Analysis of germline variants in pediatric patients diagnosed with desmoid tumors and nuchal-type fibromas.

Desmoid tumor (DT) is a fibroblastic proliferation arising in soft tissue characterized by localized infiltrative growth with an inability to metastasize but with a tendency to recurrence. Nuchal-type fibromas are benign soft tissue lesions that are usually developed in the posterior neck. The development of these neoplasms can be associated with a hereditary cancer predisposition syndrome, mainly familial adenomatous polyposis (FAP) syndrome caused by APC germline mutations. Gardner syndrome is a variant of FAP characterized by the presence of extracolonic manifestations including soft tissue tumors as DTs and nuchal-type fibromas. However, the development of these tumors could be associated with germline alterations in other genes related to colorectal cancer development. The objective of this study was to analyze germline variants in APC, MUTYH, POLD1 and POLE genes in five pediatric patients diagnosed with DTs or nuchal-type fibromas. We identified two pathogenic variants in the APC gene in two different patients diagnosed with nuchal-type fibroma and DTs and two variants of uncertain significance in POLD1 in two patients diagnosed with nuchal-type fibroma. Two patients had family history of colorectal cancer, however, only one of them showed an APC germline pathogenic variant. The analysis of germline variants and genetic counseling is essential for pediatric patients diagnosed with DTs or nuchal-type fibromas and their relatives.

Incidental finding at methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA): how to proceed?

Introduction: Since the advent of new generation sequencing, professionals are aware of the possibility of obtaining findings unrelated to the pathology under study. However, this possibility is usually forgotten in the case of studies aimed at a single gene or region. We report a case of a 16-month-old girl with clinical suspicion of Silver-Russell syndrome (SRS). Methods: Following the international SRS consensus, methylation alterations and copy number variations (CNVs) at 11p15 region and maternal uniparental disomy of chromosome 7 were analysed and discarded by MS-MLPA. Results: Unexpectedly, the 11p15 region MS-MLPA showed a decrease in the signal of a copy number reference probe. Deletions affecting a single probe are inconclusive. So, we faced the ethical dilemma of whether it was appropriate to confirm this alteration with independent techniques and to offer a diagnostic possibility that was in no way related to clinical suspicion. Fortunately, in this particular case, the informed consent had not been specific to a particular pathology but to any disorder associated with growth failure. Performed alternative studies allowed the final diagnosis of 22q deletion syndrome. Conclusion: We demonstrate the importance of informing patients about the possibility of obtaining incidental findings in genetic techniques (not only in next generation sequencing) during pre-test genetic counselling consultations. In addition, we highlight the relevance of including in the informed consent the option of knowing these unexpected incidental findings as in some cases, this will help to elucidate the definitive diagnosis and provide the correct follow-up and treatment.

Clinical description, molecular delineation and genotype-phenotype correlation in 340 patients with KBG syndrome: addition of 67 new patients.

BACKGROUND: KBG syndrome is a highly variable neurodevelopmental disorder and clinical diagnostic criteria have changed as new patients have been reported. Both loss-of-function sequence variants and large deletions (copy number variations, CNVs) involving ANKRD11 cause KBG syndrome, but no genotype-phenotype correlation has been reported. METHODS: 67 patients with KBG syndrome were assessed using a custom phenotypical questionnaire. Manifestations present in >50% of the patients and a 'phenotypical score' were used to perform a genotype-phenotype correlation in 340 patients from our cohort and the literature. RESULTS: Neurodevelopmental delay, macrodontia, triangular face, characteristic ears, nose and eyebrows were the most prevalentf (eatures. 82.8% of the patients had at least one of seven main comorbidities: hearing loss and/or otitis media, visual problems, cryptorchidism, cardiopathy, feeding difficulties and/or seizures. Associations found included a higher phenotypical score in patients with sequence variants compared with CNVs and a higher frequency of triangular face (71.1% vs 42.5% in CNVs). Short stature was more frequent in patients with exon 9 variants (62.5% inside vs 27.8% outside exon 9), and the prevalence of intellectual disability/attention deficit hyperactivity disorder/autism spectrum disorder was lower in patients with the c.1903_1907del variant (70.4% vs 89.4% other variants). Presence of macrodontia and comorbidities were associated with larger deletion sizes and hand anomalies with smaller deletions. CONCLUSION: We present a detailed phenotypical description of KBG syndrome in the largest series reported to date of 67 patients, provide evidence of a genotype-phenotype correlation between some KBG features and specific ANKRD11 variants in 340 patients, and propose updated clinical diagnostic criteria based on our findings.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

RNA Analysis and Clinical Characterization of a Novel Splice Variant in the NSD1 Gene Causing Familial Sotos Syndrome.

