SERPINA11 related novel serpinopathy - A perinatal lethal disorder.

SERPINA11 is a hitherto poorly characterised gene belonging to Clade A of the SERPIN superfamily, with unknown expression pattern and functional significance. We report a perinatal lethal phenotype in two foetuses from the same family associated with a biallelic loss of function variant in SERPINA11, and provide functional evidence to support its candidature as a Mendelian disorder. The SERPINA11 variant-associated foetal phenotype is characterised by gross and histopathological features of extracellular matrix disruption. Western blot and immunofluorescence analyses revealed SERPINA11 expression in multiple mouse tissues, with pronounced expression in the bronchiolar epithelium. We observed a significant decrease in SERPINA11 immunofluorescence in the affected foetal lung compared with a healthy gestation-matched foetus. Protein expression data from HEK293T cell lines following site-directed mutagenesis support the loss of function nature of the variant. Transcriptome analysis from the affected foetal liver indicated the possibility of reduced SERPINA11 transcript abundance. This novel serpinopathy appears to be a consequence of the loss of inhibition of serine proteases involved in extracellular matrix remodelling, revealing SERPINA11 as a protease inhibitor critical for embryonic development.

Comparison of three scoring criteria to assess recovery from general anesthesia in the postanesthesia care unit in the indian population.

Background: Different discharge criteria are available for shifting patients out from postanesthesia care room following surgery. This study was done to compare the three-scoring system namely traditional time-based criteria, Fast track criteria and modified Aldrete score, in Indian population patients who recover after general anesthesia in postanesthesia care unit (PACU). Materials and Methods: Three hundred and seventy-five patients scheduled for general anesthesia were included in this study. Induction of anesthesia was done with intravenous (IV) propofol and maintained with sevoflurane inhalation with oxygen and nitrous oxide. Reversal of residual neuromuscular blockade was done with IV neostigmine and glycopyrrolate. Patients were shifted to PACU following tracheal extubation and recovery was assessed using the traditional time-based criteria, fast track criteria, and modified Aldrete score. Results: As per modified Aldrete score, mean time of shift out is 19 min with median of 15 min and standard deviation of 21.7 min. As per fast-track score, mean time of shift out is 187 min with median of 30 min and standard deviation of 243.7 min. As per the time-based criteria, mean time of shift out is 222 min with median of 240 min and standard deviation of 136.8 min. While using modified Aldrete score, majority of patients had a shorter stay in PACU and faster time to shift out as compared to fast-track criteria and traditional time-based criteria. Conclusion: Modified Aldrete score when compared to fast-track scoring and time-based criteria shows early recovery and reduces the length of stay in PACU.

Résumé Contexte: Différents critères de sortie sont disponibles pour faire sortir les patients de la salle de soins post-anesthésie après une intervention chirurgicale. Cette étude a été réalisée pour comparer le système de notation à trois, à savoir les critères traditionnels basés sur le temps, les critères accélérés et le score Aldrete modifié, en Inde. Population de patients qui se rétablissent après une anesthésie générale en unité de soins post-anesthésiques (USPA). Matériels et méthodes: Trois cent et soixante-quinze patients devant subir une anesthésie générale ont été inclus dans cette étude. L'induction de l'anesthésie a été réalisée par voie intraveineuse (IV) propofol et maintenu avec inhalation de sévoflurane avec de l'oxygène et du protoxyde d'azote. L'inversion du bloc neuromusculaire résiduel a été réalisée avec néostigmine IV et glycopyrrolate. Les patients ont été transférés vers une USPA après l'extubation trachéale et la récupération a été évaluée à l'aide du critères traditionnels basés sur le temps, critères accélérés et score d'Aldrete modifié. Résultats: Selon le score d'Aldrete modifié, temps moyen de sortie est de 19 min avec une médiane de 15 min et un écart type de 21,7 min. Selon le score accéléré, le temps moyen de sortie est de 187 minutes avec une médiane de 30 min et écart type de 243,7 min. Selon les critères temporels, le temps moyen de changement de poste est de 222 minutes avec une médiane de 240 minutes et écart type de 136,8 min. En utilisant le score d'Aldrete modifié, la majorité des patients ont eu un séjour plus court en USPA et un temps de changement plus rapide. Par rapport aux critères accélérés et aux critères traditionnels basés sur le temps. Conclusion: Score d'Aldrete modifié par rapport au traitement accéléré la notation et les critères basés sur le temps montrent une récupération précoce et réduisent la durée du séjour en USPA. Mots-clés: Critères accélérés, score d'Aldrete modifié, unité de soins post-anesthésie, critères de sortie post-anesthésie, sortie basée sur le temps.

