Motion estimation of subcellular structures from fluorescence microscopy images.

We present an automatic image processing framework to study moving intracellular structures from live cell fluorescence microscopy. The system includes the identification of static and dynamic structures from time-lapse images using data clustering as well as the identification of the trajectory of moving objects with a probabilistic tracking algorithm. The method has been successfully applied to study mitochondrial movement in neurons. The approach provides excellent performance under different experimental conditions and is robust to common sources of noise including experimental, molecular and biological fluctuations.

The EGR2 gene is involved in axonal Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: A three-generation family affected by axonal Charcot-Marie-Tooth disease (CMT) was investigated with the aim of discovering genetic defects and to further characterize the phenotype. METHODS: The clinical, nerve conduction studies and muscle magnetic resonance images of the patients were reviewed. A whole exome sequencing was performed and the changes were investigated by genetic studies, in silico analysis and luciferase reporter assays. RESULTS: A novel c.1226G>A change (p.R409Q) in the EGR2 gene was identified. Patients presented with a typical, late-onset axonal CMT phenotype with variable severity that was confirmed in the ancillary tests. The in silico studies showed that the residue R409 is an evolutionary conserved amino acid. The p.R409Q mutation, which is predicted as probably damaging, would alter the conformation of the protein slightly and would cause a decrease of gene expression. CONCLUSIONS: This is the first report of an EGR2 mutation presenting as an axonal CMT phenotype with variable severity. This study broadens the phenotype of the EGR2-related neuropathies and suggests that the genetic testing of patients suffering from axonal CMT should include the EGR2 gene.

The HLA-DQ2 genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease.

OBJECTIVE: Intestinal dysbiosis has been associated with coeliac disease (CD), but whether the alterations are cause or consequence of the disease is unknown. This study investigated whether the human leukocyte antigen (HLA)-DQ2 genotype is an independent factor influencing the early gut microbiota composition of healthy infants at family risk of CD. DESIGN: As part of a larger prospective study, a subset (n=22) of exclusively breastfed and vaginally delivered infants with either high genetic risk (HLA-DQ2 carriers) or low genetic risk (non-HLA-DQ2/8 carriers) of developing CD were selected from a cohort of healthy infants with at least one first-degree relative with CD. Infant faecal microbiota was analysed by 16S rRNA gene pyrosequencing and real time quantitative PCR. RESULTS: Infants with a high genetic risk had significantly higher proportions of Firmicutes and Proteobacteria and lower proportions of Actinobacteria compared with low-risk infants. At genus level, high-risk infants had significantly less Bifidobacterium and unclassified Bifidobacteriaceae proportions and more Corynebacterium, Gemella, Clostridium sensu stricto, unclassified Clostridiaceae, unclassified Enterobacteriaceae and Raoultella proportions. Quantitative real time PCR also revealed lower numbers of Bifidobacterium species in infants with high genetic risk than in those with low genetic risk. In high-risk infants negative correlations were identified between Bifidobacterium species and several genera of Proteobacteria (Escherichia/Shigella) and Firmicutes (Clostridium). CONCLUSIONS: The genotype of infants at family risk of developing CD, carrying the HLA-DQ2 haplotypes, influences the early gut microbiota composition. This finding suggests that a specific disease-biased host genotype may also select for the first gut colonisers and could contribute to determining disease risk.

A novel locus for a hereditary recurrent neuropathy on chromosome 21q21.

Hereditary recurrent neuropathies are uncommon. Disorders with a known molecular basis falling within this group include hereditary neuropathy with liability to pressure palsies (HNPP) due to the deletion of the PMP22 gene or to mutations in this same gene, and hereditary neuralgic amyotrophy (HNA) caused by mutations in the SEPT9 gene. We report a three-generation family presenting a hereditary recurrent neuropathy without pathological changes in either PMP22 or SEPT9 genes. We performed a genome-wide mapping, which yielded a locus of 12.4 Mb on chromosome 21q21. The constructed haplotype fully segregated with the disease and we found significant evidence of linkage. After mutational screening of genes located within this locus, encoding for proteins and microRNAs, as well as analysis of large deletions/insertions, we identified 71 benign polymorphisms. Our findings suggest a novel genetic locus for a recurrent hereditary neuropathy of which the molecular defect remains elusive. Our results further underscore the clinical and genetic heterogeneity of this group of neuropathies.

