Progressive liver, kidney, and heart degeneration in children and adults affected by TULP3 mutations.

Organ fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-ß signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.

The ciliary transition zone protein TMEM218 synergistically interacts with the NPHP module and its reduced dosage leads to a wide range of syndromic ciliopathies.

Mutations in genes that lead to dysfunctional cilia can cause a broad spectrum of human disease phenotypes referred to as ciliopathies. Many ciliopathy-associated proteins are localized to the evolutionary conserved ciliary transition zone (TZ) subdomain. We identified biallelic missense and nonsense mutations in the gene encoding the transmembrane protein TMEM218 in unrelated patients with features related to Bardet-Biedl, Joubert and Meckel-Gruber syndrome (MKS) and characterized TMEM218 as a major component of the ciliary TZ module. Co-immunoprecipitation assays resulted in the physical interaction of TMEM218 with the MKS module member TMEM67/Meckelin that was significantly reduced by the TMEM218 missense change harboured by one of our patients. We could further validate its pathogenicity by functional in vivo analysis in zebrafish (Danio rerio) as a well-established vertebrate model for ciliopathies. Notably, ciliopathy-related phenotypes were most prominent by genetic interactions with the NPHP module component Nphp4. Conclusively, we describe TMEM218 as a new disease gene for patients with a wide spectrum of syndromic ciliopathy phenotypes and provide evidence for a synergistic interaction of TMEM218 and the NPHP module crucial for proper ciliary function.

Congenital Hyperinsulinism in Humans and Insulin Secretory Dysfunction in Mice Caused by Biallelic DNAJC3 Variants.

The BiP co-chaperone DNAJC3 protects cells during ER stress. In mice, the deficiency of DNAJC3 leads to beta-cell apoptosis and the gradual onset of hyperglycemia. In humans, biallelic DNAJC3 variants cause a multisystem disease, including early-onset diabetes mellitus. Recently, hyperinsulinemic hypoglycemia (HH) has been recognized as part of this syndrome. This report presents a case study of an individual with HH caused by DNAJC3 variants and provides an overview of the metabolic phenotype of individuals with HH and DNAJC3 variants. The study demonstrates that HH may be a primary symptom of DNAJC3 deficiency and can persist until adolescence. Additionally, glycemia and insulin release were analyzed in young DNACJ3 knockout (K.O.) mice, which are equivalent to human infants. In the youngest experimentally accessible age group of 4-week-old mice, the in vivo glycemic phenotype was already dominated by a reduced total insulin secretion capacity. However, on a cellular level, the degree of insulin release of DNAJC3 K.O. islets was higher during periods of increased synthetic activity (high-glucose stimulation). We propose that calcium leakage from the ER into the cytosol, due to disrupted DNAJC3-controlled gating of the Sec61 channel, is the most likely mechanism for HH. This is the first genetic mechanism explaining HH solely by the disruption of intracellular calcium homeostasis. Clinicians should screen for HH in DNAJC3 deficiency and consider DNAJC3 variants in the differential diagnosis of congenital hyperinsulinism.

Hyperinsulinemic Hypoglycemia Associated with a CaV1.2 Variant with Mixed Gain- and Loss-of-Function Effects.

The voltage-dependent L-type calcium channel isoform CaV1.2 is critically involved in many physiological processes, e.g., in cardiac action potential formation, electromechanical coupling and regulation of insulin secretion by beta cells. Gain-of-function mutations in the calcium voltage-gated channel subunit alpha 1 C (CACNA1C) gene, encoding the CaV1.2 α1-subunit, cause Timothy syndrome (TS), a multisystemic disorder that includes autism spectrum disorders and long QT (LQT) syndrome. Strikingly, TS patients frequently suffer from hypoglycemia of yet unproven origin. Using next-generation sequencing, we identified a novel heterozygous CACNA1C mutation in a patient with congenital hyperinsulinism (CHI) and associated hypoglycemic episodes. We characterized the electrophysiological phenotype of the mutated channel using voltage-clamp recordings and in silico action potential modeling experiments. The identified CaV1.2L566P mutation causes a mixed electrophysiological phenotype of gain- and loss-of-function effects. In silico action potential modeling supports that this mixed electrophysiological phenotype leads to a tissue-specific impact on beta cells compared to cardiomyocytes. Thus, CACNA1C variants may be associated with non-syndromic hyperinsulinemic hypoglycemia without long-QT syndrome, explained by very specific electrophysiological properties of the mutated channel. We discuss different biochemical characteristics and clinical impacts of hypoglycemia in the context of CACNA1C variants and show that these may be associated with significant morbidity for Timothy Syndrome patients. Our findings underline that the potential of hypoglycemia warrants careful attention in patients with CACNA1C variants, and such variants should be included in the differential diagnosis of non-syndromic congenital hyperinsulinism.

