathway may help clinicians to guide in the diagnosis and management of CMs, as well as to emphasize the crucial role of multidisciplinary expert centers in the management of these patients. This pathway is available on the VASCERN website (http://vascern.eu/).

The clinical management of lymphatic malformations presents challenges due to their rarity, variable presentation, and the potential for recurrence. Dissemination of the latest knowledge in lymphatic malformations is needed to streamline referral pathways and possibly improve patient outcomes. This paper provides a narrative review of the latest developments in aetiopathogenesis, diagnosis and management of lymphatic malformations. Multidisciplinary recommendations from the European Reference Network on Rare Multisystemic Vascular Diseases, Vascular Anomalies working group sets the basis for this review. A literature search was conducted to include the latest research based on authors' preferences. Understanding the aetiopathogenesis of lymphatic malformations is important, as new genetic insights can dictate management strategies. Advancements in diagnostics allow for better differentiation between malformation types and imaging of anatomical infiltrations, where treatment options differ and are more commonly multimodal. Further systematic studies are needed on larger study populations, such as prospective trials and meta-analyses, to improve evidence-based practice.

BACKGROUND:Vascular anomalies caused by somatic (postzygotic) variants are clinically and genetically heterogeneous diseases with overlapping or distinct entities. The genetic knowledge in this field is rapidly growing, and genetic testing is now part of the diagnostic workup alongside the clinical, radiological and histopathological data. Nonetheless, access to genetic testing is still limited, and there is significant heterogeneity across the approaches used by the diagnostic laboratories, with direct consequences on test sensitivity and accuracy. The clinical utility of genetic testing is expected to increase progressively with improved theragnostics, which will be based on information about the efficacy and safety of the emerging drugs and future molecules. The aim of this study was to make recommendations for optimising and guiding the diagnostic genetic testing for somatic variants in patients with vascular malformations. RESULTS:Physicians and lab specialists from 11 multidisciplinary European centres for vascular anomalies reviewed the genes identified to date as being involved in non-hereditary vascular malformations, evaluated gene-disease associations, and made recommendations about the technical aspects for identification of low-level mosaicism and variant interpretation. A core list of 24 genes were selected based on the current practices in the participating laboratories, the ISSVA classification and the literature. In total 45 gene-phenotype associations were evaluated: 16 were considered definitive, 16 strong, 3 moderate, 7 limited and 3 with no evidence. CONCLUSIONS:This work provides a detailed evidence-based view of the gene-disease associations in the field of vascular malformations caused by somatic variants. Knowing both the gene-phenotype relationships and the strength of the associations greatly help laboratories in data interpretation and eventually in the clinical diagnosis. This study reflects the state of knowledge as of mid-2023 and will be regularly updated on the VASCERN-VASCA website (VASCERN-VASCA, https://vascern.eu/groupe/vascular-anomalies/ ).

Background: De novo variants are a common cause to rare intellectual disability syndromes, associated with low recurrence risk. However, when such variants occur pre-zygotically in parental germ cells, the recurrence risk might be higher. Still, the recurrence risk estimates are mainly based on empirical data and the prevalence of germline mosaicism is often unknown. Methods: To establish the prevalence of mosaicism in parents of children with intellectual disability syndromes caused by de novo variants, we performed droplet digital PCR on DNA extracted from blood (43 trios), and sperm (31 fathers). Results: We detected low-level mosaicism in sperm-derived DNA but not in blood in the father of a child with Kleefstra syndrome caused by an EHMT1 variant. Additionally, we found a higher level of paternal mosaicism in sperm compared to blood in the father of a child with Gillespie syndrome caused by an ITPR1 variant. Conclusion: By employing droplet digital PCR, we detected paternal germline mosaicism in two intellectual disability syndromes. In both cases, the mosaicism level was higher in sperm than blood, indicating that analysis of blood alone may underestimate germline mosaicism. Therefore, sperm analysis can be clinically useful to establish the recurrence risk for parents and improve genetic counselling.

