Clinical phenotype of hereditary spastic paraplegia due to KIF1C gene mutations across life span.

Hereditary spastic paraplegias (HSPs) are a group of genetic disorders resulting in pyramidal tract impairment, predominantly in lower limbs. KIF1C gene has recently been identified as one of the genetic causes of HSP and associated with pure or complicated HSP. We present three patients with complicated HSP from two unrelated families, who had early onset progressive cerebellar signs and developed pyramidal tract signs during follow-up. Whole exome sequencing in these patients followed by segregation analysis identified novel truncating KIF1C mutations (c.463C> T; p.R155∗ and c.2478delA; p.Ala828Argfs∗13). Neuroimaging findings showed cerebral and upper cervical spinal atrophy, bilateral symmetrical pyramidal tract involvement, and focal cerebral white matter lesions. Patients with KIF1C mutations may present with cerebellar signs and pyramidal findings may emerge later, therefore complicated HSP should be considered in the differential diagnosis of unidentified cases with cerebellar dysfunction.

A patient with mitochondrial disorder due to a novel mutation in MRPS22.

MRPS22 gene defect is a very rare newly discovered mitochondrial disorder. We report a 4-month-old severely affected male infant with MRPS22 mutation. Whole exome sequencing revealed a novel homozygous splicing mutation c.339 + 5 G > A in MRPS22 gene. He has mild dysmorphism, hypotonia, developmental delay but not hypertrophic cardiomyopathy and tubulopathy which differ from other majority of reported patients. Therefore, hypertrophic cardiomyopathy and tubulopathy may not be considered as constant features of MRPS22. With this case report, we also present first symmetrical bilateral brainstem and medial thalamic lesions, and cerebellar and cerebral atrophy on a brain MR imaging follow-up of ten months.

A System Architecture for Efficient Transmission of Massive DNA Sequencing Data.

The DNA sequencing data analysis pipelines require significant computational resources. In that sense, cloud computing infrastructures appear as a natural choice for this processing. However, the first practical difficulty in reaching the cloud computing services is the transmission of the massive DNA sequencing data from where they are produced to where they will be processed. The daily practice here begins with compressing the data in FASTQ file format, and then sending these data via fast data transmission protocols. In this study, we address the weaknesses in that daily practice and present a new system architecture that incorporates the computational resources available on the client side while dynamically adapting itself to the available bandwidth. Our proposal considers the real-life scenarios, where the bandwidth of the connection between the parties may fluctuate, and also the computing power on the client side may be of any size ranging from moderate personal computers to powerful workstations. The proposed architecture aims at utilizing both the communication bandwidth and the computing resources for satisfying the ultimate goal of reaching the results as early as possible. We present a prototype implementation of the proposed architecture, and analyze several real-life cases, which provide useful insights for the sequencing centers, especially on deciding when to use a cloud service and in what conditions.

Coffin-Siris syndrome with café-au-lait spots, obesity and hyperinsulinism caused by a mutation in the ARID1B gene.

Coffin-Siris syndrome (CSS) (MIM 135900) is characterized by developmental delay, severe speech impairment, distinctive facial features, hypertrichosis, aplasia or hypoplasia of the distal phalanx or nail of the fifth digit and agenesis of the corpus callosum. Recently, it was shown that mutations in the ARID1B gene are the main cause of CSS, accounting for 76% of identified mutations. Here, we report a 15 year-old female patient who was admitted to our clinic with seizures, speech problems, dysmorphic features, bilaterally big, large thumb, café-au-lait (CAL) spots, obesity and hyperinsulinism. First, the patient was thought to have an association of neurofibromatosis and Rubinstein Taybi syndrome. Because of the large size of the NF1 gene for neurofibromatosis and CREBBP gene for Rubinstein Taybi syndrome, whole exome sequence analysis (WES) was conducted and a novel ARID1B mutation was identified. The proband WES test identified a novel heterozygous frameshift mutation c.3394_3395insTA in exon 13 of ARID1B (NM_017519.2) predicting a premature stop codon p.(Tyr1132Leufs*67). Sanger sequencing confirmed the heterozygous c.3394_3395insTA mutation in the proband and that it was not present in her parents indicating de novo mutation. Further investigation and new cases will help to understand this phenomenon better.

Loss-of-Function Mutations in ELMO2 Cause Intraosseous Vascular Malformation by Impeding RAC1 Signaling.

Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2 (which translates extracellular signals into cellular movements) that are causative for autosomal-recessive intraosseous vascular malformation (VMOS) in five different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial, and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant downregulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically normal, suggesting that there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2 originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones.