Phylogenetic Cluster Analysis Identifies Virological and Behavioral Drivers of Human Immunodeficiency Virus Transmission in Men Who Have Sex With Men.

BACKGROUND: Identifying local outbreaks and their drivers is a key step toward curbing human immunodeficiency virus (HIV) transmission and potentially achieving HIV elimination. Such outbreaks can be identified as transmission clusters extracted from phylogenetic trees constructed of densely sampled viral sequences. In this study, we combined phylogenetic transmission clusters with extensive data on virological suppression and behavioral risk of cluster members to quantify the drivers of ongoing transmission over 10 years. METHODS: Using the comprehensive Swiss HIV Cohort Study and its drug-resistance database, we reconstructed phylogenetic trees for each year between 2007 and 2017. We identified HIV transmission clusters dominated by men who have sex with men (MSM) and determined their annual growth. We used Poisson regression to assess if cluster growth was associated with a per-cluster infectivity and behavioral risk score. RESULTS: Both infectivity and behavioral risk scores were significantly higher in growing MSM transmission clusters compared to nongrowing clusters (P ≤ .01). The fraction of transmission clusters without infectious members acquiring new infections increased significantly over the study period. The infectivity score was significantly associated with per-capita incidence of MSM transmission clusters in 8 years, while the behavioral risk score was significantly associated with per-capita incidence of MSM transmission clusters in 3 years. CONCLUSIONS: We present a phylogenetic method to identify hotspots of ongoing transmission among MSM. Our results demonstrate the effectiveness of treatment as prevention at the population level. However, the significantly increasing number of new infections among transmission clusters without infectious members highlights a relative shift from diagnosed to undiagnosed individuals as drivers of HIV transmission in Swiss MSM.

The underestimated burden of monogenic kidney disease in adults waitlisted for kidney transplantation.

PURPOSE: Chronic kidney disease (CKD) is a major health-care burden. Increasing evidence suggests that a considerable proportion of patients are affected by a monogenic kidney disorder. METHODS: In this study, the kidney transplantation waiting list at the Charité was screened for patients with undetermined cause of CKD. By next-generation sequencing (NGS) we targeted all 600 genes described and associated with kidney disease or allied disorders. RESULTS: In total, 635 patients were investigated. Of these, 245 individuals had a known cause of CKD (38.5%) of which 119 had a proven genetic disease (e.g., ADPKD, Alport). The other 340 patients (53.5%) were classified as undetermined diagnosis, of whom 87 had kidney failure (KF) onset <40 years. To this latter group genetic testing was offered as well as to those patients (n = 29) with focal segmental glomerulosclerosis (FSGS) and all individuals (n = 21) suspicious for thrombotic microangiopathy (TMA) in kidney biopsy. We detected diagnostic variants in 26 of 126 patients (20.6%) of which 14 of 126 (11.1%) were pathogenic or likely pathogenic. In another 12 of 126 (9.5%) patients, variants of unknown significance (VUS) were detected. CONCLUSION: Our study demonstrates the diagnostic value of comprehensive genetic testing among patients with undetermined CKD.

Loss of CBY1 results in a ciliopathy characterized by features of Joubert syndrome.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.

Cancer incidence and spectrum among children with genetically confirmed Beckwith-Wiedemann spectrum in Germany: a retrospective cohort study.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a cancer predisposition syndrome caused by defects on chromosome 11p15.5. The quantitative cancer risks in BWS patients depend on the underlying (epi)genotype but have not yet been assessed in a population-based manner. METHODS: We identified a group of 321 individuals with a molecularly confirmed diagnosis of BWS and analysed the cancer incidence up to age 15 years and cancer spectrum by matching their data with the German Childhood Cancer Registry. RESULTS: We observed 13 cases of cancer in the entire BWS cohort vs 0.4 expected. This corresponds to a 33-fold increased risk (standardised incidence ratio (SIR) = 32.6; 95% confidence interval = 17.3-55.7). The specific cancers included hepatoblastoma (n = 6); nephroblastoma (n = 4); astrocytoma (n = 1); neuroblastoma (n = 1) and adrenocortical carcinoma (n = 1). The cancer SIR was highest in patients with a paternal uniparental disomy of 11p15.5 (UPDpat). A high cancer risk remained when cases of cancer diagnosed prior to the BWS diagnosis were excluded. CONCLUSIONS: This study confirms an increased cancer risk in children with BWS. Our findings suggest that the highest cancer risk is associated with UPDpat. We were unable to confirm an excessive cancer risk in patients with IC1 gain of methylation (IC1-GOM) and this finding requires further investigation.

