Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Development, behaviour and sensory processing in Marshall-Smith syndrome and Malan syndrome: phenotype comparison in two related syndromes.

BACKGROUND: Ultrarare Marshall-Smith and Malan syndromes, caused by changes of the gene nuclear factor I X (NFIX), are characterised by intellectual disability (ID) and behavioural problems, although questions remain. Here, development and behaviour are studied and compared in a cross-sectional study, and results are presented with genetic findings. METHODS: Behavioural phenotypes are compared of eight individuals with Marshall-Smith syndrome (three male individuals) and seven with Malan syndrome (four male individuals). Long-term follow-up assessment of cognition and adaptive behaviour was possible in three individuals with Marshall-Smith syndrome. RESULTS: Marshall-Smith syndrome individuals have more severe ID, less adaptive behaviour, more impaired speech and less reciprocal interaction compared with individuals with Malan syndrome. Sensory processing difficulties occur in both syndromes. Follow-up measurement of cognition and adaptive behaviour in Marshall-Smith syndrome shows different individual learning curves over time. CONCLUSIONS: Results show significant between and within syndrome variability. Different NFIX variants underlie distinct clinical phenotypes leading to separate entities. Cognitive, adaptive and sensory impairments are common in both syndromes and increase the risk of challenging behaviour. This study highlights the value of considering behaviour within developmental and environmental context. To improve quality of life, adaptations to environment and treatment are suggested to create a better person-environment fit.

A novel lamin A/C mutation in a Dutch family with premature atherosclerosis.

OBJECTIVE: We report a novel lamin A/C (LMNA) mutation, p.Glu223Lys, in a family with extensive atherosclerosis, diabetes mellitus and steatosis hepatis. METHODS: Sequence analysis of LMNA (using Alamut version 2.2), co-segregation analysis, electron microscopy, extensive phenotypic evaluation of the mutation carriers and literature comparison were used to determine the loss of function of this mutation. RESULTS: The father of three siblings died at the age of 45 years. The three siblings and the brother and sister of the father were referred to the cardiovascular genetics department, because of the premature atherosclerosis and dysmorphic characteristics observed in the father at autopsy. The novel LMNA mutation, p.Glu223Lys, was identified in the proband and his two sons. Clinical evaluation revealed atherosclerosis, insulin resistance and hypertension in the proband and dyslipidemia and hepatic steatosis in all the patients with the mutation. CONCLUSION: Based on the facts that in silico analysis predicts a possibly pathogenic mutation, the mutation co-segregates with the disease, only fibroblasts from mutation carriers show nuclear blebbing and a similar phenotype was reported to be due to missense mutations in LMNA we conclude that we deal with a pathogenic mutation. We conclude that the phenotype is similar to Dunnigan-type familial partial lipodystrophy.

Late rather than early responses of human dendritic cells highlight selective induction of cytokines, chemokines and growth factors by probiotic bacteria.

The probiotic properties of commensal bacteria including lactobacilli and bifidobacteria are likely to be determined at least in part by their effects on dendritic cells. Like traditional immune stimulants such as lipopolysaccharides (LPS), probiotic bacteria promote maturation of cultured human dendritic cells (DC) by inducing elevated expression of MHC-II and co-stimulatory molecules. Different effects have been reported on cytokine induction, especially of major regulatory cytokines such as TNF-α, IL-12 and IL-10. Yet, these previous analyses have failed to reveal consistent differences between such effects of probiotics on the one hand, and of LPS on the other. Selective response markers for probiotics, however, would be important for our understanding of their biological properties and for a rational selection of strains for in vivo studies. In this study, we compared in detail both early and late effects on cultured human DC of 4 different probiotics with those of LPS. At the early stages of stimulation, all stimuli induced qualitatively very similar responses in DC at the level of surface markers and secretion of cytokines and chemokines. A lower immune stimulatory effect was observed by Bifidobacterium animalis BB-12 as compared to lactobacilli. Late responses, on the other hand, tended to diverge. Microarray transcript profiling for 268 cytokines, chemokines, growth factors and their receptors after 2 days of culture revealed various transcripts to be selectively induced by certain probiotics but not LPS. Our data indicate that late rather than early DC responses may be helpful to clarify the divergent biological effects of probiotics on human innate immune responses.

[From gene to disease; pseudoxanthoma elasticum and the ABCC6 gene]. / Van gen naar ziekte; pseudoxanthoma elasticum en het ABCC6-gen.

