Epigenetics in ENS development and Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) is a neurocristopathy caused by a failure of the enteric nervous system (ENS) progenitors derived from neural crest cells (NCCs), to migrate, proliferate, differentiate or survive to and within the gastrointestinal tract, resulting in aganglionosis in the distal colon. The formation of the ENS is a complex process, which is regulated by a large range of molecules and signalling pathways involving both the NCCs and the intestinal environment. This tightly regulated process needs correct regulation of the expression of ENS specific genes. Alterations in the expression of these genes can have dramatic consequences. Several mechanisms that control the expression of genes have been described, such as DNA modification (epigenetic mechanisms), regulation of transcription (transcription factor, enhancers, repressors and silencers), post-transcriptional regulation (3'UTR and miRNAs) and regulation of translation. In this review, we focus on the epigenetic DNA modifications that have been described so far in the context of the ENS development. Moreover we describe the changes that are found in relation to the onset of HSCR.

Atypical fundoscopic manifestation with good visual prognosis in familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

PURPOSE: Pathogenic variants in the CLDN19 gene are responsible for Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC) with ocular pathology (MIM *248190). Our objective was to delineate the ophthalmological and genetic manifestations of a patient with FHHNC and a pathogenic variant in CLDN19. CASE REPORT: A 25-year-old woman presented with renal involvement and a best-corrected visual acuity of 20/25 in the right eye and finger-counting ability in the left eye. The patient exhibited high myopia, convergent strabismus, and chorioretinal atrophic plaques in the perifoveal and peripapillary areas. We conducted a comprehensive ophthalmological examination, including refraction, fundoscopy, color and autofluorescence retinography, optical coherence tomography, and electrophysiology tests. Additionally, next-generation sequencing was performed using Illumina NextSeq500. We identified a homozygous missense variant, c.59G>A p.Gly20Asp, in the CLDN19 gene as the cause of renal and ocular manifestations. CONCLUSION: FHHNC is associated with various ocular alterations. The unique retinal disorders described in this article suggest a more favorable visual prognosis compared to those previously reported in the literature. Determining the phenotypic diversity of this disease may aid in the diagnosis and management of future cases.

[Intrauterine myelomeningocele repair: experience of the fetal medicine and therapy program of the Virgen de Rocío University Hospital]. / Corrección intrauterina de mielomeningocele: experiencia del programa de medicina y terapia fetal del Hospital Universitario Virgen del Rocío.

The most frequent form of spina bifida is myelomeningocele. There is no optimal postnatal treatment for this defect. In addition to the motor or sensory deficits, which depend on the location of the lesion, the defect is usually associated with Chiari ii malformation in affected children. Myelomeningocele has high mortality and, in up to 80% to 90% of patients, can be accompanied by hydrocephalus, which causes severe neurocognitive impairment and requires the patient to be shunted for survival. Intrauterine repair of fetal malformations employing open access through hysterotomy has become a therapeutic option due to improved anesthetic and surgical techniques and instrumentation, which have allowed this type of intervention to become relatively frequent. Anesthetic treatment should focus on both the mother and fetus and the hemodynamic factors regulating placental flow, uterine dynamics, blood loss and fetal well-being must remain well-controlled. Within our Program for Fetal Medicine and Therapy, 21 open fetal interventions have been performed: 17 EXIT procedures and 4 procedures for the intrauterine correction of fetal myelomeningocele. We describe our experience of the intrauterine repair of fetal myelomeningocele through open fetal surgery.

Contribution of RET, NTRK3 and EDN3 to the expression of Hirschsprung disease in a multiplex family.

BACKGROUND: Hirschsprung disease (HSCR) is a developmental disorder caused by a defect in the neural crest neuroblast migration process. It is considered to be a paradigm of complex disorders, with many loci contributing to manifestation of the disease. Although HSCR commonly appears as a sporadic trait, approximately 20% of HSCR cases are familial, with complex patterns of inheritance. METHOD: A multiplex HSCR family with an additive model of inheritance, in which the contribution of three genes (RET, NTRK3, EDN3) leads to the HSCR phenotype is reported. RESULTS AND DISCUSSION: The findings suggest that both RET and NTRK3 mutations acting together are necessary and sufficient for the appearance of the disease, and that the EDN3 mutation is acting as a phenotype-modifier factor in the context of this family, as two different HSCR phenotypes are seen among the affected members: a short segment form, and a total colonic aganglionosis. The results therefore support the complex additive model of inheritance previously proposed for Hirschsprung disease.