Comparative meta-analysis of Kabuki syndrome with and without hyperinsulinaemic hypoglycaemia.

BACKGROUND AND OBJECTIVE: Kabuki syndrome (KS), caused by pathogenic variants in KMT2D or KDM6A, is associated with hyperinsulinaemic hypoglycaemia (HH) in 0.3%-4% of patients. We characterized the clinical, biochemical and molecular data of children with KS and HH compared to children with KS without HH in a multicentre meta-analysis. METHODS: Data of seven new and 17 already published children with KS and HH were compared to 373 recently published KS patients without HH regarding molecular and clinical characteristics. RESULTS: Seven new patients were identified with seven different pathogenic variants in KDM6A (n = 4) or KMT2D (n = 3). All presented with HH on the first day of life and were responsive to diazoxide. KS was diagnosed between 9 months and 14 years of age. In the meta-analysis, 24 KS patients with HH had a significantly higher frequency of variants in KDM6A compared to 373 KS patients without HH (50% vs 11.5%, P < .001), and KDM6A-KS was more likely to be associated with HH than KMT2D-KS (21.8% vs. 3.5%, P < .001). Sex distribution and other phenotypic features did not differ between KS with and without HH. CONCLUSION: The higher incidence of HH in KDM6A-KS compared to KMT2D-KS indicates that KDM6A loss of function variants predispose more specifically to beta cell dysfunction compared to KMT2D variants. As difficulties to assign syndromic characteristics to KS in early infancy often lead to delayed diagnosis, genetic testing for KS should be considered in children with HH, especially in the presence of other extrapancreatic/syndromic features.

Genotype-Informed Versus Empiric Management Of VirEmia (GIVE MOVE): study protocol of an open-label randomised clinical trial in children and adolescents living with HIV in Lesotho and Tanzania.

BACKGROUND: Globally, the majority of people living with HIV have no or only limited access to HIV drug resistance testing to guide the selection of antiretroviral drugs. This is of particular concern for children and adolescents, who experience high rates of treatment failure. The GIVE MOVE trial assesses the clinical impact and cost-effectiveness of routinely providing genotypic resistance testing (GRT) to children and adolescents living with HIV who have an unsuppressed viral load (VL) while taking antiretroviral therapy (ART). METHODS: GIVE MOVE is an open-label randomised clinical trial enrolling children and adolescents (≥6 months to <19 years) living with HIV with a VL ≥400 copies/mL (c/mL) while taking first-line ART. Recruitment takes place at sites in Lesotho and Tanzania. Participants are randomised in a 1:1 allocation to a control arm receiving the standard of care (3 sessions of enhanced adherence counselling, a follow-up VL test, continuation of the same regimen upon viral resuppression or empiric selection of a new regimen upon sustained elevated viremia) and an intervention arm (GRT to inform onward treatment). The composite primary endpoint is the occurrence of any one or more of the following events during the 36 weeks of follow-up period: i) death due to any cause; ii) HIV- or ART-related hospital admission of ≥24 h duration; iii) new clinical World Health Organisation stage 4 event (excluding lymph node tuberculosis, stunting, oral or genital herpes simplex infection and oesophageal candidiasis); and iv) no documented VL <50 c/mL at 36 weeks follow-up. Secondary and exploratory endpoints assess additional health-related outcomes, and a nested study will assess the cost-effectiveness of the intervention. Enrolment of a total of 276 participants is planned, with an interim analysis scheduled after the first 138 participants have completed follow-up. DISCUSSION: This randomised clinical trial will assess if the availability of resistance testing improves clinical outcomes in children and adolescents with elevated viremia while taking ART. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov ( NCT04233242 ; registered 18.01.2020). More information: www.givemove.org .

PEBRA trial - effect of a peer-educator coordinated preference-based ART service delivery model on viral suppression among adolescents and young adults living with HIV: protocol of a cluster-randomized clinical trial in rural Lesotho.

