OPA1 Dominant Optic Atrophy: Diagnostic Approach in the Pediatric Population.

A clinical and genetic study was conducted with pediatric patients and their relatives with optic atrophy 1 (OPA1) mutations to establish whether there is a genotype-phenotype correlation among the variants detected within and between families. Eleven children with a confirmed OPA1 mutation were identified during the study period. The main initial complaint was reduced visual acuity (VA), present in eight patients of the cohort. Eight of eleven patients had a positive family history of optic atrophy. The mean visual acuity at the start of the study was 0.40 and 0.44 LogMAR in the right and left eye, respectively. At the end of the study, the mean visual acuity was unchanged. Optical coherence tomography during the first visit showed a mean retinal nerve fiber layer thickness of 81.6 microns and 80.5 microns in the right and left eye, respectively; a mean ganglion cell layer of 52.5 and 52.4 microns, respectively, and a mean central macular thickness of 229.5 and 233.5 microns, respectively. The most common visual field defect was a centrocecal scotoma, and nine out of eleven patients showed bilateral temporal disc pallor at baseline. Sequencing of OPA1 showed seven different mutations in the eleven patients, one of which, NM_130837.3: c.1406_1407del (p.Thr469LysfsTer16), has not been previously reported. Early diagnosis of dominant optic atrophy is crucial, both for avoiding unnecessary consultations and/or treatments and for appropriate genetic counseling.

Whole Exome Sequencing of 20 Spanish Families: Candidate Genes for Non-Syndromic Pediatric Cataracts.

Non-syndromic pediatric cataracts are defined as opacification of the crystalline lens that occurs during the first years of life without affecting other organs. Given that this disease is one of the most frequent causes of reversible blindness in childhood, the main objective of this study was to propose new responsible gene candidates that would allow a more targeted genetic approach and expand our genetic knowledge about the disease. We present a whole exome sequencing (WES) study of 20 Spanish families with non-syndromic pediatric cataracts and a previous negative result on an ophthalmology next-generation sequencing panel. After ophthalmological evaluation and collection of peripheral blood samples from these families, WES was performed. We were able to reach a genetic diagnosis in 10% of the families analyzed and found genes that could cause pediatric cataracts in 35% of the cohort. Of the variants found, 18.2% were classified as pathogenic, 9% as likely pathogenic, and 72.8% as variants of uncertain significance. However, we did not find conclusive results in 55% of the families studied, which suggests further studies are needed. The results of this WES study allow us to propose LONP1, ACACA, TRPM1, CLIC5, HSPE1, ODF1, PIKFYVE, and CHMP4A as potential candidates to further investigate for their role in pediatric cataracts, and AQP5 and locus 2q37 as causal genes.

Whole-Exome Sequencing of 21 Families: Candidate Genes for Early-Onset High Myopia.

High myopia is the most severe and pathological form of myopia. It occurs when the spherical refractive error exceeds -6.00 spherical diopters (SDs) or the axial length (AL) of the eye is greater than 26 mm. This article focuses on early-onset high myopia, an increasingly common condition that affects children under 10 years of age and can lead to other serious ocular pathologies. Through the genetic analysis of 21 families with early-onset high myopia, this study seeks to contribute to a better understanding of the role of genetics in this disease and to propose candidate genes. Whole-exome sequencing studies with a panel of genes known to be involved in the pathology were performed in families with inconclusive results: 3% of the variants found were classified as pathogenic, 6% were likely pathogenic and the remaining 91% were variants of uncertain significance. Most of the families in this study were found to have alterations in several of the proposed genes. This suggests a polygenic inheritance of the pathology due to the cumulative effect of the alterations. Further studies are needed to validate and confirm the role of these alterations in the development of early-onset high myopia and its polygenic inheritance.

Broadening the phenotypic spectrum of EVEN-PLUS syndrome through identification of HSPA9 pathogenic variants in the original EVE dysplasia family and two sibs with milder facial phenotype.