A novel point variant in NTRK3, R645C, suggests a role of this gene in the pathogenesis of Hirschsprung disease.

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the myenteric and submucosal plexuses due to a defect in the migration process of neural crest neuroblasts. Manifestation of the disease has been linked to the dysfunction of two principal signalling pathways involved in the enteric nervous system (ENS) formation: the RET-GDNF and the EDN3-EDNRB receptor systems. However, the NTF3/NTRK3 signalling pathway plays an essential role in the development of the ENS suggesting a potential role for those genes in the pathogenesis of HSCR. We have sought to evaluate the candidature of the NTRK3 gene, which encodes the TrkC receptor, as a susceptibility gene for Hirschsprung disease. Using dHPLC technology we have screened the NTRK3 coding region in 143 Spanish HSCR patients. A total of four previously described polymorphisms and 12 novel sequence variants were detected. Of note, the novel R645C mutation was detected in 2 affected siblings of a HSCR family also carrying a RET splicing mutation. Using bioinformatics tools we observed that the presence of an additional cysteine residue might implicate structural alterations in the mutated protein. We propose haploinsufficiency as the most probable mechanism for the NTRK3 R645C mutation. NTRK3 and RET mutations in this family only appear together in the HSCR patients, suggesting that they per se are necessary but not sufficient to produce the phenotype. In addition, it is quite probable that the contribution of other still unidentified modifier genes, may be responsible for the different phenotypes (length of aganglionosis) in the two affected members.

Hirschsprung disease, associated syndromes and genetics: a review.

Hirschsprung disease (HSCR, aganglionic megacolon) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. In the last decades, the development of surgical approaches has importantly decreased mortality and morbidity which allowed the emergence of familial cases. Isolated HSCR appears to be a non-Mendelian malformation with low, sex-dependent penetrance, and variable expression according to the length of the aganglionic segment. While all Mendelian modes of inheritance have been described in syndromic HSCR, isolated HSCR stands as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene with both rare coding sequence mutations and/or a frequent variant located in an enhancer element predisposing to the disease. Hitherto, 10 genes and five loci have been found to be involved in HSCR development.

LncRNA LUCAT1 as a novel prognostic biomarker for patients with papillary thyroid cancer.

In recent years, long non-coding RNAs have emerged as a novel class of regulators of cancer biological processes. While they are dysregulated in many cancer types, little is known about their expression and functional profiles. This study has been focused on the determination of the role of a specific lncRNA in papillary thyroid cancer. Quantitative reverse transcription PCR was performed to detect the expression levels of 84 lncRNAs in 61 papillary thyroid carcinoma tissues and their adjacent non-tumor tissues. The highest fold-change was obtained for lung cancer associated transcript 1 LUCAT1, and thus, this study determines the expression and biological implication of lncRNA LUCAT1 through different in vitro and ex vivo approaches in this tumor. LUCAT1 was specifically located at the cell nucleus in tumoral regions of patient tissues. Furthermore, LUCAT1 knockdown significantly reduced both cell proliferation and invasion ex vivo and induced cell-cycle arrest and apoptosis. These facts were corroborated by an enhanced expression of P21, P57, P53 and BAX, and a reduced expression of EZH2 and HDAC1. In addition, a significant decrease was observed on DNMT1 and NRF2 genes, helping to clarify the role of LUCAT1 on PTC. Our study reveals the involvement of LUCAT1 in PTC development, through acting in cell-cycle regulation, proliferation, epigenetic modifications through LUCAT1/ CDK1/ EZH2/ P57/ P21/ HDAC1/ DNMT1/ P53/ BAX axis and apoptosis, via extrinsic pathway activating caspases. These findings indicate that LUCAT1 is maybe a potential therapeutic target and molecular biomarker for PTC.

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II.

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3' end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.

Mutation profile of the MYO7A gene in Spanish patients with Usher syndrome type I.

Usher syndrome type I is the most severe form of Usher syndrome. It is an autosomal recessive disorder characterized by profound congenital sensorineural deafness, retinitis pigmentosa, and vestibular abnormalities. Mutations in the myosin VIIA gene (MYO7A) are responsible for Usher syndrome type 1B (USH1B). This gene is thought to bear greatest responsibility for USH1 and, depending on the study, has been reported to account for between 24% and 59% of USH1 cases. In this report a mutation screening of the MYO7A gene was carried out in a series of 48 unrelated USH1 families using single strand conformation polymorphism analysis (SSCP) and direct sequencing of those fragments showed an abnormal electrophoretic pattern. Twenty-five mutations were identified in 23 out of the 48 families studied (47.9%). Twelve of these mutations were novel, including five missense mutations, three premature stop codons, three frameshift, and one putative splice-site mutation. Based on our results we can conclude there is an absence of hot spot mutations in the MYO7A gene and that this gene plays a major role in Usher syndrome.

