Piya Lahiry

Kinase mutations in human disease: interpreting genotype–phenotype relationships

Protein kinases are one of the largest families of evolutionarily related proteins and comprise one of the most abundant gene families in humans. Here we survey kinase gene mutations from the perspective of human disease phenotypes and further analyse the structural features of mutant kinases, including mutational hotspots. Our evaluation of the genotype–phenotype relationship across 915 human kinase mutations — that underlie 67 single-gene diseases, mainly inherited developmental and metabolic disorders and also certain cancers — enhances our understanding of the role of kinases in development, kinase dysfunction in pathogenesis and kinases as potential targets for therapy.

Micromethods for the Characterization of Lipid A-Core and O-Antigen Lipopolysaccharide

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Phenomics: Expanding the Role of Clinical Evaluation in Genomic Studies

With advances in high-throughput genotyping technologies, the rate-limiting step of large-scale genetic investigations has become the collection of sensitive and specific phenotype information in large samples of study participants. Clinicians play a pivotal role for successful genetic studies because sound clinical acumen can substantially increase study power by reducing measurement error and improving diagnostic precision for translational research. Phenomics is the systematic measurement and analysis of qualitative and quantitative traits, including clinical, biochemical, and imaging methods, for the refinement and characterization of a phenotype. Phenomics requires deep phenotyping, the collection of a wide breadth of phenotypes with fine resolution, and phenomic analysis, composed of constructing heat maps, cluster analysis, text mining, and pathway analysis. In this article, we review the components of phenomics and provide examples of their application to genomic studies, specifically for implicating novel disease processes, reducing sample heterogeneity, hypothesis generation, integration of multiple types of data, and as an extension of Mendelian randomization studies.

A Multiplex Human Syndrome Implicates a Key Role for Intestinal Cell Kinase in Development of Central Nervous, Skeletal, and Endocrine Systems

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.

Exome sequencing identifies NFS1 deficiency in a novel Fe-S cluster disease, infantile mitochondrial complex II/III deficiency

Iron‐sulfur (Fe‐S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe‐S proteins, to assist in various key biochemical pathways. Mutations in Fe‐S proteins often disrupt Fe‐S cluster assembly leading to a spectrum of severe disorders such as Friedreichs ataxia or iron‐sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe‐S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

Common variants APOC3, APOA5, APOE and PON1 are associated with variation in plasma lipoprotein traits in Greenlanders

Objectives. We undertook studies of the association between common genomic variations in APOC3, APOA5, APOE and PON1 genes and variation in biochemical phenotypes in a sample of Greenlanders. Study Design. Genetic association study of quantitative lipoprotein traits. Methods. In a sample of 1,310 adult Greenlanders, fasting plasma lipid, lipoprotein and apolipoprotein (apo) concentrations were assessed for association with known functional genomic variants of APOC3, APOA5, APOE and PON1. For significantly associated polymorphisms, between-genotype differences were examined in closer detail. Results. We found that (1) the APOE restriction isotype was associated with variation in plasma total and LDL cholesterol and apo B (all p & .0001); (2) the APOC3 promoter genotype was associated with variation in plasma triglycerides, HDL cholesterol and apo A-I (all p & .002); (3) the APOA5 codon 19 genotype was associated with variation in plasma triglycerides (p = .027); and (4) the PON1 codon 192 genotype was associated with variation in total and LDL cholesterol and apo B (all p & .05). Conclusions. Taken together, our results suggest that common genetic variations in APOC3, APOA5, APOE and PON1 are associated with significant variation in intermediate traits in plasma lipoprotein metabolism in Greenlanders; the associations are similar to those observed for these variants in other populations.