BackgroundGermline pathogenic variants in DICER1 cause DICER1 syndrome, an autosomal dominant, pleiotropic tumour predisposition syndrome with variable expressivity and reduced penetrance for specific dysplastic and neoplastic lesions. Recently, a syndrome with the acronym GLOW (Global developmental delay, Lung cysts, Overgrowth, Wilms tumour) was described in two children with mosaic missense mutations in hotspot residues of the DICER1 RNase IIIb domain.MethodsWhole genome sequencing, exome sequencing, Sanger sequencing, digital PCR and a review of Wilms tumours with DICER1 RNase III domain mutations were performed.ResultsA de novo heterozygous c.4031C>T (p.S1344L) variant in the sequence encoding the RNase IIIa domain of DICER1 was detected. Clinical investigations revealed a phenotype that resembles the GLOW subphenotype of DICER1 syndrome.ConclusionThe phenotypic overlap between patients with p.S1344L mutation and GLOW syndrome provide clinical support for recent discoveries that RNase IIIa-Ser1344 site mutations impede miRNA-5p biogenesis analogous to DICER1 hotspot mutations in the RNase IIIb domain. We show that an individual with a heterozygous germline p.S1344L mutation has a severe form of DICER1 syndrome (‘DICER1 syndrome plus’), with notable features of intellectual disability, macrocephaly, physical abnormalities, Wilms tumour and a well-differentiated fetal adenocarcinoma of the lung.

PIK3CA-related overgrowth spectrum is a group of rare genetic disorders with asymmetric overgrowth caused by somatic mosaic PIK3CA mutations. Here, we report clinical data and molecular findings from two patients with congenital muscular upper limb overgrowth and aberrant anatomy. During debulking surgery, numerous ectopic muscles were found in the upper limbs of the patients. DNA sequencing, followed by digital polymerase chain reaction, was performed on DNA extracted from biopsies from hypertrophic ectopic muscles and identified the somatic mosaic PIK3CA hotspot mutations c.3140A > G, p.(His1047Arg) and c.1624G > A, p.(Glu542Lys) in a male (patient 1) and a female (patient 2) patient, respectively. Patient 1 had four ectopic muscles and unilateral isolated muscular overgrowth while patient 2 had 13 ectopic muscles and bilateral isolated muscular overgrowth of both upper limbs, indicating that her mutation occurred at early pre-somitic mesoderm state. The finding of PIK3CA mutations in ectopic muscles highlights the importance of PIK3CA in cell fate in early human embryonic development. Moreover, our findings provide evidence that the disease phenotype depends on the timing of PIK3CA mutagenesis during embryogenesis and confirm the diagnostic entity PIK3CA-related muscular overgrowth with ectopic accessory muscles.

Here, we report a novel mosaic mutation in the PORCN gene in a male Goltz syndrome patient. We also compare the phenotypes of all reported males with a confirmed molecular diagnosis. This report serves to further clarify the phenotype of Goltz syndrome and suggests that expression in males varies.

All antipsychtics currently in clinical use are antagonists or weak partial agonists at the dopamine D2 receptor (D2R). Antipsychotic medication is associated with adverse effects such as extrapyramidal symptoms (EPS). The lower EPS liability of newer, so-called atypical antipsychotics, typified by clozapine, has been proposed to reflect their faster rates of dissociation from the D2R, as compared to older, typical antipsychotics such as haloperidol. This hypothesis has received increasing attention in recent years, and several pharmaceutical companies have endeavored to developed their own "fast off"-antipsychotics. However, previous studies have measured dissociation of radiolabeled antipsychotics or used modified G proteins to study receptor activation-induced calcium release, which confers certain limitations in terms of temporal resolution. We have examined antagonist dissociation in living cells, employing an assay based on the activation of G protein coupled potassium channels. This assay uses native G proteins and has higher temporal resolution than previous studies. Our preliminary data suggest that there may be larger differences between different atypical antipsychotics than has previously been appreciated. Furthermore, the differences between atypical and typical drugs appear to relate mainly to the differential hydrophilicities of these drugs.

The lower liability of atypical antipsychotics to produce side-effects correlates with their faster rates of dissociation from the dopamine D2 receptor. Recent studies indicate that the novel D2 ligands, ACR16 and OSU6162, act as antagonists with similarly high dissociation rates. However, those studies measured dissociation of radiolabeled ligand from membrane preparations or used modified G proteins to study calcium release. We examined relative antagonist dissociation rates in living cells, using a time-resolved assay based on activation of G protein-coupled potassium channels (GIRK) by native G proteins. GIRK responses to dopamine receptor activation were studied using two-electrode voltage clamp in Xenopus oocytes expressing D2 receptors and GIRK. First, dopamine was applied, resulting in a "baseline" response. Next, antagonist was washed in, in the continued presence of dopamine. After attaining steady-state response inhibition, antagonist was washed out, still in the presence of dopamine. Response recovery was recorded over six minutes, and the time-course and relative amplitude of recovery were taken as measures of antagonist dissociation. Significant differences in response recovery T1/2 and recovery amplitudes were observed between the different D2 receptor antagonists: In experiments with haloperidol, risperidone, and aripiprazole, virtually no response recovery was observed. With clozapine and quetiapine, similar recovery amplitudes and recovery time courses were observed (T1/2 ∼ 40 s). ACR16 and OSU6162 behaved as antagonists, lacking detectable efficacy in the GIRK assay. These compounds washed out with similar time courses (T1/2 ∼ 8 s); significantly faster than the other antagonists. ACR16 and OSU6162 appear to dissociate faster than clozapine and quetiapine, a finding which has not been reported earlier. Such very rapid dissociation might be relevant to the low incidence of side effects reported from clinical trials with these compounds.

