Bhaswati Pandit

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes ∼60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non–HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome–associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

The rat STSL locus: characterization, chromosomal assignment, and genetic variations in sitosterolemic hypertensive rats

BackgroundElevated plant sterol accumulation has been reported in the spontaneously hypertensive rat (SHR), the stroke-prone spontaneously hypertensive rat (SHRSP) and the Wistar-Kyoto (WKY) rat. Additionally, a blood pressure quantitative trait locus (QTL) has been mapped to rat chromosome 6 in a New Zealand genetically hypertensive rat strain (GH rat). ABCG5 and ABCG8 (encoding sterolin-1 and sterolin-2 respectively) have been shown to be responsible for causing sitosterolemia in humans. These genes are organized in a head-to-head configuration at the STSL locus on human chromosome 2p21.MethodsTo investigate whether mutations in Abcg5 or Abcg8 exist in SHR, SHRSP, WKY and GH rats, we initiated a systematic search for the genetic variation in coding and non-coding region of Abcg5 and Abcg8 genes in these strains. We isolated the rat cDNAs for these genes and characterized the genomic structure and tissue expression patterns, using standard molecular biology techniques and FISH for chromosomal assignments.ResultsBoth rat Abcg5 and Abcg8 genes map to chromosome band 6q12. These genes span ~40 kb and contain 13 exons and 12 introns each, in a pattern identical to that of the STSL loci in mouse and man. Both Abcg5 and Abcg8 were expressed only in liver and intestine. Analyses of DNA from SHR, SHRSP, GH, WKY, Wistar, Wistar King A (WKA) and Brown Norway (BN) rat strains revealed a homozygous G to T substitution at nucleotide 1754, resulting in the coding change Gly583Cys in sterolin-1 only in rats that are both sitosterolemic and hypertensive (SHR, SHRSP and WKY).ConclusionsThe rat STSL locus maps to chromosome 6q12. A non-synonymous mutation in Abcg5, Gly583Cys, results in sitosterolemia in rat strains that are also hypertensive (WKY, SHR and SHRSP). Those rat strains that are hypertensive, but not sitosterolemic (e.g. GH rat) do not have mutations in Abcg5 or Abcg8. This mutation allows for expression and apparent apical targeting of Abcg5 protein in the intestine. These rat strains may therefore allow us to study the pathophysiological mechanisms involved in the human disease of sitosterolemia.

Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB.

Sitosterolaemia in Switzerland: molecular genetics links the US Amish-Mennonites to their European roots

Sitosterolaemia is a rare autosomal recessive disease characterized by increased intestinal absorption of plant sterols, decreased hepatic excretion into bile and elevated concentrations in plasma phytosterols. Homozygous or compound heterozygous loss of function mutations in either of the ATP‐binding cassette (ABC) proteins ABCG5 and ABCG8 explain the increased absorption of plant sterols. Here we report a Swiss index patient with sitosterolaemia, who presented with the classical symptoms of xanthomas, but also had mitral and aortic valvular heart disease. Her management over the last 20 years included a novel therapeutic approach of high‐dose cholesterol feeding that was semi‐effective. Mutational and extended haplotype analyses showed that our patient shared this haplotype with that of the Amish‐Mennonite sitosterolaemia patients, indicating they are related ancestrally.

Identification of Two Differentially Expressed Mitochondrial Genes in a Methotrexate-Resistant Chinese Hamster Cell Strain Derived from V79 Cells Using RNA Fingerprinting by Arbitrary Primed Polymerase Chain Reaction

Abstract Chaudhuri, K., Banerjee, R., Pandit, B., Mukherjee, A., Das, S., Sengupta, S., Roychoudury, S. and Bhattacharyya, N. P. Identification of Two Differentially Expressed Mitochondrial Genes in a Methotrexate-Resistant Chinese Hamster Cell Strain Derived from V79 Cells Using RNA Fingerprinting by Arbitrary Primed Polymerase Chain Reaction. Radiat. Res. 160, 77–85 (2003). To identify genes that are differentially expressed in a methotrexate (MTX)-resistant cell strain designated as M5 that exhibits resistance to γ radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was about twofold higher in the M5 cell strain compared to V79 cells. Moreover, the expression of both the subunits decreased in γ-irradiated Chinese hamster V79 cells. Cytochrome oxidase, another enzyme of the mitochondrial electron transport chain encoded in the mitochondrial genome, was also found to be overexpressed in M5 cells. All three genes are under the control of the same promoter. However, no amplification of DNA was observed. These data indicate that the alterations in mitochondrial gene expression may be involved in the recovery of irradiated cells, which may arise from transcriptional modulation of the mitochondria from the nucleus.

Increased expression of genes in a radioresistant cell strain: modulation of hnRNP E2, Hsp90 and SSBP2 genes in γ-irradiated Chinese hamster V79 cells

The methotrexate-resistant cell strain M5, derived from Chinese hamster lung fibroblast cell line V79, provides a good model to study differential gene expression in response to stress. Genes coding for human homologues of myosin phosphatase (AF458589), filamin-like protein (AAA92644), phosphate transporter 1 (AAH199441) and hnRNP E2 (NP_035172.1) were upregulated in M5 as determined by RAP-PCR. The Gene Expression Array (GEArray) analysis showed increased expression of caspase-6 and Hsp90 in M5 cells compared to that observed in V79 cells. Genomic amplification of SSBP2 as well as its transcriptional upregulation was also detected in M5 cells. Following γ-irradiation, expressions of SSBP2, Hsp90 and hnRNP E2 increased steadily in V79 cells. However, expression of hnRNP K and hnRNP A2 genes decreased at first and then increased, while hnRNP C1 gene expression remained unaffected up to the time studied. Thus, SSBP2, Hsp90 and hnRNP E2 are involved in the radiation response of V79 cells.