Joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity.

BACKGROUND: Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterised by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS. METHODS: We sequenced 27 JS-associated genes in 440 affected individuals (375 families) from a cohort of 532 individuals (440 families) with JS, using molecular inversion probe-based targeted capture and next-generation sequencing. Variant pathogenicity was defined using the Combined Annotation Dependent Depletion algorithm with an optimised score cut-off. RESULTS: We identified presumed causal variants in 62% of pedigrees, including the first B9D2 mutations associated with JS. 253 different mutations in 23 genes highlight the extreme genetic heterogeneity of JS. Phenotypic analysis revealed that only 34% of individuals have a 'pure JS' phenotype. Retinal disease is present in 30% of individuals, renal disease in 25%, coloboma in 17%, polydactyly in 15%, liver fibrosis in 14% and encephalocele in 8%. Loss of CEP290 function is associated with retinal dystrophy, while loss of TMEM67 function is associated with liver fibrosis and coloboma, but we observe no clear-cut distinction between JS subtypes. CONCLUSIONS: This work illustrates how combining advanced sequencing techniques with phenotypic data addresses extreme genetic heterogeneity to provide diagnostic and carrier testing, guide medical monitoring for progressive complications, facilitate interpretation of genome-wide sequencing results in individuals with a variety of phenotypes and enable gene-specific treatments in the future.

Search for ReCQL4 mutations in 39 patients genotyped for suspected Rothmund-Thomson/Baller-Gerold syndromes.

Three overlapping conditions, namely Rothmund-Thomson (RTS), Baller-Gerold (BGS) and RAPADILINO syndromes, have been attributed to RECQL4 mutations. Differential diagnoses depend on the clinical presentation, but the numbers of known genes remain low, leading to the widespread prescription of RECQL4 sequencing. The aim of our study was therefore to determine the best clinical indicators for the presence of RECQL4 mutations in a series of 39 patients referred for RECQL4 molecular analysis and belonging to the RTS (27 cases) and BGS (12 cases) spectrum. One or two deleterious RECQL4 mutations were found in 10/27 patients referred for RTS diagnosis. Clinical and molecular reevaluation led to a different diagnosis in 7/17 negative cases, including Clericuzio-type poikiloderma with neutropenia, hereditary sclerosing poikiloderma, and craniosynostosis/anal anomalies/porokeratosis. No RECQL4 mutations were found in the BGS group without poikiloderma, confirming that RECQL4 sequencing was not indicated in this phenotype. One chromosomal abnormality and one TWIST mutation was found in this cohort. This study highlights the search for differential diagnoses before the prescription of RECQL4 sequencing in this clinically heterogeneous group. The combination of clinically defined subgroups and next-generation sequencing will hopefully bring to light new molecular bases of syndromes with poikiloderma, as well as BGS without poikiloderma.

Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy.

Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

PLP1 gene analysis in 88 patients with leukodystrophy.

Pelizaeus-Merzbacher disease (PMD) is caused in most cases by either duplications or point mutations in the PLP1 gene. This disease, a dysmyelinating disorder affecting mainly the central nervous system, has a wide clinical spectrum and its causing mutations act through different molecular mechanisms. Eighty-eight male patients with leukodystrophy were studied. PLP1 gene analysis was performed by the Multiplex Ligation-dependent Probe Amplification technique and DNA sequencing, and, in duplicated cases of PLP1, gene dosage was completed by using array-CGH. We have identified 21 patients with mutations in the PLP1 gene, including duplications, short and large deletions and several point mutations in our cohort. A customized array-CGH at the Xq22.2 area identified several complex rearrangements within the PLP1 gene region. Mutations found in the PLP1 gene are the cause of PMD in around 20% of the patients in this series.

Association of common copy number variants at the glutathione S-transferase genes and rare novel genomic changes with schizophrenia.

Copy number variants (CNVs) are a substantial source of human genetic diversity, influencing the variable susceptibility to multifactorial disorders. Schizophrenia is a complex illness thought to be caused by a number of genetic and environmental effects, few of which have been clearly defined. Recent reports have found several low prevalent CNVs associated with the disease. We have used a multiplex ligation-dependent probe amplification-based (MLPA) method to target 140 previously reported and putatively relevant gene-containing CNV regions in 654 schizophrenic patients and 604 controls for association studies. Most genotyped CNVs (95%) showed very low (<1%) population frequency. A few novel rare variants were only present in patients suggesting a possible pathogenic involvement, including 1.39 Mb overlapping duplications at 22q11.23 found in two unrelated patients, and duplications of the somatostatin receptor 5 gene (SSTR5) at 16p13.3 in three unrelated patients. Furthermore, among the few relatively common CNVs observed in patients and controls, the combined analysis of gene copy number genotypes at two glutathione S-transferase (GST) genes, GSTM1 (glutathione S-transferase mu 1) (1p13.3) and GSTT2 (glutathione S-transferase theta 2) (22q11.23), showed a statistically significant association of non-null genotypes at both loci with an additive effect for increased vulnerability to schizophrenia (odds ratio of 1.92; P=0.0008). Our data provide complementary evidences for low prevalent, but highly penetrant chromosomal variants associated with schizophrenia, as well as for common CNVs that may act as susceptibility factors by disturbing glutathione metabolism.

