Beyond the kidney biopsy: genomic approach to undetermined kidney diseases.

Background: According to data from large national registries, almost 20%-25% of patients with end-stage kidney disease have an undetermined kidney disease (UKD). Recent data have shown that monogenic disease-causing variants are under-diagnosed. We performed exome sequencing (ES) on UKD patients in our center to improve the diagnosis rate. Methods: ES was proposed in routine practice for patients with UKD including kidney biopsy from January 2019 to December 2021. Mutations were detected using a targeted bioinformatic customized kidney gene panel (675 genes). The pathogenicity was assessed using American College of Medical Genetics guidelines. Results: We included 230 adult patients, median age 47.5 years. Consanguinity was reported by 25 patients. A family history of kidney disease was documented in 115 patients (50%). Kidney biopsies were either inconclusive in 69 patients (30.1%) or impossible in 71 (30.9%). We detected 28 monogenic renal disorders in 75 (32.6%) patients. Collagenopathies was the most common genetic kidney diagnosis (46.7%), with COL4A3 and COL4A4 accounting for 80% of these diagnoses. Tubulopathies (16%) and ciliopathies (14.7%) yielded, respectively, the second and third genetic kidney diagnosis category and UMOD-associated nephropathy as the main genetic findings for tubulopathies (7/11). Ten of the 22 patients having ES "first" eventually received a positive diagnosis, thereby avoiding 11 biopsies. Among the 44 patients with glomerular, tubulo-interstitial or vascular nephropathy, 13 (29.5%) were phenocopies. The diagnostic yield of ES was higher in female patients (P = .02) and in patients with a family history of kidney disease (P < .0001), reaching 56.8% when the patient had both first- and second-degree family history of renal disease. Conclusion: Genetic diagnosis has provided new clinical insights by clarifying or reclassifying kidney disease etiology in over a third of UKD patients. Exome "first" may have a significant positive diagnostic yield, thus avoiding invasive kidney biopsy; moreover, the diagnostic yield remains elevated even when biopsy is impossible or inconclusive. ES provides a clinical benefit for routine nephrological healthcare in patients with UKD.

Update of the UMD-VHL database: classification of 164 challenging variants based on genotype-phenotype correlation among 605 entries.

BACKGROUND: The von Hippel-Lindau (VHL) disease is a hereditary tumour syndrome caused by germline mutations in VHL tumour suppressor gene. The identification of VHL variants requires accurate classification which has an impact on patient management and genetic counselling. METHODS: The TENGEN (French oncogenetics network of neuroendocrine tumors) and PREDIR (French National Cancer Institute network for Inherited predispositions to kidney cancer) networks have collected VHL genetic variants and clinical characteristics of all VHL-suspected patients analysed from 2003 to 2021 by one of the nine laboratories performing VHL genetic testing in France. Identified variants were registered in a locus-specific database, the Universal Mutation Database-VHL database (http://www.umd.be/VHL/). RESULTS: Here we report the expert classification of 164 variants, including all missense variants (n=124), all difficult interpretation variants (n=40) and their associated phenotypes. After initial American College of Medical Genetics classification, first-round classification was performed by the VHL expert group followed by a second round for discordant and ambiguous cases. Overall, the VHL experts modified the classification of 87 variants including 30 variants of uncertain significance that were as (likely)pathogenic variants for 19, and as likely benign for 11. CONCLUSION: Consequently, this work has allowed the diagnosis and influenced the genetic counselling of 45 VHL-suspected families and can benefit to the worldwide VHL community, through this review.

Diagnosis of Kidney Diseases of Unknown Etiology Through Biopsy-Genetic Analysis.