Monogenic conditions and central nervous system anomalies: A prospective study, systematic review and meta-analysis.

OBJECTIVES: Determine the incremental diagnostic yield of prenatal exome sequencing (pES) over chromosome microarray (CMA) or G-banding karyotype in fetuses with central nervous system (CNS) abnormalities. METHODS: Data were collected via electronic searches from January 2010 to April 2022 in MEDLINE, Cochrane, Web of Science and EMBASE. The NHS England prenatal exome cohort was also included. Incremental yield was calculated as a pooled value using a random-effects model. RESULTS: Thirty studies were included (n = 1583 cases). The incremental yield with pES for any CNS anomaly was 32% [95%CI 27%-36%; I2 = 72%]. Subgroup analysis revealed apparent incremental yields in; (a) isolated CNS anomalies; 27% [95%CI 19%-34%; I2 = 74%]; (b) single CNS anomaly; 16% [95% CI 10%-23%; I2 = 41%]; (c) more than one CNS anomaly; 31% [95% Cl 21%-40%; I2 = 56%]; and (d) the anatomical subtype with the most optimal yield was Type 1 malformation of cortical development, related to abnormal cell proliferation or apoptosis, incorporating microcephalies, megalencephalies and dysplasia; 40% (22%-57%; I2 = 68%). The commonest syndromes in isolated cases were Lissencephaly 3 and X-linked hydrocephalus. CONCLUSIONS: Prenatal exome sequencing provides a high incremental diagnostic yield in fetuses with CNS abnormalities with optimal yields in cases with multiple CNS anomalies, particularly those affecting the midline, posterior fossa and cortex.

Proband only exome sequencing in 403 Indian children with neurodevelopmental disorders: Diagnostic yield, utility and challenges in a resource-limited setting.

Whole exome sequencing is recommended as the first tier test for neurodevelopmental disorders (NDDs) with trio being an ideal option for the detection of de novo variants. Cost constraints have led to adoption of sequential testing i.e. proband-only whole exome followed by targeted testing of parents. The reported diagnostic yield for proband exome approach ranges between 31 and 53%. Typically, these study designs have aptly incorporated targeted parental segregation before concluding a genetic diagnosis to be confirmed. The reported estimates however do not accurately reflect the yield of proband only standalone whole -exome, a question commonly posed to the referring clinician in self pay medical systems like India. To assess the utility of standalone proband exome (without follow up targeted parental testing), we retrospectively evaluated 403 cases of neurodevelopmental disorders referred for proband-only whole exome sequencing at Neuberg Centre for Genomic Medicine (NCGM), Ahmedabad during the period of January 2019 and December 2021. A diagnosis was considered confirmed only upon the detection of Pathogenic/Likely Pathogenic variants in concordance with patient's phenotype as well as established inheritance pattern. Targeted parental/familial segregation analysis was recommended as a follow up test where applicable. The diagnostic yield of the proband-only standalone whole exome was 31.5%. Only 20 families submitted samples for follow up targeted testing, and a genetic diagnosis was confirmed in twelve cases increasing the yield to 34.5%. To understand factors leading to poor uptake of sequential parental testing, we focused on cases where an ultra-rare variant was detected in hitherto described de novo dominant neurodevelopmental disorder. A total of 40 novel variants in genes associated with de novo autosomal dominant disorders could not be reclassified as parental segregation was denied. Semi-structured telephonic interviews were conducted upon informed consent to comprehend reasons for denial. Major factors influencing decision making included lack of definitive cure in the detected disorders; especially when couples not planning further conception and financial constraints to fund further targeted testing. Our study thus depicts the utility and challenges of proband-only exome approach and highlights the need for larger studies to understand factors influencing decision making in sequential testing.

