Background: Type 1 diabetes (T1D) shares clinical characteristics with other forms of diabetes, particularly monogenic diabetes such as maturity-onset diabetes of the young (MODY). Differential diagnosis is complicated by the existence of intermediate phenotypes. We aimed to delineate the phenotypic continuum between T1D and monogenic diabetes. Methods: The multicentric GENEPEDIAB study included patients aged 6 months to 18 years diagnosed with diabetes and treated for either T1D or monogenic diabetes. Analyses comprised glycemic variability, continuous glucose monitoring metrics, application of the DIAMODIA criteria, and genetic investigations. Results: A gradient was observed across T1D, atypical diabetes (Adia), and MODY cohorts for several glycemic parameters. T1D patients exhibited values furthest from treatment targets, whereas MODY patients showed better glycemic control. Stratification of the Adia cohort according to the number of positive DIAMODIA criteria further supported this trend, as demonstrated by glycemic measures and multiple correspondence analysis. Genetic analyses did not identify a uniform causative variant in the Adia cohort; however, several rare variants, including variants of uncertain significance and likely pathogenic variants in diabetes-related genes, were detected. Conclusions: These findings showed, in our specific cohort of pediatric patients, the existence of a phenotypic gradient between T1D and monogenic diabetes, with atypical diabetes occupying an intermediate position, including when stratified by DIAMODIA criteria.

Anti-epidermal growth factor receptor (EGFR) therapy (cetuximab) shows a limited clinical benefit for patients with locally advanced or recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), due to the frequent occurrence of secondary resistance mechanisms. Here we report that cetuximab-resistant HNSCC cells display a peroxisome proliferator-activated receptor alpha (PPARα)-mediated lipid metabolism reprogramming, with increased fatty acid uptake and oxidation capacities, while glycolysis is not modified. This metabolic shift makes cetuximab-resistant HNSCC cells particularly sensitive to a pharmacological inhibition of either carnitine palmitoyltransferase 1A (CPT1A) or PPARα in 3D spheroids and tumor xenografts in mice. Importantly, the PPARα-related gene signature, in human clinical datasets, correlates with lower response to anti-EGFR therapy and poor survival in HNSCC patients, thereby validating its clinical relevance. This study points out lipid metabolism rewiring as a non-genetic resistance-causing mechanism in HNSCC that may be therapeutically targeted to overcome acquired resistance to anti-EGFR therapy. Resistance to anti-EGFR therapy is a clinical issue for patients with advanced head and neck cancers. Here, the authors show that therapy-resistant cancer cells enhance fatty acid metabolism, which can be therapeutically targeted by inhibiting peroxisome proliferator-activated receptor alpha (PPARα).

This study describes the results of whole exome sequencing in the etiological investigation and genetic counseling of families presenting with non-syndromic oral clefts with vertical transmission recorded in the Brazilian Database on Craniofacial Anomalies. Whole exome sequencing was performed in 18 families presenting with non-syndromic oral clefts with vertical transmission, and variant filtering was used to identify rare, and also possibly pathogenic variants in genes associated with oral clefts. Overall, our study identified seven families (38.88%) presented with segregating variants possibly related to oral clefts in our cohort: SETX, NOTCH1, FRAS1, ARHGAP29, KMT2D, ANKRD11, SIX1, BMP6, LRP2 and TFAP2A. In another family, all affected members (5.55%) presented a rare variant in FAM193A, which has no recognized function yet, but has prediction of deleterious effect. Our study highlights oral clefts clinical and etiological heterogeneity and shows the complexity of using whole exome sequencing for genetic counseling in non-syndromic oral clefts with vertical transmission.

The genetic basis of nonsyndromic orofacial cleft (NsOFC) remains elusive, although associations have been identified with various genetic loci. NsOFC has a less pronounced genetic background than syndromic orofacial cleft (SyOFC), albeit Mendelian inheritance has been identified. Our hypothesis was that genes related to spliceosome function may contribute to NsOFC pathophysiology, as they do for some syndromic cases. Exome sequencing was conducted on 224 unrelated NsOFC probands. We performed filtering and analyses of predicted pathogenicity of rare variants using Highlander. We focused on 26 genes encoding spliceosome proteins. Subsequently, bioinformatic tools, such as AlphaFold, and PyMol, were applied to generate three-dimensional structures to interpret the effects of the identified variants on protein structure and interaction domains. We found six likely damaging variants: three heterozygous missense variants in small nuclear ribonucleoprotein U5 200 kDa subunit (SNRNP200) in three multiplex NsOFC families, and two missense and one splice site variant in splicing factor 3b subunit 1 (SF3B1), 4 (SF3B4), and 2 (SF3B2) in two posterior CP families and a complete CP patient, respectively. These results suggest that variants in the spliceosome complex genes, observed in 2.7% of NsOFC cases in our cohort, may contribute to disease susceptibility as potential risk factors.

