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1.  A generalizable pre-clinical research approach for orphan disease therapy 
With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.
doi:10.1186/1750-1172-7-39
PMCID: PMC3458970  PMID: 22704758
Orphan disease therapy; Preclinical drug development; Generalizable screening methods; Translational toolbox
2.  Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease 
Disease Models & Mechanisms  2013;6(3):780-792.
SUMMARY
Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.
doi:10.1242/dmm.010942
PMCID: PMC3634660  PMID: 23519028
3.  Saturation of the Human Phenome 
Current Genomics  2010;11(7):482-499.
The phenome is the complete set of phenotypes resulting from genetic variation in populations of an organism. Saturation of a phenome implies the identification and phenotypic description of mutations in all genes in an organism, potentially constrained to those encoding proteins. The human genome is believed to contain 20-25,000 protein coding genes, but only a small fraction of these have documented mutant phenotypes, thus the human phenome is far from complete. In model organisms, genetic saturation entails the identification of multiple mutant alleles of a gene or locus, allowing a consistent description of mutational phenotypes for that gene. Saturation of several model organisms has been attempted, usually by targeting annotated coding genes with insertional transposons (Drosophila melanogaster, Mus musculus) or by sequence directed deletion (Saccharomyces cerevisiae) or using libraries of antisense oligonucleotide probes injected directly into animals (Caenorhabditis elegans, Danio rerio). This paper reviews the general state of the human phenome, and discusses theoretical and practical considerations toward a saturation analysis in humans. Throughout, emphasis is placed on high penetrance genetic variation, of the kind typically asociated with monogenic versus complex traits.
doi:10.2174/138920210793175886
PMCID: PMC3048311  PMID: 21532833
Human genome; phenome; genetics; saturation mutagenesis.
4.  Compound heterozygosity in sodium channel Nav1.7 in a family with hereditary erythermalgia 
Molecular Pain  2008;4:21.
Hereditary erythermalgia is a painful and debilitating genetic disorder associated with mutations in voltage-gated sodium channel Nav1.7. We have previously reported a Canadian family segregating erythermalgia consistently with a dominant genetic etiology. Molecular analysis of the proband from the family detected two different missense mutations in Nav1.7. In the present study we have performed a long-term follow-up clinical study of disease progression in three affected family members. A more extensive molecular study has also been completed, analyzing the segregation of the two missense variants in the family. The two variants (P610T, L858F) segregate independently with respect to clinical presentation. Detailed genotype/phenotype correlation suggests that one of the two variants (L858F) is causal for erythermalgia. The second variant (P610T) may modify the phenotype in the proband. This is the second reported study of potential compound heterozygosity for coding polymorphisms in Nav1.7, the first being in a patient with paroxysmal extreme pain disorder.
doi:10.1186/1744-8069-4-21
PMCID: PMC2430949  PMID: 18518989
5.  Mutations in a novel serine protease PRSS56 in families with nanophthalmos 
Molecular Vision  2011;17:1850-1861.
Purpose
Nanophthalmos is a rare genetic ocular disorder in which the eyes of affected individuals are abnormally small. Patients suffer from severe hyperopia as a result of their markedly reduced axial lengths, but otherwise are capable of seeing well unlike other more general forms of microphthalmia. To date one gene for nanophthalmos has been identified, encoding the membrane-type frizzled related protein MFRP. Identification of additional genes for nanophthalmos will improve our understanding of normal developmental regulation of eye growth.
Methods
We ascertained a cohort of families from eastern Canada and Mexico with familial nanophthalmos. We performed high density microsatellite and high density single nucleotide polymorphism (SNP) genotyping to identify potential chromosomal regions of linkage. We sequenced coding regions of genes in the linked interval by traditional PCR-based Sanger capillary electrophoresis methods. We cloned and sequenced a novel cDNA from a putative causal gene to verify gene structure.
Results
We identified a linked locus on chromosome 2q37 with a peak logarithm (base 10) of odds (LOD) score of 4.7. Sequencing of coding exons of all genes in the region identified multiple segregating variants in one gene, recently annotated as serine protease gene (PRSS56), coding for a predicted trypsin serine protease-like protein. One of our families was homozygous for a predicted pathogenic missense mutation, one family was compound heterozygous for two predicted pathogenic missense mutations, and one family was compound heterozygous for a predicted pathogenic missense mutation plus a frameshift leading to obligatory truncation of the predicted protein. The PRSS56 gene structure in public databases is based on a virtual transcript assembled from overlapping incomplete cDNA clones; we have now validated the structure of a full-length transcript from embryonic mouse brain RNA.
Conclusions
PRSS56 is a good candidate for the causal gene for nanophthalmos in our families.
PMCID: PMC3137557  PMID: 21850159
6.  Application of Homozygosity Haplotype Analysis to Genetic Mapping with High-Density SNP Genotype Data 
PLoS ONE  2009;4(4):e5280.