Pseudoxanthoma elasticum (PXE) is a hereditary disease of the connective tissue characterized by progressive dystrophic mineralization of elastic fibres. PXE patients have skin lesions, may experience loss of visual acuity and cardiovascular complications. The inheritance pattern of PXE is almost always autosomal recessive. In less than 2% of the families, PXE may be inherited in an autosomal dominant fashion. PXE is caused by mutations in the ABCC6 (MRP6) gene. The R1141X mutation is by far the most common mutation; it has been identified in 19 patients, or 30% of all PXE-patients in the Netherlands. The molecular pathology of PXE is complicated by yet unknown factors causing a variable clinical expression of the disease. In 80% of the 110 PXE patients the authors studied, at least one ABCC6 mutation was found. Molecular diagnostics of PXE is especially useful to confirm the clinical diagnosis.

[Changing perception of hereditary eye diseases]. / Veranderende visie op erfelijke oogaandoeningen.

The authors present the cases of two parents with Usher syndrome type I who appeared to have normal offspring, and two families, one with autosomal dominant retinoblastoma and a RB1-gene mutation and one with primary open angle glaucoma and a myocilin gene mutation, in whom DNA-analysis was used to see whether check-ups were needed. The field of ophthalmogenetics comprises many disorders, both congenital and those with a later onset. Mendelian, mitochondrial, as well as multifactorial heredity is seen. Recent progress in this field, especially in molecular genetics, has created new possibilities, but some situations appear to be more complex than previously assumed. Particularly if there is genetic heterogeneity or multifactorial inheritance, possibilities for counselling and DNA analysis remain limited.

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.

An unknown combination of infantile spasms, retinal lesions, facial dysmorphism and limb abnormalities.

A female patient is presented with infantile spasms, punched-out retinal lesions, facial dysmorphism, short upper arms, short thumbs, left lower limb hypoplasia with foot deformity, a hemivertebra, atrial septal defect, growth retardation and severe developmental delay. There is some similarity to patients with Aicardi syndrome (AS), but the retinal lesions in our patient are different and she does not have agenesis of the corpus callosum, one of the diagnostic features of AS. She might represent an atypical form of this syndrome with additional features, usually not present in AS. As there is no diagnostic test for AS yet, this diagnosis cannot be confirmed nor rejected with certainty. However, it might be more likely that our patient has another, possibly unique, condition.

Mutations in ABCC6 cause pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. PXE patients frequently experience visual field loss and skin lesions, and occasionally cardiovascular complications. Histopathological findings reveal calcification of the elastic fibres and abnormalities of the collagen fibrils. Most PXE patients are sporadic, but autosomal recessive and dominant inheritance are also observed. We previously localized the PXE gene to chromosome 16p13.1 (refs 8,9) and constructed a physical map. Here we describe homozygosity mapping in five PXE families and the detection of deletions or mutations in ABCC6 (formerly MRP6) associated with all genetic forms of PXE in seven patients or families.

Tolerance controls encephalitogenicity of alphaB-crystallin in the Lewis rat.

The myelin-associated protein, alphaB-crystallin, is considered a candidate autoantigen in multiple sclerosis (MS). In the present study, we examined the potential of alphaB-crystallin to induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Attempts to induce EAE with either bovine, rat or murine alphaB-crystallin or alphaB-crystallin peptides consistently failed. Immunization with either autologous rat or murine alphaB-crystallin did not trigger any antigen-specific T cell response. Immunization with bovine alphaB-crystallin or a synthetic peptide representing the cryptic epitope 49-64 did trigger T cell responses but these failed to crossreact with autologous rat alphaB-crystallin. Examination of lymphoid tissues of the Lewis rat revealed constitutive expression of alphaB-crystallin in thymus, spleen, and peripheral lymphocytes. Our data show that in Lewis rats, constitutive lymphoid expression of alphaB-crystallin is associated with a state of nonresponsiveness to autologous alphaB-crystallin that effectively controls the development of EAE in response to this myelin antigen.

Mistaken self, a novel model that links microbial infections with myelin-directed autoimmunity in multiple sclerosis.

Several findings indicate that infectious events play a role in the pathogenesis of multiple sclerosis (MS). At the same time, T-cell autoimmunity to myelin antigens is widely believed to be crucial to the development of MS lesions. Several mechanisms have been put forward to explain the presumed link between microbial infections and myelin-directed autoimmunity. These include molecular mimicry, bystander activation including epitope spreading and superantigenic activation of T cells. Evidence that either one of these mechanisms actually occurs in MS patients, however, is still weak. Also, none of the above mechanisms explain why MS is unique to humans. We propose an alternative link between microbial infection and myelin autoimmunity, which we refer to as 'mistaken self'. In this mechanism, peripheral microbial infections of lymphoid cells prime the human T-cell repertoire not only to microbial antigens but also to the stress protein alpha B-crystallin that is expressed de novo in infected lymphoid cells. Subsequently, stress-induced accumulation of this self antigen in oligodendocytes/myelin can provoke pro-inflammatory responses as the recruited memory T-cell repertoire then mistakes the self protein for a microbial antigen. In this paper we review the currently available evidence that 'mistaken self' centering on alpha B-crystallin represents a powerful source of anti-myelin autoimmunity in a way that is unique to humans.