The pathophysiological basis of non-syndromic orofacial cleft (NsOFC) is still largely unclear. However, exome sequencing (ES) has led to identify several causative genes, often with reduced penetrance. Among these, the Rho GTPase activating protein 29 (ARHGAP29) has been previously implicated in 7 families with NsOFC. We investigated a cohort of 224 NsOFCs for which no genetic pathogenic variant had been identified by diagnostic testing. We used ES and bioinformatic variant filtering and identified four novel putative pathogenic variants in ARHGAP29 in four families. One was a missense variant leading to the substitution of the first methionine with threonine, two were heterozygous frameshift variants leading to a premature termination codon, and one was a nonsense variant. All variants were predicted to result in loss of function, either through mRNA decay, truncated ARHGAP29, or abnormal N-terminal initiation of translation of ARHGAP29. The truncated ARHGAP29 proteins would lack the important RhoGAP domain. The variants were either absent or rare in the control population databases, and the loss of intolerance score (pLI) of ARHGAP29 is 1.0, suggesting that ARHGAP29 haploinsufficiency is not tolerated. Phenotypes ranged from microform cleft lip (CL) to complete bilateral cleft lip and palate (CLP), with one unaffected mutation carrier. These results extend the mutational spectrum of ARHGAP29 and show that it is an important gene underlying variable NsOFC phenotypes. ARHGAP29 should be included in diagnostic genetic testing for NsOFC, especially familial cases, as it may be mutated in ∼4% of them (4/97 in our cohort) with high penetrance (89%).

Common capillary malformations are red vascular skin lesions, most commonly associated with somatic activating GNAQ or GNA11 mutations. We focused on capillary malformations lacking such a mutation to identify previously unreported genetic causes. We used targeted next-generation sequencing on 82 lesions. Bioinformatic analysis allowed the identification of 9 somatic pathogenic variants in PIK3R1 and PIK3CA, encoding for the regulatory and catalytic subunits of phosphoinositide 3-kinase, respectively. Recharacterization of these lesions unraveled a common phenotype: a pale capillary malformation associated with visible dilated veins. Primary endothelial cells from 2 PIK3R1-mutated lesions were isolated, and PI3k-Akt-mTOR and RAS-RAF-MAPK signaling were assessed by western blot. This unveiled an abnormal increase in Akt phosphorylation, effectively reduced by PI3K pathway inhibitors, such as mTOR, Akt, and PIK3CA inhibitors. The effects of mutant PIK3R1 were further studied using zebrafish embryos. Endothelium-specific expression of PIK3R1 mutants resulted in abnormal development of the posterior capillary-venous plexus. In summary, capillary malformation associated with visible dilated veins emerges as a clinical entity associated with somatic pathogenic variants in PIK3R1 or PIK3CA (nonhotspot). Our findings suggest that the activated Akt signaling can be effectively reversed by PI3K pathway inhibitors. In addition, the proposed zebrafish model holds promise as a valuable tool for future drug screening aimed at developing patient-tailored treatments.

Capillary malformations (CMs) are the most common type of vascular anomalies, affecting around 0.3% of newborns. They are usually caused by somatic pathogenic variants in GNAQ or GNA11. PIK3CA and PIK3R1, part of the phosphoinositide 3-kinase-protein kinase B-mammalian target of rapamycin pathway, are mutated in fainter CMs such as diffuse CM with overgrowth and megalencephaly CM. In this study, we present two young patients with a CM-like phenotype associated with cerebral anomalies and severe epilepsy. Pathogenic variants in PIK3CA and PIK3R1, as well as GNAQ and GNA11, were absent in affected cutaneous tissue biopsies. Instead, we identified two somatic pathogenic variants in the AKT3 gene. Subsequent analysis of the DNA obtained from surgically resected brain tissue of one of the two patients confirmed the presence of the AKT3 variant. Focal cortical dysplasia was also detected in this patient. Genetic analysis thus facilitated workup to reach a precise diagnosis for these patients, associating the vascular anomaly with the neurological symptoms. This study underscores the importance of searching for additional signs and symptoms to guide the diagnostic workup, especially in cases with atypical vascular malformations. In addition, it strongly emphasizes the significance of genotype-phenotype correlation studies in guiding clinicians' informed decision-making regarding patient care.

