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Over a relatively short period of time, the clinical geneticist’s “toolbox” has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

Background: Guanylin (GN) and uroguanylin (UGN) are endogenous ligands for the intestinal receptor guanylate cyclase C (GC-C), an important regulator of intestinal fluid homeostasis. Gene expression and protein levels of GN are suppressed in inflamed intestinal tissue from patients with inflammatory bowel disease (IBD), but knowledge about plasma levels of guanylins in these conditions is sparse. We aimed to investigate the fasting plasma levels of the prohormones proGN and proUGN in patients with Crohn’s Disease (CD) and relate these to levels found in persons with other diarrheal conditions, as well as persons with normal bowel habits. Methods: Plasma from patients with CD, patients with Familial GUCY2C Diarrheal Disease (FGDS), diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls (HC) was analyzed using ELISA assays. Results: Significantly lower fasting plasma levels of proguanylins were found in CD and FGDS patients, compared to HC. In CD patients, plasma proGN levels correlated negatively with Harvey Bradshaw Index and with number of stools/24 h. Conclusion: Our data indicate that diarrhea may be a determinant for levels of proGN in plasma, and should be further explored in studies of different diarrheal disorders. acceptedVersion

Rare sequence variants in the non-coding part of the BRCA genes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1 c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant’s effect on mRNA splicing. Mean age at breast (n = 12) and ovarian (n = 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1 c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1 c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1 c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.

Background With 25% prevalence of Crohn's disease, Familial GUCY2C diarrhea syndrome (FGDS) is a monogenic disorder potentially suited to study initiating factors in inflammatory bowel disease (IBD). We aimed to characterize the impact of an activating GUCY2C mutation on the gut microbiota in patients with FGDS controlling for Crohn's disease status and to determine whether changes share features with those observed in unrelated patients with IBD. Methods Bacterial DNA from fecal samples collected from patients with FGDS (N = 20), healthy relatives (N = 11), unrelated healthy individuals (N = 263), and IBD controls (N = 46) was subjected to sequencing of the V3-V4 region of the 16S rRNA gene to determine gut microbiota composition. Food frequency questionnaires were obtained from patients with FGDS and their relatives. Results Compared with healthy controls, FGDS displayed prominent changes in many microbial lineages including increase in Enterobacteriaceae, loss of Bifidobacterium and Faecalibacterium prausnitzii but an unchanged intraindividual (alpha) diversity. The depletion of F. prausnitzii is in line with what is typically observed in Crohn's disease. There was no significant difference in the dietary profile between the patients and related controls. The gut microbiota in related and unrelated healthy controls was also similar, suggesting that diet and familial factors do not explain the gut microbiota alterations in FGDS. Conclusions The findings support that the activating mutation in GUCY2C creates an intestinal environment with a major influence on the microbiota, which could contribute to the increased susceptibility to IBD in patients with FGDS.

Contains fulltext : 174194.pdf (Publisher’s version ) (Open Access) Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.

Rare sequence variants in the non-coding part of the BRCA genes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1 c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant’s effect on mRNA splicing. Mean age at breast (n = 12) and ovarian (n = 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1 c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1 c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1 c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.

Background: Reported concentrations of serotonin in platelet-poor plasma (PPP) in healthy subjects vary widely due to different pre-analytical procedures. Aim: To examine how different pre-analytical conditions affect the measured concentration of serotonin in PPP. Method: Six pre-analytical protocols were compared for preparation of PPP from EDTA whole blood for quantification of serotonin from nine healthy individuals. Three combinations of centrifugation with a mild centrifugation of gel-free EDTA tubes followed by a stronger centrifugation were compared to single-stage centrifugation of EDTA tubes with separator gel and heat shock treatment of blood prior to centrifugation. All samples were analysed using the same enzyme linked immunosorbent assay (ELISA) method. Results: Findings show that two consecutive centrifugations; first a mild centrifugation at 100 or 200×g followed by centrifugation at 4500 or 14500×g resulted in the lowest serotonin concentration in PPP. Conclusion: Two successive centrifugations to produce PPP for serotonin analysis; first a mild centrifugation to avoid mechanical stress on the platelets, and next a stronger centrifugation to remove platelets, is superior to the use of gel tubes and heat shock treatment. Keywords: Serotonin, 5-HT, 5-Hydroxytryptamine, Plasma, Platelet-poor plasma, Pre-analytical procedure

