Genome-wide expression of low temperature response genes in Rosa hybrida L.

Plants respond to low temperature stress during cold acclimation, a complex process involving changes in physiological and biochemical modifications. The rose serves as a good model to investigate low temperature responses in perennial ornamentals. In this study, a heterologous apple microarray is used to investigate genome-wide expression profiles in Rosa hybrida subjected to low temperature dark treatment. Transcriptome profiles are determined in floral buds at 0h, 2h, and 12h of low temperature treatment (4 °C). It is observed that a total of 134 transcripts are up-regulated and 169 transcripts are down-regulated in response to low temperature. Interestingly, a total of eight up-regulated genes, including those coding for two cytochrome P450 proteins, two ankyrin repeat family proteins, two metal ion binding proteins, and two zinc finger protein-related transcription factors, along with a single down-regulated gene, coding for a dynamin-like protein, are detected. Transcript profiles of 12 genes known to be involved in cold stress response are also validated using qRT-PCR. Furthermore, expression patterns of the AP2/ERF gene family of transcription factors are investigated in both floral buds and leaves. Overall, AP2/ERFs genes are more rapidly induced in leaves than in floral buds. Moreover, differential expression of several AP2/ERF genes are detected earlier in vegetative rather than in reproductive tissues. These findings highlight important roles of various low temperature response genes in mediating cold acclimation, thereby allowing roses to adapt to low temperatures, but without adversely affecting flower bud development and subsequent flowering, while vegetative tissues undergo early adaptation to low temperatures.

Exome sequencing identifies a novel and a recurrent BBS1 mutation in Pakistani families with Bardet-Biedl syndrome.

PURPOSE: To determine the genetic cause of Bardet-Biedl syndrome (BBS) in two consanguineous Pakistani families. METHODS: Clinical characterization of the affected individuals in both families was performed with ophthalmic examination, electroretinography, electrocardiography, and liver and renal profiling. Seventeen genes are known to be associated with BBS, so exome sequencing was preferred over candidate gene sequencing. One affected individual from both families was selected for exome sequencing. Segregation of the identified variants was confirmed with Sanger sequencing. RESULTS: Retinitis pigmentosa, obesity, and learning difficulties were present in the affected individuals in both families. In family A, a sixth finger (polydactyly) of the proband's sister was removed by a surgical operation leaving a scar on the little finger. Polydactyly was also present in both affected individuals from family B. All diagnostic symptoms were characteristic of BBS in both families. In both affected individuals from family A, exome sequencing identified a novel homozygous mutation (c.47+1G>T) in BBS1 that inactivates the splice donor site at the end of exon 1. In family B, a previously reported mutation, c.442G>A; p.(Asp148Asn), was detected. CONCLUSIONS: Exome sequencing is an efficient and cost-effective technique for identifying mutations in genetically heterogeneous diseases. In addition, intrafamilial phenotypic variability in family A argues for the modifying effect of other still unknown modifier alleles.

The genetic spectrum of familial hypercholesterolemia in Pakistan.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the genes coding for the low density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type-9 (PCSK9) or apo-lipoprotein B-100 (APOB). The aim of the present work was to determine the genetic basis of dyslipidemia in 11 unrelated Pakistani families. METHODS: High resolution melting (HRM), sequencing and restriction fragment length polymorphism (RFLP). RESULTS: Probands were screened for the promoter and all coding regions, including intron/exon boundaries, of LDLR and PCSK9 and part of exon 26 of APOB including p.(R3527Q). Two families were identified with previously unreported LDLR mutations (c.1019_1020delinsTG, p.(C340L) and c.1634G>A, p.(G545E)). Both probands had tendon xanthomas or xanthelasma and/or a history of cardiovascular disease. Co-segregation with hypercholesterolemia was demonstrated in both families. In silico studies predicted these variations to be damaging. In two families, novel PCSK9 variations were identified (exon2; c.314G>A, p.(R105Q) and exon3; c.464C>T, p.(P155L)). In silico studies suggested both were likely to be damaging, and family members carrying the p.(105Q) allele had lower total cholesterol levels, suggesting this is a loss-of-function mutation. For c.464C>T p.(P155L) the small number of relatives available precluded any strong inference. CONCLUSION: This report brings to seven the number of different LDLR mutations reported in FH patients from Pakistan and, as expected in this heterogeneous population, no common LDLR mutation has been identified.