EVEN-PLUS syndrome is a rare autosomal recessive disorder caused by biallelic pathogenic variants in the mitochondrial chaperone called mortalin, encoded by HSPA9. This genetic disorder, presenting with several overlapping features with CODAS syndrome, is characterized by the involvement of the Epiphyses, Vertebrae, Ears, and Nose (EVEN), PLUS associated findings. Only five individuals presenting with the EVEN-PLUS phenotype and biallelic variants in HSPA9 have been published. Here, we expand the phenotypic and molecular spectrum associated with this disorder, reporting two sibs with a milder phenotype and compound heterozygous pathogenic variants (a recurrent variant and a novel one). Also, we confirm a homozygous pathogenic variant in the family originally reported as EVE dysplasia.

Next-Generation Sequencing Screening of 43 Families with Non-Syndromic Early-Onset High Myopia: A Clinical and Genetic Study.

Early-onset high myopia (EoHM) is a disease that causes a spherical refraction error of ≥-6 diopters before 10 years of age, with potential multiple ocular complications. In this article, we report a clinical and genetic study of 43 families with EoHM recruited in our center. A complete ophthalmological evaluation was performed, and a sample of peripheral blood was obtained from proband and family members. DNA was analyzed using a customized next-generation sequencing panel that included 419 genes related to ophthalmological disorders with a suspected genetic cause, and genes related to EoHM pathogenesis. We detected pathogenic and likely pathogenic variants in 23.9% of the families and detected variants of unknown significance in 76.1%. Of these, 5.7% were found in genes related to non-syndromic EoHM, 48.6% in genes associated with inherited retinal dystrophies that can include a syndromic phenotype, and 45.7% in genes that are not directly related to EoHM or retinal dystrophy. We found no candidate genes in 23% of the patients, which suggests that further studies are needed. We propose a systematic genetic analysis for patients with EoHM because it helps with follow-up, prognosis and genetic counseling.

Delineation of the clinical and radiological features of Stuve-Wiedemann syndrome childhood survivors, four new cases and review of the literature.

Stuve-Wiedemann syndrome (SWS; MIM 601559) is a rare autosomal recessive disease caused by mutations in the leukemia inhibitor factor receptor gene (LIFR). Common clinical and radiological findings are often observed, and high neonatal mortality occurs due to respiratory distress and hyperthermic episodes. Despite initially considered as a lethal disorder during the newborn period, in recent years, several SWS childhood survivors have been reported. We report a detailed clinical and radiological characterization of four unrelated childhood SWS molecularly confirmed patients and review 22 previously reported childhood surviving cases. We contribute to the definition of the childhood survival phenotype of SWS, emphasizing the evolving phenotype, characterized by skeletal abnormalities with typical radiological findings, distinctive dysmorphic features, and dysautonomia. Based on the typical features and clinical course, early diagnosis is possible and crucial to plan appropriate management and prevent potential complications. Genetic confirmation is advisable in order to improve genetic counseling to the patients and their families.

Loss of function BMP4 mutation supports the implication of the BMP/TGF-ß pathway in the etiology of combined pituitary hormone deficiency.

Despite BMP4 signaling being critical to Rathke's pouch induction and maintenance during early stages of pituitary development, its implication in the etiology of combined pituitary hormone deficiency (CPHD) and other clinical presentations of congenital hypopituitarism has not yet been definitely demonstrated. We report here the first CPHD patient with a de novo pathogenic loss-of-function variant in BMP4. A 6-year-old boy, with macrocephaly, myopia/astigmatism, mild psychomotor retardation, anterior pituitary hypoplasia and ectopic posterior pituitary, clinically diagnosed with growth hormone deficiency, and central hypothyroidism, was referred for genetic analysis of CPHD. Targeted NGS analysis with a custom panel (n = 310 genes) identified a novel heterozygous de novo nonsense variant, NM_001202.5:c.794G > A, p.(Trp265*) in BMP4, which introduces a premature stop codon in the BMP4 pro-domain, impairing the transcription of the TGF-ß mature peptide domain. Additional relevant variants in other genes implicated in pituitary development signaling pathways such as SMAD4 and E2F4 (BMP/TGF-pathway), ALMS1 (NOTCH-pathway), and TSHZ1 (Prokineticin-pathway), were also identified. Our results support the implication of the BMP/TGF-ß signaling pathway in the etiology of CPHD and suggest that oligogenic contribution of additional inherited variants may modify the phenotypic expressivity of BMP4 pathogenic variants.