Familial GUCY2C diarrhoea syndrome (FGDS) is caused by an activating mutation in the GUCY2C gene encoding the receptor guanylate cyclase C in enterocytes. Activation leads to increased secretion of fluid into the intestinal lumen. Twenty percent of the patients have increased risk of Crohn's disease and intestinal obstruction (CD, 20%) and the condition resembles irritable bowel syndrome with diarrhoea. We aimed to describe fluid content, contractility, peristaltic activity and bowel wall thickness in the intestine in fasting FGDS patients, using ultrasound, with healthy volunteers serving as controls.Twenty-three patients with FGDS and 22 healthy controls (HC) were examined with a Logiq E9 scanner in a fasting state. Bowel wall thickness was measured and fluid-filled small bowel loops were counted using three-dimensional (3D) magnetic positioning navigation. The HC ingested 500 ml PEG solution, an electrolyte balanced, non-absorbable solution, in order to investigate the contractions of the small bowel.The fasting 23 FGDS patients had significantly higher number of fluid-filled small bowel segments compared to 22 fasting HC, p 0.001. A high number of non-occlusive contractions in the ileum was observed, which was significant when compared to HC after ingesting PEG solution, p 0.016. An increase in intestinal wall thickness or other signs of CD were not observed.FGDS is characterised by multiple, fluid-filled small bowel loops with incomplete contractions and fluid stagnation in fasting state. These findings may play a role in the increased risk of bowel obstruction as well as IBS-like symptoms observed in these patients.

Background Mitochondria play an important role in iron metabolism and haematopoietic cell homeostasis. Recent studies in mice showed that a mutation in the catalytic subunit of polymerase gamma (POLG) was associated with haematopoietic dysfunction including anaemia. The aim of this study was to analyse the frequency of anaemia in a large cohort of patients with POLG related disease. Methods We conducted a multi-national, retrospective study of 61 patients with confirmed, pathogenic biallelic POLG mutations from six centres, four in Norway and two in the United Kingdom. Clinical, laboratory and genetic data were collected using a structured questionnaire. Anaemia was defined as an abnormally low haemoglobin value adjusted for age and sex. Univariate survival analysis was performed using log-rank test to compare differences in survival time between categories. Results Anaemia occurred in 67% (41/61) of patients and in 23% (14/61) it was already present at clinical presentation. The frequency of anaemia in patients with early onset disease including Alpers syndrome and myocerebrohepatopathy spectrum (MCHS) was high (72%) and 35% (8/23) of these had anaemia at presentation. Survival analysis showed that the presence of anaemia was associated with a significantly worse survival (P = 0.004). Conclusion Our study reveals that anaemia can be a feature of POLG-related disease. Further, we show that its presence is associated with significantly worse prognosis either because anaemia itself is impacting survival or because it reflects the presence of more serious disease. In either case, our data suggests anaemia is a marker for negative prognosis.

Introduction Increased intestinal hydration by activation of the epithelial enzyme linked receptor guanylate cyclase C (GC-C) is a pharmacological principle for treating constipation. Activating mutations in the GUCY2C gene encoding GC-C cause Familial GUCY2C diarrhea syndrome (FGDS) which has been diagnosed with severe dysmotility. Aim To investigate gut motility and hormones before and after a meal in FGDS patients and compare with healthy controls (HC). Subjects and methods Bristol stool chart and stool frequency was assessed. Before and after a meal occlusive and non-occlusive contractions were obtained using ultrasound. A wireless motility capsule (WMC) recorded gut transit time, pH, contractions and pressure. Plasma levels of selected gut hormones were measured at different time points. Results The FGDS patients had 4 (range 1–10) loose stools/day and prolonged total gut transit time compared to HC, 55.5 h vs 28.5 h, respectively,with significantly increased colon transit time. In FGDS patients, pH in duodenum, small bowel and colon was increased and the number of contractions and the intraluminal pressure were significantly decreased, measured by WMC. Ultrasound showed in small bowel increased number of non-occlusive contractions in the FGDS patients. Serotonin (5-HT) plasma levels in the HC peaked 30 min after the meal, while the FGDS patients had no response. Conclusion Despite having diarrhea, the FGDS patients have prolonged transit time through the gut compared to HC, particularly in colon. The reduced number of intestinal contractions and lack of 5-HT release after a meal in FGDS patients surprisingly resemble colonic motility disturbances seen in patients with constipation.

