A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). -- Case Presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G > A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A > G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G > A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A > G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. --Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor. The possible phenotypical implications of this mutation are discussed.

Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.

These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.

Lynx1 and the β2V287L mutation affect the stoichiometry of the α4β2 nicotinic acetylcholine receptor

GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent α4 and β2 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of I-125-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with I-125-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the I-125 methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than I-125-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Epitope mapping of antibodies to alpha-synuclein in LRRK2 mutation carriers, idiopathic Parkinson disease patients, and healthy controls

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1-60) or C-terminal (a.a. 109-140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls' studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD patholog

Perspectivas crítica s sobre a mensuração da pobreza e desigualdade no Brasil: uma reflexão a partir do IDH

O presente estudo tem como objetivo desenvolver uma reflexão sobre o uso dos indicadores sociais sintéticos no sentido de formular e implementar políticas públicas na área social, especificamente em relação ao caso brasileiro, tomando como referência principal o Índice de Desenvolvimento Humano (IDH). Nossa análise partiu da premissa de que, pelos menos nas últimas três décadas, os indicadores sociais têm sido amplamente utilizados no Brasil com o objetivo de justificar e orientar ações públicas direcionadas para as tentativas de equacionamento das mais variadas questões sociais, tais como a pobreza e a concentração de renda. Principalmente a partir dos anos 1990 os dados e apontamentos de alguns indicadores sociais sintéticos, especialmente o IDH, parecem que vêm sendo utilizados como justificativa principal ou até mesmo única para a priorização de determinadas políticas públicas voltadas para a área social. A partir dessa reflexão, nossa análise buscou avaliar, com um olhar mais apurado sobre o IDH, a existência de falhas e limites inerentes aos indicadores sintéticos, que poderiam estar comprometendo sua eficácia no sentido de refletir dados mais aproximados com a realidade social brasileira. Assim, pretendemos apontar que a prática de priorização de políticas a partir dos dados desses indicadores sintéticos, que parece ser constante no Brasil, acaba se tornando bastante preocupante ou, algumas vezes, talvez inadequada. Do ponto de vista metodológico, este estudo dependeu basicamente de uma pesquisa bibliográfica e de uma pesquisa documental, a partir das quais decompomos nossas análises em reflexões sobre a formação e evolução dos indicadores sociais, sobre os aspectos filosóficos e metodológicos do IDH e sobre as possíveis falhas e limites inerentes aos indicadores, com base na análise do IDH e dos índices parciais que o compõem (índices de Renda, Educação e Longevidade). A seguir, para mostrar que a prática de priorização de políticas sociais a partir de indicadores parece constante no Brasil, ilustramos os casos selecionados de determinados estados e municípios brasileiros.

A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory

Neuropsin (kallikrein 8, ELKS) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only express

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Moderate mutation rate in the SARS coronavirus genome and its implications

Background: The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) an

mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample f

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in d

Evidence of gene conversion in the evolutionary process of the codon 41/42 (-CTTT) mutation causing beta-thalassemia in southern China

The 4-bp deletion (-CTTT) at codon 41/42 (CD41/42) of the human beta-globin gene represents one of the most common beta-thalassemia mutations in East Asia and Southeast Asia, which is historically afflicted with endemic malaria, thus hypothetically evolvi