Genetics of the Charcot-Marie-Tooth disease in the Spanish Gypsy population: the hereditary motor and sensory neuropathy-Russe in depth.

Four private mutations responsible for three forms demyelinating of Charcot-Marie-Tooth (CMT) or hereditary motor and sensory neuropathy (HMSN) have been associated with the Gypsy population: the NDRG1 p.R148X in CMT type 4D (CMT4D/HMSN-Lom); p.C737_P738delinsX and p.R1109X mutations in the SH3TC2 gene (CMT4C); and a G>C change in a novel alternative untranslated exon in the HK1 gene causative of CMT4G (CMT4G/HMSN-Russe). Here we address the findings of a genetic study of 29 Gypsy Spanish families with autosomal recessive demyelinating CMT. The most frequent form is CMT4C (57.14%), followed by HMSN-Russe (25%) and HMSN-Lom (17.86%). The relevant frequency of HMSN-Russe has allowed us to investigate in depth the genetics and the associated clinical symptoms of this CMT form. HMSN-Russe probands share the same haplotype confirming that the HK1 g.9712G>C is a founder mutation, which arrived in Spain around the end of the 18th century. The clinical picture of HMSN-Russe is a progressive CMT disorder leading to severe weakness of the lower limbs and prominent distal sensory loss. Motor nerve conduction velocity was in the demyelinating or intermediate range.

Ancient origin of the CTH alelle carrying the c.200C>T (p.T67I) variant in patients with cystathioninuria.

Hereditary cystathioninuria is due to mutations in the CTH gene that encodes for cystathionase, a pyridoxal-5'-phosphate (PLP) dependent enzyme. To date, mutations in this gene have been described in 10 unrelated cystathioninuric patients. Enzyme assays have showed that mutated cystathionase exhibits lower activity than controls. As cystathioninuria is usually accompanied by a wide variety of symptoms, it has been questioned whether it is a disease or just a biochemical finding not associated with the clinical picture of these patients. This is the first report of Spanish patients with cystathioninuria and mild to severe neurological symptoms in childhood. After oral pyridoxine therapy biochemical parameters have normalized but clinical amelioration was not evident. All patients were homozygotes for the c.200C>T (p.T67I) variant which is the most prevalent inactivating mutation in the CTH gene. To further investigate the history of the alleles carrying the c.200C>T transition in Europe, we also constructed the haplotypes on the CTH locus in our Spanish patients as well as in a clinical series of cystathioninuric patients from the Czech Republic harboring the same nucleotide change. We suggest that the CTH p.T67I substitution could have an ancient common origin, which probably occurred in the Neolithic Era and spread throughout Europe.

Interplay between human leukocyte antigen genes and the microbial colonization process of the newborn intestine.

Coeliac disease (CD) development involves genetic (HLA-DQ2/DQ8) and environmental factors. Herein, the influence of the HLA-DQ genotype on the gut colonization process of breast-fed children was determined. A cohort of 20 newborns, with at least one first-degree relative with CD, were classified according to their HLA-DQ genotype into high, intermediate and low genetic risk groups, showing 24-28%, 7-8% and less than 1% probability to develop CD, respectively. Faecal microbiota was analysed at 7 days, 1 and 4 months of children's age by fluorescence in situ hybridization. When considering all data, Gram-negative bacteria and Bacteroides-Prevotella group proportions were higher (P<0.05) in the high than in the intermediate and low genetic risk groups. E. coli, Streptococcus-Lactococcus, E. rectale-C. coccoides, sulphate-reducing bacteria, C. lituseburense and C. histolyticum group proportions were also significantly higher (P<0.05) in the high than in the low genetic risk group. Correlations between these bacterial groups and the genetic risk were also detected (P<0.05). In addition, the number and type of CD relative seemed to influence (P<0.050) these bacterial proportions in children at CD risk. At 4 months of age, similar relationships were established between the high genetic risk to develop CD and the proportions of Streptococcus-Lactococcus (P<0.05), E. rectale-C. coccoides (P<0.05), C. lituseburense (P<0.05), C. histolyticum (P<0.05), Bacteroides-Prevotella (P<0.10) groups and total Gram-negative bacteria (P<0.05). The results suggest a relationship between HLA-DQ genes and the gut microbial colonization process that could lead to a change in the way this disorder is investigated.

Allelic distribution and the effect of haplotype combination for HLA type II loci in the celiac disease population of the Valencian community (Spain).