BackgroundRecent diabetes subclassifications have improved the differentiation between patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus despite several overlapping features, yet without considering genetic forms of diabetes. We sought to facilitate the identification of monogenic diabetes by creating a new tool that we validated in a pediatric maturity-onset diabetes of the young (MODY) cohort. MethodsWe first created the DIAgnose MOnogenic DIAbetes (DIAMODIA) criteria based on the pre-existing, but incomplete, MODY calculator. This new score is composed of four strong and five weak criteria, with patients having to display at least one weak and one strong criterion. ResultsThe effectiveness of the DIAMODIA criteria was evaluated in two patient cohorts, the first consisting of patients with confirmed MODY diabetes (n=34) and the second of patients with T1DM (n=390). These DIAMODIA criteria successfully detected 100% of MODY patients. Multiple correspondence analysis performed on the MODY and T1DM cohorts enabled us to differentiate MODY patients from T1DM. The three most relevant variables to distinguish a MODY from T1DM profile were: lower insulin-dose adjusted A1c score ≤9, glycemic target-adjusted A1c score ≤4.5, and absence of three anti-islet cell autoantibodies. ConclusionWe validated the DIAMODIA criteria, as it effectively identified all monogenic diabetes patients (MODY cohort) and succeeded to differentiate T1DM from MODY patients. The creation of this new and effective tool is likely to facilitate the characterization and therapeutic management of patients with atypical diabetes, and promptly referring them for genetic testing which would markedly improve clinical care and counseling, as well.

The pathophysiological basis of non-syndromic cleft lip and/or palate (NsCL/P) is still largely unclear. However, exome sequencing (ES) has allowed to associate several genes with NsCL/P, often with reduced penetrance. Among these genes, the Rho GTPase activating protein 29 (ARHGAP29) has been previously implicated in 7 families with NsCL/P. We investigated a cohort of 224 NsCLPs for which no genetic mutation had been identified by diagnostic testing. We used ES and bioinformatic variant filtering and identified four novel likely pathogenic/pathologic variants in ARHGAP29 in four multiplex families. One was a missense variant leading to the substitution of the first methionine with threonine, two were heterozygous frameshift variants leading to a premature termination codon, and the last one was a nonsense variant. All variants were predicted to result in loss of function, either through NMD-mediated mRNA decay, or by truncated ARHGAP29, or by non-translation or abnormal N-terminal initiation of translation of ARHGAP29. The truncated ARHGAP29 proteins would lack the important RhoGAP domain. The variants were not present in the control population databases, and the loss of intolerance score (pLI) of ARHGAP29 is 1.0, suggesting that ARHGAP29 haploinsufficiency is not tolerated. Phenotypes ranged from microform CL to complete bilateral CLP, with one unaffected mutation carriers. These results extend the mutational spectrum of ARHGAP29 and show that it is an important gene underlying variable NsCL/P phenotypes. ARHGAP29 should be included in diagnostic genetic testing for NsCL/P, especially familial cases, as it may be mutated in ⁓2% of patients with high penetrance (88%).

Over the past years, progress made in next-generation sequencing technologies and bioinformatics have sparked a surge in association studies. Especially, genome-wide association studies (GWASs) have demonstrated their effectiveness in identifying disease associations with common genetic variants. Yet, rare variants can contribute to additional disease risk or trait heterogeneity. Because GWASs are underpowered for detecting association with such variants, numerous statistical methods have been recently proposed. Aggregation tests collapse multiple rare variants within a genetic region (e.g. gene, gene set, genomic loci) to test for association. An increasing number of studies using such methods successfully identified trait-associated rare variants and led to a better understanding of the underlying disease mechanism. In this review, we compare existing aggregation tests, their statistical features and scope of application, splitting them into the five classical classes: burden, adaptive burden, variance-component, omnibus and other. Finally, we describe some limitations of current aggregation tests, highlighting potential direction for further investigations.

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.