The objective of this study was to study the prevalence of gastrointestinal (GI) symptoms and histopathology in patients with common variable immunodeficiency (CVID) as well as linking the findings to GI infections and markers of systemic immune activation.In this cross-sectional study, we addressed GI symptoms in 103 patients and GI histopathological findings in 53 patients who underwent upper and lower endoscopic examination. The most frequent histopathological findings were linked to GI symptoms, B-cell phenotype, and markers of systemic immune activation (soluble (s)CD14, sCD25, and sCD163). Microarray analysis compared "celiac-like disease" in CVID to celiac disease. Screening for selected bacterial and viral infections in fecal samples and gut mucosal biopsies was performed.The main findings of this study were as follows: most common GI symptoms were bloating (34%), pain (30%), and diarrhea (26%). The most frequent histopathological findings were increased intraepithelial lymphocytes in the descending part of the duodenum, i.e., "celiac-like disease" (46% of patients), decreased numbers of plasma cells in GI tract mucosa (62%), and lymphoid hyperplasia (38%), none of which were associated with GI symptoms. Reduced plasma cells in GI mucosa were associated with B-cell phenotypic characteristics of CVID, and increased serum levels of sCD14 (P=0.025), sCD25 (P=0.01), and sCD163 (P=0.04). Microarray analyses distinguished between CVID patients with "celiac-like disease" and celiac disease. Positive tests for bacterial and viral infections were scarce both in fecal samples and gut mucosal biopsies, including PCR test for norovirus in biopsy specimens (0 positive tests).In conclusion, GI pathology is common in CVID, but does not necessarily cause symptoms. However, reduced plasma cells in GI mucosa were linked to systemic immune activation, "celiac-like disease" in CVID and true celiac disease appear to be different disease entities, as assessed by gene expression, and infections (including norovirus) are rarely a cause of the CVID enteropathy.

Motivation: The search for causative genetic variants in rare diseases of presumed monogenic inheritance has been boosted by the implementation of whole exome (WES) and whole genome (WGS) sequencing. In many cases, WGS seems to be superior to WES, but the analysis and visualization of the vast amounts of data is demanding. Results: To aid this challenge, we have developed a new tool—RareVariantVis—for analysis of genome sequence data (including non-coding regions) for both germ line and somatic variants. It visualizes variants along their respective chromosomes, providing information about exact chromosomal position, zygosity and frequency, with point-and-click information regarding dbSNP IDs, gene association and variant inheritance. Rare variants as well as de novo variants can be flagged in different colors. We show the performance of the RareVariantVis tool in the Genome in a Bottle WGS data set. Availability and implementation: https://www.bioconductor.org/packages/3.3/bioc/html/RareVariantVis.html Contact: tomasz.stokowy@k2.uib.no Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

Posttranslationally glycosylated proteins are important in many biological processes in humans and Congenital disorders of glycosylation (CDGs) are associated with a broad range of phenotypes. Type I CDGs are a group of rare autosomal recessive conditions. To date 17 subtypes have been enzymatically and molecularly characterized. Impaired function of the enzyme dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase encoded by the ALG8 gene, causes ALG8-CDG (CDG-Ih, OMIM #608104). This enzyme facilitates the transfer of a second glucose molecule to a growing lipid-linked oligosaccharide chain, a process that transpires in the endoplasmic reticulum (ER). We present a female patient of consanguineous parents, with pre- and postnatal growth retardation, dysmorphic features, significant developmental delay, visual impairment and an electrophoretic serum transferrin pattern indicative of a type I CDG. Type I CDG subgroup was determined by exome sequencing facilitated by homozygosity analysis. The patient was homozygous for two variants, nine nucleotides apart, in exon 8 of ALG8; c.799T > C [p.Ser267Pro] and c.808T > C [p.Phe270Leu]. Both missense mutations are predicted to affect a conserved region of an intraluminal ER loop of dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase. To our knowledge, the current report describes the ninth published case of ALG8-CDG, contributing to the further delineation of this rare and variable disorder.