BACKGROUND: Sotos syndrome is an autosomal dominant disorder characterized by overgrowth, macrocephaly, distinctive facial features and learning disabilities. Haploinsufficiency of the nuclear receptor SET domain-containing protein 1 (NSD1) gene located on chromosome 5q35 is the major cause of the syndrome. This syndrome shares characteristics with other overgrowth syndromes, which can complicate the differential diagnosis. METHODS: Genomic DNA was extracted from peripheral blood samples of members of the same family and targeted exome analysis was performed. In silico study of the variant found by next-generation sequencing was used to predict disruption/creation of splice sites and the identification of potential cryptic splice sites. RNA was extracted from peripheral blood samples of patients and functional analyses were performed to confirm the pathogenicity. RESULTS: We found a novel c.6463 + 5G>A heterozygous NSD1 gene pathogenic variant in a son and his father. Molecular analyses revealed that part of the intron 22 of NSD1 is retained due to the destruction of the splicing donor site, causing the appearance of a premature stop codon in the NSD1 protein. CONCLUSIONS: Our findings underline the importance of performing RNA functional assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives. Our work also highlights the relevance of using in silico prediction tools to detect a potential alteration in the splicing process.

Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players.

BACKGROUND: Inherited kidney diseases are one of the leading causes of chronic kidney disease (CKD) that manifests before the age of 30 years. Precise clinical diagnosis of early-onset CKD is complicated due to the high phenotypic overlap, but genetic testing is a powerful diagnostic tool. We aimed to develop a genetic testing strategy to maximize the diagnostic yield for patients presenting with early-onset CKD and to determine the prevalence of the main causative genes. METHODS: We performed genetic testing of 460 patients with early-onset CKD of suspected monogenic cause using next-generation sequencing of a custom-designed kidney disease gene panel in addition to targeted screening for c.428dupC MUC1. RESULTS: We achieved a global diagnostic yield of 65% (300/460), which varied depending on the clinical diagnostic group: 77% in cystic kidney diseases, 76% in tubulopathies, 67% in autosomal dominant tubulointerstitial kidney disease, 61% in glomerulopathies and 38% in congenital anomalies of the kidney and urinary tract. Among the 300 genetically diagnosed patients, the clinical diagnosis was confirmed in 77%, a specific diagnosis within a clinical diagnostic group was identified in 15%, and 7% of cases were reclassified. Of the 64 causative genes identified in our cohort, 7 (COL4A3, COL4A4, COL4A5, HNF1B, PKD1, PKD2 and PKHD1) accounted for 66% (198/300) of the genetically diagnosed patients. CONCLUSIONS: Two-thirds of patients with early-onset CKD in this cohort had a genetic cause. Just seven genes were responsible for the majority of diagnoses. Establishing a genetic diagnosis is crucial to define the precise aetiology of CKD, which allows accurate genetic counselling and improved patient management.

Novel APOB mutation in familial hypobetalipoproteinemia.

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The Value of Merging Medical Data from Ambulance Services and General Practice Cooperatives Using Triple Aim Outcomes.

BACKGROUND: Acute care services are currently overstretched in many high income countries. Overcrowding also plays a major role in acute care in the Netherlands. In a region of the Netherlands, the general practice cooperative (GPC) and ambulance service have begun to integrate their care, and the rapid and complete transfer of information between these two care organisations is now the basis for delivering appropriate care. The primary aim of this mixed-methods study is to evaluate the Netherlands Triage System (NTS) merger project and answering the question: What is the added value of implementing a digital NTS merger in terms of healthcare use and healthcare costs? A secondary question is: What are the experiences of patients and care professionals in different acute healthcare organisations following implementation of the digital NTS merger? METHODS: Patients who made an acute care request during the 12 months before the NTS merge intervention (control period) were compared with matched patients in the 12 months following the start of the NTS merge. Outcomes included difference in healthcare use 30 days after an acute event and patient' and care professional' experiences during the intervention period. To assess healthcare costs, we used reference prices updated to 2021. RESULTS: Compared to patients in the control period, patients in the intervention period were hospitalized less often (52.9% vs 64.4%, p = 0.061) and had fewer emergency department (ED) visits (58.7% vs 69.3%, p = 0.074) in the 30 days following the acute care request. The ED costs were significantly lower during the intervention period compared to the control period (p = 0.042). Furthermore, patients in the intervention period were very satisfied overall with the acute care network (4.63 of 5) and care professionals were fairly satisfied with the cooperation to date (2.73 of 4). CONCLUSION: The Triple Aim for acute care can be met using relatively simple interventions, but medical data merging is a prerequisite for achieving more robust results covering on the various aspects of the Triple Aim. These successes should be communicated so that a common language can be developed that will support the successful further implementation of larger scale initiatives.