Introduction: Previous studies have suggested that genetic kidney diseases in adults are often overlooked, representing up to 10% of all cases of chronic kidney disease (CKD). We present data obtained from exome sequencing (ES) analysis of patients with biopsy-proven undetermined kidney disease (UKD). Methods: ES was proposed during routine clinical care in patients with UKD from January 2020 to December 2021. We used in silico custom kidney genes panel analysis to detect pathological variations using American College of Medical Genetics guidelines in 52 patients with biopsy-proven UKD with histological finding reassessment. Results: We detected 12 monogenic renal disorders in 21 (40.4%) patients. The most common diagnoses were collagenopathies (8/21,38.1%), COL4A3 and COL4A4 accounting for 80% of these diagnoses, and ciliopathies (5/21, 23.8%). The diagnostic yield of ES was higher in female patients and patients with a family history of kidney disease (57.1% and 71%, respectively). Clinical nephropathy categories matched with the final genetic diagnoses in 72.7% of cases, whereas histological renal lesions matched with the final diagnoses in 92.3% of cases. The genetics diagnoses and histopathological findings were in complete agreement for both glomerular and tubulointerstitial cases. Interstitial inflammation without tubulitis was only observed in tubulopathies or ciliopathies. Isolated CKD, CKD with proteinuria or hematuria, and isolated proteinuria or hematuria yielded the highest diagnostic yields (54.6%, 52.6%, and 42.9%, respectively). Conclusion: ES done in patients with biopsy-proven UKD should be considered as a first-line tool for CKD patients with a family history of kidney disease. Combination of ES and kidney biopsy may have major impacts on kidney disease ontology.

Spatial Distribution of Immune Cells in Primary and Recurrent Glioblastoma: A Small Case Study.

Only a minority of patients with glioblastoma (GBM) respond to immunotherapy, and always only partially. There is a lack of knowledge on immune distribution in GBM and in its tumor microenvironment (TME). To address the question, we used paired primary and recurrent tumors from GBM patients to study the composition and the evolution of the immune landscape upon treatment. We studied the expression of a handful of immune markers (CD3, CD8, CD68, PD-L1 and PD-1) in GBM tissues in 15 paired primary and recurrent GBM. In five selected patients, we used Nanostring Digital Spatial Profiling (DSP) to obtain simultaneous assessments of multiple biomarkers both within the tumor and the microenvironment in paired primary and recurrent GBM. Our results suggest that the evolution of the immune landscape between paired primary and recurrent GBM tumors is highly heterogeneous. However, our study identifies B3-H7 and HLA-DR as potential targets in primary and recurrent GBM. Spatial profiling of immune markers from matched primary and recurrent GBM shows a nonlinear complex evolution during the progression of cancer. Nonetheless, our study demonstrated a global increase in macrophages, and revealed differential localization of some markers, such as B7-H3 and HLA-DR, between GBM and its TME.

Diagnostic interest of whole-body MRI in early- and late-onset LAMA2 muscular dystrophies: a large international cohort.

BACKGROUND: LAMA2-related muscular dystrophy (LAMA2-RD) encompasses a group of recessive muscular dystrophies caused by mutations in the LAMA2 gene, which codes for the alpha-2 chain of laminin-211 (merosin). Diagnosis is straightforward in the classic congenital presentation with no ambulation and complete merosin deficiency in muscle biopsy, but is far more difficult in milder ambulant individuals with partial merosin deficiency. OBJECTIVE: To investigate the diagnostic utility of muscle imaging in LAMA2-RD using whole-body magnetic resonance imaging (WBMRI). RESULTS: 27 patients (2-62 years, 21-80% with acquisition of walking ability and 6 never ambulant) were included in an international collaborative study. All carried two pathogenic mutations, mostly private missense changes. An intronic variant (c.909 + 7A > G) was identified in all the Chilean cases. Three patients (two ambulant) showed intellectual disability, epilepsy, and brain structural abnormalities. WBMRI T1w sequences or T2 fat-saturated images (Dixon) revealed abnormal muscle fat replacement predominantly in subscapularis, lumbar paraspinals, gluteus minimus and medius, posterior thigh (adductor magnus, biceps femoris, hamstrings) and soleus. This involvement pattern was consistent for both ambulant and non-ambulant patients. The degree of replacement was predominantly correlated to the disease duration, rather than to the onset or the clinical severity. A "COL6-like sandwich sign" was observed in several muscles in ambulant adults, but different involvement of subscapularis, gluteus minimus, and medius changes allowed distinguishing LAMA2-RD from collagenopathies. The thigh muscles seem to be the best ones to assess disease progression. CONCLUSION: WBMRI in LAMA2-RD shows a homogeneous pattern of brain and muscle imaging, representing a supportive diagnostic tool.