Association of ANRIL polymorphism (rs1333049:C>G) with myocardial infarction and its pharmacogenomic role in hypercholesterolemia.

Single nucleotide polymorphisms (SNPs) of non-coding RNA in the INK4 locus (ANRIL) have been found to be associated with myocardial infarction (MI). However, the effect of rs1333049:C>G in INK4 locus in familial hypercholesterolemia patients and on lipid profile of the patients has not been studied in Pakistan. We therefore investigated the association of SNP rs1333049:C>G with MI as well as familial hypercholesterolemia patients and also determined the effect of genotype on lipid levels in a northern Pakistani population. A case-control association study was performed in which 611 individuals (294 patients, 290 healthy controls and 27 patients from hypercholesterolemia families) were genotyped for rs1333049:C>G, using an Allele specific polymerase chain reaction. We found a significant association of rs1333049:C>G with MI (χ(2)=22.3, p<0.001). The frequency of risk genotype CC was significantly different from the healthy controls (p<0.001, χ(2)=22.3). The risk allele C was at a higher frequency in the MI patients as compared to the controls (odds ratio [OR]=1.55 (95% confidence interval [CI]=1.22-1.96), p<0.001). The logistic regression analysis for the genotype distribution resulted in strong association of risk allele C with MI under recessive model (OR=3.17 (95% CI=1.85-5.44) p<0.001). When the data were further analyzed along the lines of gender, a significant association with both males and females was observed. The pleiotropic role of rs1333049 was revealed further when CC genotype hypercholesterolemic individuals on statins were found to have a significantly lower TC, LDL-C and Tg levels as compared to the CG and GG individuals (p<0.05). The current study demonstrates a strong association of the ANRIL SNP (rs1333049) with MI as well as familial hypercholesterolemia patients in a northern Pakistani population and could be used as a useful genetic marker for the screening of MI in the general Pakistani population.

Identification and characterization of alternative exon usage linked glioblastoma multiforme survival.

BACKGROUND: Alternative exon usage (AEU) is an important component of gene regulation. Exon expression platforms allow the detection of associations between AEU and phenotypes such as cancer. Numerous studies have identified associations between gene expression and the brain cancer glioblastoma multiforme (GBM). The few consistent gene expression biomarkers of GBM that have been reported may be due to the limited consideration of AEU and the analytical approaches used. The objectives of this study were to develop a model that accounts for the variations in expression present between the exons within a gene and to identify AEU biomarkers of GBM survival. METHODS: The expression of exons corresponding to 25,403 genes was related to the survival of 250 individuals diagnosed with GBM in a training data set. Genes exhibiting AEU in the training data set were confirmed in an independent validation data set of 78 patients. A hierarchical mixed model that allows the consideration of covariation between exons within a gene and of the effect of the epidemiological characteristics of the patients was developed to identify associations between exon expression and patient survival. This general model describes all three possible scenarios: multi-exon genes with and without AEU, and single-exon genes. RESULTS: AEU associated with GBM survival was identified on 2477 genes (P-value < 5.0E-04 or FDR-adjusted P-value < 0.05). G-protein coupled receptor 98 (Gpr98) and epidermal growth factor (Egf) were among the genes exhibiting AEU with 30 and 9 exons associated with GBM survival, respectively. Pathways enriched among the AEU genes included focal adhesion, ECM-receptor interaction, ABC transporters and pathways in cancer. In addition, 24 multi-exon genes without AEU and 8 single-exon genes were associated with GBM survival (FDR-adjusted P-value < 0.05). CONCLUSIONS: The inferred patterns of AEU were consistent with in silico AS models. The hierarchical model used offered a flexible and simple way to interpret and identify associations between survival that accommodates multi-exon genes with or without AEU and single exon genes. Our results indicate that differential expression of AEU could be used as biomarker for GBM and potentially other cancers.

Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients.

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.

A novel pathogenic nonsense triple-nucleotide mutation in the low-density lipoprotein receptor gene and its clinical correlation with familial hypercholesterolemia.

