Batten disease: biochemical and molecular characterization revealing novel <Emphasis Type="Italic">PPT1</Emphasis> and <Emphasis Type="Italic">TPP1</Emphasis> gene mutations in Indian patients

Background

Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy.

Methods

The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants.

Results

Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal.

Conclusion

The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

     

Meiotic drive in fungi: Chromosomal elements that cause fratricide and distort genetic ratios

Fungal Spore killers (Sk), studied most extensively inNeurospora and to a lesser extent inPodospora, Gibberella andCochliobolus, cause the death of ascospores (= meiospores) that do not contain the killer (Skk) element. When a Spore killer is heterozygous (SkK× Sks) inNeurospora, every ascus (= meiocyte) contains four normal-sized, black, viable ascospores (SkK), and four ascospores that are tiny, unpigmented and unviable (SKs). Killing of sensitive nuclei is expressed postmeiotically, and results in gross distortion of segregation ratios forSk-linked genes. A sensitive nucleus that would otherwise die is rescued if a killer nucleus is also enclosed in the same ascospore. InNeurospora, Sk is centromere-linked (linkage group III), and when heterozygous, shows a recombination block in a 30-map-unit region spanning the centromere of linkage group III. There is no ascospore death or recombination block in killer×killer or sensitive×sensitive crosses. Spore killers are fairly common inGibberella fujikuroi andNeurospora sitophila but extremely rare inN. intermedia, and have not yet been found among natural isolates ofN. crassa.     

Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences.

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.     

Intra-fruit seed abortion in a wind dispersed tree,Dalbergia sissoo Roxb: Proximate mechanisms

Dalbergia sissoo, a tropical tree with wind-dispersed pods, exhibits a highly positively skewed distribution of seeds per pod with predominantly only one of the four or five ovules maturing into seed. The abortion cannot be attributed to lack of pollen or resources. This study examines the hypothesis that the abortion is due to an intense rivalry among the developing sibs to gain dispersal advantage. Aqueous extract of the dominant embryos at the stigmatic end that generally develop to maturity significantly inhibited the uptake of labelled sucrose by the young developing (subject) embryos in an in vitro assay system. Extracts of tissues such as subordinate (peduncular embryos), unfertilized ovules and pod coat did not cause such inhibition. Aqueous diffusate of dominant embryos also inhibited the uptake of labelled sucrose by subject embryos. The chemical substance responsible for the inhibition appears to be heat-stable and non-proteinaceous. HPLC analysis indicated the presence of two retention time peaks, different from that of standard indole acetic acid, but with considerable overlap. We hypothesize that the compound could be an indole derivative. We propose that the stigmatic embryos have a head start due to earlier fertilization and produce a chemical that either directly (by metabolically killing) or indirectly (by preventing the uptake of assimilates) kills the proximally placed peduncular embryos.     

Gait analysis parameters of healthy human subjects with asymmetric loads

This article focuses on the analysis of gait parameters, ground reaction forces (GRF), and motion signals, for the various asymmetric loads carried by healthy human subjects during walking. Eight asymptomatic human volunteers were enrolled in this study. They were asked to walk, at self-selected pace, with various weights ranging from 0 to 11.33 kg (25 lbs) in 2.26 kg (5 lbs) increments, in one hand on a wooden area equipped with a force platform. Moreover, motion data were recorded from lumbar L1 vertebrae at a frequency of 120 Hz. Three trials of data have been recorded for each subject. In order to quantify the effect of increasing loads on the GRF we define the compression area, restitution area, and coefficient of restitution (COR) for GRF curves. We observe an increase in the compression area with respect to the load and almost constant values for the COR. For motion signals analysis we employ wavelet theory. The signals obtained from the lumbar L1 sensor of the spine vertebrae show a decrease in the wavelet detail energy, for the levels 3, 4, and 5, with respect to increasing loads.     

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation X Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered.     

Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

Bilateral carcinoid tumor of the breast. Report of a case with diagnosis by fine needle aspiration cytology.

A case of bilateral carcinoid tumors of the breast was studied by cytology, histology, immunohistochemistry and electron microscopy. The preoperative aspiration cytologic findings strongly suggested a carcinoid tumor of the breast. The differential diagnosis with fine needle aspiration cytology of other breast lesions, the bilaterality of the condition and terminology are discussed.