The association between human leukocyte antigen (HLA) class II antigens and celiac disease (CD) was analyzed in a Spanish population. No association with DRB1*04 and DQB1*0302 was noted. The main associated haplotype (70.8%) was DRB1*03-DQB1*0201-DQA1*0501(DR3-DQ2), followed by DRB1*07-DQB1*0202-DQA1*0201 (DR7-DQ2) haplotype, which is associated with DRB1*11-DQB1*0301-DQA1*0505 (DR11-DQ7). The combinations of DR3-DQ2 with DR7-DQ2, and DR7-DQ2 with DR11-DQ7, present a twofold risk compared with each haplotype in homozygosis. An independence test in DR3-DQ2 haplotype found that association with CD was attributable to the whole haplotype, but for DR7-DQ2 was secondary to DQB1/DQA1. There is no need of a double gene dosage to increase the risk. CD-associated alleles typing demonstrates a very high negative predictive value to exclude CD in risk groups.

Mutations in the urocanase gene UROC1 are associated with urocanic aciduria.

Urocanase is an enzyme in the histidine pathway encoded by the UROC1 gene. This report describes the first putative mutations, p.L70P and p.R450C, in the coding region of the UROC1 gene in a girl with urocanic aciduria presenting with mental retardation and intermittent ataxia. Computed (in silico) predictions, protein expression studies and enzyme activity assays suggest that none of the mutations can produce a fully functional enzyme. The p.L70P substitution, which probably implies the disruption of an alpha-helix in the N-terminus, would alter its properties and therefore, its function. The p.R450C change would render impossible any interaction between urocanase and its substrate and would loss its enzyme activity. Consequently, these studies suggest that both mutations could alter the correct activity of urocanase, which would explain the clinical and biochemical findings described in this patient.

Genetic analyses of celiac disease in a Spanish population confirm association with CELIAC3 but not with CELIAC4.

Genetic predisposition to celiac disease (CD) is determined primarily by the human leukocyte antigen (HLA) genes (CELIAC1 region; 6p21), although many loci are involved in disease susceptibility. First, we have analysed a large series of CD patients from the Spanish Mediterranean region who had previously been characterised for the HLA complex. We have investigated how relevant regions contribute to CD susceptibility: CELIAC3 (CD28/CTLA4/ICOS region on 2q33) and CELIAC4 (19p13) as well as the tumour necrosis factor alpha (TNF-alpha) and the linfotoxin loci by case-control and association analyses. We highlight the association with the +49*A allele of cytotoxic T-lymphocyte-associated antigen 4 locus (P = 0.01), and the -308*A of TNF-alpha locus (P = 0.0008) in DQ2 individuals, although an independent role for TNF-alpha as risk factor has not been proven. Moreover, we do not confirm the association with the CELIAC4 region polymorphisms described in other populations.

The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.

Charcot-Marie-Tooth (CMT) disease type 4 (CMT4) is the name given to autosomal recessive forms of hereditary motor and sensory neuropathy (HMSN). When we began this study, three genes or loci associated with inherited peripheral neuropathies had already been identified in the European Gypsy population: HMSN-Lom (MIM 601455), HMSN-Russe (MIM 605285) and the congenital cataracts facial dysmorphism neuropathy syndrome (MIM 604168). We have carried out genetic analyses in a series of 20 Spanish Gypsy families diagnosed with a demyelinating CMT disease compatible with an autosomal recessive trait. We found the p.R148X mutation in the N-myc downstream-regulated gene 1 gene to be responsible for the HMSN-Lom in four families and also possible linkage to the HMSN-Russe locus in three others. We have also studied the CMT4C locus because of the clinical similarities and showed that in 10 families, the disease is caused by mutations located on the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene: p.R1109X in 20 out of 21 chromosomes and p.C737_P738delinsX in only one chromosome. Moreover, the SH3TC2 p.R1109X mutation is associated with a conserved haplotype and, therefore, may be a private founder mutation for the Gypsy population. Estimation of the allelic age revealed that the SH3TC2 p.R1109X mutation may have arisen about 225 years ago, probably as the consequence of a bottleneck.

Fundamentos de genética en el cáncer de mama / Rationale of genetics in breast cancer

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Unique origin and low penetrance of the 946delGAG mutation in Valencian DYT1 families.