Standardisation of pathogenicity classification for somatic alterations in solid tumours and haematologic malignancies.

BACKGROUND: The difficulty in interpreting somatic alterations is correlated with the increase in sequencing panel size. To correctly guide the clinical management of patients with cancer, there needs to be accurate classification of pathogenicity followed by actionability assessment. Here, we describe a specific detailed workflow for the classification of the pathogenicity of somatic variants in cancer into five categories: benign, likely benign, unknown significance, likely pathogenic and pathogenic. METHODS: Classification is obtained by combining a set of eight relevant criteria in favour of either a pathogenic or a benign effect (pathogenic stand-alone, pathogenic very strong, pathogenic strong, pathogenic moderate, pathogenic supporting, benign supporting, benign strong and benign stand-alone). RESULTS: Our guide is concordant with the ACMG/AMP 2015 guidelines for germline variants. Interpretation of somatic variants requires considering specific criteria, such as the disease and therapeutic context, co-occurring genomic events in the tumour when available and the use of cancer-specific variant databases. In addition, the gene role in tumorigenesis (oncogene or tumour suppressor gene) also needs to be taken into consideration. CONCLUSION: Our classification could contribute to homogenize best practices on somatic variant pathogenicity interpretation and improve interpretation consistency both within and between laboratories.

Pathogenic FBN1 Genetic Variation and Aortic Dissection in Patients With Marfan Syndrome.

BACKGROUND: Aortic risk has not been evaluated in patients with Marfan syndrome and documented pathogenic variants in the FBN1 gene. OBJECTIVES: This study sought to describe aortic risk in a population with Marfan syndrome with pathogenic variants in the FBN1 gene as a function of aortic root diameter. METHODS: Patients carrying an FBN1 pathogenic variant who visited our reference center at least twice were included, provided they had not undergone aortic surgery or had an aortic dissection before their first visit. Aortic events (aortic surgery or aortic dissection) and deaths were evaluated during the 2 years following each patient visit. The risk was calculated as the number of events divided by the number of years of follow-up. RESULTS: A total of 954 patients were included (54% women; mean age 23 years). During follow-up (9.1 years), 142 patients underwent prophylactic aortic root surgery, 5 experienced type A aortic dissection, and 12 died (noncardiovascular causes in 3, unknown etiology in 3, post-operative in 6). When aortic root diameter was <50 mm, risk for proven type A dissection (0.4 events/1,000 patient-years) and risk for possible aortic dissection (proven aortic dissection plus death of unknown cause, 0.7 events/1,000 patients-years) remained low in this population that was treated according to guidelines. Three type A aortic dissections occurred in this population during the 8,594 years of follow-up, including 1 in a patient with a tubular aortic diameter of 50 mm, but none in patients with a family history of aortic dissection. The risk for type B aortic dissection in the same population was 0.5 events/1,000 patient-years. CONCLUSIONS: In patients with FBN1 pathogenic variants who receive beta-blocker therapy and who limit strenuous exercise, aortic risk remains low when maximal aortic diameter is <50 mm. The risk of type B aortic dissection is close to the remaining risk of type A aortic dissection in this population, which underlines the global aortic risk.

The ELIXIR Human Copy Number Variations Community: building bioinformatics infrastructure for research.

