Qatar Foundation Annual Research Forum Volume 2010 Issue 1

Psoriasis is a chronic inflammatory skin disease posing a considerable world-wide health problem due to its high prevalence, associated morbidity and high health-care costs. It is a multifactorial “complex” disorder, with compelling evidence for a genetic predisposition.

Majeed syndrome is a Mendelian disorder with a consistent phenotype and its causative gene can be examined for its role in the more common bone and skin inflammatory disorders of complex etiology. Majeed syndrome is caused by mutations in LPIN2.

Many observations have implicated LPIN2 in the genetic etiology of psoriasis. Based on these observations, we hypothesize that variations in LPIN2 play a role in the susceptibility to development of psoriasis and that LPIN2 is the psoriasis susceptibility locus on 18p.

In our previous study, we identified 6 coding variants that may be associated with psoriasis due to the fact that they change evolutionary conserved amino acids and they are present in the general population at a very low allele frequency. However, the ultimate evidence of their causation of the phenotype is to prove that there is a change in protein properties or function with the molecular variations. One of the aims of the current work is to shed light on whether each identified LPIN2 mutation has an effect on the integrity of the properties of the Lipin2 protein and therefore its function.

The wide type and six different cDNA LPIN2 clones, each harboring one of the six described variations were successfully synthesised after codon optimisation for maximum expression in yeast.

The LPIN2 gene and two mutants were excised from its original construct and inserted into similarly digested pYES2. The newly formed constructs were transformed into competent cells of Saccharomyces cerevisiae for protein expression. Our preliminary analyses using SDS gel electrophoresis and Western blot indicate that the wild type and the two mutants are expressed in S. cerevisiae but at a low level. Optimization of the expression as well as expression of these genes in different expression system is being carried out. The recombinant protein of the wild type LPIN2 and the two mutants will be subjected to circular dichroism and fluorescence measurements to study the effect of each mutant on the folding and therefore the function of the protein.

Evidence in healthy sleepers suggests the glycemic index (GI) can mediate changes in sleep onset latency, given the availability of tryptophan to the brain (i.e. TRP/LNAA ratio) is increased after high GI carbohydrate-only food. However, these meals have limited clinical application given the high glycemic load (GL) and insulin responses. Therefore, we investigated the efficacy of a mixed macronutrient high GI (MHGI) compared to an isoenergetic (∼1915 kJ) low GI (MLGI) meal taken three hours prior to habitual bedtime to improve sleep quality in participants meeting research diagnostic criteria for insomnia. Four men and four women (n=8) were randomized to the MHGI or MLGI meal for two consecutive nights. Blood samples were taken prior to the meal, and 60, 120, 180 min after eating. Subjective (sleep diary) and objective (polysomnography, PSG) sleep was also measured each night. The individual 10cm visual analogue scales indicate that meal palatability was identical for both meals; which were of good taste (average 7.8cm); meal satiety was maintained until bedtime after the MHGI meal (>5cm), whereas after the MLGI meal satiety ratings in men were low (<5cm); and the average meal energetic load (kJ/kg) was greater for women (33.0 ± 4.1) than men (25.4 ± 3.8; p<0.05). Postprandial measures indicate glucose was larger after the MHGI meal but there was no difference in insulin response; the peak percentage rise in plasma TRP/LNAA from baseline after the MHGI meal (17%) was substantially but only marginally different than the MLGI (8%) meal (p = 0.12); postprandial serotonin was unaltered. The participant group self-reported (5pt Likert scale) feeling more rested after the MHGI (2.8) compared to the MLGI meal (2.3; p<0.05); also ratings were higher in women (3.0) than in men (2.6; p<0.05). There were no differences in PSG sleep variables. This study demonstrates symptoms of insomnia are improved, especially in women, after a high GI mixed macronutrient meal. Given the present data, we suggest the possibility for a physiological threshold within the postprandial plasma TRP/LNAA response that must be surpassed in order to promote measurable changes to serotonin and PSG sleep. Further studies should evaluate the potential long-term risks and benefits of habitual mixed macronutrient high GI meals to improve sleep.