Background
In families segregating a monogenic genetic disorder with a single disease gene introduction, patients share a mutation-carrying chromosomal interval with identity-by-descent (IBD). Such a shared chromosomal interval or haplotype, surrounding the actual pathogenic mutation, is typically detected and defined by multipoint linkage and phased haplotype analysis using microsatellite or SNP genotype data. High-density SNP genotype data presents a computational challenge for conventional genetic analyses. A novel non-parametric method termed Homozygosity Haplotype (HH) was recently proposed for the genome-wide search of the autosomal segments shared among patients using high density SNP genotype data.
Methodology/Principal Findings
The applicability and the effectiveness of HH in identifying the potential linkage of disease causative gene with high-density SNP genotype data were studied with a series of monogenic disorders ascertained in eastern Canadian populations. The HH approach was validated using the genotypes of patients from a family affected with a rare autosomal dominant disease Schnyder crystalline corneal dystrophy. HH accurately detected the ∼1 Mb genomic interval encompassing the causative gene UBIAD1 using the genotypes of only four affected subjects. The successful application of HH to identify the potential linkage for a family with pericentral retinal disorder indicates that HH can be applied to perform family-based association analysis by treating affected and unaffected family members as cases and controls respectively. A new strategy for the genome-wide screening of known causative genes or loci with HH was proposed, as shown the applications to a myoclonus dystonia and a renal failure cohort.
Conclusions/Significance
Our study of the HH approach demonstrates that HH is very efficient and effective in identifying potential disease linked region. HH has the potential to be used as an efficient alternative approach to sequencing or microsatellite-based fine mapping for screening the known causative genes in genetic disease study.
doi:10.1371/journal.pone.0005280
PMCID: PMC2670504  PMID: 19399176
7.  Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia 
Journal of Medical Genetics  2013;50(5):324-329.
Background
Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort.
Methods
We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing.
Results
Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes.
Conclusions
Based on our genetic results, TTC7A is the likely causal gene for MIA.
doi:10.1136/jmedgenet-2012-101483
PMCID: PMC3625823  PMID: 23423984
Gastroenterology; Genetics; Developmental; Molecular genetics
8.  Meier–Gorlin syndrome genotype–phenotype studies: 35 individuals with pre-replication complex gene mutations and 10 without molecular diagnosis 
Meier–Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, patellar aplasia/hypoplasia, and short stature. Recently, mutations in five genes from the pre-replication complex (ORC1, ORC4, ORC6, CDT1, and CDC6), crucial in cell-cycle progression and growth, were identified in individuals with MGS. Here, we report on genotype–phenotype studies in 45 individuals with MGS (27 females, 18 males; age 3 months–47 years). Thirty-five individuals had biallelic mutations in one of the five causative pre-replication genes. No homozygous or compound heterozygous null mutations were detected. In 10 individuals, no definitive molecular diagnosis was made. The triad of microtia, absent/hypoplastic patellae, and short stature was observed in 82% of individuals with MGS. Additional frequent clinical features were mammary hypoplasia (100%) and abnormal genitalia (42% predominantly cryptorchidism and hypoplastic labia minora/majora). One individual with ORC1 mutations only had short stature, emphasizing the highly variable clinical spectrum of MGS. Individuals with ORC1 mutations had significantly shorter stature and smaller head circumferences than individuals from other gene categories. Furthermore, compared with homozygous missense mutations, compound heterozygous mutations appeared to have a more severe effect on phenotype, causing more severe growth retardation in ORC4 and more frequently pulmonary emphysema in CDT1. A lethal phenotype was seen in four individuals with compound heterozygous ORC1 and CDT1 mutations. No other clear genotype–phenotype association was observed. Growth hormone and estrogen treatment may be of some benefit, respectively, to growth retardation and breast hypoplasia, though further studies in this patient group are needed.
doi:10.1038/ejhg.2011.269
PMCID: PMC3355263  PMID: 22333897
Meier–Gorlin syndrome; ear-patella-short stature syndrome; origin recognition complex; pre-replication complex; genotype–phenotype
9.  Mutation in the Gene Encoding Ubiquitin Ligase LRSAM1 in Patients with Charcot-Marie-Tooth Disease 
PLoS Genetics  2010;6(8):e1001081.
Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730–129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.
Author Summary
Sensory motor neuropathies are diseases of the peripheral nervous system, involving primarily the nerves which control our muscles. These can result from either genetic or non-genetic causes, with genetic causes usually referred to as Charcot-Marie-Tooth (CMT) disease after the three clinicians who first described the key diagnostic markers. CMT patients lose muscle function, mainly in their arms and legs, with increasing severity during their lives. There are almost two dozen known genes that can mutate to cause CMT, and these fall into a wide variety of biochemical cellular pathways. We identified a group of patients with CMT from a small rural community, with good reason to suspect a genetic basis for their disease. Using high-throughput mapping and DNA sequencing technologies developed as part of the Human Genome Project, we were able to find the likely mutated gene, which was not any of the previously known CMT genes. Based on its sequence, the gene, called LRSAM1, probably plays a role in the correct metabolism of other proteins in the cell. Among the known CMT genes, some are also involved in protein metabolism, suggesting that this is a generally important pathway in the neurons that control muscle activity.
doi:10.1371/journal.pgen.1001081
PMCID: PMC2928813  PMID: 20865121

Results 1-9 (9)