Copy number variations (CNVs) are major causative contributors both in the genesis of genetic diseases and human neoplasias. While "High-Throughput" sequencing technologies are increasingly becoming the primary choice for genomic screening analysis, their ability to efficiently detect CNVs is still heterogeneous and remains to be developed. The aim of this white paper is to provide a guiding framework for the future contributions of ELIXIR's recently established human CNV Community, with implications beyond human disease diagnostics and population genomics. This white paper is the direct result of a strategy meeting that took place in September 2018 in Hinxton (UK) and involved representatives of 11 ELIXIR Nodes. The meeting led to the definition of priority objectives and tasks, to address a wide range of CNV-related challenges ranging from detection and interpretation to sharing and training. Here, we provide suggestions on how to align these tasks within the ELIXIR Platforms strategy, and on how to frame the activities of this new ELIXIR Community in the international context.

Proposition of adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants.

In 2015, the ACMG-AMP guidelines provided a general procedure for the objective and reproducible classification of genomic variants. While the benefits of this framework are of major importance, its adaptation for locus-specific use is needed. Multiple Endocrine Neoplasia type 1 (MEN1) occurs due to inactivating mutations in the tumour suppressor gene MEN1, including 20% of missense variants. The classification of these variants may be extremely challenging. Here, we compared the interpretation of the 122 MEN1 missense variants, identified in the French population over the past 15 years by the TENGEN network (French oncogenetics network of neuroendocrine tumors) versus by using the ACMG-AMP guidelines, and analyzed the causes of discordance. A total of 59.8% of missense variants were termed as (likely)-pathogenic variants by TENGEN versus only 28.7% using ACMG-AMP guidelines. Actually, 53.4% (39/73) of TENGEN (likely)-pathogenic variants were declassified in variant of uncertain significance (VUS) by using ACMG-AMP guidelines, thereby affecting the clinical management of patients and their families. Twenty of these ACMG-AMP VUS were found in patients with a clinically authentic MEN1 disease. Here, TENGEN proposes adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants. These propositions merge both the classification systems, and are particularly interesting, as MEN1 is included in the ACMG secondary findings list for reporting in clinical genomic sequencing.

Clinical Significance of Aortic Root Modification Associated With Bicuspid Aortic Valve in Marfan Syndrome.

BACKGROUND: Both bicuspid aortic valve (BAV) and Marfan syndrome have been associated with aortic dissection risk, but it is unknown whether the presence of BAV is associated with an increased aortic risk in patients with an FBN1 gene mutation. We evaluated aortic diameters, aortic valve function, and aortic shape in Marfan syndrome patients with and without BAV and reported aortic events during follow-up. METHODS: All patients with an FBN1 gene mutation evaluated in our clinic were included. Aortic root diameters were measured, and the aortic valve was studied using echocardiography at each visit. RESULTS: Of the 1437 patients with an FBN1 gene mutation, 26 patients (1.8%) had a BAV. Both aortic root maximal diameter and normalized Z score were larger at all ages, in patients with BAV when compared with patients with tricuspid aortic valve. Prophylactic aortic root surgery tended to be performed in younger patients when BAV was present, although aortic diameter threshold was similar in the 2 populations. No aortic dissection was observed in Marfan syndrome patients with BAV. CONCLUSIONS: In patients with a FBN1 mutation, BAV is associated with larger aortic root diameter, with no difference in evolution of Z score with age. We found a trend towards prophylactic aortic root surgery at younger ages but similar aortic diameter thresholds without occurrence of aortic dissection. We did not find any evidence for lowering aortic diameter thresholds used to propose preventive aortic root surgery in the presence of BAV in patients with FBN1 mutations.

UMD-MEN1 Database: An Overview of the 370 MEN1 Variants Present in 1676 Patients From the French Population.