BACKGROUND: Despite tremendous progress in controlling the HIV epidemic in sub-Saharan Africa, HIV-related mortality continues to increase among adolescents and young people living with HIV (AYPLHIV). Globally, sub-Saharan Africa accounts for 85% of the AYPLHIV. Overall outcomes along the HIV care cascade are worse among AYPLHIV as compared to all other age groups due to various challenges in accessing and adhering to antiretroviral therapy (ART). New, innovative multicomponent packages of differentiated service delivery (DSD) models, are required to address the specific needs of AYPLHIV. This study aims to evaluate the feasibility and effectiveness of a multicomponent DSD model (PEBRA model) designed for AYPLHIV and coordinated by a peer-educator. METHODS: PEBRA (Peer-Educator Based Refill of ART) is a cluster randomized, open-label, superiority trial conducted at 20 health facilities in three districts of Lesotho, Southern Africa. The clusters (health facilities) are randomly assigned to either the PEBRA model or standard of care in a 1:1 ratio, stratified by district. AYPLHIV aged 15-24 years old in care and on ART at one of the clusters are eligible. In the PEBRA model, a peer-educator coordinates the antiretroviral therapy (ART) services - such as medication pick-up, SMS notifications and support options - according to the preferences of the AYPLHIV. The peer-educator delivers this personalized model using a tablet-based application called PEBRApp. The control clusters continue to offer standard of care: ART services coordinated by the nurse. The primary endpoint is viral suppression at 12 months. Secondary endpoints include self-reported adherence to ART, quality of life, satisfaction with care and engagement in care. The target sample size is 300 AYPLHIV. Statistical analyses are conducted and reported in line with CONSORT guidelines for cluster randomized trials. DISCUSSION: The PEBRA trial will provide evidence on the feasibility and effectiveness of an inclusive, holistic and preference-based DSD model for AYPLHIV that is coordinated by a peer-educator. Many countries in SSA have an existing peer-educator program. If proven effective, the PEBRA model and PEBRApp have the potential to be scaled up to similar settings. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03969030. Registered on 31 May 2019. More information: www.pebra.info.

A Systematic Phylogenetic Approach to Study the Interaction of HIV-1 With Coinfections, Noncommunicable Diseases, and Opportunistic Diseases.

To systematically test whether coinfections spread along the HIV-1 transmission network and whether similarities in HIV-1 genomes predict AIDS-defining illnesses and comorbidities, we analyzed the distribution of these variables on the HIV phylogeny of the densely sampled Swiss HIV Cohort Study. By combining different statistical methods, we could detect, quantify, and explain the clustering of diseases. Infectious conditions such as hepatitis C, but also Kaposi sarcoma, clustered significantly, suggesting transmission of these infections along the HIV-1 transmission network. The clustering of patients with neurocognitive complaints could not be completely explained by the clustering of patients with similar demographic risk factors, which suggests a potential impact of viral genetics. In summary, the consistent and robust signal for coinfections and comorbidities highlights the strong interaction of HIV-1 and other infections and shows the potential of combining phylogenetic methods to identify disease traits that are likely to be related to virus genetic factors.

Viral Diversity Based on Next-Generation Sequencing of HIV-1 Provides Precise Estimates of Infection Recency and Time Since Infection.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) genetic diversity increases over the course of infection and can be used to infer the time since infection and, consequently, infection recency, which are crucial for HIV-1 surveillance and the understanding of viral pathogenesis. METHODS: We considered 313 HIV-infected individuals for whom reliable estimates of infection dates and next-generation sequencing (NGS)-derived nucleotide frequency data were available. Fractions of ambiguous nucleotides, obtained by population sequencing, were available for 207 samples. We assessed whether the average pairwise diversity calculated using NGS sequences provided a more exact prediction of the time since infection and classification of infection recency (<1 year after infection), compared with the fraction of ambiguous nucleotides. RESULTS: NGS-derived average pairwise diversity classified an infection as recent with a sensitivity of 88% and a specificity of 85%. When considering only the 207 samples for which fractions of ambiguous nucleotides were available, the NGS-derived average pairwise diversity exhibited a higher sensitivity (90% vs 78%) and specificity (95% vs 67%) than the fraction of ambiguous nucleotides. Additionally, the average pairwise diversity could be used to estimate the time since infection with a mean absolute error of 0.84 years, compared with 1.03 years for the fraction of ambiguous nucleotides. CONCLUSIONS: Viral diversity based on NGS data is more precise than that based on population sequencing in its ability to predict infection recency and provides an estimated time since infection that has a mean absolute error of <1 year.