Item does not contain fulltext A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Background The trichorhinophalangeal syndrome (TRPS) is a hereditary, skeletal dysplasia which has a characteristic clinical presentation and is classified in types 1, 2 and 3, based on phenotype and genotype. Typical findings may be mild and many patients probably remain undiagnosed. Material and methods The paper is based on four case reports and provides a short review of the condition. Results Our four patients all have typical facial features, such as a large nose and thin upper lip, thin hair and short curved fingers with characteristic radiological findings. The condition is autosomal dominant and caused by a mutation in the TRPS1 gene, which codes a gene-regulating protein involved in development of hair and modulation of chondrocytes. The diagnosis can be based on clinical findings, but DNA-analysis can be of help in unclear situations. Two of our patients were diagnosed from clinical and radiological findings, but for the two others genetic examinations were done as well. There is no causal treatment, but the diagnosis can give patients an explanation of their problems, and genetic counseling for the patient and family can be offered. Orthopedic surgery and cosmetic aids are valuable for many. Interpretation In an increasingly technified medical daily life, the clinical view is still the most important tool in diagnosing patients with this condition.

tery stenosis. A kidney biopsy was performed, and considered nondiagnostic, and a new biopsy was performed in 1994. During the following years, renal function gradually deteriorated and she underwent successful deceased donor kidney transplantation in 2007. Patient 2, the daughter of patient 1, was referred to a nephrologist in 1999 at the age of 31. At that stage she had minimal proteinuria (0.4 g/24 h), no hematuria, a normal serum creatinine Biography

Objective: Clinical and genetic characterization of a neurologic disorder resembling Refsum disease in a Norwegian consanguineous family. Methods: The affected individuals comprise a brother and sister and their third cousin. The family comes from a small island community and genealogic studies showed that both sets of parents are descendants of a man born in 1585. Based on the hypothesis that this is an autosomal recessive disease and that the patients were homozygous for the same mutation (identical by descent), we used homozygosity mapping to define the genetic locus of this disorder. Results: This slowly progressive disorder starts in childhood with signs of peripheral neuropathy (pes cavus, tendoachilles contracture). Hearing loss and cataract become evident in the third decade. Subsequently, patients develop a disorder of gait due to the combination of ataxia and spasticity, and a pigment retinopathy. While the clinical picture is reminiscent of Refsum disease, affected individuals have normal phytanic and pristanic acid levels in plasma, as well as normal enzymatic activity for α-oxidation. We mapped the disease to a 15.96 Mb region on chromosome 20 (20p11.21-q12), containing approximately 200 genes (maximum lod score = 6.3). Sequencing of 23 candidate genes failed to demonstrate detrimental sequence variants. Conclusions: Our findings show that the clinical syndromes that include Refsum disease are more heterogeneous than previously recognized. We have chosen to report the clinical features and mapping of this novel disorder in the hope that this will permit identification of other families and thus proper genetic characterization.

Background Friedreich ataxia is an autosomal recessive hereditary spinocerebellar disorder, characterized by progressive limb and gait ataxia due to proprioceptive loss, often complicated by cardiomyopathy, diabetes and skeletal deformities. Friedreich ataxia is the most common hereditary ataxia, with a reported prevalence of 1:20 000 – 1:50 000 in Central Europe. Previous reports from south Norway have found a prevalence varying from 1:100 000 – 1:1 350 000; no studies are previously done in the rest of the country. Methods In this cross-sectional study, Friedreich ataxia patients were identified through colleagues in neurological, pediatric and genetic departments, hospital archives searches, patients’ associations, and National Centre for Rare Disorders. All included patients, carriers and controls were investigated clinically and molecularly with genotype characterization including size determination of GAA repeat expansions and frataxin measurements. 1376 healthy blood donors were tested for GAA repeat expansion for carrier frequency analysis. Results Twenty-nine Friedreich ataxia patients were identified in Norway, of which 23 were ethnic Norwegian, corresponding to a prevalence of 1:176 000 and 1:191 000, respectively. The highest prevalence was seen in the north. Carrier frequency of 1:196 (95 % CI = [1:752–1:112]) was found. Homozygous GAA repeat expansions in the FXN gene were found in 27/29, while two patients were compound heterozygous with c.467 T

SummaryThe autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Contains fulltext : 152770.pdf (Publisher’s version ) (Open Access) We describe a three-generation Norwegian family with a novel X-linked intellectual disability (XLID) syndrome characterized by subtle facial dysmorphism, autism and severe feeding problems. By exome sequencing we detected a rare missense variant (c.1067A>G, p.(Tyr356Cys)) in the RLIM gene, in two affected male second cousins. Sanger sequencing confirmed the presence of the variant in the four affected males (none of whom were siblings) and in three mothers available for testing. The variant was not present in 100 normal Norwegian controls, has not been reported in variant databases and is deleterious according to in silico prediction tools. The clinical phenotype and the variant co-segregate, yielding a LOD score of 3.0 for linkage to the shared region (36.09 Mb), which contains 242 genes. No other shared rare variants on the X chromosome were detected in the two affected exome-sequenced individuals, and all female carriers had an extremely skewed X-chromosome inactivation pattern. RLIM encodes RING zinc finger protein 12 (RNF12), an ubiquitin ligase that is essential for X inactivation in mice and that acts as a co-regulator of a range of transcription factors, particularly those containing a LIM homeodomain. Tyrosine in position 356 in RNF12 is located within a highly conserved domain essential for binding such transcription factors. Expression of RNF12 is widespread during embryogenesis, and is particularly high in the outer layers of the cerebral cortex. Functional studies are needed to prove a definite causal relationship between the variant and the phenotype. Subsequent reports may confirm a role for RLIM variants in patients with XLID.