Incidence and clinical profile of venous thromboembolism in hospitalized COVID-19 patients from Madrid region.

BACKGROUND: COVID-19 related in-hospital venous thromboembolism (VTE) incidence is high but data reported vary significantly. Some studies show that up to half of the events are diagnosed early after admission. OBJECTIVES: To study symptomatic VTE incidence in acute COVID-19 hospitalized patients and to describe timing of VTE diagnosis. METHODS: Multicenter cohort of 5966 patients hospitalized with acute COVID-19. Multicenter Registry of 844 hospitalized patients with acute COVID-19 and associated acute VTE. RESULTS: By the time of cohort data collection, 68 patients (1.14%) were still hospitalized, 19.8% had died, and 5.4% required ICU. During a median follow-up of 6 days (IQR, 4-12), 183 patients (3.07%; 95% CI, 2.64-3.55) presented a symptomatic VTE event. The cumulative incidences of VTE at 7, 14 and 21 days in wards [2.3% (95% CI, 1.9-2.7), 3.6% (95% CI, 3.0-4.3), and 4.3% (95% CI, 3.5-5.1)] were similar to the ones reported in ICU [2.2% (95% CI, 1.0-4.4), 2.9% (95% CI, 1.5-5.3), and 4.1% (95% CI, 2.2-6.8)], but at 30 and 60 days were higher in ICU [6.9% (95% CI, 4.2-10.5), and 12.8% (95% CI, 8.1-18.5)] than in wards. Eighty-eight VTE events (48%) were diagnosed early, within 48 h of admission. VTE was not associated with death (HR, 0.79; 95% CI, 0.55-1.12). CONCLUSIONS: Incidence of symptomatic VTE in our COVID-19 cohort is consistent with that of other real-life studies recently published. Early VTE events are, along with COVID-19, the reason for admission rather than an in-hospital complication.

Novel Variant in PLAG1 in a Familial Case with Silver-Russell Syndrome Suspicion.

Silver-Russell syndrome (SRS) is a rare growth-related genetic disorder that is mainly associated with prenatal and postnatal growth retardation. Molecular causes are not clear in all cases, the most common ones being loss of methylation on chromosome 11p15 (≈50%) and maternal uniparental disomy for chromosome 7 (upd(7)mat) (≈10%). However, pathogenic variants in genes such as CDKN1C, HMGA2, IGF2, or PLAG1 have also been described. Previously, two families and one sporadic case have been reported with PLAG1 alterations. Here, we present a case of a female with clinical suspicion of SRS (i.e., intrauterine and postnatal growth retardation, triangular face, psychomotor delay, speech delay, feeding difficulties). No alterations in methylation or copy number were detected at chromosomes 11p15 and 7 using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The custom panel study by next-generation sequencing (NGS) revealed a frameshift variant in the PLAG1 gene (NM_002655.3:c.551delA; p.(Lys184Serfs *45)). Familial studies confirmed that the variant was inherited from the mother and it was also present in other family members. New evidence of pathogenic alterations in the HMGA2-PLAG1-IGF2 pathway suggest the importance of studying and taking into account these genes as alternative molecular causes of Silver-Russell syndrome.

Prenatal and foetal autopsy findings in glutaric aciduria type II.

BACKGROUND: Glutaric aciduria type 2 is a rare, lethal disorder that affects metabolism of fatty acids caused by genetic defects in electron transfer (ETF) or in electron transfer flavoprotein dehydrogenase (ETFDH). We aimed to describe the pathological findings of 15 week old foetus, born from a consanguineous couple with 3 previous perinatal deaths. The last son died at 4 days of life and genetic analyses revealed a novel probably pathogenic variant at ETFDH (c.706dupG + c.706dupG) that codifies for a truncated protein (p.Glu236Glyfs*5 + p.Glu236Glyfs*5). CASE: During the gestation, due to the medical familial history, prenatal echography and a chorial biopsy for ETFDH-associated glutaric aciduria analysis were carried out. Sanger sequencing confirmed the presence of the homozygous familial variant in the ETFDH gene. The gestation was terminated and the foetal autopsy performed. Autopsy revealed prominent forehead, flat nasal bridge, malformed ears, intrauterine growth retardation, polycystic kidneys and steatosis in the liver, consistent with the diagnosis of glutaric aciduria type II. The comparison of present cases with the previously reported in the literature confirmed the presence of classical criteria, but also revealed the association with urogenital deformities, not previously stated. CONCLUSIONS: Clinical and foetal findings allowed the characterisation of the novel variant (c.706dupG at ETDFH) as pathogenic. Genotype-phenotype relationship is important when studying rare genetic disorders such as glutaric aciduria type II, as variants are usually family-specific, leading to a difficulty in the characterisation of their pathogenicity.