Purpose of the study: Antipsychotic medication is often associated with adverse effects such as extrapyramidal symptoms (EPS) and increased serum prolactin. There is evidence that the lower liability to produce EPS and increased prolactin attributed to newer, so-called atypical antipsychotics, is correlated with their faster rates of dissociation from the dopamine D2 receptor [1]. Recent studies have indicated that the novel D2 receptor ligands, ACR16 and (−)-OSU6162, initially described as “dopamine stabilizers,” act as antagonists with similarly high dissociation rates [2,3]. However, these previous studies of antagonist unbinding rates measured either dissociation of radiolabeled ligand from membrane preparations [1] or used modified G proteins to study receptor activation-induced calcium release in living cells [2,3]. We wanted to examine the relative kinetics of antagonist dissociation in living cells, using an assay based an activation of G protein-coupled potassium (GIRK) channels. This assay uses native G proteins and has higher temporal resolution than previously used assays. Methods used: Xenopus oocytes were injected with cRNA encoding the human dopamine D2S receptor, Regulator of G protein Signalling (RGS)-4, and GIRK1/4 channel subunits. GIRK current responses to dopamine receptor activation were recorded at −80mV using twoelectrode voltage clamp. First, dopamine (100 nM) was applied, resulting in a “baseline” GIRK response. Next, a maximally effective concentration of antagonist was washed in, in the continued presence of dopamine. After achieving steady-state response inhibition the antagonist was washed out, still in the presence of dopamine. Response recovery was recorded over six minutes, and the recovery time-course and the amplitude of the (pseudo)steady state current relative to the baseline response were taken as measures of antagonist dissociation. Summary of results: Significant differences (P< 0.05; Student’s t-test) in response recovery T1/2 (time to half-maximal recovery) and recovery amplitudes were observed between the different D2 receptor antagonists: In experiments with haloperidol, risperidone, paliperidone, aripiprazole, and bifeprunox, no response recovery was detected. With clozapine, quetiapine, and N-desmethylclozapine, similar recovery time courses were observed (T1/2 = 48±5.5 s, 60±2.2 s, and 46±3.7 s, respectively). With clozapine and quetiapine, about 50% response recovery was seen, whereas N-desmethylclozapine washout allowed 80% recovery. The “stabilizer” compounds ACR16 and (−)-OSU6162, along with the structurally related experimental antipsychotic, (−)-3-PPP, behaved as antagonists, lacking detectable efficacy in the assay. These compounds washed out with similar time courses (T1/2 = 8.2±1.8 s, 6.1±0.44 s, and 7.8±0.87 s, respectively); significantly faster than the other antagonists in the study, and allowed near-complete response recovery. Conclusions: The present data support the idea that the atypical antipsychotics, clozapine and quetiapine, dissociate faster from D2 receptors than the typical antipsychotic haloperidol, but also faster than several other atypical drugs in the study, including risperidone, paliperidone, and aripiprazole. Furthermore, the “dopamine stabilizers” ACR16 and (−)-OSU6162 appear to dissociate faster than clozapine and quetiapine, a finding which has not been reported earlier. Such very rapid dissociation might be relevant to the low incidence of side effects reported from clinical trials with these compounds.

In the present study we have investigated whether pharmacological manipulations of central l-arginine-nitric oxide (l-Arg-NO) pathway could affect blood pressure (BP) and heart rate (HR) in normotensive rats either untreated or pretreated with E. coli lipopolysaccharide (LPS). The intracerebroventricular injection (i.c.v.) of Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthesis, caused a fall of BP and HR in LPS-treated but not in control rats. Furthermore, the pressor responses to i.c.v. injection of N-methyl-d-aspartate (NMDA) were enhanced by l-Arg or LPS treatment and, in both cases, this potentiation was blocked by l-NAME. The present results show that in some experimental conditions, such as activation of NMDA receptors or LPS pretreatment, the central microinfusion of drugs affecting the l-Arg-NO pathway may interfere with BP and HR.