Mutations in the DYT1 gene cause idiopathic torsion dystonia (ITD) transmitted in families as an autosomal dominant trait with incomplete penetrance. The most common mutation, 946delGAG, has been observed in populations with different ethnic and geographic origins. We have investigated 40 individuals from 22 unrelated families with ITD originating from the Land of Valencia, Spain, for the presence of this mutation and we found 5 patients and 6 unaffected subjects from 4 families who were carriers of the mutation. This finding indicates that 18% of families may be diagnosed as DYT1 and that penetrance is reduced. We detected two different geographic and linguistic origins of the Valencian families. However, by haplotype analysis using D9S1260, D9S1261, D9S63 and D9S1262 as flanking markers, we demonstrated that all affected and unaffected carriers shared a common chromosome confirming identical origin of the mutation in the four families. We postulate a unique origin for the 946delGAG mutation in the Land of Valencia and, based on linguistic criterion, we propose that the mutation might have occurred at the beginning of the second millennium. Genetic analysis of another family from Castilla-La Mancha showed a different haplotype segregating with the disease, suggesting that at least two distinct mutational events for the 946delGAG mutation have occurred in Spain.

Mutations in GDAP1: autosomal recessive CMT with demyelination and axonopathy.

BACKGROUND: Mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) were recently shown to be responsible for autosomal recessive (AR) demyelinating Charcot-Marie-Tooth disease (CMT) type 4A (CMT4A) as well as AR axonal CMT with vocal cord paralysis. METHODS: The coding region of GDAP1 was screened for the presence of mutations in seven families with AR CMT in which the patients were homozygous for markers of the CMT4A locus at chromosome 8q21.1. RESULTS: A nonsense mutation was detected in exon 5 (c.581C>G, S194X), a 1-bp deletion in exon 6 (c.786delG, G262fsX284), and a missense mutation in exon 6 (c.844C>T, R282C). CONCLUSIONS: Mutations in GDAP1 are a frequent cause of AR CMT. They result in an early-onset, severe clinical phenotype. The range of nerve conduction velocities (NCV) is variable. Some patients have normal or near normal NCV, suggesting an axonal neuropathy, whereas others have severely slowed NCV compatible with demyelination. The peripheral nerve biopsy findings are equally variable and show features of demyelination and axonal degeneration.

[Advances in the molecular genetics of the hereditary neuropathies]. / Avances en la genética molecular de las neuropatías hereditarias.

OBJECTIVE: We reviewed current knowledge of the molecular and genetic bases of hereditary peripheral neuropathies, with special emphasis on the senso motor neuropathies and their different clinical phenotypes. DEVELOPMENT: The peripheral neuropathies show great clinical variability and genetic heterogeneity. To date 12 genes and over 20 genetic loci have been described in relation to Charcot Marie Tooth disease and related neuropathies. The commonest form is the type 1A Charcot Marie Tooth disease (CMT1A) caused by tandem duplication of a monomer of 1.5 megabases (Mb) on chromosome 17q11.2. The CMT 1A duplication is found in 70% of the patients with CMT 1. The deletion of 1.5 Mb is the most prevalent mutation (85%) in hereditary neuropathy with susceptibility to paralysis due to pressure. This monomer includes the PMP22 gene which is affected by a genetic dose effect. The different proteins encoded by the genes described are well expressed in the Schwann cell and in the nerve axon. They have different functions. There are the structural proteins of myelin, transcription factors, cytoskeleton components, molecular motors of the microtubules, proteins involved in growth and cellular differentiation or with presumed enzyme activity. CONCLUSIONS: Diagnosis of molecular pathology is important for genetic counselling. The development of new treatment for hereditary neuropathies is based on the generation of animal models for the different genes and on understanding the role of the proteins involved in axon Schwann cell interaction.