Muscle spasms as presenting feature of Nivelon-Nivelon-Mabile syndrome.

Hedgehog acyltransferase gene (HHAT)-associated Nivelon-Nivelon-Mabile syndrome (NNMS) is a rare genetic disorder of multiple system involvement with microcephaly, central nervous system malformations, skeletal dysplasia, and 46,XY sex reversal. Other variable and inconsistent features reported in this disorder are muscle spasms, facial dysmorphism, prenatal onset growth restriction, microphthalmia, and holoprosencephaly. This is the sixth postnatal reported patient with biallelic variants in HHAT gene, who presented with microcephaly, short stature, muscle hypertrophy, muscle spasms, and facial dysmorphism. The most prominent and presenting finding in this patient were muscle hypertrophy and muscle spasms which had a clinical response to phenytoin and acetazolamide treatment. Our report emphasizes the phenotypic variability of NNMS and further reiterates muscle spasms as an important clinical manifestation of this extremely rare condition.

Next Generation Sequencing in a Case of Early Onset Hydrops: Closing the Loop on the Diagnostic Odyssey!

Non-immune fetal hydrops (NIFH) is an etiologically heterogeneous condition. Cardiac anomalies are one of the common causes of NIFH. Cardiac anomalies can be isolated, multifactorial malformations or have a genetic basis. PLD1 variants have been associated with developmental defects involving the right heart. We present a NIFH with a PLD1 associated right heart malformation.We describe a spontaneously aborted 14 weeks old NIFH fetus with a rudimentary right ventricle, pulmonary valve atresia and pulmonary artery stenosis found at fetopsy. After a normal microarray, whole exome sequencing revealed a homozygous missense variant c.2023 C > T (p. Arg675Trp) in the PLD1 gene. Conclusion: Detailed fetopsy and genetic evaluation in this NIFH allowed an etiological explanation, further corroborated the association of PLD1 gene variants and developmental right heart defects, and that this defect can be associated with NIHF.

Prenatal phenotype of FBXL4-associated encephalomyopathic mitochondrial DNA depletion syndrome-13.

FBXL4 -associated encephalomyopathic mitochondrial DNA depletion syndrome-13 (MTDPS13) is a rare genetic disorder characterized by early neonatal onset of encephalopathy, seizures, lactic acidosis, hypotonia, dysmorphism, and severe global developmental delay. Prenatal phenotype of molecularly confirmed MTDPS13 has not been well studied. This is the case report of a non-consanguineously conceived fetus ascertained first at 20 weeks of gestation with multiple soft markers. Follow-up fetal ultrasonogram at 26 weeks revealed periventricular cysts, periventricular echogenicity, ventriculomegaly, thin corpus callosum, mega cisterna magna, and large cavum. Fetal MRI confirmed these findings. Postnatally, the baby had clinical and biochemical findings indicative of a mitochondriopathy and died on neonatal day 3. Whole exome sequencing on stored amniotic fluid DNA confirmed the diagnosis of encephalomyopathic mitochondrial DNA depletion syndrome-13 (MTDPS13). This report presents the prenatal phenotype of this rare mitochondriopathy, which has been recognized primarily in postnatal patients. The brain imaging findings in the reported fetus indicate that MTDPS13 is associated with progressive neurological involvement and brain tissue destructive changes starting as early as the second trimester of pregnancy. The case also raises concerns regarding the association of so-called soft markers, which were the only initial finding in this case, with severe monogenic diseases.

Prenatal phenotyping: A community effort to enhance the Human Phenotype Ontology.