Severe microcephaly, choreiform movements, cataracts and sensorineural deafness in two patients: a new syndrome?

Two unrelated male patients are described with severe microcephaly, early-onset choreiform movements, cataracts, sensorineural deafness and profound developmental delay. Our patients have much in common with the three male siblings described by Tomiwa et al., who also had cataracts, deafness and developmental delay, but much less severe microcephaly and a different type of movement disorder with later onset [Tomiwa K et al. (1987). Neuropediatrics 18:231-234]. An extensive literature search did not reveal any other reports of patients with a similar condition. We discuss the differential diagnosis.

Presentation of alpha B-crystallin to T cells in active multiple sclerosis lesions: an early event following inflammatory demyelination.

In the development of multiple sclerosis (MS), (re)activation of infiltrating T cells by myelin-derived Ags is considered to be a crucial step. Previously, alpha B-crystallin has been shown to be an important myelin Ag to human T cells. Since alpha B-crystallin is an intracellular heat shock protein, the question arises at what stage, if any, during lesional development in MS this Ag becomes available for CD4+ T cells. In 3 of 10 active MS lesions, alpha B-crystallin could be detected inside phagocytic vesicles of perivascular macrophages, colocalizing with myelin basic protein and myelin oligodendrocyte glycoprotein (MOG). Although the detectability of MOG in phagosomes is considered as a marker for very recent demyelination, MOG was detected in more macrophages and in more lesions than alpha B-crystallin. The disappearance of alpha B-crystallin from macrophages even before MOG was confirmed by in vitro studies; within 6 h after myelin-uptake alpha B-crystallin disappears from the phagosomes. Alpha B-crystallin-containing macrophages colocalized with infiltrating T cells and they were characterized by expression of MHC class II, CD40, and CD80. To examine functional presentation of myelin Ags to T cells, purified macrophages were pulsed in vitro with whole myelin membranes. These macrophages activated both myelin-primed and alpha B-crystallin-primed T cells in terms of proliferation and IFN-gamma secretion. In addition, alpha B-crystallin-pulsed macrophages activated myelin-primed T cells to the same extent as myelin-pulsed macrophages, whereas myelin basic protein-pulsed macrophages triggered no response at all. These data indicate that, in active MS lesions, alpha B-crystallin is available for functional presentation to T cells early during inflammatory demyelination.

Two cases of partial trisomy 8p and partial monosomy 21q in a family with a reciprocal translocation (8;21)(p21.1;q22.3).

We report on two mentally retarded adults with an unbalanced karyotype resulting from a familial balanced translocation between chromosomes 8 and 21, t(8;21)(p21.1;q22.3). This translocation has not been reported before. Both patients had partial trisomy 8p and partial monosomy 21q. Fluorescence in situ hybridisation (FISH) was used to determine the chromosomal breakpoints more precisely. The first patient showed mild mental retardation and facial dysmorphism, slightly resembling the earlier described trisomy 8p phenotype. He did not resemble his affected niece, who was more severely retarded, had serious epilepsy, but lacked the facial dysmorphism. Comparing the data of both patients with published reports of trisomy 8p, marked differences were found between patients with an inversion duplication (inv dup) 8p, patients with partial trisomy 8p caused by an unbalanced translocation, and our patients. Inv dup(8p) causes a recognisable phenotype, whereas the phenotype of trisomy 8p resulting from a translocation is much more variable, probably because of the accompanying monosomies. However, even the same abnormal karyotype can cause different phenotypes, as our patients show. Counselling carriers of the balanced translocation in this family, a 20-25% recurrence risk for unbalanced offspring and a 25% risk for miscarriages seem appropriate.

Characterization of a de novo unbalanced translocation t(14q18q) using microdissection and fluorescence in situ hybridization.

We report on a patient with a de novo translocation between the long arms of chromosomes 14 and 18. The translocation was studied using microdissection in combination with fluorescence in situ hybridization (micro-FISH). Five copies of the chromosomes involved in the translocation were isolated by microdissection and amplified by means of degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Reverse chromosome painting with the biotin-labeled PCR product showed that part of the q-arm of chromosome 18 had no signal. The deletion was characterized further by FISH with band-specific probes and it was concluded that the rearrangement was unbalanced: 46,XY,t(14;18)(14pter-->14q22::18q21.1-->18qter) (18pter-->18q12.2::14q22-->14qter). The patient, who presented with psychomotor retardation, mild obesity, pes equinovarus, strabismus, and facial anomalies, is compared with previously reported patients with an interstitial deletion of band 18q12.