CLAPO syndrome: identification of somatic activating PIK3CA mutations and delineation of the natural history and phenotype.

PURPOSE: CLAPO syndrome is a rare vascular disorder characterized by capillary malformation of the lower lip, lymphatic malformation predominant on the face and neck, asymmetry, and partial/generalized overgrowth. Here we tested the hypothesis that, although the genetic cause is not known, the tissue distribution of the clinical manifestations in CLAPO seems to follow a pattern of somatic mosaicism. METHODS: We clinically evaluated a cohort of 13 patients with CLAPO and screened 20 DNA blood/tissue samples from 9 patients using high-throughput, deep sequencing. RESULTS: We identified five activating mutations in the PIK3CA gene in affected tissues from 6 of the 9 patients studied; one of the variants (NM_006218.2:c.248T>C; p.Phe83Ser) has not been previously described in developmental disorders. CONCLUSION: We describe for the first time the presence of somatic activating PIK3CA mutations in patients with CLAPO. We also report an update of the phenotype and natural history of the syndrome.

Heterozygous aggrecan variants are associated with short stature and brachydactyly: Description of 16 probands and a review of the literature.

OBJECTIVE: Mutations in the aggrecan gene (ACAN) have been identified in two autosomal dominant skeletal dysplasias, spondyloepiphyseal dysplasia, Kimberley type (SEDK), and osteochondritis dissecans, as well as in a severe recessive dysplasia, spondyloepimetaphyseal dysplasia, aggrecan type. Next-generation sequencing (NGS) has aided the identification of heterozygous ACAN mutations in individuals with short stature, minor skeletal defects and mild facial dysmorphisms, some of whom have advanced bone age (BA), poor pubertal spurt and early growth cessation as well as precocious osteoarthritis. DESIGN AND METHODS: This study involves clinical and genetic characterization of 16 probands with heterozygous ACAN variants, 14 with short stature and mild skeletal defects (group 1) and two with SEDK (group 2). Subsequently, we reviewed the literature to determine the frequency of the different clinical characteristics in ACAN-positive individuals. RESULTS: A total of 16 ACAN variants were located throughout the gene, six pathogenic mutations and 10 variants of unknown significance (VUS). Interestingly, brachydactyly was observed in all probands. Probands from group 1 with a pathogenic mutation tended to be shorter, and 60% had an advanced BA compared to 0% in those with a VUS. A higher incidence of coxa valga was observed in individuals with a VUS (37% vs 0%). Nevertheless, other features were present at similar frequencies. CONCLUSIONS: ACAN should be considered as a candidate gene in patients with short stature and minor skeletal defects, particularly those with brachydactyly, and in patients with spondyloepiphyseal dysplasia. It is also important to note that advanced BA and osteoarticular complications are not obligatory conditions for aggrecanopathies/aggrecan-associated dysplasias.

FGF9 mutation causes craniosynostosis along with multiple synostoses.