Importance of routine viral load monitoring: higher levels of resistance at ART failure in Uganda and Lesotho compared with Switzerland.

Objectives: Emerging resistance to antiretroviral drugs may jeopardize the achievements of improved access to ART. We compared the prevalence of different resistance mutations in HIV-infected adults with virological failure in a cohort with regular routine viral load (VL) monitoring (Switzerland) and cohorts with limited access to VL testing (Uganda and Lesotho). Methods: We considered individuals who had genotypic resistance testing (GRT) upon virological failure (≥1000 copies/mL) and were on ART consisting of at least one NNRTI and two NRTIs. From Lesotho, individuals with two subsequent VLs ≥1000 copies/mL despite enhanced adherence counselling (n = 58) were included in the analysis. From Uganda, individuals with a single VL ≥1000 copies/mL (n = 120) were included in the analysis. From the Swiss HIV Cohort Study (SHCS), a population without history of monotherapy or dual therapy with the first GRT upon virological failure (n = 61) was selected. Results: We found that 50.8% of individuals in the SHCS, 72.5% in Uganda and 81.0% in Lesotho harboured HIV with high-level resistance to at least two drugs from their current regimen. Stanford resistance scores were higher in Uganda compared with Switzerland for all drugs used in first-line treatment except zidovudine and tenofovir (P < 0.01) and higher in Lesotho compared with Uganda for all drugs used in first-line treatment except zidovudine (P < 0.01). Conclusions: Frequent VL monitoring and possibly pretreatment GRT as done in the SHCS pays off by low levels of resistance even when treatment failure occurs. The high-level resistance patterns in Lesotho compared with Uganda could reflect a selection of strains with multiple resistance during enhanced adherence counselling.

Diagnostic strategy in segmentation defect of the vertebrae: a retrospective study of 73 patients.

BACKGROUND: Segmentation defects of the vertebrae (SDV) are non-specific features found in various syndromes. The molecular bases of SDV are not fully elucidated due to the wide range of phenotypes and classification issues. The genes involved are in the Notch signalling pathway, which is a key system in somitogenesis. Here we report on mutations identified in a diagnosis cohort of SDV. We focused on spondylocostal dysostosis (SCD) and the phenotype of these patients in order to establish a diagnostic strategy when confronted with SDV. PATIENTS AND METHODS: We used DNA samples from a cohort of 73 patients and performed targeted sequencing of the five known SCD-causing genes (DLL3, MESP2, LFNG, HES7 and TBX6) in the first 48 patients and whole-exome sequencing (WES) in 28 relevant patients. RESULTS: Ten diagnoses, including four biallelic variants in TBX6, two biallelic variants in LFNG and DLL3, and one in MESP2 and HES7, were made with the gene panel, and two diagnoses, including biallelic variants in FLNB and one variant in MEOX1, were made by WES. The diagnostic yield of the gene panel was 10/73 (13.7%) in the global cohort but 8/10 (80%) in the subgroup meeting the SCD criteria; the diagnostic yield of WES was 2/28 (8%). CONCLUSION: After negative array CGH, targeted sequencing of the five known SCD genes should only be performed in patients who meet the diagnostic criteria of SCD. The low proportion of candidate genes identified by WES in our cohort suggests the need to consider more complex genetic architectures in cases of SDV.

Parent-offspring regression to estimate the heritability of an HIV-1 trait in a realistic setup.

BACKGROUND: Parent-offspring (PO) regression is a central tool to determine the heritability of phenotypic traits; i.e., the relative extent to which those traits are controlled by genetic factors. The applicability of PO regression to viral traits is unclear because the direction of viral transmission-who is the donor (parent) and who is the recipient (offspring)-is typically unknown and viral phylogenies are sparsely sampled. METHODS: We assessed the applicability of PO regression in a realistic setting using Ornstein-Uhlenbeck simulated data on phylogenies built from 11,442 Swiss HIV Cohort Study (SHCS) partial pol sequences and set-point viral load (SPVL) data from 3293 patients. RESULTS: We found that the misidentification of donor and recipient plays a minor role in estimating heritability and showed that sparse sampling does not influence the mean heritability estimated by PO regression. A mixed-effect model approach yielded the same heritability as PO regression but could be extended to clusters of size greater than 2 and allowed for the correction of confounding effects. Finally, we used both methods to estimate SPVL heritability in the SHCS. We employed a wide range of transmission pair criteria to measure heritability and found a strong dependence of the heritability estimates to these criteria. For the most conservative genetic distance criteria, for which heritability estimates are conceptually expected to be closest to true heritability, we found estimates ranging from 32 to 46% across different bootstrap criteria. For less conservative distance criteria, we found estimates ranging down to 8%. All estimates did not change substantially after adjusting for host-demographic factors in the mixed-effect model (±2%). CONCLUSIONS: For conservative transmission pair criteria, both PO regression and mixed-effect models are flexible and robust tools to estimate the contribution of viral genetic effects to viral traits under real-world settings. Overall, we find a strong effect of viral genetics on SPVL that is not confounded by host demographics.