Pfeiffer syndrome type 2: further delineation and review of the literature.

We present 5 unrelated patients, 3 boys and 2 girls, with Pfeiffer syndrome (PS) type 2. They all had cloverleaf skull, severe proptosis, ankylosis of the elbows, broad thumbs and/or broad halluces and variable accompanying anomalies. We review the literature on all subtypes of PS. Most patients with PS type 2 died shortly after birth. Causes of death include pulmonary problems, brain abnormalities, prematurity and post-operative complications. DNA studies were performed in 3 of the 5 patients. Two of them showed a 1036T --> C mutation in the fibroblast growth factor receptor 2 (FGFR2) gene, that was earlier reported in PS and in Crouzon syndrome. Probably most, if not all, PS type 2 cases are caused by a de novo mutation in the FGFR2 gene or in another, yet unidentified gene. To date all type 2 cases have been non-familial. A low recurrence risk for parents can be advised.

Bladder rehabilitation, the effect of a cognitive training programme on urge incontinence.

OBJECTIVE: To assess the effectiveness of a 10-day inpatient treatment programme for persistent urge incontinence based on behavior modification via biofeedback of micturition behavior. METHODS: 95 patients aged 6-17 (86 girls, 9 boys) with documented and persistent urge incontinence, with or without dysfunctional voiding, mostly based on recurrent urinary tract infections, and at least a 1 year lasting failure of standard regimen and pharmacological therapy, were 'cognitively' treated. After 6 months the patients were evaluated for flow pattern, number of wet incidents, micturition frequency and urge compliants. RESULTS: 65 patients (68.4%) obtained good results, 12 (12.6%) showed average improvement, 18 patients (19%) did not improve. CONCLUSIONS: This cognitive noninvasive treatment programme seems promising in its effectiveness and compares favorably with existing biofeedback methods based on urodynamic procedures, although expensive by its inpatient status. Further study towards an outpatient implementation is needed.

Treatment of enuresis risoria in children by self-administered electric and imaginary shock.

OBJECTIVE: To treat enuresis risoria (giggle micturition) by a self-administered electric and imaginary shock and to evaluate the outcome after behavioural therapy. PATIENTS AND METHODS: Six boys and three girls with enuresis risoria were evaluated and treated. The mean age at referral was 10.4 years (range 5.7-14.2). All children had episodes of involuntary complete bladder emptying triggered by hearty laughter or giggling. The frequency ranged from four times per day to twice a week. No other voiding problems were noted. Five patients (four boys and one girl, mean age 10.2 years, range 5.7-14.2) received conditioning training which consisted largely of the self-administration of a harmless, painless electric shock to the back of one hand, at the moment when micturition was induced by laughter, leading to inhibition of the voiding reflex. Later, the electric shock was replaced by an imaginary shock. Training was undertaken on an outpatient basis. A mean of eight sessions, each of 45 min duration, was necessary to train the children. The mean follow-up was 26 months (range 12-51). RESULTS: The frequency of wetting was reduced by a mean of 89% in all children 1 year after the successful completion of the training. In three children, followed for 2 years, this result stabilized at that proportion. One of these children, followed for > 3 years, gradually reverted to the original pattern of daily wetting and another patient, with a follow-up of > 4 years, maintained an 86% reduction of wetting incidents. CONCLUSION: Until now, there was no specific treatment for enuresis risoria and not all patients outgrow the problem; this experimental conditioning programme shows promise in diminishing wetting incidents. However, the programme needs further testing in a prospective study.

Interstitial deletion of the short arm of chromosome 8: report of a patient and review of the literature.

We report a mentally retarded girl with minimal dysmorphic signs. Cytogenetic examination showed an interstitial deletion of chromosome 8: 46, XX, del(8)(p21.2p22). This deletion has not been reported before. We compare the patient with the literature data.

Coffin-Lowry syndrome: clinical aspects at different ages and symptoms in female carriers.

We present three patients with the Coffin-Lowry syndrome, two males aged 21 years and 14 months respectively, and an unrelated girl aged 11 years. In the male patients the features at different ages are reviewed. Besides, we describe the pertinent features of their affected female relatives. The contribution of the family history to making the diagnosis is stressed. The isolated female proband is much more severely affected than the female relatives of the male probands, demonstrating that the clinical picture in female carriers of Coffin-Lowry syndrome can vary considerably. The differential diagnosis of Coffin-Lowry syndrome will be discussed shortly.