Clinical guide for the diagnosis and follow-up of myotonic dystrophy type 1, MD1 or Steinert's disease. / Guía clínica para el diagnóstico y seguimiento de la distrofia miotónica tipo 1, DM1 o enfermedad de Steinert.

BACKGROUND AND OBJECTIVES: Steinert's disease or myotonic dystrophy type 1 (MD1), (OMIM 160900), is the most prevalent myopathy in adults. It is a multisystemic disorder with dysfunction of virtually all organs and tissues and a great phenotypical variability, which implies that it has to be addressed by different specialities with experience in the disease. The knowledge of the disease and its management has changed dramatically in recent years. This guide tries to establish recommendations for the diagnosis, prognosis, follow-up and treatment of the complications of MD1. MATERIAL AND METHODS: Consensus guide developed through a multidisciplinary approach with a systematic literature review. Neurologists, pulmonologists, cardiologists, endocrinologists, neuropaediatricians and geneticists have participated in the guide. RECOMMENDATIONS: The genetic diagnosis should quantify the number of CTG repetitions. MD1 patients need cardiac and respiratory lifetime follow-up. Before any surgery under general anaesthesia, a respiratory evaluation must be done. Dysphagia must be screened periodically. Genetic counselling must be offered to patients and relatives. CONCLUSION: MD1 is a multisystemic disease that requires specialised multidisciplinary follow-up.

Psychopathology and personality traits in psychotic patients and their first-degree relatives.

Personality dimensions have been associated with symptoms dimensions in schizophrenic patients (SP). In this paper we study the relationships between symptoms of functional psychoses and personality dimensions in SP and their first-degree relatives (SR), in other psychotic patients (PP) and their first-degree relatives (PR), and in healthy controls in order to evaluate the possible clinical dimensionality of these disorders. Twenty-nine SP, 29 SR, 18 PP, 18 PR and 188 controls were assessed using the temperament and character inventory (TCI-R). Current symptoms were evaluated with positive and negative syndrome scale (PANSS) using the five-factor model described previously (positive [PF], negative [NF], disorganized [DF], excitement [EF] and anxiety/depression [ADF]). Our TCI-R results showed that patients had different personality dimensions from the control group, but in relatives, these scores were not different from controls. With regard to symptomatology, we highlight the relations observed between harm avoidance (HA) and PANSS NF, and between self-transcendence (ST) and PANSS PF. From a personality traits-genetic factors point of view, schizophrenia and other psychosis may be initially differentiated by temperamental traits such as HA. The so-called characterial traits like ST would be associated with the appearance of psychotic symptoms.

Salbutamol tolerability and efficacy in patients with spinal muscular atrophy type II.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by homozygous deletions or loss-of-function mutations in SMN1, which result in a degeneration of motor neurons in the spinal cord and brain stem. Even without a randomized placebo-controlled trial, salbutamol has been offered to patients with SMA in the neuromuscular clinics of most of hospitals for many years. We describe the response to salbutamol in 48 patients with SMA type II who were not taking any other medication. We investigate the changes over an eighteen-month period in motor functional scales and we analyze side effects and subjective response to treatment. Our results suggest that oral administration of salbutamol might be helpful in the maintenance of motor function in patients with SMA type II. An apparent beneficial effect was observed in functional scales of children under the age of 6, especially during the first 6 months of therapy. The majority of patients of all ages referred some kind of subjective positive effect associated with therapy intake. Salbutamol seemed safe and was well tolerated without serious side effects.

Germline promoter hypermethylation in BRCA1 and BRCA2 genes is not present in hereditary breast cancer patients.

PURPOSE: Germline promoter hypermethylation of BRCA1 and BRCA2 genes is an alternative event of gene silencing that has not been widely investigated in hereditary breast and ovarian cancer (HBOC) syndrome. METHODS: We analyzed germline BRCA promoter hypermethylation in HBOC patients with and without BRCA mutations and control subjects, using a recently developed BRCA methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. RESULTS: Neither the patients tested nor the control subjects showed germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed. CONCLUSIONS: Despite the results achieved at somatic levels by other researchers, these were not confirmed in our study at the germline level. Our results show the need to establish more predictive CpG sites in the BRCA promoter regions to optimize the MS-MLPA assay for the detection of germline hypermethylation as an effective pre-screening tool for whole-BRCA genetic analysis in HBOC, because we can not rule out the existence of germline promoter hypermethylation in BRCA.