Novel deletion in 11p15.5 imprinting center region 1 in a patient with Beckwith-Wiedemann syndrome provides insight into distal enhancer regulation and tumorigenesis.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an early-onset overgrowth disorder with a high risk for embryonal tumors. It is mainly caused by dysregulation of imprinted genes on chromosome 11p15.5; however, the driving forces in the development of tumors are not fully understood. PROCEDURE: We report on a female patient presenting with macrosomia, macroglossia, organomegaly and extensive bilateral nephroblastomatosis. Adjuvant chemotherapy was initiated; however, the patient developed hepatoblastoma and Wilms tumor at 5 and 12 months of age, respectively. Subsequent radiofrequency ablation of the liver tumor and partial nephrectomy followed by consolidation therapy achieved complete remission. RESULTS: Molecular genetic analysis revealed a maternally derived large deletion of the complete H19-differentially methylated region (H19-DMR; imprinting control region-1 [ICR1]), the whole H19 gene itself as well as large parts of the distal enhancer region within the imprinting cluster-1 (IC1). Extended analysis showed highly elevated insulin-like growth factor 2 (IGF2) expression, possibly explaining at least in part the distinct BWS features and tumor manifestations. CONCLUSIONS: This study of a large maternal deletion encompassing the H19 gene and complete ICR1 is the first to demonstrate transcriptional consequences on IGF2 in addition to methylation effects resulting in severe overgrowth and occurrence of multiple tumors in a BWS patient. Studying this deletion helps to clarify the complex molecular processes involved in BWS and provides further insight into tumorigenesis.

Combined NGS approaches identify mutations in the intraflagellar transport gene IFT140 in skeletal ciliopathies with early progressive kidney Disease.

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

An expanded clinical spectrum of hypoinsulinaemic hypoketotic hypoglycaemia.

BACKGROUND: Hypoketotic hypoglycaemia with suppressed plasma fatty acids and detectable insulin suggests congenital hyperinsulinism (CHI). Severe hypoketotic hypoglycaemia mimicking hyperinsulinism but without detectable insulin has recently been described in syndromic individuals with mosaic genetic activation of post-receptor insulin signalling. We set out to expand understanding of this entity focusing on metabolic phenotypes. METHODS: Metabolic profiling, candidate gene and exome sequencing were performed in six infants with hypoketotic, hypoinsulinaemic hypoglycaemia, with or without syndromic features. Additional signalling studies were carried out in dermal fibroblasts from two individuals. RESULTS: Two infants had no syndromic features. One was mistakenly diagnosed with CHI. One had mild features of megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, one had non-specific macrosomia, and two had complex syndromes. All required intensive treatment to maintain euglycaemia, with CHI-directed therapies being ineffective. Pathogenic PIK3CA variants were found in two individuals - de novo germline c.323G>A (p.Arg108His) in one non-syndromic infant and postzygotic mosaic c.2740G>A (p.Gly914Arg) in the infant with MCAP. No causal variants were proven in the other individuals despite extensive investigation, although rare variants in mTORC components were identified in one. No increased PI3K signalling in fibroblasts of two individuals was seen. CONCLUSIONS: We expand the spectrum of PI3K-related hypoinsulinaemic hypoketotic hypoglycaemia. We demonstrate that pathogenic germline variants activating post-insulin-receptor signalling may cause non-syndromic hypoinsulinaemic hypoketotic hypoglycaemia closely resembling CHI. This distinct biochemical footprint should be sought and differentiated from CHI in infantile hypoglycaemia. To facilitate adoption of this differential diagnosis, we propose the term "pseudohyperinsulinism".