Developmental and epileptic encephalopathy in two siblings with a novel, homozygous missense variant in SCN1B.

Developmental and epileptic encephalopathies are genetic disorders in which both the developmental disability and the frequent epileptic activity are the effect of a specific gene variant. While heterozygous variants in SCN1B have been described in families with generalized epilepsy with febrile seizures plus, Type 1, only three cases of homozygous, missense variants in SCN1B have been reported in association with autosomal recessive inheritance of a severe developmental and epileptic encephalopathy. We present two siblings who are homozygous for a novel, missense variant in SCN1B, c.265C>T, predicting p.Arg89Cys. The proband is an 11-year-old female with infantile-onset, fever-induced, intractable generalized tonic-clonic seizures, myoclonic seizures, and developmental slowing and autism spectrum disorder occurring later in the course of the disease. Her 4-year-old brother had a similar epilepsy phenotype, but still displays normal development. This variant has not been previously reported in the homozygous state in control databases. The variant was predicted to be damaging and occurred in the vicinity of other epileptic encephalopathy-associated missense variants that are biallelic and located in the extracellular immunoglobulin loop domain of the protein, which mediates interaction of the beta-1 subunit with cellular adhesion molecules. Our report is the first set of siblings with homozygosity for the p.Arg89Cys variant in SCN1B and further implicates biallelic mutations in this gene as a cause of epileptic encephalopathy mimicking Dravet syndrome. Interestingly, the phenotype we observed was milder compared to that previously described in patients with recessive SCN1B mutations.

Orbital and peri-orbital amyloidosis: a report of four cases.

Our objective was to describe our experience with orbital amyloidosis, and illustrate the different forms of presentation. This was a retrospective case series of four patients with biopsy-proven orbital amyloi- dosis, over the period from 2014 to 2016. We describe its diagnostic and clinical characteristics, management and systemic study. The series comprised three women and one man of mean age 52 ± 9.4 years. Affected sites were the lacrimal gland, tarsal conjunctiva, lacrimal sac and orbit. In three of the four patients, calcifications were observed. Three patients had associated ptosis. The patient with orbital involvement suffered an unusual vascular complication during surgery and systemic disease was detected. Management included debulking and complete resection of the lesion. In conclusion, orbital amyloidosis presents as a wide variety of forms. Its diagnosis is biopsy-based. Calcifications in biopsy specimens or images should raise suspicion of amyloidosis. It is important to always check for systemic amyloidosis.

Transcaruncular rectus tendon fixation to the orbit and frontalis flap for complete third nerve palsy.

PURPOSE: To describe our experience and outcomes managing complete third cranial nerve palsy. METHODS: This was a retrospective analysis of the clinical records of 7 consecutive patients treated at our centre for unilateral third nerve palsy over the period 2010-2016. We describe our surgical approach using a frontalis muscle flap to correct the eyelid ptosis associated with medial fixation of the rectus muscle tendon to the orbit to correct the horizontal deviation. RESULTS: The seven patients, four women and three men, were of mean age of 44 ± 19 years [18-75 years]. Follow up was 29 ± 31 months [5-82 months]. In the preoperative exam, exotropia in prism diopters (PD) was -70 ± -28 PD [-30 to -90 PD]. At the end of follow up, this was reduced to -11 ± -14 PD [0 to -30 PD]. Preoperative marginal reflex distance 1 (MRD1) was -4 ± 1 mm [-3 to -5 mm] and palpebral fissure height (PFH) was 0.5 ± 1 mm [0-2 mm]. Surgical undercorrection was the target in all patients due to the absent or poor Bell's phenomenon. At the end of follow up, MRD1 was 2.5 ± 0.5 mm [2-3 mm] and PFH was 7 ± 1 mm [6-8 mm]. Cosmetic and functional results were good in all patients. CONCLUSIONS: Medial fixation of the rectus muscle tendon to the orbit associated with a frontalis muscle flap is a valid option for the treatment of exotropia and ptosis in patients with third cranial nerve palsy.

Primary Intraocular Malignant Rhabdoid Tumor Without Extrascleral Compromise.

Primary intraocular malignant rhabdoid tumor is classified as a malignant extrarenal rhabdoid tumor. It is extremely rare, highly aggressive, and, so far, only one case (in a newborn) has been described in the medical literature. The authors report a second case of primary intraocular malignant rhabdoid tumor, this time without extrascleral involvement and in a teenager, and describe its histological, immunohistochemical, and radiological characteristics along with clinical correlations. [J Pediatr Ophthalmol. 2018;55:e7-e9.].