Neuron-derived orphan receptor-1 (NOR-1) is induced by thrombin and mediates vascular endothelial cell growth.

BACKGROUND AND AIM: Neuron-derived orphan receptor-1 (NOR-1) is a transcription factor overexpressed in human atherosclerotic plaques that is involved in vascular smooth muscle cell (VSMC) proliferation. The aim of this study was to analyze the role of NOR-1 in thrombin-induced endothelial cell growth. RESULTS: Thrombin induced an early and transient up-regulation of NOR-1 in human umbilical vein endothelial cells (HUVEC). NOR-1 up-regulation by thrombin is dependent on multiple pathways, including cytosolic Ca(2+), activation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) pathways [both extracellular-regulated kinase (ERK) and p38 MAPK], and downstream activation of cAMP response element binding protein (CREB). The critical role of CREB in the induction of NOR-1 by thrombin was demonstrated using a dominant-negative of CREB. By site-direct mutagenesis we identified two CRE sites present at -79 and -53 bp in the NOR-1 promoter involved in the up-regulation of NOR-1 by thrombin. Inhibition of thrombin receptor PAR-1 abolished CREB activation, NOR-1 up-regulation and DNA synthesis (used as an index of cell proliferation). TRAP-6 mimicked both NOR-1 up-regulation and CREB activation induced by thrombin, while PPACK (an irreversible thrombin inhibitor) prevented such an effect. Direct inhibition of thrombin-induced NOR-1 up-regulation, using antisense oligonucleotides or siRNA against NOR-1, reduced DNA synthesis and endothelial cell re-growth after injury in an in vitro model of wound repair. CONCLUSIONS: These results indicate that NOR-1 up-regulation plays a key role in thrombin-induced endothelial cell growth. Strategies aimed to block NOR-1 could be useful to prevent vascular effects triggered by thrombin.

Sustained and selective attention deficits as vulnerability markers to psychosis.

The first descriptions of schizophrenia emphasized attention problems patients with schizophrenia have but recent results evidence that other psychotic disorders share them. We compared the performance in sustained and selective attention between psychotic patients (P), their healthy first degree relatives (R) and healthy volunteers (C) to prove whether these alterations could be an endophenotype of vulnerability to psychosis. We also compared the performance of schizophrenic patients (SZP) and that of patients with other functional psychoses (OP) in order to prove whether these alterations are specific of any psychotic disorder. Seventy-six P, 70 R and 39 C were included in the study. A selective attention index, comprising TMT A and B and Stroop Test, and a sustained attention index comprising the Continuous Performance Test were calculated. We conducted an univariant general linear model to compare three group performances in these indexes, with age, sex and years of education as a covariables. We found significant differences between the indexes when we compared P, R and C. No differences in performance were found between SZP and OP. Our data showed that sustained and selective attention alterations could be a vulnerability factor to psychotic disorders in general, but they were not specific of schizophrenia.

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. SUMMARY: Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

Guía clínica para el diagnóstico y seguimiento de la distrofia miotónica tipo 1, DM1 o enfermedad de Steinert / Clinical guide for the diagnosis and follow-up of myotonic dystrophy type 1, MD1 or Steinert's disease

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Complex patterns of male germline instability and somatic mosaicism in myotonic dystrophy type 1.

The genetic basis of myotonic dystrophy type 1 (DM1) is the expansion of a CTG repeat in the 3' untranslated region of DM1PK. Once into the disease range, the repeat becomes highly unstable and is biased toward expansion in both somatic and germline tissues. Intergenerational differences usually reveal an increase in allele length, concordant with the clinical anticipation characteristic of DM1, but there have also been cases with intergenerational contractions of the repeat length, accompanied by apparent anticipation. In order to gain a better understanding of this intergenerational behaviour, we have obtained semen samples from six DM males and used single molecule analyses to compare the allele distributions present in their sperm and blood with those of their offspring. We have confirmed that the male germline mutational pathway is distinct from that of the soma, but the extent of variation is highly variable from one individual to another and not obviously correlated with progenitor allele length. Nonetheless, in all cases the alleles present in the father's sperm overlap with those observed in their offspring. These data also provide further indications that the interpretation of intergenerational transmissions by standard analyses is frequently compromised by the masking of germline differences by age-dependent somatic expansion in the parent.