Technological advances in both genome sequencing and prenatal imaging are increasing our ability to accurately recognize and diagnose Mendelian conditions prenatally. Phenotype-driven early genetic diagnosis of fetal genetic disease can help to strategize treatment options and clinical preventive measures during the perinatal period, to plan in utero therapies, and to inform parental decision-making. Fetal phenotypes of genetic diseases are often unique and at present are not well understood; more comprehensive knowledge about prenatal phenotypes and computational resources have an enormous potential to improve diagnostics and translational research. The Human Phenotype Ontology (HPO) has been widely used to support diagnostics and translational research in human genetics. To better support prenatal usage, the HPO consortium conducted a series of workshops with a group of domain experts in a variety of medical specialties, diagnostic techniques, as well as diseases and phenotypes related to prenatal medicine, including perinatal pathology, musculoskeletal anomalies, neurology, medical genetics, hydrops fetalis, craniofacial malformations, cardiology, neonatal-perinatal medicine, fetal medicine, placental pathology, prenatal imaging, and bioinformatics. We expanded the representation of prenatal phenotypes in HPO by adding 95 new phenotype terms under the Abnormality of prenatal development or birth (HP:0001197) grouping term, and revised definitions, synonyms, and disease annotations for most of the 152 terms that existed before the beginning of this effort. The expansion of prenatal phenotypes in HPO will support phenotype-driven prenatal exome and genome sequencing for precision genetic diagnostics of rare diseases to support prenatal care.

Clinical and Molecular Spectrum of Degenerative Cerebellar Ataxia: A Single Centre Study.

Background: Cerebellar ataxia is a disabling neurological symptom with extreme clinical and etiological heterogeneity. Objective: To study the clinical and molecular characteristics in patients with degenerative cerebellar ataxia. Materials and Methods: In this study, 150 South-Indian patients with degenerative cerebellar ataxia underwent a phenotype guided, sequential tiered testing. Phenotypic features studied included cerebellar symptoms, pyramidal and extrapyramidal features, and ophthalmic and systemic findings. Tier one included conventional tests such as short PCR/fragment analysis for spinocerebellar ataxia (SCA) subtypes 1, 2, 3, 6, 7, 8, 12, 17, and 36 and TP-PCR for Friedreich ataxia (FA). Tier two testing comprised next-generation sequencing (NGS)-based strategies reserved for select undiagnosed cases. Results: The clinical features were highly overlapping and had limited specificity, except in autosomal recessive ataxias and SCA 34. The overall diagnostic yield of our study was 49.3%. SCA 1, 2, and 3 were noted in 13 (12.6%), 12 (11.6%) and 14 (13.5%), respectively, out of the 103 tested, and FA was noted in 17/55 (30.9%) patients. SCA subtypes 6, 7, 8, 12, 17, and 36 were absent in the cohort studied. Targeted Sanger sequencing and NGS revealed some rare diagnoses in 17 among the 18 patients tested. Whole exome sequencing uncovered a novel genotype-phenotype association in a sibling-pair with ataxia, dysmorphism, and retinopathy. Conclusion: SCA 1, 2, 3 and FRDA were the most common causes of ataxia. SCA 6, 7, 8, 12, 17, and 36 were absent in the cohort studied. NGS testing revealed several rare forms of ataxia. Clinical features based testing is cost-effective, achieves good genotype-phenotype correlation, and prioritizes variants for further studies.

Fetal phenotypes of Mendelian disorders: A descriptive study from India.

OBJECTIVE: Exome sequencing (ES)-based diagnosis of Mendelian diseases in the fetus is limited by paucity of phenotypic information. This study reports the comprehensive phenotypes of some fetuses with Mendelian disorders. METHODS: Next generation technology-based sequencing of all coding regions of the genome (Exome sequencing) or targeted gene sequencing using Sanger or next generation platforms was performed in a cohort of deeply phenotyped, cytogenetically normal fetuses with morphological defects. Prenatal ultrasonographic phenotypes and postmortem details including dysmorphology, histopathology, and radiography were ascertained. Novel candidate genes, novel/unusual findings, and unusual genotypes in cases with confirmed Mendelian disorders are described. RESULTS: Of the 102 fetuses sequenced, 45 (44%) achieved definitive diagnosis of a Mendelian disorder with 50 pathogenic/likely pathogenic variants. The majority (87%) were autosomal recessive, 69% families were consanguineous, and 54% variants were novel. Dysmorphic syndromes, skeletal dysplasias, and metabolic disorders were the commonest disease categories, ciliopathies and dystroglycanopathies, commonest molecular categories. We describe the first fetal description of six monogenic diseases, and nine cases with novel histological findings. Nineteen cases had novel/unusual findings. CONCLUSION: This cohort demonstrates how deep fetal phenotypes of some Mendelian disorders can show novel/unusual findings, which have important implications for prenatal diagnosis of these conditions.