Background: Only 15-20% of recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) patients derive long-term benefit from nivolumab or pembrolizumab. We developed a circulating tumour DNA (ctDNA) tumour-agnostic assay aimed at the early prediction of single agent programmed cell death 1 (PD1) inhibitor efficacy in R/M SCCHN.Patients and methods: Our tumour-agnostic assay included 37 genes frequently mutated in R/M SCCHN and two HPV16 genes. Primary endpoint was the concordance between ctDNA kinetics (ActDNA) and the best overall response according to Response Evaluation Criteria in Solid Tumors version 1.1. ActDNA was defined as the difference in mean variant allele frequency (VAF) between the on-treatment sample harvested 6-10 weeks (FU1) after PD1 inhibitor initiation and the pre-treatment plasma sample (ActDNA = mean FU1 VAF -mean pre-treatment VAF).Results: ctDNA was detected in 35/44 (80%) of the pre-treatment plasma samples. The concordance between ActDNA and imaging response was observed in 74%. Median progression-free survival was 8.6 months in the favourable ActDNA group and 2.5 months in the unfavourable ActDNA group (p = 0.057). Median overall survival (OS) was 18.1 and 8.2 months in the favourable and unfavourable ActDNA groups, respectively (p = 0.13). In patients with PD-L1 expressing SCCHN (Combined Positive Score >= 1), OS was significantly better in patients with favourable ActDNA compared with patients with unfavourable ActDNA: median OS was 41.5 and 8.4 months (p = 0.033), respectively.Conclusions: Tumour-agnostic ctDNA analysis for human papillomavirus (HPV)-negative and HPV-positive R/M SCCHN is feasible. ctDNA kinetics show promising results in predicting the efficacy of PD1 inhibitors in R/M SCCHN.

Background Primary lymphoedema (PL) is a chronic, debilitating disease caused by developmental and functional defects of the lymphatic system. It is marked by an accumulation of interstitial fluid, fat and tissue fibrosis. There is no cure. More than 50 genes and genetic loci have been linked to PL. We sought to study systematically cell polarity signalling protein Cadherin Epidermal Growth Factor Laminin G Seven-pass G-type Receptor 1 (CELSR1) variants linked to PL. Methods We investigated 742 index patients from our PL cohort using exome sequencing. Results We identified nine variants predicted to cause CELSR1 loss of function. Four of them were tested for nonsense-mediated mRNA decay, but none was observed. Most of the truncated CELSR1 proteins would lack the transmembrane domain, if produced. The affected individuals had puberty/late-onset PL on lower extremities. The variants had a statistically significant difference in penetrance between female patients (87%) and male patients (20%). Eight variant carriers had a kidney anomaly, mostly in the form of ureteropelvic junction obstruction, which has not been associated with CELSR1 before. CELSR1 is located in the 22q13.3 deletion locus of the Phelan-McDermid syndrome. As variable renal defects are often seen in patients with the Phelan-McDermid syndrome, CELSR1 may be the long-sought gene for the renal defects. Conclusion PL associated with a renal anomaly suggests a CELSR1-related cause.

The development of high-throughput next-generation sequencing technologies and large-scale genetic association studies produced numerous advances in the biostatistics field. Various aggregation tests, i.e. statistical methods that analyze associations of a trait with multiple markers within a genomic region, have produced a variety of novel discoveries. Notwithstanding their usefulness, there is no single test that fits all needs, each suffering from specific drawbacks. Selecting the right aggregation test, while considering an unknown underlying genetic model of the disease, remains an important challenge. Here we propose a new ensemble method, called Excalibur, based on an optimal combination of 36 aggregation tests created after an in-depth study of the limitations of each test and their impact on the quality of result. Our findings demonstrate the ability of our method to control type I error and illustrate that it offers the best average power across all scenarios. The proposed method allows for novel advances in Whole Exome/Genome sequencing association studies, able to handle a wide range of association models, providing researchers with an optimal aggregation analysis for the genetic regions of interest.

