Advantage of Whole Exome Sequencing over Allele-Specific and Targeted Segment Sequencing in Detection of Novel TULP1 Mutation in Leber Congenital Amaurosis.

BACKGROUND: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions. MATERIALS AND METHODS: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants. RESULTS: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C > T, p.Arg361*) in TULP1, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines. CONCLUSIONS: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.

AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N-Acetylaspartate.

BACKGROUND: Whole exome sequencing (WES) offers a powerful diagnostic tool to rapidly and efficiently sequence all coding genes in individuals presenting for consideration of phenotypically and genetically heterogeneous disorders such as suspected mitochondrial disease. Here, we report results of WES and functional validation in a consanguineous Indian kindred where two siblings presented with profound developmental delay, congenital hypotonia, refractory epilepsy, abnormal myelination, fluctuating basal ganglia changes, cerebral atrophy, and reduced N-acetylaspartate (NAA). METHODS: Whole blood DNA from one affected and one unaffected sibling was captured by Agilent SureSelect Human All Exon kit and sequenced on the Illumina HiSeq2000. Mutations were validated by Sanger sequencing in all family members. Protein from wild-type and mutant fibroblasts was isolated to assess mutation effects on protein expression and enzyme activity. RESULTS: A novel SLC25A12 homozygous missense mutation, c.1058G>A; p.Arg353Gln, segregated with disease in this kindred. SLC25A12 encodes the neuronal aspartate-glutamate carrier 1 (AGC1) protein, an essential component of the neuronal malate/aspartate shuttle that transfers NADH and H(+) reducing equivalents from the cytosol to mitochondria. AGC1 activity enables neuronal export of aspartate, the glial substrate necessary for proper neuronal myelination. Recombinant mutant p.Arg353Gln AGC1 activity was reduced to 15% of wild type. One prior reported SLC25A12 mutation caused complete loss of AGC1 activity in a child with epilepsy, hypotonia, hypomyelination, and reduced brain NAA. CONCLUSIONS: These data strongly suggest that SLC25A12 disease impairs neuronal AGC1 activity. SLC25A12 sequencing should be considered in children with infantile epilepsy, congenital hypotonia, global delay, abnormal myelination, and reduced brain NAA.

Mutations in PDGFRB cause autosomal-dominant infantile myofibromatosis.

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.

Serotonergic mechanisms in human allergic contact dermatitis.

Expression of serotonin (5-hydroxytryptamine; 5-HT), 5-HT receptors 1A (5-HT1AR) and 2A, and serotonin transporter protein (SERT) was studied in positive epicutaneous reactions to nickel sulphate in nickel-allergic patients, at 72 h post-challenge with the antigen. In addition, the effects of 5-HT2AR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), and the selective serotonin reuptake inhibitors (SSRIs) citalopram and fluoxetine, were tested on nickel-stimulated peripheral blood mononuclear cells from nickel-allergic patients, regarding their proliferation and interleukin (IL)-2 production, as well as the effect of these SSRIs on a murine Langerhans' cell-like line (XS52), regarding its IL-1beta production. Serotonin-positive platelets were increased in the inflamed skin compared with control skin. A decrease (p <0.01) in 5-HT1AR-positive mononuclear cells was evident in the eczematous skin compared with control skin, whereas 5-HT2AR- and SERT-positive cells were increased (p <0.001 for both) in the eczematous skin. Treatment of nickel-stimulated peripheral blood mononuclear cells with 5x10(-5) mol/l of DOI inhibited (p <0.01) the proliferation of nickel-stimulated peripheral blood mononuclear cells, while no effect was found regarding IL-2 production. Citalopram at 10(-6) mol/l tended to inhibit the production of IL-1beta by the XS52 cell line. These results indicate the implication of the serotonergic system in the contact allergic reaction.