Ancestral RET haplotype associated with Hirschsprung's disease shows linkage disequilibrium breakpoint at -1249.

BACKGROUND: Hirschsprung disease (HSCR) is a complex disorder with traditional germline mutations in RET in up to 30% of familial cases and in 3% of sporadic cases in a population-based series. We have previously demonstrated that an ancestral haplotype at the 5' end of RET (haplotype 0) was strongly associated with a large subset of isolated HSCR cases and that a putative low penetrance susceptibility locus was encompassed within this ancestral haplotype, anchored by exon 2 SNP A45A. OBJECTIVE: To determine the 5' extent of the HSCR-associated ancestral haplotype by defining the linkage disequilibrium breakpoint in search for the low penetrance susceptibility locus. METHODS: Systematic screening of the region upstream of the anchoring A45A SNP, comprising RET intron 1, exon 1, and promoter in 117 population-based HSCR cases and 100 controls. Dual luciferase assay to determine differential activities between SNP combinations near a transcription start site. RESULTS: New SNP's were found which formed upstream haplotypes, anchored by A45A, in linkage disequilibrium with HSCR (2 = 76.96, p<0.00000001). Linkage disequilibrium appeared to break at the -1249C/T SNP. Further, the HSCR-associated genotype (00) was found in >60% of HSCR but only 2% of controls. Because only 2 variants, -200A>G and -196C>A, lie within the promoter region and are in proximity to the transcriptional start site (at -195), we modelled these combinations into constructs for luciferase reporter assay. The HSCR-associated SNP combination showed the lowest activity and the control-associated combination, the highest. CONCLUSIONS: Our observations seem to discard the existence of a HSCR-causing mutation as it is conceived in the traditional sense, but strengthen the idea of a specific combination of variants conferring susceptibility to the disease in a low penetrance fashion. The data derived from our functional "in vitro" studies would suggest that the HSCR-associated haplotype 0 may result in a lower level of expression of the RET gene [corrected]

Polymorphisms in the promoter regions of FAS and FASL genes as candidate genetic factors conferring susceptibility to endometriosis.

Although the pathophysiological mechanisms leading to endometriosis remain unknown, several hypothesis have been proposed, including a dysregulation of the normal apoptotic process which takes place in the endometrium. One of the apoptotic pathways playing a crucial role in the programmed cell death within the endometrium is the Fas-FasL system. In this study we have performed a case-control analysis in order to evaluate three polymorphisms located within FAS (-1377G>A and -670A>G) and FASL (-843C>T) genes, as susceptibility factors for endometriosis. We have analysed a series of women with endometriosis compared respectively to a group of women without symptoms of the disease, and to a group of confirmed unaffected women. The genotyping of the three variants was carried out by Fluorescence Resonance Energy Transfer (FRET) technology, and statistical analysis was performed using chi2 test with Yates correction. Our results show that the differences in the distribution of the polymorphic variants were not statistically significant when the group of patients was compared to the other groups. Thus, it seems to indicate that the variants here analysed are not involved in the pathogenesis of the disease in our population. However this does not let us to completely exclude such genes as potential candidates for the disease. A complete genetic analysis of the genes involved in the intricate regulatory system of the apoptosis may lead to the identification of susceptibility factors for the disease and a better understanding of its etiology.

RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR), which may be sporadic or familial, occurs in 1:5000 live births and presents with functional intestinal obstruction secondary to aganglionosis of the hindgut. Germline mutations of the RET proto-oncogene are believed to account for up to 50% of familial cases and up to 30% of isolated cases in most series. However, these series are highly selected for the most obvious and severe cases and large familial aggregations. Population based studies indicate that germline RET mutations account for no more than 3% of isolated HSCR cases. Recently, we and others have noted that specific polymorphic sequence variants, notably A45A (exon 2), are over-represented in isolated HSCR. PURPOSE: In order to determine if it is the variant per se, a combination thereof, or another locus in linkage disequilibrium which predisposes to HSCR, we looked for association of RET haplotype(s) and disease in HSCR cases compared to region matched controls. METHODS: Seven loci across RET were typed and haplotypes formed for HSCR cases, their unaffected parents, and region matched controls. Haplotype and genotype frequencies and distributions were compared among these groups using the transmission disequilibrium test and standard case-control statistic. RESULTS: Twelve unique haplotypes, labelled A-L, were obtained. The distributions of haplotypes between cases and controls (chi(11)(2) =81.4, p<<0.0001) and between cases and non-transmitted parental haplotypes were significantly different (chi(2)(11)=53.1, p<0.0001). Genotypes comprising pairs of haplotypes were formed for cases and controls. There were 38 different genotypes among cases and controls combined. Inspection of the genotypes in these two groups showed that the genotype distribution between cases and controls was distinct (chi(37)(2)=93. 8, p<<0.0001). For example, BB, BC, BD, and CD, all of which contain at least one allele with the polymorphic A45A, are prominently represented among HSCR cases, together accounting for >35% of the case genotypes, yet these four genotypes were not represented among the population matched normal controls. Conversely, AA, AG, DD, GG, and GJ, none of which contains A45A, are commonly represented in the controls, together accounting for 43% of the control genotypes, and yet they are never seen among the HSCR cases. CONCLUSIONS: Our data suggest that genotypes comprising specific pairs of RET haplotypes are associated with predisposition to HSCR either in a simple autosomal recessive manner or in an additive, dose dependent fashion.

Mutations P51U and G122E in retinal transcription factor NRL associated with autosomal dominant and sporadic retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinopathy. RP is genetically heterogeneous with autosomal dominant, autosomal recessive and X-linked forms. Autosomal dominant retinitis pigmentosa (adRP) accounts for about 20-25% of all RP cases. At least ten adRP loci have so far been mapped. However, mutations causing adRP have been identified only in four retina-specific genes: RHO (encoding rhodopsin) in approximately 20% of adRP families, peripherin/RDS (3-5% of adRP) and recently RP1 (Pierce et al., 1999, Sulivan et al., 1999) and NRL gene. Only one mutation in the NRL gene causing adRP has so far been reported (Bessant et al., 1999). Here we report a novel mutation Pro51Leu in an adRP Spanish family supporting that mutation in NRL is the cause of adRP. A second missense mutation Gly122Glu has been observed in a simplex RP patient that may represent a sporadic case of retinitis pigmentosa. Hum Mutat 17:520, 2001.

Molecular analysis of the ret and GDNF genes in a family with multiple endocrine neoplasia type 2A and Hirschsprung disease.

The clinical association between multiple endocrine neoplasia type 2 (MEN2) and Hirschsprung disease (HSCR) is infrequent. Germline mutations of the ret protooncogene are the underlying cause of the MEN2 syndromes and a proportion of cases of HSCR. In this report, we describe a new kindred in which the MEN2 and HSCR phenotypes are associated with a single C620S point mutation at one of the cysteine codons of the extracellular domain of the ret protooncogene. We also speculate about the role of a silent mutation in exon 2 of this same gene (A45A), present in a homozygous state in the patient with both MEN2A and HSCR. To investigate the contribution of GDNF to the phenotype observed in this kindred, we scanned the coding region of GDNF in the patient with MEN2/HSCR, but no mutation was found.

Mutation analysis of the RPGR gene reveals novel mutations in south European patients with X-linked retinitis pigmentosa.

The RPGR (retinitis pigmentosa GTPase regulator) gene has been shown to be mutated in 10-20% of patients with X-linked retinitis pigmentosa (XLRP), a severe form of inherited progressive retinal degeneration. A total of 29 different RPGR mutations have been identified in northern European and United States patients. We have performed mutation analysis of the RPGR gene in a cohort of 49 southern European males affected with XLRP. By multiplex SSCA and automatic direct sequencing of all 19 RPGR exons, seven different and novel mutations were identified in eight of the 49 families; these include three splice site mutations, two microdeletions, and two missense mutations. RNA analysis showed that the three splice site defects resulted in the generation of aberrant RPGR transcripts. Six of these mutations were detected in the conserved amino-terminal region of RPGR protein, containing tandem repeats homologous to the RCC1 protein, a guanine nucleotide-exchange factor for Ran-GTPase. Several exonic and intronic sequence variations were also detected. None of the RPGR mutations reported in other populations were identified in our series. Our results are consistent with the notions of heterogeneity and minority causation of XLRP by mutations in RPGR in Caucasian populations.

PCR mutagenesis-based method for generation of positive controls for SSCP analysis.