Hereditary spastic paraplegia with recessive trait caused by mutation in KLC4 gene.

We report an association between a new causative gene and spastic paraplegia, which is a genetically heterogeneous disorder. Clinical phenotyping of one consanguineous family followed by combined homozygosity mapping and whole-exome sequencing analysis. Three patients from the same family shared common features of progressive complicated spastic paraplegia. They shared a single homozygous stretch area on chromosome 6. Whole-exome sequencing revealed a homozygous mutation (c.853_871del19) in the gene coding the kinesin light chain 4 protein (KLC4). Meanwhile, the unaffected parents and two siblings were heterozygous and one sibling was homozygous wild type. The 19 bp deletion in exon 6 generates a stop codon and thus a truncated messenger RNA and protein. The association of a KLC4 mutation with spastic paraplegia identifies a new locus for the disease.

Robustness of Massively Parallel Sequencing Platforms.

The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications.

Comparative transcriptome profiling approach to glean virulence and immunomodulation-related genes of Fasciola hepatica.

BACKGROUND: Fasciola hepatica causes chronic liver disease, fasciolosis, leading to significant losses in the livestock economy and concerns for human health in many countries. The identification of F. hepatica genes involved in the parasite's virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, to identify the parasite's putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult F. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. In brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms (Dugesiidae species and Caenorhabditis elegans) or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution (directional selection) and cytokine signaling relation were conducted. RESULTS: NGS of the whole transcriptome resulted in 19,534,766 sequence reads, yielding a total of 40,260 transcripts (N50 = 522 bp). A number of the parasite transcripts (n = 1,671) were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. Of these, a group of the virulence-related genes (n = 62), not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors (i.e. MHC I class, TGF-ß receptor, toll/interleukin-1 receptor, T-cell receptor, TNF receptor, and IL-18 receptor accessory protein), cytokines (i.e. TGF-ß, interleukin-4/interleukin-13 and TNF-α), cluster of differentiations (e.g. CD48 and CD147) or molecules associated with other immunomodulatory mechanisms (such as regulation of macrophage activation). Some of the genes (n = 5) appeared to be under positive selection (Ka/Ks > 1), imitating proteins associated with cytokine signaling (through sequence homologies with thrombospondin type 1, toll/interleukin-1 receptor, TGF-ß receptor and CD147). CONCLUSIONS: With a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of F. hepatica (n = 62), which are firstly described here, could be employed for the development of better strategies (including RNAi) in the battle against both zoonotically and economically important disease, fasciolosis.

Whole-exome sequencing revealed two novel mutations in Usher syndrome.

Usher syndrome is a clinically and genetically heterogeneous autosomal recessive inherited disorder accompanied by hearing loss and retinitis pigmentosa (RP). Since the associated genes are various and quite large, we utilized whole-exome sequencing (WES) as a diagnostic tool to identify the molecular basis of Usher syndrome. DNA from a 12-year-old male diagnosed with Usher syndrome was analyzed by WES. Mutations detected were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined by in silico analysis. A maternally inherited deleterious frameshift mutation, c.14439_14454del in exon 66 and a paternally inherited non-sense c.10830G>A stop-gain SNV in exon 55 of USH2A were found as two novel compound heterozygous mutations. Both of these mutations disrupt the C terminal of USH2A protein. As a result, WES revealed two novel compound heterozygous mutations in a Turkish USH2A patient. This approach gave us an opportunity to have an appropriate diagnosis and provide genetic counseling to the family within a reasonable time.

Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.

Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.

Design and characterisation of a thin-film electrode array with shared reference/counter electrodes for electrochemical detection.

In the current study, a novel electrode array and integrated microfluidics have been designed and characterised in order to create a sensor chip which is not only easy, rapid and cheaper to produce but also have a smaller imprint and good electrochemical sensing properties. The current study includes the assessment of the effects of an Au quasi-reference electrode and the use of shared reference/counter electrodes for the array, in order to obtain a small array that can be produced using a fine metal mask. In the study, it is found that when Au is used as the quasi-reference electrode, the arrays with shared reference and counter electrodes result in faster electron transfer kinetics and prevent the potential change with respect to scan rate, and hence is advantageous with respect to conventional electrodes. In addition, the resulting novel electrode array has been shown to result in higher current density (10.52 µA/cm(2); HRP detection assay) and measured diffusion coefficient (14.40×10(-12) cm(2)/s; calculated from the data of cyclic voltammetry with 1mM potassium ferricyanide) with respect to conventional electrodes tested in the study. Using the new electrode arrays, the detection limits obtained from horse radish peroxidase (HRP) and bisphenol A assays were 12.5 ng/ml (2.84×10(-10) M ) and 10 ng/ml (44×10(-9) M), respectively. Performing the HRP detection assay in a flow injection system using array integrated microfluidics provided 25 times lower detection limit (11.36×10(-12) M), although Ti has been used as electrode material instead of Au. In short, incorporation of this new electrode array to lab-on-a-chip or MEMs (micro-electro mechanic systems) technologies may pave the way for easy to use automated biosensing devices that could be used for a variety of applications from diagnostics to environmental monitoring, and studies will continue to move forward in this direction.