Analysis of endocannabinoid signaling elements and related proteins in lymphocytes of patients with Dravet syndrome.

Cannabidiol (CBD) reduces seizures in childhood epilepsy syndromes including Dravet syndrome (DS). A formulation of CBD has obtained orphan drug designation for these syndromes and clinical trials are currently underway. The mechanism responsible for CBD effects is not known, although it could involve targets sensitive to CBD in other neurological disorders. We believe of interest to investigate whether these potential targets are altered in DS, in particular whether the endocannabinoid system is dysregulated. To this end, lymphocytes from patients and controls were used for analysis of gene expression of transmitter receptors and transporters, ion channels, and enzymes associated with CBD effects, as well as endocannabinoid genes. Plasma endocannabinoid levels were also analyzed. There were no differences between DS patients and controls in most of the CBD targets analyzed, except an increase in the voltage-dependent calcium channel α-1h subunit. We also found that cannabinoid type-2 (CB 2) receptor gene expression was elevated in DS patients, with no changes in other endocannabinoid-related receptors and enzymes, as well as in plasma levels of endocannabinoids. Such elevation was paralleled by an increase in CD70, a marker of lymphocyte activation, and certain trends in inflammation-related proteins (e.g., peroxisome proliferator-activated receptor-γ receptors, cytokines). In conclusion, together with changes in the voltage-dependent calcium channel α-1h subunit, we found an upregulation of CB 2 receptors, associated with an activation of lymphocytes and changes in inflammation-related genes, in DS patients. Such changes were also reported in inflammatory disorders and may indirectly support the occurrence of a potential dysregulation of the endocannabinoid system in the brain.

Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants.

Craniosynostosis, caused by the premature fusion of one or more of the cranial sutures, can be classified into non-syndromic or syndromic and by which sutures are affected. Clinical assignment is a difficult challenge due to the high phenotypic variability observed between syndromes. During routine diagnostics, we screened 182 Spanish craniosynostosis probands, implementing a four-tiered cascade screening of FGFR2, FGFR3, FGFR1, TWIST1 and EFNB1. A total of 43 variants, eight novel, were identified in 113 (62%) patients: 104 (92%) detected in level 1; eight (7%) in level 2 and one (1%) in level 3. We subsequently screened additional genes in the probands with no detected mutation: one duplication of the IHH regulatory region was identified in a patient with craniosynostosis Philadelphia type and five variants, four novel, were identified in the recently described TCF12, in probands with coronal or multisuture affectation. In the 19 Saethre-Chotzen syndrome (SCS) individuals in whom a variant was detected, 15 (79%) carried a TWIST1 variant, whereas four (21%) had a TCF12 variant. Thus, we propose that TCF12 screening should be included for TWIST1 negative SCS patients and in patients where the coronal suture is affected. In summary, a molecular diagnosis was obtained in a total of 119/182 patients (65%), allowing the correct craniosynostosis syndrome classification, aiding genetic counselling and in some cases provided a better planning on how and when surgical intervention should take place and, subsequently the appropriate clinical follow up.

Achondroplasia with 47, XXY karyotype: a case report of the neonatal diagnosis of an extremely unusual association.

BACKGROUND: The association of achondroplasia and Klinefelter syndrome is extremely rare. To date, five cases have been previously reported, all of them diagnosed beyond the postnatal period, and only one was molecularly characterized. We describe the first case of this unusual association diagnosed in the neonatal period, the clinical findings and the molecular studies undertaken. CASE PRESENTATION: The boy was born at term with clinical and radiological features indicating the diagnosis of achondroplasia or hypochondroplasia combined with the prenatal karyotype of Klinefelter syndrome (47,XXY). Neonatal FGFR3 mutation screening showed that the newborn was heterozygous for the classic achondroplasia G340R mutation. Microsatellite marker analysis showed that the sex chromosome aneuploidy had arisen from a non-disjunction error in paternal meiosis I, with a recombination event in the pseudoautosomal region 1 (PAR1). CONCLUSION: Specific mutation analysis is appropriate to confirm the clinical diagnosis of achondroplasia for appropriate diagnosis, prognosis, and genetic counseling, especially when the karyotype does not explain the abnormal prenatal sonographic findings. In the present case, a recombination event was observed in the PAR1 region, although recombinational events in paternally derived Klinefelter syndrome cases are much rarer than expected.

Identification of the first recurrent PAR1 deletion in Léri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer.

BACKGROUND: SHOX, located in the pseudoautosomal region 1 (PAR1) of the sexual chromosomes, encodes a transcription factor implicated in human growth. Defects in SHOX or its enhancers have been observed in ∼60% of Leri-Weill dyschondrosteosis (LWD) patients, a skeletal dysplasia characterised by short stature and/or the characteristic Madelung deformity, and in 2-5% of idiopathic short stature (ISS). To identify the molecular defect in the remaining genetically undiagnosed LWD and ISS patients, this study screened previously unanalysed PAR1 regions in 124 LWD and 576 ISS probands. METHODS: PAR1 screening was undertaken by multiplex ligation dependent probe amplification (MLPA). Copy number alterations were subsequently confirmed and delimited by locus-specific custom-designed MLPA, array comparative genomic hybridisation (CGH) and breakpoint junction PCR/sequencing. RESULTS: A recurrent PAR1 deletion downstream of SHOX spanning 47543 bp with identical breakpoints was identified in 19 LWD (15.3%) and 11 ISS (1.9%) probands, from 30 unrelated families. Eight evolutionarily conserved regions (ECRs 1-8) identified within the deleted sequence were evaluated for SHOX regulatory activity by means of chromosome conformation capture (3C) in chicken embryo limbs and luciferase reporter assays in human U2OS osteosarcoma cells. The 3C assay indicated potential SHOX regulatory activity by ECR1, which was subsequently confirmed to act as a SHOX enhancer, operating in an orientation and position independent manner, in human U2OS cells. CONCLUSIONS: This study has identified the first recurrent PAR1 deletion in LWD and ISS, which results in the loss of a previously uncharacterised SHOX enhancer. The loss of this enhancer may decrease SHOX transcription, resulting in LWD or ISS due to SHOX haploinsufficiency.

Mild isolated craniosynostosis due to a novel FGFR3 mutation, p.Ala334Thr.

Craniosynostosis is the premature fusion of one or more sutures of the skull, which can be syndromic or isolated. Mutations in FGFR1, FGFR2, or FGFR3, among others, are often responsible for these syndromic cases. The associated of FGFR3 mutations with craniosynostosis has been restricted to three mutations, the common p.Pro250Arg in Muenke syndrome, p.Ala391Glu in Crouzon syndrome with acanthosis nigricans, and p.Pro250Leu identified in a family with isolated craniosynostosis. Other FGFR3 mutations result in various skeletal dysplasias: achondroplasia, hypochondroplasia, and thanatophoric dysplasia. Here, we report a novel mutation in exon 8 (IIIc) of FGFR3, p.Ala334Thr, in a young boy with mild craniosynostosis. The mutation segregated with mild craniosynostosis in the family and was absent in 188 normal controls. Alanine 334 is evolutionarily conserved in vertebrates and is located at the amino terminus of the ßF loop in the FGFR3c isoform. The mutation is predicted to alter the protein tertiary structure which may impair its binding to its ligand, FGF1. The identification of a mutation in these clinically heterogeneous disorders can aid recurrence risk assessments. Although the implementation of a stepwise screening strategy is useful in diagnostics, mutations in unscreened regions of genes associated with craniosynostosis may explain a small proportion of craniosynostosis cases.

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia.

We report the clinical and molecular characteristics of 12 Spanish families with multiple members affected with Léri-Weill dyschondrosteosis (LWD) or Langer mesomelic dysplasia (LMD), who present the SHOX (short stature homeobox gene) mutation p.A170P (c.508G>C) in heterozygosity or homozygosity, respectively. In all studied families, the A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed by microsatellite analysis, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. Mutation screening in 359 Eastern-European Gypsies failed to identify any carriers. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. We have also identified a novel mutation at the same residue, c. 509C>A (p.A170D), in two unrelated Spanish LWD families, which similar to A170P mutation impedes nuclear localization of SHOX. In conclusion, we have identified A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals.

Identification of the first de novo PAR1 deletion downstream of SHOX in an individual diagnosed with Léri-Weill dyschondrosteosis (LWD).

Léri-Weill dyschondrosteosis (LWD, MIM 127300), is a dominantly inherited skeletal dysplasia with disproportionate short stature, mesomelic limb shortening, and the characteristic Madelung deformity. Two regions of the pseudoautosomal region 1 (PAR1) have been shown to be involved in LWD, SHOX (short-stature homeobox-containing gene) and the downstream enhancer region. We report our genetic findings of a young girl clinically diagnosed with LWD. We analyzed the proband and her family using MLPA and microsatellite analysis. We identified a deletion, 726-866 kb in size, of the downstream SHOX enhancer region in the proband. Neither parent carried the deletion. Microsatellite analysis showed that the deleted allele was of paternal origin. The mutation is more likely to have arisen from a de novo event but paternal gonadal mosaicism cannot be excluded. In conclusion, we report the clinical and molecular details of the first case of a de novo deletion of the downstream PAR1 region in an LWD individual. De novo deletions of SHOX and the downstream enhancer region must be therefore considered in cases of isolated LWD.

Genetic analysis of the vitamin D receptor gene in two epithelial cancers: melanoma and breast cancer case-control studies.

BACKGROUND: Vitamin D serum levels have been found to be related to sun exposure and diet, together with cell differentiation, growth control and consequently, cancer risk. Vitamin D receptor (VDR) genotypes may influence cancer risk; however, no epidemiological studies in sporadic breast cancer (BC) or malignant melanoma (MM) have been performed in a southern European population. In this study, the VDR gene has been evaluated in two epithelial cancers BC and MM. METHODS: We have conducted an analysis in 549 consecutive and non-related sporadic BC cases and 556 controls, all from the Spanish population, and 283 MM cases and 245 controls. Genotyping analyses were carried out on four putatively functional SNPs within the VDR gene. RESULTS: An association with the minor allele A of the non-synonymous SNP rs2228570 (rs10735810, FokI, Met1Thr) was observed for BC, with an estimated odds ratio (OR) of 1.26 (95% CI = 1.02-1.57; p = 0.036). The synonymous variant rs731236 (TaqI) appeared to be associated with protection from BC (OR = 0.80, 95%CI = 0.64-0.99; p = 0.047). No statistically significant associations with MM were observed for any SNP. Nevertheless, sub-group analyses revealed an association between rs2228570 (FokI) and absence of childhood sunburns (OR = 0.65, p = 0.003), between the 3'utr SNP rs739837 (BglI) and fair skin (OR = 1.31, p = 0.048), and between the promoter SNP rs4516035 and the more aggressive tumour location in head-neck and trunk (OR = 1.54, p = 0.020). CONCLUSION: In summary, we observed associations between SNPs in the VDR gene and BC risk, and a comprehensive analysis using clinical and tumour characteristics as outcome variables has revealed potential associations with MM. These associations required confirmation in independent studies.

Evaluating HapMap SNP data transferability in a large-scale genotyping project involving 175 cancer-associated genes.

One of the many potential uses of the HapMap project is its application to the investigation of complex disease aetiology among a wide range of populations. This study aims to assess the transferability of HapMap SNP data to the Spanish population in the context of cancer research. We have carried out a genotyping study in Spanish subjects involving 175 candidate cancer genes using an indirect gene-based approach and compared results with those for HapMap CEU subjects. Allele frequencies were very consistent between the two samples, with a high positive correlation (R) of 0.91 (P<<1x10(-6)). Linkage disequilibrium patterns and block structures across each gene were also very similar, with disequilibrium coefficient (r (2)) highly correlated (R=0.95, P<<1x10(-6)). We found that of the 21 genes that contained at least one block larger than 60 kb, nine (ATM, ATR, BRCA1, ERCC6, FANCC, RAD17, RAD50, RAD54B and XRCC4) belonged to the GO category "DNA repair". Haplotype frequencies per gene were also highly correlated (mean R=0.93), as was haplotype diversity (R=0.91, P<<1x10(-6)). "Yin yang" haplotypes were observed for 43% of the genes analysed and 18% of those were identical to the ancestral haplotype (identified in Chimpazee). Finally, the portability of tagSNPs identified in the HapMap CEU data using pairwise r (2) thresholds of 0.8 and 0.5 was assessed by applying these to the Spanish and current HapMap data for 66 genes. In general, the HapMap tagSNPs performed very well. Our results show generally high concordance with HapMap data in allele frequencies and haplotype distributions and confirm the applicability of HapMap SNP data to the study of complex diseases among the Spanish population.