Clinical and Molecular Description of 16 Families With Heterozygous IHH Variants.

CONTEXT: Heterozygous variants in the Indian hedgehog gene (IHH) have been reported to cause brachydactyly type A1 and mild hand and feet skeletal anomalies with short stature. Genetic screening in individuals with short stature and mild skeletal anomalies has been increasing over recent years, allowing us to broaden the clinical spectrum of skeletal dysplasias. OBJECTIVE: The objective of this article is to describe the genotype and phenotype of 16 probands with heterozygous variants in IHH. PATIENTS AND METHODS: Targeted next-generation sequencing or Sanger sequencing was performed in patients with short stature and/or brachydactyly for which the genetic cause was unknown. RESULTS: Fifteen different heterozygous IHH variants were detected, one of which is the first reported complete deletion of IHH. None of the patients showed the classical phenotype of brachydactyly type A1. The most frequently observed clinical characteristics were mild to moderate short stature as well as shortening of the middle phalanx on the fifth finger. The identified IHH variants were demonstrated to cosegregate with the short stature and/or brachydactyly in the 13 probands whose family members were available. However, clinical heterogeneity was observed: Two short-statured probands showed no hand radiological anomalies, whereas another 5 were of normal height but had brachydactyly. CONCLUSIONS: Short stature and/or mild skeletal hand defects can be caused by IHH variants. Defects in this gene should be considered in individuals with these findings, especially when there is an autosomal dominant pattern of inheritance. Although no genotype-phenotype correlation was observed, cosegregation studies should be performed and where possible functional characterization before concluding that a variant is causative.

Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment.

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.

Cri-du-chat syndrome mimics Silver-Russell syndrome depending on the size of the deletion: a case report.

BACKGROUND: Silver-Russell Syndrome (SRS) is a rare growth-related genetic disorder mainly characterized by prenatal and postnatal growth failure. Although molecular causes are not clear in all cases, the most common mechanisms involved in SRS are loss of methylation on chromosome 11p15 (≈50%) and maternal uniparental disomy for chromosome 7 (upd(7)mat) (≈10%). CASE PRESENTATION: We present a girl with clinical suspicion of SRS (intrauterine and postnatal growth retardation, prominent forehead, triangular face, mild psychomotor delay, transient neonatal hypoglycemia, mild hypotonia and single umbilical artery). Methylation and copy number variations at chromosomes 11 and 7 were studied by methylation-specific multiplex ligation-dependent probe amplification and as no alterations were found, molecular karyotyping was performed. A deletion at 5p15.33p15.2 was identified (arr[GRCh37] 5p15.33p15.2(25942-11644643)× 1), similar to those found in patients with Cri-du-chat Syndrome (CdCS). CdCS is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-), whose main feature is a high-pitched mewing cry in infancy, accompanied by multiple congenital anomalies, intellectual disability, microcephaly and facial dysmorphism. CONCLUSIONS: The absence of some CdCS features in the current patient could be due to the fact that in her case the critical regions responsible do not lie within the identified deletion. In fact, a literature review revealed a high degree of concordance between the clinical manifestations of the two syndromes.

Formin Homology 2 Domain Containing 3 (FHOD3) Is a Genetic Basis for Hypertrophic Cardiomyopathy.

BACKGROUND: The genetic cause of hypertrophic cardiomyopathy remains unexplained in a substantial proportion of cases. Formin homology 2 domain containing 3 (FHOD3) may have a role in the pathogenesis of cardiac hypertrophy but has not been implicated in hypertrophic cardiomyopathy. OBJECTIVES: This study sought to investigate the relation between FHOD3 mutations and the development of hypertrophic cardiomyopathy. METHODS: FHOD3 was sequenced by massive parallel sequencing in 3,189 hypertrophic cardiomyopathy unrelated probands and 2,777 patients with no evidence of cardiomyopathy (disease control subjects). The authors evaluated protein-altering candidate variants in FHOD3 for cosegregation, clinical characteristics, and outcomes. RESULTS: The authors identified 94 candidate variants in 132 probands. The variants' frequencies were significantly higher in patients with hypertrophic cardiomyopathy (74 of 3,189 [2.32%]) than in disease control subjects (18 of 2,777 [0.65%]; p < 0.001) or in the gnomAD database (1,049 of 138,606 [0.76%]; p < 0.001). FHOD3 mutations cosegregated with hypertrophic cardiomyopathy in 17 families, with a combined logarithm of the odds score of 7.92, indicative of very strong segregation. One-half of the disease-causing variants were clustered in a small conserved coiled-coil domain (amino acids 622 to 655); odds ratio for hypertrophic cardiomyopathy was 21.8 versus disease control subjects (95% confidence interval: 1.3 to 37.9; p < 0.001) and 14.1 against gnomAD (95% confidence interval: 6.9 to 28.7; p < 0.001). Hypertrophic cardiomyopathy patients carrying (likely) pathogenic mutations in FHOD3 (n = 70) were diagnosed after age 30 years (mean 46.1 ± 18.7 years), and two-thirds (66%) were males. Of the patients, 82% had asymmetric septal hypertrophy (mean 18.8 ± 5 mm); left ventricular ejection fraction <50% was present in 14% and hypertrabeculation in 16%. Events were rare before age 30 years, with an annual cardiovascular death incidence of 1% during follow-up. CONCLUSIONS: FHOD3 is a novel disease gene in hypertrophic cardiomyopathy, accounting for approximately 1% to 2% of cases. The phenotype and the rate of cardiovascular events are similar to those reported in unselected cohorts. The FHOD3 gene should be routinely included in hypertrophic cardiomyopathy genetic testing panels.

A kidney-disease gene panel allows a comprehensive genetic diagnosis of cystic and glomerular inherited kidney diseases.

Molecular diagnosis of inherited kidney diseases remains a challenge due to their expanding phenotypic spectra as well as the constantly growing list of disease-causing genes. Here we develop a comprehensive approach for genetic diagnosis of inherited cystic and glomerular nephropathies. Targeted next generation sequencing of 140 genes causative of or associated with cystic or glomerular nephropathies was performed in 421 patients, a validation cohort of 116 patients with previously known mutations, and a diagnostic cohort of 207 patients with suspected inherited cystic disease and 98 patients with glomerular disease. In the validation cohort, a sensitivity of 99% was achieved. In the diagnostic cohort, causative mutations were found in 78% of patients with cystic disease and 62% of patients with glomerular disease, mostly familial cases, including copy number variants. Results depict the distribution of different cystic and glomerular inherited diseases showing the most likely diagnosis according to perinatal, pediatric and adult disease onset. Of all the genetically diagnosed patients, 15% were referred with an unspecified clinical diagnosis and in 2% genetic testing changed the clinical diagnosis. Therefore, in 17% of cases our genetic analysis was crucial to establish the correct diagnosis. Complex inheritance patterns in autosomal dominant polycystic kidney disease and Alport syndrome were suspected in seven and six patients, respectively. Thus, our kidney-disease gene panel is a comprehensive, noninvasive, and cost-effective tool for genetic diagnosis of cystic and glomerular inherited kidney diseases. This allows etiologic diagnosis in three-quarters of patients and is especially valuable in patients with unspecific or atypical phenotypes.

Comparing the Sexual Reproductive Success of Two Exotic Trees Invading Spanish Riparian Forests vs. a Native Reference.

A widely accepted hypothesis in invasion ecology is that invasive species have higher survival through the early stages of establishment than do non-invasive species. In this study we explore the hypothesis that the sexual reproductive success of the invasive trees Ailanthus altissima (Mill.) Swingle and Robinia pseudoacacia L. is higher than that of the native Fraxinus angustifolia Vahl., all three species coexisting within the riparian forests of Central Spain. We compared different stages of the early life cycle, namely seed rain, seed infestation by insects, seed removal by local fauna, seed germination under optimal conditions and seedling abundance between the two invasive trees and the native, in order to assess their sexual reproductive success. The exotic species did not differ from the native reference (all three species displaying high seed rain and undergoing seed losses up to 50% due to seed removal by the local fauna). Even if the exotic R. pseudoacacia showed a high percentage of empty and insect-parasited seeds along with a low seedling emergence and the exotic A. altissima was the species with more viable seeds and of higher germinability, no differences were found regarding these variables when comparing them with the native F. angustifolia. Unsuitable conditions might have hampered either seedling emergence and survival, as seedling abundance in the field was lower than expected in all species -especially in R. pseudoacacia-. Our results rather suggest that the sexual reproductive success was not higher in the exotic trees than in the native reference, but studies focusing on long-term recruitment would help to shed light on this issue.

Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

BACKGROUND: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). METHODS: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). RESULTS: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. CONCLUSIONS: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.