Frequency and stability of the myotonic dystrophy type 1 premutation.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is associated with the expansion of an unstable CTG repeat. Larger alleles are associated with a more severe form of the disease and almost always increase in length from one generation to the next, accounting for the clinical anticipation characteristic of DM1. As such, expanded alleles are rapidly lost from the population. However, the incidence of the disease appears to remain constant. It was the authors' aim to determine the frequency and germline stability of the DM1 premutation alleles that give rise to new DM1 families. METHODS: The authors measured the size of the DM1 CTG repeat in blood DNA derived from a large number of individuals in DM1 families, including distant and unaffected relatives. RESULTS: It was determined that DM1 premutation alleles can be identified both in distant relatives of DM1 probands and more rarely in unaffected spouses. These premutation alleles are not directly associated with a clinical phenotype in the carrier but are highly unstable and liable to expand in succeeding generations, particularly when transmitted by a man. In addition, the authors observed occasional expansion-biased instability of alleles within the high end of the normal size range. CONCLUSIONS: Individuals carrying premutation alleles are at high risk of having affected offspring within a limited number of generations. Such data indicate that premutation alleles cannot be the long-term source of new DM1 families, which must ultimately arise from mutations of alleles within the upper normal size range.

Prevalence of myotonic dystrophy in Guipúzcoa (Basque Country, Spain).

Prevalence figures for inherited neuromuscular disorders are important both for health care planning purposes and for evaluating the need for DNA diagnostic services for eugenic approaches. We screened for the prevalence of myotonic dystrophy (MyD) through extensive inquiry of neurologic and primary health services of Guipúzcoa (Basque Country, northern Spain) between 1989 and 1991. Typical adult-onset and neonatal cases and relatives at risk; suffering from a partial syndrome, were included. In the latter, molecular typing was performed with DNA probes close to the MyD gene to demonstrate the MyD gene carrier status. The high prevalence detected (26.5 cases per 100,000 population) could be explained by methodological factors, but intrinsic factors, such as a possible founder genetic effect or the quick growth of the Guipúzcoa population since the last century may contribute to one of the highest MyD prevalence in the world. In the future, the methodological basis for epidemiologic surveys of MyD must combine molecular technology with more-extensive family inquiries.

Anticipation is not associated with CAG repeat expansion in parent-offspring pairs of patients affected with schizophrenia.

With the rationale that a disease that presents with anticipation could be associated with expansion of trinucleotide repeats, we selected parent-offspring pairs of schizophrenia patients with earlier age at onset in the filial generation to measure the expansion of CAG repeats using the repeat expansion detection (RED) method. Intergenerational comparisons were made for age at onset, length of CAG repeats, and clinical variables. Although the patients from the filial generation became affected 13 years earlier than the parents (P < 0.0005), we did not find larger CAG repeats in the offspring. No association was found between size of CAG repeat and age at onset or with any other clinical variable. Overall, the frequency of patients with CAG repeats longer than 40 was 32%, which was similar to that observed in control subjects (27%). It is particularly noteworthy that in 86% of the pairs, the mother was the affected parent. In this Spanish sample with parent-offspring pairs presenting schizophrenia with clinical anticipation and apparent female bias of transmission, neither the phenomenon of anticipation nor disease status was associated with the expansion of CAG repeats.

Prediction of myotonic dystrophy clinical severity based on the number of intragenic [CTG]n trinucleotide repeats.

We carried out a genotype-phenotype correlation study, based on clinical findings in 465 patients with myotonic dystrophy (DM), in order to assess [CTG] repeat number as a predictive test of disease severity. Our analysis showed that the DM subtypes defined by strict clinical criteria fall into three different classes with a log-normal distribution. This distribution is useful in predicting the probability of specific DM phenotypes based on triplet [CTG] number. This study demonstrates that measurement of triplet expansions in patients' lymphocyte DNA is highly valuable and accurate for prognostic assessment.

Association study of schizophrenia with polymorphisms at six candidate genes.

Clinical studies have shown that there is a genetic contribution to the pathogenesis of schizophrenia. The molecular mechanisms of effective antipsychotic drugs and recent advances in neural development suggest that several dopamine receptor, serotonin receptor and neurotrophic factor genes might be involved in the disorder. In this study, we assessed the associations between schizophrenia and polymorphisms in the D2 and D3 dopamine receptor (DRD2, DRD3), the serotonin 2A receptor (5HTR2A), the brain-derived neurotrophic factor (BDNF), the ciliary neurotrophic factor (CNTF) and the neurotrophin-3 (NT-3) genes. Our results suggest that the polymorphisms at the DRD3, 5HTR2A, CNTF and BDNF gene loci are unlikely to make our sample more genetically susceptible to schizophrenia. However, we found significant differences in microsatellite allele frequencies between schizophrenic and control groups for DRD2 in the whole sample and for DRD2 and NT-3 only in women. Therefore, clinical differences in the presentation of schizophrenia between gender might be related to genetic factors.