APOC1 T45S polymorphism is associated with reduced obesity indices and lower plasma concentrations of leptin and apolipoprotein C-I in aboriginal Canadians

Apolipoprotein (apo) C-I is a constituent of chylomicrons, very low density lipoprotein, and high density lipoprotein. The role of apo C-I in human metabolism is incompletely defined. We took advantage of a naturally occurring amino acid polymorphism that is present in aboriginal North Americans, namely apo C-I T45S. We assessed the hypothesis that metabolic traits, including obesity-related and lipoprotein-related traits, would differ between carriers and noncarriers of apo C-I T45S. A genotyping assay was developed for APOC1 T45S and genotypes were determined in a sample of 410 Canadian Oji-Cree subjects. The allele frequency of the apo C-I S45 allele was ∼8% in this sample. We observed the apo C-I S45 allele was significantly associated with 1) lower percent body fat (P < 0.05), 2) lower waist circumference (P = 0.058), 3) lower serum leptin levels (P < 0.05), and 4) lower plasma apo C-I levels (P < 0.0001), using a newly developed ELISA-based method. Taken together, these results suggest that at the whole human phenotype level, apo C-I is associated with the complex metabolic trait of obesity as well as with serum leptin levels.

Transcriptional Profiling of Endocrine Cerebro-Osteodysplasia Using Microarray and Next-Generation Sequencing

Background Transcriptome profiling of patterns of RNA expression is a powerful approach to identify networks of genes that play a role in disease. To date, most mRNA profiling of tissues has been accomplished using microarrays, but next-generation sequencing can offer a richer and more comprehensive picture. Methodology/Principal Findings ECO is a rare multi-system developmental disorder caused by a homozygous mutation in ICK encoding intestinal cell kinase. We performed gene expression profiling using both cDNA microarrays and next-generation mRNA sequencing (mRNA-seq) of skin fibroblasts from ECO-affected subjects. We then validated a subset of differentially expressed transcripts identified by each method using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, we used gene ontology (GO) to identify critical pathways and processes that were abnormal according to each technical platform. Methodologically, mRNA-seq identifies a much larger number of differentially expressed genes with much better correlation to qRT-PCR results than the microarray (r2 = 0.794 and 0.137, respectively). Biologically, cDNA microarray identified functional pathways focused on anatomical structure and development, while the mRNA-seq platform identified a higher proportion of genes involved in cell division and DNA replication pathways. Conclusions/Significance Transcriptome profiling with mRNA-seq had greater sensitivity, range and accuracy than the microarray. The two platforms generated different but complementary hypotheses for further evaluation.

MG-137 Autosomal recessive disorders are common in the old order amish population of southwestern ontario

Background The Old Order Amish in Milverton, Ontario, represent a genetically isolated group which originated with 12 founding families in 1824. Due to inbreeding and founder effect, certain genetic disorders have become highly prevalent in Ontario Amish. Objective This study aims to describe the spectrum of genetic disorders observed in the Old Order Amish of Southwestern Ontario (population size ˜ 2500). Design and method Over the past 25 years, we have documented the clinical phenotype, and molecular aetiology of genetic disorders in Old Order Amish children. Results A disorder characterised by acute encephalopathy with fever, visual hallucinations, hearing loss, and retinal dystrophy was observed in 7 out of about 220 families, caused by mutation in the histidyl t-RNA synthetase (HARS) gene. Acute respiratory distress syndrome may lead to sudden death. This disorder was first reported in the Amish in Pennsylvania. Other disorders observed in the Ontario Amish but not in American Amish include endocrine-osteodysplasia syndrome (ICK), cerebral atrophy (TMPRSS4), cystinosis (CTNS), sodium diarrhoea (SPINT2), juvenile-onset glaucoma (CYP1B1), epidermolysis bullosa simplex, and Fraser syndrome (FRAS1). Lethal disorders include a central hypoventilation syndrome, a disorder with oligohydramnios and renal hypoplasia, and ichthyosis-microcephaly. In total, the causative mutations have been found for 8 disorders, while another 5 disorders are presumed autosomal recessive. Knowledge of these disorders enabled molecular diagnosis of a child affected concurrently with two conditions within 5 days of birth. Conclusion Awareness of genetic disorders common in this population will aid in early diagnosis and management, and avoid the diagnostic odyssey.