Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts

Background: Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration

Identification of gene expression changes associated with the initiation of diapause in the brain of the cotton bollworm, Helicoverpa armigera

<p>Abstract</p> <p>Background</p> <p>Diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. <it>Helicoverpa armigera </it>(Har) undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. Using suppression subtractive hybridization (SSH), we investigated differentially expressed genes in diapause- and nondiapause-destined pupal brains at diapause initiation.</p> <p>Results</p> <p>We constructed two SSH libraries (forward, F and reverse, R) to isolate genes that are up-regulated or down-regulated at diapause initiation. We obtained 194 unique sequences in the F library and 115 unique sequences in the R library. Further, genes expression at the mRNA and protein level in diapause- and nondiapause-destined pupal brains were confirmed by RT-PCR, Northern blot or Western blot analysis. Finally, we classified the genes and predicted their possible roles at diapause initiation.</p> <p>Conclusion</p> <p>Differentially expressed genes at pupal diapause initiation are possibly involved in the regulation of metabolism, energy, stress resistance, signaling pathways, cell cycle, transcription and translation.</p

Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC

Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma

IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT–PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins

Early-onset ocular ochronosis in a girl with alkaptonuria (aku) and a novel mutation in homogentisate 1,2-dioxygenase (HGD)

Alkaptonuria (AKU) is a disorder of phenylalanine/tyrosine metabolism due to a defect in the enzyme homogentisate 1,2-dioxygenase (HGD). This recessive disease is caused by mutations in the HGD gene. We report a 14-year-old girl who was referred after presenting black urine. Careful examination revealed ochronosis of the conjunctiva. There was no affection of the cardiac valves. Elevated excretion of homogentisic acid in urine was found. Sequence analysis of the HGD gene from genomic DNA revealed that the patient is a compound heterozygote with a previously described mutation (c.473C > T, p.Pro158Leu), and a novel one (c.821C > T, p.Pro274Leu). Her mother is heterozygous for the novel mutation, while the brother is heterozygous for the previously described mutation. In summary, we describe an alkaptonuric patient with ocular ochronosis and a novel HGD mutation, c.821C > T, p.Pro274Leu

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA,based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild,type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild,type sequences. Our results suggest that the wild,type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense,and silent mutations, and to understand the structure-function relationship in the corresponding protein. (C) 2004 Wiley-Liss, Inc