Microcephalic primordial dwarfism with predominant Meier-Gorlin phenotype, ichthyosis, and multiple joint deformities-Further expansion of DONSON Cell Cycle-opathy phenotypic spectrum.

We report a patient with microcephalic primordial dwarfism with predominant Meier-Gorlin syndrome phenotype with ichthyosis and disabling multiple joint deformities in addition to classic features of the syndrome. The patient was a 10.5-year-old girl referred in view of short stature, joint deformities, and facial dysmorphism. There was history of intrauterine growth restriction and collodion like skin abnormality at birth. She had normal developmental milestones and intellect. On clinical evaluation, anthropometry was suggestive of proportionate short stature and microcephaly. There was abnormal posture due to spine and peripheral joint deformities, along with ichthyosis, facial, and digital dysmorphism. Skeletal radiographs showed radial subluxation, acetabular dysplasia and hip dislocation, bilateral knee joint dislocation, absent patellae, slender long bones with delayed bone age, and subluxation of small joints of hands and feet. Work up for metabolic bone disease and peripheral blood karyotype was normal. Whole exome sequencing revealed a pathogenic homozygous variant c.C1297T (p.Pro433Ser) in the exon 8 of DONSON gene. This report further expands the genotypic-phenotypic spectrum of the group of disorders known as Cell Cycle-opathies.

Fetal presentation of chondrodysplasia with joint dislocations, GPAPP type, caused by novel biallelic IMPAD1 variants.

Biallelic IMPAD1 pathogenic variants leads to deficiency of GPAPP (Golgi 3-prime phosphoadenosine 5-prime phosphate 3-prime phosphatase) protein and clinically causes chondrodysplasia, which is characterized by short stature with short limbs, craniofacial malformations, cleft palate, hand and foot anomalies, and various radiographic skeletal manifestations. Here we describe prenatal presentation of GPAPP deficiency caused by novel biallelic pathogenic variants, 2 base pair duplication in exon 2 of IMAPD1 gene in a patient of Asian-Indian origin. Further we report on diagnostic clues of prenatal presentation of GPAPP deficiency through ultrasonography, fetal MRI, and postmortem findings. We also provide evidence of pathophysiology of underlying GPAPP deficiency in the form of disorganization and dysplastic chondrocytes and reduced sulfation of glycoproteins through histopathology of cartilage similar to that described in mice IMPAD1 homozygous mutant model.

Recurrent Vein of Galen Aneurysmal Malformation as a Presentation of Hereditary Hemorrhagic Telangiectasia.

Introduction: Vein of Galen malformation (VGM) results from an aneurysmal aberration with an arteriovenous shunting of blood and is the most frequent arteriovenous malformation in infants and fetuses. The congenital malformation develops during weeks 6-11 of fetal development. Infants often die from high-output congestive heart failure. VGM is mostly considered as a sporadic condition with minimal recurrence risk in subsequent pregnancies. Mendelian forms of VGM have rarely been described as infrequent phenotypic presentations of 2 disorders: capillary malformation-arteriovenous malformation syndrome (RASA1, EPHB4) and hereditary hemorrhagic telangiectasia (ENG, ACVRL1, and SMAD4), both showing autosomal dominant inheritance. Case Presentation: Here, we report on a consanguineous couple with recurrent VGM in 2 pregnancies. Both partners were found to be affected by hereditary hemorrhagic telangiectasia due to a known pathogenic heterozygous c.790G>A (p.Asp264Asn) variant in ENG. Fetal DNA was unavailable, however in view of the mild phenotype in the couple, along with the severe prenatal presentation in 2 pregnancies, the fetus was presumed to be homozygous for the ENG variant. A subsequent pregnancy revealed a fetus heterozygous for the variant, which had an uneventful perinatal course. Conclusion: This report highlights a severe perinatal lethal phenotype due to biallelic variants in a gene hitherto known to cause an autosomal dominant disorder.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Role of whole exome sequencing for unidentified genetic syndromes.

PURPOSE OF REVIEW: The current review seeks to provide a comprehensive update on the revolutionary technology of whole exome sequencing (WES) which has been used to interrogate abnormal foetal phenotypes since the last few years, and is changing the paradigms of prenatal diagnosis, facilitating accurate genetic diagnosis and optimal management of pregnancies affected with foetal abnormalities, as well enabling delineation of novel Mendelian disorders. RECENT FINDINGS: WES has contributed to identification of more than 1000 Mendelian genes and made rapid strides into clinical diagnostics in recent years. Diagnostic yield of WES in postnatal cohorts has ranged from 25 to 50%, and this test is now a first tier investigation for various clinical presentations. Various abnormal perinatal phenotypes have also been investigated using WES since 2014, with diagnostic yields ranging from 8.5 to 80%. Studies in foetal phenotypes have been challenging and guidelines in this cohort are still evolving. SUMMARY: WES has proven to be a disrupting technology, enabling genetic diagnosis for pregnancies complicated by previously unexplained foetal abnormalities, and revealing a significant contribution of single gene disorders in these, thereby changing clinical diagnostic paradigms. The application of this technology in perinatal cohorts is also providing interesting insights into single gene defects presenting as previously unknown genetic syndromes, hence contributing to expansion of Mendelian genetics to encompass various foetal phenotypes.

A synonymous variant in a non-canonical exon of CDC45 disrupts splicing in two affected sibs with Meier-Gorlin syndrome with craniosynostosis.

Disruption of the initiation of DNA replication is significantly associated with Meier-Gorlin syndrome (MGORS), an autosomal recessive condition of reduced growth, microtia and patellar a/hypoplasia. Biallelic mutations in CDC45, a member of the pre-initiation complex in DNA replication, cause a spectrum of phenotypes ranging from MGORS with craniosynostosis, through to isolated short stature and craniosynostosis. Here we report two affected sibs with MGORS and craniosynostosis, with biallelic variants in CDC45 identified by 10X Chromium whole genome sequencing. One variant is a frameshift mutation, predicted to be pathogenic, and is inherited in trans with a synonymous variant in a non-canonical exon (exon 7) of CDC45. An in vitro splicing assay showed that while the canonical CDC45 exon 6-exon 8 transcript (with skipping of exon 7; numbering as per NM001178010.2) remained as the predominant transcript, the variant allele induced the use of novel splice acceptor sites in intron 6, all of which produced transcripts harbouring premature stop codons. This perturbation of canonical splicing provides evidence that this synonymous variant is indeed a deleterious alteration in this family. This report adds to the initial patient cohort in which several synonymous variants were also described, further highlighting the contribution of this variant type in CDC45. It also reiterates the true potential pathogenicity of synonymous variants, which is a mutation type that is commonly ignored in variant prioritization strategies.

Validation of the American Joint Committee on Cancer Staging in Squamous Cell Carcinoma of the Vermilion Lip.

BACKGROUND: The vermilion lip is a unique anatomical junction between cutaneous and mucosal surfaces. Squamous cell carcinoma (SCC) of the vermilion lip (vlSCC) was previously classified as oral SCC (oSCC) under the American Joint Committee on Cancer (AJCC) 7th edition (AJCC7), but has been recategorized as a cutaneous SCC of the head and neck (HNcSCC) in the AJCC 8th edition (AJCC8). We investigated the locoregional control (LRC), disease-free survival (DFS), and overall survival (OS) for the various pathological T categories and disease stages of vlSCC as per AJCC8. METHODS: We performed a retrospective cohort study of 297 patients diagnosed with vlSCC between January 2004 and February 2019. For this study, vlSCC cases were staged according to both AJCC7 and AJCC8. Kaplan-Meier survival curves and Cox regression models were used to analyze differences in LRC, DFS, and OS between each pT category and disease stage, and log-rank tests were performed for subgroup analysis. RESULTS: Restaging of vlSCC using the AJCC8 resulted in 19% of patients being upstaged to pT3, and 16% being upstaged to stage III. No patients were downstaged in pT stage or overall stage. CONCLUSIONS: Our study shows that when the AJCC8 HNcSCC staging system is applied to vlSCC, there are important aberrations leading to unwarranted upstaging of pT1 and redundancy of pT2. Understanding of these limitations are important in considering treatment escalation.

Missense mutations in CASK, coding for the calcium-/calmodulin-dependent serine protein kinase, interfere with neurexin binding and neurexin-induced oligomerization.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.