Frontometaphyseal dysplasia is an X-linked trait primarily characterized by a skeletal dysplasia comprising hyperostosis of the skull and modeling anomalies of the tubular bones. Extraskeletal features include tracheobronchial, cardiac, and urological malformations. A proportion of individuals have missense mutations or small deletions in the X-linked gene, FLNA. We report here our experience with comprehensive screening of the FLNA gene in a group of 23 unrelated probands (11 familial instances, 12 simplex cases; total affected individuals 32) with FMD. We found missense mutations leading to substitutions in the actin-binding domain and within filamin repeats 9, 10, 14, 16, 22, and 23 of filamin A in 13/23 (57%) of individuals in this cohort. Some mutations present with a male phenotype that is characterized by a severe skeletal dysplasia, cardiac, and genitourinary malformations that leads to perinatal death. Although no phenotypic feature consistently discriminates between females with FMD who are heterozygous for FLNA mutations and those in whom no FLNA mutation can be identified, there is a difference in the degree of skewing of X-inactivation between these two groups. This observation suggests that locus heterogeneity may exist for this disorder.

Background A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism. Methods and results With a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 – Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP’s ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients’ fibroblasts compared to controls. Conclusions These results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease. Electronic supplementary material The online version of this article (doi:10.1186/s13023-014-0146-0) contains supplementary material, which is available to authorized users.

We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h-1), followed by reduction of CH3Cbl (to < 20%). Both enzyme activity and cofactor levels recovered rapidly when the cells were subsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there was a reduction of cellular AdoCbl and holo-MCM activity (measured in the absence of exogenous AdoCbl) to about 50% of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These findings confirm irreversible damage of MS by nitrous oxide; hence, synthesis of the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger hydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH3Cbl are associated with reversible changes in both MCM holoactivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl cofactors during depletion and repletion.

We compared the metabolic response of a methionine(Met)-dependent (P60) human glioma cell line with that of a Met-independent variant (P60H) when cultured in a homocysteine (Hcy) medium and exposed to N2O. In Hcy medium (without Met), remethylation of Hcy in P60H cells was enhanced and supported growth, whereas remethylation was low in P60 cells, which failed to thrive under these conditions. Both cell types seemed to contain adequate amounts of folates and total cobalamin (Cbl). P60 cells showed increased total and methylcobalamin (CH3Cbl) content after the shift to a Hcy medium, but the high, stable level of CH3Cbl detected in P60H cells was not attained. Further metabolic differences were induced by N2O exposure, which markedly reduced Met-synthase activity in cell-free extracts in both cell lines and completely blocked intact-cell Hcy remethylation in P60, whereas Hcy remethylation was only partly inhibited in P60H cells cultured in Met medium. The residual Hcy remethylation in P60H cells may be related to only a moderate depletion of CH3Cbl. The resulting high CH3Cbl level relative to Met-synthase activity during N2O exposure was even higher in Hcy medium. These findings in P60H cells probably reflect increased provision of Cbl to support Hcy remethylation under metabolic strain. The inability of P60 to furnish CH3Cbl to the enzyme may explain both the Met-dependent phenotype and the increased sensitivity of Hcy remethylation to N2O exposure in these cells.

Plasma total homocysteine (tHcy) determination is used in the diagnosis of homocystinuria, in cobalamin and folate deficiency and in cardiovascular risk assessment. However, determination of tHcy includes many pitfalls which complicate the assessment of homocysteine status. In the present article, we review basic knowledge for a rational use of plasma tHcy in diagnostic as well as scientific work. The subjects dealt with are procedures for sample handling and processing, the principles of tHcy analyses, and genetic and acquired determinants of the plasma tHcy concentration.

