Anna M. Rose

CNOT3 Is a Modifier of PRPF31 Mutations in Retinitis Pigmentosa with Incomplete Penetrance

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA–mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.

Aetiology and management of malnutrition in HIV-positive children

Worldwide, more than 3 million children are infected with HIV and, without treatment, mortality among these children is extremely high. Both acute and chronic malnutrition are major problems for HIV-positive children living in resource-limited settings. Malnutrition on a background of HIV represents a separate clinical entity, with unique medical and social aetiological factors. Children with HIV have a higher daily calorie requirement than HIV-negative peers and also a higher requirement for micronutrients; furthermore, coinfection and chronic diarrhoea due to HIV enteropathy play a major role in HIV-associated malnutrition. Contributory factors include late presentation to medical services, unavailability of antiretroviral therapy, other issues surrounding healthcare provision and food insecurity in HIV-positive households. Treatment protocols for malnutrition have been greatly improved, yet there remains a discrepancy in mortality between HIV-positive and HIV-negative children. In this review, the aetiology, prevention and treatment of malnutrition in HIV-positive children are examined, with particular focus on resource-limited settings where this problem is most prevalent.

Epistasis and immunity: the role of genetic interactions in autoimmune diseases

Autoimmune disorders are a complex and varied group of diseases that are caused by breakdown of self‐tolerance. The aetiology of autoimmunity is multi‐factorial, with both environmental triggers and genetically determined risk factors. In recent years, it has been increasingly recognized that genetic risk factors do not act in isolation, but rather the combination of individual additive effects, gene–gene interactions and gene–environment interactions determine overall risk of autoimmunity. The importance of gene–gene interactions, or epistasis, has been recently brought into focus, with research demonstrating that many autoimmune diseases, including rheumatic arthritis, autoimmune glomerulonephritis, systemic lupus erythematosus and multiple sclerosis, are influenced by epistatic interactions. This review sets out to examine the basic mechanisms of epistasis, how epistasis influences the immune system and the role of epistasis in two major autoimmune conditions, systemic lupus erythematosus and multiple sclerosis.

Transcriptional regulation of PRPF31 gene expression by MSR1 repeat elements causes incomplete penetrance in retinitis pigmentosa

PRPF31-associated retinitis pigmentosa presents a fascinating enigma: some mutation carriers are blind, while others are asymptomatic. We identify the major molecular cause of this incomplete penetrance through three cardinal features: (1) there is population variation in the number (3 or 4) of a minisatellite repeat element (MSR1) adjacent to the PRPF31 core promoter; (2) in vitro, 3-copies of the MSR1 element can repress gene transcription by 50 to 115-fold; (3) the higher-expressing 4-copy allele is not observed among symptomatic PRPF31 mutation carriers and correlates with the rate of asymptomatic carriers in different populations. Thus, a linked transcriptional modifier decreases PRPF31 gene expression that leads to haploinsufficiency. This result, taken with other identified risk alleles, allows precise genetic counseling for the first time. We also demonstrate that across the human genome, the presence of MSR1 repeats in the promoters or first introns of genes is associated with greater population variability in gene expression indicating that copy number variation of MSR1s is a generic controller of gene expression and promises to provide new insights into our understanding of gene expression regulation.

Dominant PRPF31 Mutations Are Hypostatic to a Recessive CNOT3 Polymorphism in Retinitis Pigmentosa: A Novel Phenomenon of "Linked Trans-Acting Epistasis"

Mutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease.

A 112 kb Deletion in Chromosome 19q13.42 Leads to Retinitis Pigmentosa

PURPOSE. This study sets out to identify novel mutations in PRPF31 in a cohort of autosomal dominant retinitis pigmentosa (adRP) patients with a history of nonpenetrance in the family. METHODS. Twenty-one patients with history of nonpenetrant autosomal dominant retinitis pigmentosa were selected; all underwent full ophthalmic examination. Multiplex ligation-dependent probe analysis (MLPA) was performed and, where a deletion was found, further family members were recruited. An individual suspected to harbor a large deletion was used as a positive control. Analysis of single nucleotide polymorphisms in the upstream region was used to determine the extent of the deletion, and the breakpoint was then characterized by PCR and sequencing. RESULTS. In one family, multiplex ligation-dependent probe analysis revealed a novel large deletion in 19q13.4 encompassing exons 1 to 13 of the PRPF31 gene. The mutation was characterized as a deletion of 112 kilobase (kb), encompassing over 90% of PRPF31 and five upstream genes: TFPT, OSCAR, NDUFA3, TARM-1, and VSTM-1. The breakpoint in the positive control family was also characterized. The mechanism of deletion in both families was Alu-mediated nonallelic homologous recombination. CONCLUSIONS. This study describes two large deletions, one in a previously reported family and one in a new family: the latter represents the largest deletion yet described on chromosome 19 and the first report of the involvement of VSTM-1. Remarkably, heterozygous deletion of this large region (encompassing six genes) produces little or no other clinical disease besides retinitis pigmentosa.

Expression of PRPF31 and TFPT: regulation in health and retinal disease

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

Epistasis: The key to understanding immunological disease?

Epistasis is fast becoming central to the understanding of the complex relationship between genotype and phenotype observed in autoimmune disease. A study in this issue of the European Journal of Immunology uses in‐depth analysis of genome‐wide mapping by polymorphic microsatellite markers to shed light on the genomic control of autoimmunity and self‐tolerance.

A Rare Case of Orbital Haemangiopericytoma Arising in Childhood

Abstract Haemangiopericytoma (HPC) is a rare soft tissue tumour of fibroblastic origin and is part of the solitary fibrous tumour spectrum. The tumour is generally considered to be benign, but can behave clinically as if sarcomatous -- with relentless infiltrative local growth. HPC generally presents in adulthood (median age 45 years for orbital disease) and is equally frequent in both sexes. HPC can arise in any site in the body and presents as a slowly growing, painless mass. We report a case of a 20 year old African male seen at Kikuyu Eye Unit, Kenya, with a 12 year history of a gradually enlarging, painless orbital mass. The patient underwent skin-sparing orbital exenteration with complete tumour excision; histology confirmed diagnosis of HPC.

Alveolar soft-part sarcoma of the orbit

Alveolar soft-part sarcoma (ASPS) is a rare soft tissue tumour of uncertain cellular origin. It accounts for only 1% of all sarcomas, which themselves represent only a small proportion of human tumours. ASPS can arise in any soft tissue of the body, but there is an unexplained predilection for the right side. The most common site for paediatric ASPS is in the head and neck region, although involvement of the orbit is rare, with fewer than 30 reported cases. A case of a 5-year-old Kenyan boy with left-sided orbital ASPS is reported and the difficulties of diagnosing rare tumours are discussed.