Context: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the MEN1 gene characterized by a broad spectrum of clinical manifestations, of which the most frequent are primary hyperparathyroidism, pituitary adenomas, and neuroendocrine tumors. Objective: The aim of this work was to facilitate interpretation of variants and improve the genetic counseling and medical care of families of patients with MEN1. Design, Setting, and Patients: The TENGEN network (Oncogenetics Network of Neuroendocrine Tumors) has interpreted and collected all allelic variants and clinical characteristics of the MEN1-positive patients identified through genetic testing performed in the French population from 1997 to 2015. Patients and their variants were registered in the locus-specific UMD-MEN1 database (www.umd.be/MEN1/). Main Outcomes: Variant classification, age-related penetrance, and odds ratios. Results: A total of 370 distinct variants reported in 1676 patients, including 181 unpublished variants, have been registered. This database analysis revealed a low frequency (6.6%) of benign or likely benign missense variants in MEN1. Eight families (1.9%) had members with familial isolated hyperparathyroidism and harbored the same mutations as that found in families with authentic MEN1. An association existed between large rearrangements and an earlier onset of the disease, whereas no difference was observed between truncating and nontruncating variants. Conclusion: The UMD-MEN1 database provides an exhaustive overview of the MEN1 variants present in the French population. For each variant, a classification is publicly available. Clinical data collections allow the determination of genotype-phenotype correlation and age-related penetrance of lesions in the cohort.

Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men.

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs∗4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111∗) and chr12: g.122395032_122395033delCT (p.Leu530Valfs∗4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

VarAFT: a variant annotation and filtration system for human next generation sequencing data.

With the rapidly developing high-throughput sequencing technologies known as next generation sequencing or NGS, our approach to gene hunting and diagnosis has drastically changed. In <10 years, these technologies have moved from gene panel to whole genome sequencing and from an exclusively research context to clinical practice. Today, the limit is not the sequencing of one, many or all genes but rather the data analysis. Consequently, the challenge is to rapidly and efficiently identify disease-causing mutations within millions of variants. To do so, we developed the VarAFT software to annotate and pinpoint human disease-causing mutations through access to multiple layers of information. VarAFT was designed both for research and clinical contexts and is accessible to all scientists, regardless of bioinformatics training. Data from multiple samples may be combined to address all Mendelian inheritance modes, cancers or population genetics. Optimized filtration parameters can be stored and re-applied to large datasets. In addition to classical annotations from dbNSFP, VarAFT contains unique features at the disease (OMIM), phenotypic (HPO), gene (Gene Ontology, pathways) and variation levels (predictions from UMD-Predictor and Human Splicing Finder) that can be combined to optimally select candidate pathogenic mutations. VarAFT is freely available at: http://varaft.eu.

Single-cell genetic analysis validates cytopathological identification of circulating cancer cells in patients with clear cell renal cell carcinoma.

CONTEXT: Circulating Rare Cells (CRC) are non-haematological cells circulating in blood. They include Circulating Cancer Cells (CCC) and cells with uncertain malignant features (CRC-UMF) according to cytomorphology. Clear cell renal cell carcinomas frequently bear a mutated Von Hippel-Lindau (VHL) gene. AIM: To match blind genetic analysis of CRC and tumor samples with CRC cytopathological diagnosis. RESULTS: 29/30 patients harboured CRC (20 harboured CCC, 29 CRC-UMF) and 25/29 patients carried VHL mutations in their tumour. 205 single CRC (64 CCC, 141 CRC-UMF) provided genetic data. 57/57 CCC and 104/125 CRC-UMF from the 25 patients with VHL-mutated tumor carried the same VHL mutation detected in the tumor. Seven CCC and 16 CRC-UMF did not carry VHL mutations but were found in patients with wild-type VHL tumor tissue. CONCLUSIONS: All the CCC and 83,2% (104/125) of the CRC-UMF were found to carry the same VHL mutation identified in the corresponding tumorous tissue, validating cytopathological identification of CCC in patients with clear cell renal cell carcinoma. METHODS: The blood of 30 patients with clear cell renal cell carcinoma was treated by ISET® for CRC isolation, cytopathology and single-cell VHL mutations analysis, performed blindly and compared to VHL mutations of corresponding tumor tissues and leukocytes.

[OISO, automatic treatment of patients management in oncogenetics]. / OISO, traitement informatisé de la prise en charge en oncogénétique clinique.