Craniosynostosis is commonly caused by mutations in fibroblast growth factor receptors (FGFRs), highlighting the essential role of FGF-mediated signaling in skeletal development. We set out to identify the molecular defect in a family referred for craniosynostosis and in whom no mutation was previously detected. Using next-generation sequencing, we identified a novel missense mutation in FGF9. Modeling based upon the crystal structure and functional studies confirmed its pathogenicity showing that it impaired homodimerization and FGFR3 binding. Only one FGF9 mutation has been previously reported in a multigeneration family with multiple synostoses (SYNS3) but no signs of craniosynostosis. In contrast, our family has a greater phenotypic resemblance to that observed in the Fgf9 spontaneous mouse mutant, elbow-knee-synostosis, Eks, with both multiple synostoses and craniosynostosis. We have demonstrated for the first time that mutations in FGF9 cause craniosynostosis in humans and confirm that FGF9 mutations cause multiple synostoses.

Early severe scoliosis in a patient with atypical progressive pseudorheumatoid dysplasia (PPD): Identification of two WISP3 mutations, one previously unreported.

Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive disorder characterized by spondyloepiphyseal dysplasia associated with pain and stiffness of multiple joints, enlargement of the interphalangeal joints, normal inflammatory parameters, and absence of extra-skeletal manifestations. Homozygous or compound heterozygous WISP3 mutations cause PPD. We report two siblings from a non-consanguineous Ecuadorian family with a late-onset spondyloepiphyseal dysplasia. Mutation screening was undertaken in the two affected siblings using a customized skeletal dysplasia next generation sequencing (NGS) panel and confirmed by Sanger sequencing. Two compound heterozygous mutations were identified in WISP3 exon 2, c.[190G>A];[197G>A] (p.[(Gly64Arg)];[(Ser66Asn)]) in the two siblings, both of which had been inherited. The p. (Gly64Arg) mutation has not been previously described whilst the p. (Ser66Asn) mutation has been reported in two PPD families. The two siblings presented with atypical PPD, as they presented during late childhood, yet the severity was different between them. The progression was particularly aggressive in the male sibling who suffered severe scoliosis by the age of 13 years. This case reaffirms the clinical heterogeneity of this disorder and the clinical utility of NGS to genetically diagnose skeletal dysplasias, enabling adequate management, monitorization, and genetic counseling. © 2016 Wiley Periodicals, Inc.

Two novel POC1A mutations in the primordial dwarfism, SOFT syndrome: Clinical homogeneity but also unreported malformations.

Primordial dwarfism encompasses rare conditions characterized by severe intrauterine growth retardation and growth deficiency throughout life. Recently, three POC1A mutations have been reported in six families with the primordial dwarfism, SOFT syndrome (Short stature, Onychodysplasia, Facial dysmorphism, and hypoTrichosis). Using a custom-designed Next-generation sequencing skeletal dysplasia panel, we have identified two novel homozygous POC1A mutations in two individuals with primordial dwarfism. The severe growth retardation and the facial profiles are strikingly similar between our patients and those described previously. However, one of our patients was diagnosed with severe foramen magnum stenosis and subglottic tracheal stenosis, malformations not previously associated with this syndrome. Our findings confirm that POC1A mutations cause SOFT syndrome and that mutations in this gene should be considered in patients with severe pre- and postnatal short stature, symmetric shortening of long bones, triangular facies, sparse hair and short, thickened distal phalanges.

Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

Chimeras taking shape: potential functions of proteins encoded by chimeric RNA transcripts.

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans.

The potential clinical impact of the release of two drafts of the human proteome.

The authors have carried out an investigation of the two "draft maps of the human proteome" published in 2014 in Nature. The findings include an abundance of poor spectra, low-scoring peptide-spectrum matches and incorrectly identified proteins in both these studies, highlighting clear issues with the application of false discovery rates. This noise means that the claims made by the two papers - the identification of high numbers of protein coding genes, the detection of novel coding regions and the draft tissue maps themselves - should be treated with considerable caution. The authors recommend that clinicians and researchers do not use the unfiltered data from these studies. Despite this these studies will inspire further investigation into tissue-based proteomics. As long as this future work has proper quality controls, it could help produce a consensus map of the human proteome and improve our understanding of the processes that underlie health and disease.

A new overgrowth syndrome is due to mutations in RNF125.

Overgrowth syndromes (OGS) are a group of disorders in which all parameters of growth and physical development are above the mean for age and sex. We evaluated a series of 270 families from the Spanish Overgrowth Syndrome Registry with no known OGS. We identified one de novo deletion and three missense mutations in RNF125 in six patients from four families with overgrowth, macrocephaly, intellectual disability, mild hydrocephaly, hypoglycemia, and inflammatory diseases resembling Sjögren syndrome. RNF125 encodes an E3 ubiquitin ligase and is a novel gene of OGS. Our studies of the RNF125 pathway point to upregulation of RIG-I-IPS1-MDA5 and/or disruption of the PI3K-AKT and interferon signaling pathways as the putative final effectors.

A Novel Missense Variant in the NKX2-1 Homeodomain Prevents Transcriptional Rescue by TAZ.

Background: Brain-lung-thyroid syndrome (BLTS) is caused by NKX2-1 haploinsufficiency, resulting in chorea/choreoathetosis, respiratory problems, and hypothyroidism. Genes interacting with NKX2-1 mutants influence its phenotypic variability. We report a novel NKX2-1 missense variant and the modifier function of TAZ/WWTR1 in BLTS. Methods: A child with BLTS underwent next-generation sequencing panel testing for thyroid disorders. His family was genotyped for NKX2-1 variants and screened for germline mosaicism. Mutant NKX2-1 was generated, and transactivation assays were performed on three NKX2-1 target gene promoters. DNA binding capacity and protein-protein interaction were analyzed. Results: The patient had severe BLTS and carried a novel missense variant c.632A>G (p.N211S) in NKX2-1, which failed to bind to specific DNA promoters, reducing their transactivation. TAZ cotransfection did not significantly increase transcription of these genes, although the variant retained its ability to bind to TAZ. Conclusions: We identify a novel pathogenic NKX2-1 variant that causes severe BLTS and is inherited through germline mosaicism. The mutant lacks DNA-binding capacity, impairing transactivation and suggesting that NKX2-1 binding to DNA is essential for TAZ-mediated transcriptional rescue.

wKinMut: an integrated tool for the analysis and interpretation of mutations in human protein kinases.

BACKGROUND: Protein kinases are involved in relevant physiological functions and a broad number of mutations in this superfamily have been reported in the literature to affect protein function and stability. Unfortunately, the exploration of the consequences on the phenotypes of each individual mutation remains a considerable challenge. RESULTS: The wKinMut web-server offers direct prediction of the potential pathogenicity of the mutations from a number of methods, including our recently developed prediction method based on the combination of information from a range of diverse sources, including physicochemical properties and functional annotations from FireDB and Swissprot and kinase-specific characteristics such as the membership to specific kinase groups, the annotation with disease-associated GO terms or the occurrence of the mutation in PFAM domains, and the relevance of the residues in determining kinase subfamily specificity from S3Det. This predictor yields interesting results that compare favourably with other methods in the field when applied to protein kinases.Together with the predictions, wKinMut offers a number of integrated services for the analysis of mutations. These include: the classification of the kinase, information about associations of the kinase with other proteins extracted from iHop, the mapping of the mutations onto PDB structures, pathogenicity records from a number of databases and the classification of mutations in large-scale cancer studies. Importantly, wKinMut is connected with the SNP2L system that extracts mentions of mutations directly from the literature, and therefore increases the possibilities of finding interesting functional information associated to the studied mutations. CONCLUSIONS: wKinMut facilitates the exploration of the information available about individual mutations by integrating prediction approaches with the automatic extraction of information from the literature (text mining) and several state-of-the-art databases.wKinMut has been used during the last year for the analysis of the consequences of mutations in the context of a number of cancer genome projects, including the recent analysis of Chronic Lymphocytic Leukemia cases and is publicly available at http://wkinmut.bioinfo.cnio.es.