Smell is an essential sense that allows animals to find food and mates while avoiding predators. In humans smell is considered an aesthetic sensory modality, but olfactory disorders may presage neurological disease including Parkinson's, Alzheimer's, and schizophrenia. The odorant receptors (ORs) comprise the largest gene family in mammals and endow an animal with the ability to smell. Critical to the development and function of olfaction is the regulation of OR gene transcription, with each sensory neuron selecting just one OR for expression, at random, from only one allele. While recent experiments have brought the outlines of this remarkable process into focus,the core mechanism has remained obscure. Using a genetic approach in mice we present data that supports a model of single OR gene choice initiated by infrequent, stochastic transcriptional activation and governed by feedback suppression mechanism.

In the face extreme requirements for diversity, cells and organisms have evolved stochastic processes of gene regulation. Such mechanisms may allow for the maximal exploration of critical biochemical, genetic, or cellular spaces and maximize the informational output of the genome. The pursuit of the solution to this question of gene regulation has captivated the biomedical research community - not only in the area of neurobiology. The elucidation of this problem will shed light on the establishment and function of this sensory system and also further our understanding of the regulation of the largest gene family in mammals. Additionally, these findings will have central relevance for other examples of stochastic gene regulation such as the expression of the lymphocyte antigen receptors, X chromosome inactivation, for diverse disease-related processes such as trypanosome vsg and malaria var gene switching, and the functional (epigenetic) loss of heterozygosity in cancer; all of which likely depend on complex transcriptional processes.

Autoinflammatory diseases are a group of disorders characterized by seemingly unprovoked inflammation in the absence of high-titer autoantibodies or antigen-specific T cells. They include familial Mediterranean fever; the tumor necrosis factor receptor–associated periodic syndrome; the hyper-IgD syndrome; a syndrome of pyogenic arthritis, pyoderma gangrenosum and acne; the cryopyrin-associated periodic syndromes; chronic recurrent multifocal osteomyelitis and others. A new autoinflammatory syndrome of skin and bone caused by recessive mutations in IL1RN, the gene encoding the interleukin-1–receptor antagonist, has been recently described and has been named deficiency of the interleukin-1-receptor antagonist, or DIRA.

Three unrelated patients with symptoms suggestive of DIRA were referred to our laboratory. The three patients had skin and bone inflammation since birth manifested as pustulosis and chronic recurrent multifocal osteomyelitis. The course of the disease was progressive with chronic sequelae. Two patients were from Brazil and the third is from Palestine. We identified a novel homozygous inframe deletion of 15 bases (c.213-228delAGATGTGGTACCCAT; p.72-77delDVVPI) in the two unrelated patients from Brazil. In the Palestinian patient, a homozygous nonsense mutation (c.160C>T; p.Q54X) was identified. This mutation has been described before in a family from Lebanon, which probably reflects on a founder effect in Middle Eastern populations.

Primary ciliary dyskinesia is a heterogeneous autosomal recessive genetic disorder that leads to ultrastructural and functional defects of cilia. This leads to recurrent and persistent respiratory infections, sinusitis, otitis media, and male infertility. In a fraction of patients situs inversus is present. Primary ciliary dyskinesia can result from mutation in at least nine different genes. However, these mutations are responsible for the disease in 40 percent of patients. These genes provide instructions for making proteins that form the inner structure of cilia and produce the force needed for motility.

We identified two large inbred Qatari families with multiple individuals affected by primary ciliary dyskinesia. As a first step we excluded all known genes associated with the disorder. We then performed whole genome genotyping using 200K SNP chips on an Illumina platform followed by homozygosity mapping. In one family two significant homozygous regions were identified, a 35 Mb region on the long arm of chromosome 3 and a 46 Mb region on the long arm of chromosome 5. In the second family single homozygous regions was identified on the short arm of chromosome 5 spanning 3.2Mb. Candidate genes were prioritized based on conservation through evolution and expression in cilia. Examination of candidate genes by resequencing is currently being performed.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism due to deficiency in the phenylalanine hydroxylase gene (PAH). This study describes the distribution of PAH mutations in nine probands from Libya with the diagnosis of phenylketonuria and hyperphenylalaninemia. Molecular genetics screening was done at the Shafallah Medical Genetics Center laboratory by resequencing and analysis of the entire coding sequences, exon flanking regions and splice sites of PAH. Genomic DNA was isolated from dry blood spots (n=9) by organic extraction technique and purified using centrifugal column filter device. The 13 exons , exon-intron boundaries and splice sites of the PAH were amplified by polymerase chain reaction using in-house designed primers and optimized conditions. The DNA sequencing reactions were carried out by automated sequencer using BigDye Terminator chemistry. Two homologous PAH mutations were found in 7/9 probands and were c.838G>A/p.E280K (missense) and c.1055delG (frame shift). The frame shift mutation produces a truncated protein of 399 amino acid length. Two Probands were homozygous for the missense mutation, three were homozygous for the frame shift mutation and two were compound heterozygous for both mutations. This initial study should be followed by an analysis of phenotype – genotype correlation pattern to better understand the disease process.

Rett syndrome is a neurodevelopmental disorder that leads to regression in language and motor skills. In most cases, it is caused by genetic mutations in the methyl-CpG-binding protein 2 gene (MECP2). Rett Syndrome occurs almost exclusively in girls and may be easily misdiagnosed, because its spectrum of clinical characteristics is overlapping with characteristics of other disorders such as autism, ataxic cerebral palsy, atypical Angelman's syndrome, spinocerebellar degeneration, etc. A 16-year-old girl with behavior within the autistic spectrum disorder, moderate to severe intellectual delay and subtle dysmorphic features enrolled in the Shafallah Center for children with special needs, School Unit 1. She is shy, with no sustained eye contact and has secondary seizures. She can use her hands in eating, drinking, painting in the class and she can hold a pencil between the index finger and thumb to do lines and circles, her history shows no regression. The clinical characteristics are closer to the autistic spectrum disorders than to the Rett syndrome.

In order to avoid misdiagnosis between Autism and Rett syndrome, a genetic testing of the MECP2 was realized in the Shafallah Medical Genetics Center, by sequencing and Multilocus Ligation Probe Amplification (MLPA). The sequencing analysis showed no variations in both directions, while the MLPA analysis detected a deletion of 0.6-2 kb in exon 4 of MECP2. The tests were repeated twice and the result was confirmed.

The molecular testing result supports a Rett syndrome diagnosis, while the clinical characteristics of the patient are not the typical Rett syndrome features. The case demonstrates that the spectrum of the clinical characteristics of the Rett syndrome is broader than is generally considered. This fact makes the diagnosis of Rett syndrome difficult by relying only in the clinical manifestations. Cases like this enforce the role of the molecular testing as a strong diagnostic tool.

Egypt has the highest prevalence of antibodies to hepatitis C virus (HCV) in the world, estimated nationally at 14.7%. An estimated 9.8% are chronically infected. Numerous HCV prevalence studies in Egypt have published various estimates from different Egyptian communities, suggesting that Egypt, relative to the other nations of the world, might be experiencing intense ongoing HCV transmission. More importantly, a new national study provided an opportunity to apply established epidemiologic models to estimate incidence. Validated mathematical models for estimating incidence from age-specific prevalence were used. All previous prevalence studies of HCV in Egypt were reviewed and used to estimate incidence provided that there was sufficient age-specific data required by the models. All reports of anti-HCV antibody prevalence were much higher than any other single national estimate. Age was the strongest and most consistently associated factor to HCV prevalence and HCV RNA positivity.It was not possible to establish a prior reference point for HCV prevalence or incidence to compare with the 2009 incidence estimates. The modeled incidence from the national study and collectively from the modeled incidence from the previous community studies was 6.9/1,000 [95% confidence interval (CI), 5.5–7.4] per person per year and 6.6/1,000 (95% CI, 5.1–7.0) per person per year, respectively. Projected to the age structure of the Egyptian population, more than 500,000 new HCV infections per year were estimated. Iatrogenic transmission is the most likely, underlining exposure to the ongoing transmission. The study demonstrates the urgency to reduce HCV transmission in Egypt.