Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S) SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare

Legius Syndrome in Fourteen Families

Legius syndrome presents as an autosomal dominant condition characterized by café-au-lait macules with or without freckling and sometimes a Noonan-like appearance and/or learning difficulties. It is caused by germline loss-of-function SPRED1 mutations and is a member of the RAS-MAPK pathway syndromes. Most mutations result in a truncated protein and only a few inactivating missense mutations have been reported. Since only a limited number of patients has been reported up until now, the full clinical and mutational spectrum is still unknown. We report mutation data and clinical details in fourteen new families with Legius syndrome. Six novel germline mutations are described. The Trp31Cys mutation is a new pathogenic SPRED1 missense mutation. Clinical details in the 14 families confirmed the absence of neurofibromas, and Lisch nodules, and the absence of a high prevalence of central nervous system tumors. We report white matter T2 hyperintensities on brain MRI scans in 2 patients and a potential association between postaxial polydactyly and Legius syndrome. © 2010 Wiley-Liss, Inc

Pathogenesis of vestibular schwannoma in ring chromosome 22

<p>Abstract</p> <p>Background</p> <p>Ring chromosome 22 is a rare human constitutional cytogenetic abnormality. Clinical features of neurofibromatosis type 1 and 2 as well as different tumour types have been reported in patients with ring chromosome 22. The pathogenesis of these tumours is not always clear yet.</p> <p>Methods</p> <p>We report on a female patient with a ring chromosome 22 presenting with severe mental retardation, autistic behaviour, café-au-lait macules and facial dysmorphism. Peripheral blood lymphocytes were karyotyped and array CGH was performed on extracted DNA. At the age of 20 years she was diagnosed with a unilateral vestibular schwannoma. Tumour cells were analyzed by karyotyping, array CGH and <it>NF2 </it>mutation analysis.</p> <p>Results</p> <p>Karyotype on peripheral blood lymphocytes revealed a ring chromosome 22 in all analyzed cells. A 1 Mb array CGH experiment on peripheral blood DNA showed a deletion of 5 terminal clones on the long arm of chromosome 22. Genetic analysis of vestibular schwannoma tissue revealed loss of the ring chromosome 22 and a somatic second hit in the <it>NF2 </it>gene on the remaining chromosome 22.</p> <p>Conclusion</p> <p>We conclude that tumours can arise by the combination of loss of the ring chromosome and a pathogenic <it>NF2 </it>mutation on the remaining chromosome 22 in patients with ring chromosome 22. Our findings indicate that patients with a ring 22 should be monitored for NF2-related tumours starting in adolescence.</p

Correction: Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype–phenotype correlation

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Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype–phenotype correlation

Purpose: Neurofibromatosis type 1 (NF1) is characterized by a highly variable clinical presentation, but almost all NF1-affected adults present with cutaneous and/or subcutaneous neurofibromas. Exceptions are individuals heterozygous for the NF1 in-frame deletion, c.2970_2972del (p.Met992del), associated with a mild phenotype without any externally visible tumors. Methods: A total of 135 individuals from 103 unrelated families, all carrying the constitutional NF1 p.Met992del pathogenic variant and clinically assessed using the same standardized phenotypic checklist form, were included in this study. Results: None of the individuals had externally visible plexiform or histopathologically confirmed cutaneous or subcutaneous neurofibromas. We did not identify any complications, such as symptomatic optic pathway gliomas (OPGs) or symptomatic spinal neurofibromas; however, 4.8% of individuals had nonoptic brain tumors, mostly low-grade and asymptomatic, and 38.8% had cognitive impairment/learning disabilities. In an individual with the NF1 constitutional c.2970_2972del and three astrocytomas, we provided proof that all were NF1-associated tumors given loss of heterozygosity at three intragenic NF1 microsatellite markers and c.2970_297

The NCFC syndromes: clinical, molecular and cognitive aspects in selected syndromes

De neuro-cardio-facio-cutane (NCFC) syndromen vormen een groep fenotypis ch overlappende aandoeningen die zich presenteren met een variabele graa d van cognitieve beperkingen, hartafwijkingen, gelaatsdysmorfieën en hui dproblemen. De meeste van deze syndromen gaan gepaard met een verhoogd r isico op kanker. De laatste jaren is het duidelijk geworden dat deze syn dromen veroorzaakt worden door mutaties in genen van de RAS-MAPKinase si gnaaltransductiecascade. Deze cascade speelt een rol in celgroei en -dif ferentiatie en is gekend voor zijn rol in kanker en meer recent ook in c ognitie. In deze thesis werd geprobeerd het gekende mutatie- en fenotypisch spect rum van enkele van deze syndromen, namelijk Costello, Noonan en CFC synd room uit te breiden. We beschrijven een nieuwe HRAS mutatie in een pa tiënt met Costello syndroom die geen effect heeft op enzymatische activi teit, maar wel op guanine-nucleotide dissociatie. Ook beschrijven we het fenotype in patiënten met Noonan syndroom en SOS1, RAF1, KRAS en NRAS mutaties en in patiënten met CFC syndroom en BRAF en MEK2 mutaties. Niet eerder gerapporteerde mutaties in SOS1, KRAS, NR AS, BRAF en MEK2 werden gevonden. Daarnaast beschrijven we het fenotype in Legius syndroom, aanvankelijk neurofibromatose type1-achtig syndroom genoemd, een nieuw lid van deze groep van NCFC syndromen. Legius syndroom wordt veroorzaakt door mutatie s in SPRED1, een negatieve regulator van de RAS-MAPKinase cascade, en patiënten presenteren zich met café-au-lait vlekken met of zonder freck ling, doch zonder de andere typische symptomen van neurofibromatose type 1. Leerproblemen werden meermaals gerapporteerd in patiënten met Legius syndroom. We bestudeerden het leergedrag in een Spred1 knockout muiz enmodel en stelden vast dat deze muizen een verrassend gelijkaardig feno type vertonen met Nf1 heterozygote muizen zowel wat betreft leren als synaptische plasticiteit. Tenslotte bestudeerden we de intelligentie en het gedrag in een groep patiënten met Legius syndroom en niet-aangetast e siblings. Patiënten met Legius syndroom hebben een normaal totaal IQ, wat niet verschilt van de siblings, maar wel een lager performaal IQ. In de toekomst willen we proberen de leerproblemen in het Spred1 muiz enmodel om te keren door behandeling met statines, een behandeling die r eeds effectief gebleken is bij het Nf1 muizenmodel. Indien we een pos itief effect van statines op het leergedrag van Spred1 muizen kunnen aantonen, versterkt dit de theoretische basis voor klinische studies met deze medicatie in kinderen met neurofibromatose type 1 en misschien bij uitbreiding de andere NCFC syndromen.TABLE OF CONTENTS General introduction 15 Abstract 16 RAS genes and cancer 17 Germline mutations in components of the RAS-MAPK pathway 19 Neurofibromatosis type 1 25 Costello syndrome 26 Noonan syndrome 27 LEOPARD syndrome 32 Cardiofaciocutaneous syndrome 32 Legius syndrome 33 Conclusions 35 Objectives 38 References 39 Chapter 1: Mutation analysis in Costello syndrome: Functional and structural characterization of the HRAS Lys117Arg mutation 47 Abstract 48 Introduction 49 Materials and methods 50 Results 55 Discussion 60 Acknowledgements 63 References 64 Supplementary material 68 Chapter 2: Tumor spectrum in children with Noonan syndrome and SOS1 or RAF1 mutations 69 Abstract 70 Introduction 71 Patients and methods 71 Results 73 Discussion 84 Acknowledgements 88 References 89 Chapter 3: KRAS and NRAS mutations in Noonan syndrome 95 Abstract 96 Introduction 97 Materials and methods 99 Results 102 Discussion 109 References 112 Chapter 4: Mutation analysis in cardiofaciocutaneous syndrome 117 Abstract 118 Introduction 119 Patients and methods 121 Results 121 Discussion 128 References 129 Chapter 5: A new syndrome caused by heterozygous germline SPRED1 mutations 133 Abstract 134 5A: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype 135 Abstract 135 Introduction 136 Methods 136 Results 137 Discussion 143 References 144 5B: Clinical and mutational spectrum of Neurofibromatosis type 1-like Syndrome 147 Abstract 147 Introduction 149 Methods 150 Results 151 Discussion 161 References 164 Chapter 6: SPRED1 is required for synaptic plasticity and hippocampus-dependent learning 167 Abstract 168 Introduction 169 Materials and methods 169 Results 174 Discussion 181 Acknowledgements 184 References 185 Supplementary material 187 Chapter 7: Legius syndrome in eleven families 191 Abstract 192 Introduction 193 Materials and methods 194 Results 196 Discussion 207 References 209 Chapter 8: Intelligence and behavior in patients with Legius syndrome 211 Abstract 212 Introduction 213 Materials and methods 214 Results and discussion 215 Acknowledgements 221 References 222 Supplementary material 224 General discussion and future perspectives 227 References 235 Samenvatting 238 Summary 240 Professional career Ellen Denayer 241nrpages: 238status: publishe

What's new in the neuro-cardio-facial-cutaneous syndromes?

The RAS-MAPKinase pathway is a signal transduction cascade which has been studied extensively during the last decades for its role in human oncogenesis. Activation of this cascade is controlled by cycling of the RAS protein between an inactive and an active state and by phosphorylation of downstream proteins. The signalling cascade regulates cell proliferation, differentiation and survival. Disturbed RAS signalling in malignancies is caused by acquired somatic mutations in RAS genes or other components of this pathway. Recently, germline mutations in genes coding for different components of the RAS signalling cascade have been recognized as the cause of several phenotypically overlapping disorders, recently referred to as the neuro-cardio-facial-cutaneous syndromes. Neurofibromatosis type 1, Noonan, LEOPARD, Costello and cardiofaciocutaneous syndromes all present with variable degrees of psychomotor delay, congenital heart defects, facial dysmorphism, short stature, skin abnormalities and a predisposition for malignancy. These findings point to important roles for this evolutionary conserved pathway in oncogenesis, development, cognition and growth. Conclusion: it has become obvious in recent years that the neuro-cardio-facial-cutaneous syndromes all share a common genetic and pathophysiologic basis. Dysregulation of the RAS-MAPKinase pathway is caused by germline mutations in genes involved in this pathway. Undoubtedly more genes causing related syndromes will be discovered in the near future since there are still a substantial number of genes in the pathway that are not yet associated with a known syndrome.status: publishe

Legius Syndrome and its Relationship with Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is the most common disorder characterized by multiple café-au-lait macules. Most individuals with this autosomal dominant disorder also have other features, such as skinfold freckling, iris Lisch nodules and benign or malignant peripheral nerve sheath tumours. Legius syndrome is a less frequent autosomal dominant disorder with similar multiple café-au-lait macules and skinfold freckling. Legius syndrome is not characterized by an increased risk of tumours, and a correct diagnosis is important. In young children with a sporadic form of multiple café-au-lait macules with or without freckling and no other manifestations of NF1 these 2 conditions cannot be differentiated based on clinical examination. Molecular analysis of the NF1 and SPRED1 genes is usually needed to differentiate the 2 conditions. Other less frequent conditions with café-au-lait macules are Noonan syndrome with multiple lentigines, constitutional mismatch repair deficiency and McCune-Albright syndrome.status: publishe