Avances en la genética molecular de las neuropatías hereditarias / Advances in the molecular genetics of the hereditary neuropathies

Objetivo. Se muestra una revisión del estado actual de los conocimientos sobre las bases genéticas y moleculares de las neuropatías periféricas hereditarias, haciendo especial hincapié en las neuropatías sensitivomotoras y sus distintos fenotipos clínicos. Desarrollo. Las neuropatías periféricas exhiben una gran variabilidad clínica y heterogeneidad genética. Hasta el momento se han descrito 12 genes y más de 20 loci génicos asociados a la enfermedad de Charcot-MarieTooth y neuropatías relacionadas. La forma más frecuente es la enfermedad de Charcot-Marie-Tooth tipo 1A (CMT1A), causada por la duplicación en tándem de un monómero de 1,5 megabases (Mb) en el cromosoma 17q11.2. La duplicación CMT1A se encuentra en el 70 por ciento de los enfermos de CMT1. La deleción de 1,5 Mb es la mutación más prevalente (85 por ciento) en la neuropatía hereditaria con susceptibilidad a la parálisis por presión. Este monómero incluye el gen PMP22 , que se ve afectado por un efecto de dosis génica. Las diferentes proteínas codificadas por los genes descritos se expresan en la célula de Schwann o en el axón neuronal, y tienen distintas funciones; hay proteínas estructurales de mielina, factores de transcripción, componentes del citoesqueleto, motores moleculares de los microtúbulos, y proteínas involucradas en el crecimiento y diferenciación celular, o con supuesta actividad enzimática. Conclusiones. La determinación de la patología molecular es importante para el consejo genético. El desarrollo de nuevas terapias para el tratamiento de las neuropatías hereditarias se fundamenta en la generación de modelos animales para los diferentes genes y en el conocimiento del papel de las proteínas en la interacción axón-célula de Schwann (AU)

[Results of a program of presymptomatic diagnosis of Huntington's disease: evaluation of a 6 year period]. / Resultados de un programa de diagnóstico presintomático en la enfermedad de Huntington: evaluación de un período de 6 años.

OBJECTIVE: To know the uptake of predictive testing of Huntington's disease, the characteristics of the applicants, as well as the consequences for them. METHODS: Prospective observational study between January of 1994 and December of 1999 of the predictive testing applicants who entered in the protocol consisted of: informative interview, psychiatric interview, blood extraction for molecular study, as well as outcome and follow-up interviews. RESULTS: There were 87 applicants with a 50% risk. The mean age of the applicants was 28 years (SD = 7). Thirty one per cent already had children in the moment of predictive testing. The application rate according to the estimate population with 50% risk for the Comunidad Valenciana is 13,4%. The rate varies depending on the access to the information of the population in risk, being of 24,7% when they have direct access and of 8,3% when they do not have it (p < 0,01). Forty per cent did not come to the post-outcome visit, the positive or negative result for the mutation not influencing over it. Only 6,8% had some adverse event in the six years of follow-up all being slight. CONCLUSIONS: The application rate is determined by the access to the information of the population in risk. The fulfilment of the protocol designed for presymptomatic diagnosis of Huntington's disease keeps the adverse events presentation to a minimum.

Resultados de un programa de diagnóstico presintomático en la enfermedad de Huntington: evaluación de un período de 6 años / Results of a program of presymptomatic diagnosis of Huntington's disease: evaluation of a 6 year period

FUNDAMENTO Y OBJETIVOS: Determinar la demanda y las características de los solicitantes de diagnóstico presintomático en la enfermedad de Huntington, así como sus posibles consecuencias. MATERIAL Y MÉTODO: Estudio observacional prospectivo entre enero de 1994 y diciembre de 1999 de los solicitantes de diagnóstico presintomático que entraban en el protocolo consistente en: entrevista informativa, entrevista psiquiátrica, extracción de sangre para un estudio molecular, entrevista para establecer los resultados y entrevistas de seguimiento. RESULTADOS: Solicitaron diagnóstico presintomático 87 individuos con antecedentes familiares de enfermedad de Huntington, un 61 por ciento mujeres y un 39 por ciento varones. La edad media de los solicitantes fue de 28 años (DE 7). El 31 por ciento ya tenían hijos en el momento de solicitar el diagnóstico. La tasa de solicitud según la población estimada en riesgo, del 50 por ciento para la Comunidad Valenciana, es del 13,4 por ciento. Esta tasa varía según el acceso a la información de la población en riesgo, siendo del 24,7 por ciento cuando tiene acceso directo y del 8,3 por ciento cuando no lo tiene (p < 0,01). No vuelven a la visita posresultado el 39,7 por ciento sin que influya el resultado positivo o negativo para la mutación. Sólo un 6,8 por ciento han tenido algún efecto adverso durante los 6 años de seguimiento y todos han sido leves. CONCLUSIONES: La tasa de solicitud está determinada por el acceso a la información de la población en riesgo. El seguimiento del protocolo diseñado para el diagnóstico (AU)