AIM: The aim of this study was to determine the genetic basis of familial hypercholesterolemia in a Pakistani family with a history of myocardial infarction and premature coronary artery disease. RESULTS: Direct sequencing of the low-density lipoprotein receptor gene resulted in the identification of a novel missense mutation c.264G>C (p.R88S) in exon 3 and a novel nonsense triple-nucleotide polymorphism (TNP) c.887-889GCA>AGC (p.C296X) in exon 6, the latter being probably the disease-causing mutation in this family. Both of these mutations were not present in the probands of 14 familial hypercholesterolemia families, 100 myocardial infarction patients, as well as 150 normolipidemic ethnically matched control individuals. CONCLUSIONS: The identification of the novel nonsense TNP is the first report of a nonsense pathogenic TNP in low-density lipoprotein receptor or any other gene and only the fourth report of a pathogenic TNP of any type, which emphasizes the importance of screening for TNPs in patients and in familial studies that might otherwise be missed if only analyzed on single-nucleotide polymorphism arrays.

Role of tissue plasminogen activator and plasminogen activator inhibitor polymorphism in myocardial infarction.

A case-control association study on 229 Myocardial Infarction (MI) patients and 217 healthy controls was carried out to determine the role of tissue-plasminogen activator (t-PA) (Alu-repeat insertion (I)/deletion (D)) and plasminogen activator inhibitor (PAI-1) (4G/5G insertion/deletion) polymorphisms with MI in the Pakistani population. In MI patients the genotype distribution of the PAI-1 gene was not found to be different when compared with the unaffected controls (P>0.05, χ2=1.03). The risk allele 4G was also not associated with MI (P>0.05, χ2=0.46, odds ratio (OR)=1.1 (95% confidence interval (CI)=0.84-1.43), P>0.05). Similarly, the genotype frequencies of t-PA I/I, I/D and D/D were not different from the unaffected controls (P>0.05, χ2=1.60), and the risk allele "I" was not found to be associated with MI (P>0.05, χ2=1.35, OR=0.86 (95% CI=0.66-1.11), P>0.05). However, when the data were distributed along the lines of gender a significant association of the 4G/4G PAI-1 genotype was observed with only the female MI patients (P<0.05, z-test=2.21). When the combined genotypes of both the polymorphisms were analyzed, a significant association of MI was observed with the homozygous DD/4G4G genotype (P<0.01, z-test=2.61), which was specifically because of the female samples (P=0.01, z-test=2.53). In addition smoking (P<0.001, χ2=13.52, OR=3.45 (95% CI=1.77-6.94)), diabetes (P<0.001, χ2=22.45, OR=8.89 (95% CI=2.96-29.95)), hypertension (OR=7.76 (95% CI=2.88-22.68), P<0.001) family history (P<0.001, χ2=13.72, OR=3.7 (95% CI=1.71-8.18)) and lower HDL levels (P<0.05) were found to be significantly associated with the disease. In conclusion the PAI-1 gene polymorphism was found to have a gender specific role in the female MI patients.

Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia.

PURPOSE: To identify the genetic defect in two Pakistani families with autosomal recessive achromatopsia. METHODS: Two families (RP26 and RP44) were originally diagnosed with retinal dystrophy based upon their medical history. To localize the causative genes in these families, homozygosity mapping was performed using Affymetrix 10K single nucleotide polymorphism (SNP) arrays. Sequence analysis was used to find the mutations in candidate genes cyclic nucleotide-gated channel alpha-3 (CNGA3; family RP26) and cyclic nucleotide-gated channel beta-3 (CNGB3; family RP44). Control individuals were analyzed by allele-specific PCR for the CNGA3 mutation and BstXI restriction analysis for the CNGB3 mutation. After genetic analysis, clinical diagnosis was re-evaluated by electroretinography and color vision testing. During the course of this study, selected affected members of family RP26 were given pink glasses as supportive therapy. RESULTS: Sequence analysis of the positional candidate genes identified a novel missense mutation in CNGA3 (c.822G>T; p.R274S) in family RP26, and a novel CNGB3 frameshift mutation (c.1825delG; p.V609WfsX9) in family RP44. Clinical re-evaluation after genetic analysis revealed that both families have segregating autosomal recessive achromatopsia. CONCLUSIONS: Genetic analysis of two Pakistani families with retinal disease enabled the establishment of the correct diagnosis of achromatopsia. Two novel mutations were identified in CNGA3 and CNGB3 that are both specifically expressed in cone photoreceptors. Re-evaluation of the clinical status revealed that both families had achromatopsia. The use of pink glasses in patients was helpful in reducing photophobia and enabled rod-mediated vision.

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia.

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300-500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.

A homozygous p.Glu150Lys mutation in the opsin gene of two Pakistani families with autosomal recessive retinitis pigmentosa.

PURPOSE: To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families. METHODS: A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene. RESULTS: In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families. CONCLUSIONS: In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry.