Pharmacogenomics and pharmacoepigenomics in pediatric medicine.

In the past several years, human genetics studies have progressed from monogenic to complex and common diseases because of the advancement in technologies. There is increased knowledge of the pharmacokinetics and pharmacogenomics of the drugs in adults as well as in children. These technological developments provided new diagnostic, prognostic, and therapeutic opportunities. We are now in a position to address many additional ambitious questions. For instance, in clinical medicine, interindividual variation in drug response is a major problem. Some of the heterogeneity of drug safety and efficacy among individuals can be explained by pharmacogenomics. It has also the potential to improve the treatment in both adults and children. In pediatrics however, there is ontogeny and metabolic capacity in children is different compared to adults. Several specific developmental changes may underlie some of the variability in drug response seen in children. They may also be responsible for adverse drug reactions (ADRs). Therefore, much of the diversity in drug effects cannot be explained by studying the genomic diversity alone. It is necessary to include the effect of growth (involves variations in gene expression) along with genetic differences when explaining the variability in treatment response. In this respect epigenomics may expand the scope of pharmacogenomics towards optimization of drug therapy. Future studies must focus on periods of maturation of the drug-metabolizing enzymes and polymorphisms in their genes by using candidate gene approach, gene expression analysis, genome-wide haplotype mapping, and proteomics. The integration of genetic data and clinical phenotypes along with the role of other factors is necessary to evaluate both efficacy and ADRs of any drug. It may require extensive genetic epidemiological studies spanning over many years.

Genetic susceptibility to primary angle closure glaucoma (PACG).

Glaucoma is a group of heterogeneous optic neuropathy and is the second leading cause of irreversible blindness worldwide. The two most common clinical types of glaucoma include primary open-angle (POAG) and primary angle-closure glaucoma (PACG). PACG is characterized by the closure of angles between iris and trabecular meshwork (iridocorneal angles) mainly because of anatomic abnormalities. The condition is more prevalent in Chinese, Asian Indians, and Eskimos. Because of an unusually high incidence of PACG among siblings of affected patients, it was suggested that genetic factors were involved in its pathology and the action of a large number of grouped or independently inherited genes along with environmental factors result in anatomical abnormalities of PACG. In PACG, the genetic basis is not well understood. Genome-wide association studies have identified several candidate genes in relation to PACG in several different populations. However, they are not reproduced from population to population or the results are controversial. This may indicate that the involvement of genetic abnormality in the pathogenesis of PACG is complex. The availability of spontaneously occurring large animal models such as dogs may provide an opportunity to identify genes responsible for the pathophysiology of PACG in the future. This article summarizes the current status of genetic investigations on PACG which is the most common cause of blindness worldwide.

Retinal ganglion cell (RGC) death in glaucomatous beagles is not associated with mutations in p53 and NTF4 genes.

BACKGROUND: Glaucoma in humans is a second leading cause of irreversible vision loss in the world and can affect all age groups as well as all populations. The precise mechanism of retinal ganglion cell (RGC) death and progressive degeneration of optic nerve in glaucoma is not understood. It has been suggested that apoptosis is the common pathway that leads to the death of RGCs in glaucoma and that neurotrophin 4 (NTF4) protein plays a role in the protection of RGCs by activating tyrosine kinase receptors. Additionally, one previous study suggested that p53 codon 72 polymorphism (R72P) might have a greater susceptibility to apoptosis in some ethnic population. Glaucoma also occurs in dogs, and the primary glaucoma in beagles is inherited as an autosomal recessive trait. Although recently a candidate gene has been isolated, the mechanism underlying RGC death is not understood. METHOD: To understand whether the same p53 and NTF4 pathway mechanism is involved in a beagle model of glaucoma, we have isolated NTF4 gene from dog and analyzed both p53 and NTF4 genes for mutations in glaucomatous animals. RESULTS: Our analyses failed to identify any disease-causing mutations in both genes with the exception of two polymorphisms in NTF4 gene. However, these are not pathogenic changes because they are also present in normal animals and are not segregated with the disease. CONCLUSION: These results suggest that impaired neurotrophin signaling or compromised trophic support to the retina and p53-mediated apoptosis may not be the underlying mechanism of RGCs death in a beagle model of glaucoma.

Pharmacogenomics and its importance in pediatric medicine.

Individual variation in drug response and adverse drug reactions (ADRs) are a serious problem in medicine. This individual variation in drug response could be due to multiple factors but there is strong evidence that genetic factors play a significant role in drug response variability and toxicity. Although substantial studies that link genetic variants to inter-individual difference in drug response in adults have been reported, such studies are comparatively rare in pediatric medicine. The ultimate goal of medical research is to improve human health in every disease and every patient. Many diseases such as asthma, autism, epilepsy, juvenile rheumatoid arthritis and attention-deficit hyperactivity disorder develop during childhood. Human development is a rapidly changing process. In children, there are differences in absorption, distribution, excretion and metabolizing capabilities of a drug compared with adults. Therefore, many pharmacological and toxicological actions of drugs in children are not predictable from adult experience. It is also possible that children may experience a different range of ADRs that may have long-term implications for their development. Therefore, an improved understanding of the drug transformation pathways for all age groups is necessary. Such studies could provide insight into the susceptibility of a child to ADRs. The availability of the complete sequence of human genome and the biochip technology may help in identifying the polymorphic variations in drug related genes. In this regard, pharmacogenetic and pharmacogenomic studies may play an important role in providing markers of increased risk or susceptibility. Based on this genetic information, children at risk can be identified before therapy is initiated and pediatric ADRs may be minimized. In this short article, an attempt has been made to emphasize the importance of pharmacogenomics in pediatrics.

Pharmacogenomics in ophthalmology.

Inter-individual variation in drug response and adverse drug reactions (ADRs) are well known in medicine. This individual variation in drug response could be at least, in part, due to genetic diversity among individuals. Although substantial studies that connect genetic variants to inter-individual variation in drug response have been documented in several diseases such as cancer and heart diseases, such studies are slowly progressing in ophthalmology. In recent years, advancement in technologies has led to the identification of genes associated with several eye disorders. At the same time, some small-scale studies have demonstrated the association of various genotypes or haplotypes with response to drug therapies. However, its integration into clinical practice in ophthalmology is not possible at present. This is because there are many challenging questions that remain to be addressed. For instance, in the case of complex disorders a single gene study is not enough. Multiple genes, environmental factors, multiple single nucleotide polymorphisms (SNPs), and rare or low frequency variants may contribute to the disease and they must be considered. The functional aspects of many genetic variants are not known. This raises questions of their biological importance and their clinical usefulness. In addition, there are legal, ethical, and social issues that need to be regulated. Moreover, physicians and patients must be educated about the limitation and sensitivity of genetic testing. At present pharmacogenetic studies in ophthalmology are still in their infancy and do not suggest that a pharmacogenetic basis of drug development in ophthalmology is a concept that can yield immediate results, but can become a reality in the future. In this article an attempt has been made to summarize some of the recent small-scale pharmacogenetic studies on two major eye disorders, age-related macular degeneration (AMD) and glaucoma.

Genetic susceptibility to advanced retinopathy of prematurity (ROP).

Retinopathy of prematurity (ROP) is a vascular vitreoretinopathy that affects infants with short gestational age and low birth-weight. The condition is a multifactorial disease and is clinically similar to familial exudative vitreoretinopathy (FEVR), which is a bilateral hereditary eye disorder affecting full-term infants. Both of them are characterized by the abnormal vessel growth in the vitreous that can lead to vitreoretinal traction, retinal detachment and other complications resulting in blindness. Despite the recent advances in diagnosis and treatment, ROP remains a major cause of childhood blindness in developed countries. The etiology of pathogenesis of advanced ROP is currently unknown. In the past, many causative factors such as length of time exposed to supplemental oxygen, excessive ambient light exposure and hypoxia have been suggested but evidence for these as independent risk factors in recent years is not compelling. It is not clear why ROP in a subset of infants with low birth-weight progresses to a severe stage (retinal detachment) despite timely intervention whereas in other infants with similar clinical characteristics ROP regresses spontaneously. Recent research with candidate gene approach, higher concordance rate in monozygotic twins and other clinical and experimental animal studies, suggest a strong genetic predisposition to ROP besides environmental factors such as prematurity. Three genes, which are involved in the Wnt signaling pathway, are mutated in both FEVR and in a small percentage of ROP disorder. However, none of the genetic factors identified thus far in ROP, account for a substantial number of patient population. Future studies involving genomics, bioinformatics and proteomics may provide a better understanding of the pathophysiology and management of ROP.

Genetic diversity and medicinal drug response in eye care.

BACKGROUND: Individual variation in drug response and adverse drug reactions are a serious problem in medicine. This inter-individual variation in drug response could be due to multiple factors such as disease determinants, environmental and genetic factors. Much has been published in the literature in recent years about the potential of pharmacogenetic testing and individualized medicine. The development of personalized medicine is truly an exciting area of research. METHODS: This pharmacogenetic concept in ophthalmology has existed for more than a century. Although substantial studies that link genetic variants to inter-individual difference in drug response have been reported in several diseases such as cancer and heart diseases, such studies are progressing slowly in the eye field. In this short article, an attempt has been made to summarize these results. RESULTS: Recently, there have been some small-scale studies that seem to associate the drug response to the genotype of patients in two major eye disorders, namely age-related macular degeneration (ARMD) and glaucoma. CONCLUSION: These studies are still in their infancy, and do not suggest that a pharmacogenetic basis of drug development is a credible concept and can become reality in the future. This is because most drug responses involve a large number of genes that have several polymorphisms and it is unlikely that any one single gene dictates the drug response. Therefore, a polygenic approach, whole genome single nucleotide polymorphism (SNP) analysis and a molecular understanding of disease itself may provide a better insight in the future about genetic predisposing factors for adverse drug reactions.

Common polymorphisms of the CFH, LOC 387715/ARMS2 and HTRA1 genes may not influence the intra-familial variability of X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis (XLRS) is the leading cause of juvenile macular degeneration in males and is rare in females. Previous studies have shown that there is a marked intra- and inter-familial variation in disease severity and progression. This suggests that additional factors, such as genetic modifiers and environmental elements, influence disease severity. In order to understand the contribution of genetic modifiers, we aimed to ascertain whether common variants of the CFH, LOC 387715/ARMS2 and HTRA1 genes, which are major risk factors in age-related macular degeneration, contribute to the phenotypic variability of the XLRS disorder. Two unrelated XLRS families were selected, one harboring the missense mutation and the second a nonsense mutation in the RS gene. Both families exhibited variations in clinical phenotype. Genomic DNA from family members were analyzed for the above three genes using the polymerase chain reaction-based restriction fragment length polymorphism method. Our analyses revealed that both families were wild-type with respect to the LOC 387715/ARMS2 and HTRA1 genes. In one family (but not the other), the most severely affected and unaffected individuals were heterozygous for the CFH polymorphisms, while the less severely affected individual was wild-type. However, this alteration did not necessarily influence disease severity. Although we cannot completely rule out the role of the above genes in determining the phenotypic variability of the disorder, and though the statistical significance of the results could not be assessed due to the small scale of the study, it is unlikely that common polymorphisms of the CFH, LOC 387715/ARMS2 and HTRA1 genes serve as disease modifiers of the XLRS disorder.

SNPs: impact on gene function and phenotype.

Single nucleotide polymorphism (SNP) is the simplest form of DNA variation among individuals. These simple changes can be of transition or transversion type and they occur throughout the genome at a frequency of about one in 1,000 bp. They may be responsible for the diversity among individuals, genome evolution, the most common familial traits such as curly hair, interindividual differences in drug response, and complex and common diseases such as diabetes, obesity, hypertension, and psychiatric disorders. SNPs may change the encoded amino acids (nonsynonymous) or can be silent (synonymous) or simply occur in the noncoding regions. They may influence promoter activity (gene expression), messenger RNA (mRNA) conformation (stability), and subcellular localization of mRNAs and/or proteins and hence may produce disease. Therefore, identification of numerous variations in genes and analysis of their effects may lead to a better understanding of their impact on gene function and health of an individual. This improved knowledge may provide a starting point for the development of new, useful SNP markers for medical testing and a safer individualized medication to treat the most common devastating disorders. This will revolutionize the medical field in the future. To illustrate the effect of SNPs on gene function and phenotype, this minireview focuses on evidences revealing the impact of SNPs on the development and progression of three human eye disorders (Norrie disease, familial exudative vitreoretinopathy, and retinopathy of prematurity) that have overlapping clinical manifestations.

Copy number variation and susceptibility to human disorders (Review).

A large number of analyses of a new form of genetic variation, known as copy number variation (CNV), have been published recently as a new tool for understanding the genetic basis of complex traits such as diabetes, asthma, Crohn's disease, autism and bipolar disorder. Through the use of different types of genome-wide scanning procedures, CNVs have been shown to be associated with several complex and common disorders, including nervous system disorders. One of the common features of the regions associated with the complex and common disorders identified thus far is the presence of CNVs and segmental duplications. Segmental duplications lead to genome instability. Because of their location and nature (several contain genes), many CNVs have functional consequences, such as gene dosage alteration, the disruption of genes and the modulation of the activities of other genes. Therefore, these genetic variations have an influence on phenotypes, the susceptibility of an individual to disease, drug response and human genome evolution. These types of variants (gain and loss of DNA) are not restricted to humans, having also been identified in other organisms. Our current knowledge regarding CNVs and their heritability is still rudimentary, due to their location in regions of complex genomic structure and to the technical limitations of association studies. Future advances in the technology will aid in the construction of a new CNV map, used to find the genes underlying common diseases and to understand familial genetic conditions, severe developmental defects in humans and other organisms, and genome evolution.

Persistent hyperplastic primary vitreous: congenital malformation of the eye.

Persistent hyperplastic primary vitreous (PHPV), also known as persistent fetal vasculature, is a rare congenital developmental malformation of the eye, caused by the failure of regression of the primary vitreous. It is divided into anterior and posterior types and is characterized by the presence of a vascular membrane located behind the lens. The condition can be of an isolated type or can occur with other ocular disorders. Most cases of PHPV are sporadic, but it can be inherited as an autosomal dominant or recessive trait. Inherited PHPV also occurs in several breeds of dogs and cats. In a limited number of cases, Norrie disease and FZD4 genes are found to be mutated in unilateral and bilateral PHPV. These genes when mutated also cause Norrie disease pseudoglioma and familial exudative vitreoretinopathy that share some of the clinical features with PHPV. Mice lacking arf and p53 tumour suppressor genes as well as Norrie disease pseudoglioma and LRP5 genes suggest that these genes are needed for hyaloid vascular regression. These experiments also indicate that abnormalities in normal apoptosis and defects in Wnt signalling pathway may be responsible for the pathogenesis of PHPV. Identification of other candidate genes in the future may provide a better understanding of the pathogenesis of the condition that may lead to a better therapeutic approach and better management.

Evaluation of the common variants of the ABCA4 gene in families with Stargardt disease and autosomal recessive retinitis pigmentosa.

Stargardt disease (STGD) is one of the most common autosomal recessive retinal dystrophies with an estimated incidence of one in 10,000. It affects the central retina (macula). Retinitis pigmentosa (RP) comprises a large and exceptionally heterogeneous group of hereditary disorders of the retina. It is caused by the loss of photoreceptors. The condition is a degenerative disorder characterized by retinal pigment deposits and has an estimated incidence of one in 4,000. Although, to date, 45 known loci have been identified, none of them independently account for a substantial portion of RP. Recently, the photoreceptor cell-specific ATP-binding cassette transporter (ABCA4) gene was found to be mutated in patients with STGD as well as autosomal recessive RP. In order to further understand the contribution of this gene to the susceptibility to STGD and RP, we analyzed three unrelated STGD families and one autosomal recessive RP family specifically for the more common variants (A1038V, G1961E, 2588G-->C, R943Q or 2828G-->A) in the ABCA4 gene. Our analyses employing standard techniques such as polymerase chain reaction, restriction fragment length polymorphism, and direct DNA sequencing of amplified products were able to identify one common variant (R943Q) in all three STGD families but not in the RP family. All three affected STGD individuals, however, were heterozygous for this variation, and this alteration did not segregate with the disease and was also present in the normal controls. Similar analysis of other common variants revealed no pathogenic mutations in the STGD and RP families. It is likely that the variant identified in this study represents a rare polymorphism (non-pathogenic). Although, at present we cannot eliminate the possibility of this gene as a candidate gene, future extensive studies on this as well as other candidate genes may uncover the susceptibility gene for these recessive forms of the disorders in these families.

SNPs in disease gene mapping, medicinal drug development and evolution.

Single nucleotide polymorphism (SNP) technologies can be used to identify disease-causing genes in humans and to understand the inter-individual variation in drug response. These areas of research have major medical benefits. By establishing an association between the genetic make-up of an individual and drug response it may be possible to develop a genome-based diet and medicines that are more effective and safer for each individual. Additionally, SNPs can be used to understand the molecular mechanisms of sequence evolution. It has been found that throughout the given gene, the rate, type and site of nucleotide substitutions as well as the selection pressure on codons is not uniform. The residues that evolve under strong selective pressures are found to be significantly associated with human disease. Deleterious mutations that affect biological function of proteins are effectively being rejected by natural selection from the gene pool. If substituted nucleotides are fixed during evolution then they may have selection advantages, they may be neutral, or they may be deleterious and cause pathology. Therefore, it is possible that disease-associated SNPs (or pathology) and evolution can be related to one another.

Assessment of the contribution of the LOC387715 gene polymorphism in a family with exudative age-related macular degeneration and heterozygous CFH variant (Y402H).

Age-related macular degeneration (AMD) is a common cause of visual impairment in the elderly population in developed countries. The etiology of AMD is not completely understood but environmental and genetic factors have been implicated in the disease. Recently it has been documented that variations in the complement factor H (CFH) and LOC 387715 genes are the major risk factors that predispose individuals to dry and wet AMD. To investigate further the genetic contribution to AMD, we have analyzed the LOC 387715 gene in a non-smoking family with an exudative AMD and a heterozygous mutation (Y402H) in the CFH gene. Direct sequencing of the amplified product of exon 1 of the LOC 387715 gene identified a previously reported missense mutation (A69S) in this family. The affected individual is homozygous for the mutation and this sequence alteration was not identified in six age-matched controls. On the basis of this and other results it is tempting to speculate that the combined effect of variants in the CFH and LOC 387715 genes may contribute to the AMD phenotype in this family. Further studies on these and other susceptibility genes may provide clues on variable phenotypes, new preventive strategies and treatment options for AMD.

Lack of association of the VEGF gene promoter (-634 G-->C and -460 C-->T) polymorphism and the risk of advanced retinopathy of prematurity.

BACKGROUND: Recently, it has been reported that genetic polymorphism (-634 G-->C and -460 C-->T) in the promoter region of the vascular endothelial growth factor (VEGF) gene can influence the progression of retinopathy of prematurity (ROP). In order to evaluate its general applicability as a screening procedure in clinics and to replicate the above result, we have undertaken the following study. METHODS: We have analyzed a cohort of 61 patients with advanced ROP (stage 4 and 5) along with 61 normal controls for the VEGF gene promoter polymorphism. For this purpose, blood samples were collected from each patient and leukocyte DNA was isolated. Genomic DNA was amplified by the polymerase chain reaction (PCR) method with two pairs of primers designed to amplify separately the promoter region (containing -634 G-->C and -460 C-->T polymorphism) of the VEGF gene. The amplified product was subjected to restriction enzyme digestion. The base change in the restriction site was further confirmed by a BigDye terminator cycle sequencing of the amplified product. RESULTS: Our analysis suggests that there is no significant difference in allelic frequency of the VEGF gene between normal subjects and patients with advanced ROP in our cohort. CONCLUSION: Our results do not support the association of the VEGF gene promoter polymorphism and the risk of advanced ROP. In order to adapt this method for the identification of high-risk infants in clinics in the future, a large-scale study involving a mixed ethnically diverse population is much needed.

Developmental dyslexia: an update.

Dyslexia is the most common and carefully studied of the learning disabilities in school-age children. It is characterized by a marked impairment in the development of reading skills, and affects a large number of people (5-10%). Reading difficulties may also arise from poor vision, emotional problems, decreased hearing ability, and behavioral disorders, such as attention-deficit hyperactivity (ADHD). Although many areas of the brain are involved in reading, analysis of postmortem brain specimens by a variety of imaging techniques most consistently suggests that deficiency within a specific component of the language system - the phonologic module - in the temporo-parietal-occipital brain region underlies dyslexia. It is a highly familial and heritable disorder with susceptibility loci on chromosomes 1, 2, 3, 6, 11, 13, 15 and 18. Recently, four candidate genes (KIAA 0319, DYX1C1, DCDC2 and ROBO1) are shown to be associated with dyslexia. Although some of these results are controversial because of the genetic heterogeneity of the disorder, the available evidence suggests that dyslexia could be due to the abnormal migration and maturation of neurons during early development. Interestingly, in spite of genetic heterogeneity, the pathology appears to involve common phonological coding deficits. The condition can be managed by a highly structured educational training exercise.

Further support for the common variants in complement factor H (Y402H) and LOC387715 (A69S) genes as major risk factors for the exudative age-related macular degeneration.

In developed countries, age-related macular degeneration (ARMD) is a common cause of blindness in the elderly. It is a clinically complex and genetically heterogeneous disorder. The etiology of the disorder may involve interactions between genetic and environmental factors. Recently it has been reported that a polymorphism in the complement factor H (CFH) and LOC387715 gene may determine the susceptibility of individuals to ARMD. In order to replicate and to determine the frequency of this polymorphism in ARMD patients, we have analyzed two unrelated families having exudative ARMD. Our analysis has identified the same common polymorphism (Y402H) in the CFH gene in one family and the A69S polymorphism in the LOC387715 gene in the second family. These results further support the notion that CFH and LOC387715 genes are the major risk factors for ARMD.