TMCO1 deficiency causes autosomal recessive cerebrofaciothoracic dysplasia.

Cerebrofaciothoracic dysplasia (CFT) (OMIM #213980) is a multiple congenital anomaly and intellectual disability syndrome involving the cranium, face, and thorax. The characteristic features are cranial involvement with macrocrania at birth, brachycephaly, various CT/MRI findings including hypoplasia of corpus callosum, enlargement of septum pellicidum, and diffuse hypodensity of the grey matter, flat face, hypertelorism, cleft lip and cleft palate, low-set, posteriorly rotated ears, short neck, and multiple costal and vertebral anomalies. The underlying genetic defect remains unknown. Using combination of homozygosity mapping and whole-exome sequencing, we identified a homozygous nonsense founder mutation, p.Arg87Ter (c.259 C>T), in the human transmembrane and coiled-coil domains protein 1 (TMCO1) in four out of five families of Turkish origin. The entire critical region on chromosome 1q24 containing TMCO1 was excluded in the fifth family with characteristic findings of CFT providing evidence for genetic heterogeneity of CFT spectrum. Another founder TMCO1 mutation has recently been reported to cause a unique genetic condition, TMCO1-defect syndrome (OMIM #614132). TMCO1-defect syndrome shares many features with CFT. This study supports the fact that "TMCO1-defect syndrome," initially thought to represent a distinct disorder, indeed belongs to the genetically heterogeneous CFT dysplasia spectrum.

HomSI: a homozygous stretch identifier from next-generation sequencing data.

UNLABELLED: In consanguineous families, as a result of inheriting the same genomic segments through both parents, the individuals have stretches of their genomes that are homozygous. This situation leads to the prevalence of recessive diseases among the members of these families. Homozygosity mapping is based on this observation, and in consanguineous families, several recessive disease genes have been discovered with the help of this technique. The researchers typically use single nucleotide polymorphism arrays to determine the homozygous regions and then search for the disease gene by sequencing the genes within this candidate disease loci. Recently, the advent of next-generation sequencing enables the concurrent identification of homozygous regions and the detection of mutations relevant for diagnosis, using data from a single sequencing experiment. In this respect, we have developed a novel tool that identifies homozygous regions using deep sequence data. Using *.vcf (variant call format) files as an input file, our program identifies the majority of homozygous regions found by microarray single nucleotide polymorphism genotype data. AVAILABILITY AND IMPLEMENTATION: HomSI software is freely available at www.igbam.bilgem.tubitak.gov.tr/softwares/HomSI, with an online manual.

Mutation in MEOX1 gene causes a recessive Klippel-Feil syndrome subtype.

BACKGROUND: Klippel-Feil syndrome (KFS) is characterized by the developmental failure of the cervical spine and has two dominantly inherited subtypes. Affected individuals who are the children of a consanguineous marriage are extremely rare in the medical literature, but the gene responsible for this recessive trait subtype of KFS has recently been reported. RESULTS: We identified a family with the KFS phenotype in which their parents have a consanguineous marriage. Radiological examinations revealed that they carry fusion defects and numerical abnormalities in the cervical spine, scoliosis, malformations of the cranial base, and Sprengel's deformity. We applied whole genome linkage and whole-exome sequencing analysis to identify the chromosomal locus and gene mutated in this family. Whole genome linkage analysis revealed a significant linkage to chromosome 17q12-q33 with a LOD score of 4.2. Exome sequencing identified the G > A p.Q84X mutation in the MEOX1 gene, which is segregated based on pedigree status. Homozygous MEOX1 mutations have reportedly caused a similar phenotype in knockout mice. CONCLUSIONS: Here, we report a truncating mutation in the MEOX1 gene in a KFS family with an autosomal recessive trait. Together with another recently reported study and the knockout mouse model, our results suggest that mutations in MEOX1 cause a recessive KFS phenotype in humans.