This study describes genomic findings among individuals with both orofacial clefts (OC) and microphthalmia/anophthalmia/coloboma (MAC) recorded in the Brazilian Database on Craniofacial Anomalies (BDCA). Chromosomal microarray analysis (CMA) and Whole Exome Sequencing (WES) were performed in 17 individuals with OC-MAC. Clinical interpretation of molecular findings was based on data available at the BDCA and on re-examination. No copy number variants (CNVs) classified as likely pathogenic or pathogenic were detected by CMA. WES allowed a conclusive diagnosis in six individuals (35.29%), two of them with variants in the CHD7 gene, and the others with variants in the TFAP2A , POMT1 , PTPN11 , and TP63 genes with the following syndromes: CHARGE, CHD7 -spectrum, Branchiooculofacial, POMT1 -spectrum, LEOPARD, and ADULT. Variants of uncertain significance (VUS) possibly associated to the phenotypes were found in six other individuals. Among the individuals with VUSes, three individuals presented variants in genes associated to defects of cilia structure and/or function, including DYNC2H1 , KIAA0586 , WDR34 , INTU , RPGRIP1L , KIF7 , and LMNA . These results show that WES was the most effective molecular approach for OC-MAC in this cohort. This study also reinforces the genetic heterogeneity of OC-MAC, and the importance of genes related to ciliopathies in this phenotype.

Only 15-20% of recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) patients derive long-term benefit from nivolumab or pembrolizumab. We developed a ctDNA tumor-agnostic assay aimed at the early prediction of single agent PD-1 inhibitor efficacy in R/M SCCHN. Our tumor-agnostic assay included 37 genes frequently mutated in R/M SCCHN and two HPV16 genes. Primary endpoint was the concordance between ctDNA kinetics (ΔctDNA) and best overall response (BOR) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECISTv1.1). ΔctDNA was defined as the difference in mean variant allele frequency (VAF) between the on-treatment sample harvested 6-10 weeks (FU1) after PD-1 inhibitor initiation and the pre-treatment plasma sample (ΔctDNA = mean FU1 VAF - mean pre-treatment VAF). ctDNA was detected in 35/44 (80%) of the pre-treatment plasma samples. The concordance between ΔctDNA and imaging response was observed in 74%. Median PFS was 8.6 months in the favorable ΔctDNA group and 2.5 months in the unfavorable ΔctDNA group (p=0.057). Median OS was 18.1 and 8.2 months in the favorable and unfavorable ΔctDNA groups, respectively (p=0.13). In patients with PD-L1 expressing SCCHN (Combined Positive Score ≥1), OS was significantly better in patients with favorable ΔctDNA compared with patients with unfavorable ΔctDNA: median OS was 8.4 and 41.5 months (p=0.033), respectively. Tumor-agnostic ctDNA analysis for HPV-negative and HPV-positive R/M SCCHN is feasible. ctDNA kinetics show promising results in predicting the efficacy of PD-1 inhibitors in R/M SCCHN.

Background: Forty to fifty percent of patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN) relapse despite multimodal treatment. Circulating tumor DNA (ctDNA) has the potential to detect minimal residual disease (MRD) after curative-intent therapy and to identify earlier which patients will progress. We developed a tumor-agnostic plasma ctDNA assay to detect MRD in unselected LA SCCHN with the aim of predicting progression-free survival (PFS) and overall survival without the need for tumor sequencing.Patients and methods: A 26-gene next-generation sequencing panel was constructed that included the most frequently mutated genes in SCCHN and two HPV-16 genes. MRD was assessed in each patient through an in-house informatic workflow informed by somatic mutations identified in the corresponding pre-treatment plasma sample. The presence of MRD was defined as the detection of ctDNA in one plasma sample collected within 1-12 weeks of the end of curative treatment. The primary endpoint was the PFS rate at 2 years. At least 32 patients were planned for inclusion with the hypothesis that PFS at 2 years was >80% in MRD-negative patients and <30% in MRD-positive patients (a = 0.05, b = 0.9).Results: We sequenced DNA from 116 plasma samples derived from 53 LA SCCHN patients who underwent curative-intent treatment. ctDNA was detected in 41/53 (77%) patients in the pre-treatment samples. Out of these 41 patients, 17 (41%) were MRD positive after treatment. The 2-year PFS rate was 23.53% (9.9% to 55.4%) and 86.6% (73.4% to 100%) in MRD-positive and MRD-negative patients, respectively (P < 0.05). Median survival was 28.37 months (14.30 months-not estimable) for MRD-positive patients and was not reached for the MRD-negative cohort (P = 0.011).Conclusions: Our ctDNA assay detects MRD in LA SCCHN and predicts disease progression and survival without the need for tumor sequencing, making this approach easily applicable in daily practice.