We have developed a primer-mediated PCR mutagenesis-based method for the generation of positive controls to test the sensitivity of single-strand conformation polymorphism (SSCP) or any other PCR-based mutation screening method. This technique is based on the incorporation of a third longer primer, containing a mismatched base, into the PCR along with the two wild-type primers normally used to amplify DNA fragments for SSCP analysis. The longer mismatch primer (LMP) shares the sequence of one of the wild-type primers and also contains 5 to 10 additional bases, which include the mismatched base. The resulting PCR product is identical in length and sequence to the wild-type template with the exception that the LMP base mismatch is incorporated into nearly 100% of the product. We have observed an altered SSCP mobility pattern in all cases where positive controls have been generated using this technique. We believe that the use of such in vitro-generated controls can contribute to the interpretation of band patterns and to the optimization of experimental conditions for SSCP to facilitate maximum detection of sequence variants.

Fluorescence resonance energy transfer analysis of CCR-V64I and SDF1-3'a polymorphisms: prevalence in southern Spain hiv type 1+ cohort and noninfected population.

The relationship between host genotype and AIDS, as well as the different genotype frequencies observed in different populations, have become important topics in HIV research. Therefore, the development of methods that provide faster and reliable results may contribute to further development and knowledge of those topics. We present the results of genotyping SDF1-3'A and CCR2-V64I in 440 HIV-1-infected people and 100 noninfected controls from southern Spain, using a novel method based on real-time PCR with LightCycler technology and fluorescence resonance energy transfer. Frequencies obtained were 23.8% for SDF1-3'A and 9.5% for CCR2-V64I for both HIV+ cohort and general population. Both polymorphisms are in accordance with the Hardy-Weinberg equilibrium law and no differences between patients and controls have been observed.

Megalocornea-mental retardation syndrome: an additional case.

In 1975, Neuhaüser (Z Kinderheilk 120:1-8) reported on a recessively inherited entity comprising mental retardation, megalocornea, and seizures. The megalocornea-mental retardation (MMR) syndrome (MIM 249310) is a rare entity. There have been 19 previously published cases and the clinical differences observed between reported patients have raised questions regarding the nosology of the syndrome and the issue of heterogeneity versus variability. We report on a new case: a 2 6/12-year-old boy, first child of nonconsanguineous healthy parents with megalocornea (corneal diameter > or = 13 mm), delayed psychomotor development and hypotonia, plus minor facial anomalies.

G106R rhodopsin mutation is also present in Spanish ADRP patients.

A large family affected with autosomal dominant retinitis pigmentosa (ADRP) with a sectorial phenotype showed a previously described (G to A) mutation in the rhodopsin gene resulting in the substitution of a glycine residue by an arginine in codon 106 of rhodopsin. This mutation shows some unusual characteristics, such as initial pathology of the inferior retina, superior visual field with normal disc and retinal vessels, and ERG findings that show a modest reduction in both cone and rod amplitudes with normal implicit times. The Gly 106 Arg mutation has been previously reported in American and British patients. Its presence in a Spanish ADRP family confirms that it and its homogeneous associated phenotype are geographically widespread.

Prevalence of 2314delG mutation in Spanish patients with Usher syndrome type II (USH2).

The Usher syndrome (USH) is a group of autosomal recessive diseases characterized by congenital sensorineural hearing loss and retinitis pigmentosa. Three clinically distinct forms of Usher syndrome have so far been recognized and can be distinguished from one another by assessing auditory and vestibular function. Usher syndrome type II (USH2) patients have congenital moderate-to-severe nonprogressive hearing loss, retinitis pigmentosa, and normal vestibular function. Genetic linkage studies have revealed genetic heterogeneity among the three types of USH, with the majority of USH2 families showing linkage to the USH2A locus in 1q41. The USH2A gene (MIM 276901) has been identified: three mutations, 2314delG, 2913delG, and 4353-54delC, were initially reported in USH2A patients, the most frequent of which is the 2314delG mutation. It has been reported that this mutation can give rise to typical and atypical USH2 phenotypes. USH2 cases represent 62% of all USH cases in the Spanish population, and 95% of these cases have provided evidence of linkage to the USH2A locus. In the present study, the three reported mutations were analyzed in 59 Spanish families with a diagnosis of USH type II. The 2314delG was the only mutation identified in our population: it was detected in 25% of families and 16% of USH2 chromosomes analyzed. This study attempts to estimate the prevalence of this common mutation in a homogeneous Spanish population.