INTRODUCTION: Oncogenetics is a long-term process, which requires a close relation between patients and medical teams, good familial links allowing lifetime follow-up. Numerous documents are exchanged in between the medical team, which has to frequently interact. We present here a new tool that has been conceived specifically for this management. METHODS: The tool has been developed according to a model-view-controler approach with the relational system PostgreSQL 9.3. The web site used PHP 5.3, HTML5 and CSS3 languages, completed with JavaScript and jQuery-AJAX functions and two additional modules, FPDF and PHPMailer. RESULTS: The tool allows multiple interactions, clinical data management, mailing and emailing, follow-up plannings. Requests are able to follow all patients and planning automatically, to send information to a large number of patients or physicians, and to report activity. DISCUSSION: The tool has been designed for oncogenetics and adapted to its different aspects. The CNIL delivered an authorization for use. Secured web access allows the management at a regional level. Its simple concept makes it evolutive according to the constant updates of genetic and clinical management of patients.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2, TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

Exome Sequencing Identifies Two Variants of the Alkylglycerol Monooxygenase Gene as a Cause of Relapses in Visceral Leishmaniasis in Children, in Sudan.

Background: Visceral leishmaniasis (kala-azar, KA) is the most severe form of leishmaniasis, characterized by fever, weight loss, hepatosplenomegaly, and lymphadenopathy. During an outbreak of KA in Babar El Fugara (Sudan), 5.7% of cured patients displayed relapses, with familial clustering in half the cases. Methods: We performed whole-exome sequencing on 10 relapsing individuals and 11 controls from 5 nuclear families. Results: Rare homozygous and compound-heterozygous nonsense (c.1213C > T, rs139309795, p.Arg405*) and missense (c.701A > G, rs143439626, p.Lys234Arg) mutations of the alkylglycerol monooxygenase (AGMO) gene were associated with KA relapse in 3 families. Sequencing in additional family members confirmed the segregation of these mutations with relapse and revealed an autosomal dominant mode of transmission. These mutations were detected heterozygous in 2 subjects among 100 unrelated individuals with KA who never relapsed after cure, suggesting incomplete penetrance of AGMO deficiency. AGMO is expressed in hematopoietic cells, and is strongly expressed in the liver. AGMO modulates PAF production by mouse macrophages, suggesting that it may act through the PAF/PAF receptor pathway previously shown to have anti-Leishmania activity. Conclusions: This is the first demonstration that relapses after a first episode of KA are due to differences in human genetic susceptibility and not to modifications of parasite pathogenicity.

New advances in DPYD genotype and risk of severe toxicity under capecitabine.

BACKGROUND: Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine. METHODS: Two-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. DPYD exome, along with flanking intronic regions 3'UTR and 5'UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement. RESULTS: Among the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient (among which, F100L and A26T, both pathogenic in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3-4 toxicity (sensitivity 16.7%, positive predictive value (PPV) 71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3-4 toxicity (PPV 72.7%, RR 7.6, p<0.001), and was significantly associated with grade 4 toxicity (sensitivity 60%, PPV 27.3%, RR 31.4, p = 0.001), suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR. CONCLUSIONS: Exploring an extended set of deleterious DPYD variants improves the performance of DPYD genotyping for predicting both grade 3-4 and grade 4 toxicities (digestive and/or neurologic and/or hematotoxicities) related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.

Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL).

BACKGROUND: Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. PATIENTS AND METHODS: We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. RESULTS: Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing of POLD1 exon 15 revealed the recurrent in-frame deletion (c.1812_1814del, p.S605del). CONCLUSION: Our work highlights that mutations in different POLD1 domains can lead to phenotypic variability, ranging from dominantly inherited cancer predisposition syndromes, to mild MDPL phenotypes without lifespan reduction, to very severe MDPL syndromes with major premature aging features. These results also suggest that POLD1 gene testing should be considered in patients presenting with severe progeroid features.