We investigated total, free and protein-bound plasma homocysteine, cysteine and cysteinylglycine in 13 subjects aged 24-29 y after a breakfast at 0900 h containing 15-18 g of protein and a dinner at 1500 h containing approximately 50 g of protein. Twelve subjects had normal fasting homocysteine (mean +/- SD, 7.6 +/- 1.1 mumol/L) and methionine concentrations (22.7 +/- 3.5 mumol/L) and were included in the statistical analyses. Breakfast caused a small but significant increase in plasma methionine (22.2 +/- 20.6%) and a brief, nonsignificant increase followed by a significant decline in free homocysteine. However, changes in total and bound homocysteine were small. After dinner, there was a marked increase in plasma methionine by 16.7 +/- 8.9 mumol/L (87.9 +/- 49%), which was associated with a rapid and marked increase in free homocysteine (33.7 +/- 19.6%, 4 h after dinner) and a moderate and slow increase in total (13.5 +/- 7.5%, 8 h) and protein-bound (12.6 +/- 9.4%, 8 h) homocysteine. After both meals, cysteine and cysteinylglycine concentrations seemed related to changes in homocysteine, because there were parallel fluctuations in the free:bound ratios of all three thiols. Dietary changes in plasma homocysteine will probably not affect the evaluation of vitamin deficiency states associated with moderate to severe hyperhomocysteinemia but may be of concern in the risk assessment of cardiovascular disease in patients with mild hyperhomocysteinemia. Synchronous fluctuations in the free:bound ratio of the plasma aminothiol compounds indicate that biological effects of homocysteine may be difficult to separate from effects due to associated changes in other aminothiol compounds.

The trichorhinophalangeal syndrome (TRPS) is a hereditary, skeletal dysplasia which has a characteristic clinical presentation and is classified in types 1, 2 and 3, based on phenotype and genotype. Typical findings may be mild and many patients probably remain undiagnosed.The paper is based on four case reports and provides a short review of the condition.Our four patients all have typical facial features, such as a large nose and thin upper lip, thin hair and short curved fingers with characteristic radiological findings. The condition is autosomal dominant and caused by a mutation in the TRPS1 gene, which codes a gene-regulating protein involved in development of hair and modulation of chondrocytes. The diagnosis can be based on clinical findings, but DNA-analysis can be of help in unclear situations. Two of our patients were diagnosed from clinical and radiological findings, but for the two others genetic examinations were done as well. There is no causal treatment, but the diagnosis can give patients an explanation of their problems, and genetic counseling for the patient and family can be offered. Orthopedic surgery and cosmetic aids are valuable for many.In an increasingly technified medical daily life, the clinical view is still the most important tool in diagnosing patients with this condition.

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked disease caused by truncating mutations in WTX. Females exhibit sclerotic striations on the long bones, cranial sclerosis, and craniofacial dysmorphism. Males with OSCS have significant skeletal sclerosis, do not have striations but do display a more severe phenotype commonly associated with gross structural malformations, patterning defects, and significant pre- and postnatal lethality. The recent description of mutations in WTX underlying OSCS has led to the identification of a milder, survivable phenotype in males. Individuals with this presentation can have, in addition to skeletal sclerosis, Hirschsprung disease, joint contractures, cardiomyopathy, and neuromuscular anomalies. A diagnosis of OSCS should be considered in males with macrocephaly, skeletal sclerosis that is most marked in the cranium and the absence of metaphyseal striations. The observation of striations in males may be indicative of a WTX mutation in a mosaic state supporting the contention that this sign in females is indicative of the differential lyonization of cells in the osteoblastic lineage.

The kinetics of plasma homocysteine were determined in 13 healthy subjects after peroral administration and in one person after intravenous injection. Various forms of homocysteine completely dissolved in an aqueous solution were rapidly absorbed after peroral administration, and the bioavailability was estimated to be 0.53. The volume of distribution was 0.66 L/kg. The area under the plasma concentration curve (AUC0-48 h) was proportional to the administered dose (33.5-134 mumol/kg body wt), and showed small interindividual variations. Plasma homocysteine showed first-order elimination kinetics for at least 6 h. The half-life (t1/2) was 223 +/- 45 min, and there was a significant correlation between t1/2 values determined on two different occasions in the same individual. The transient hyperhomocysteinemia was associated with an increase in plasma methionine, which probably reflects intracellular remethylation of homocysteine. Less than 2% of the administered homocysteine dose was recovered in the urine. These findings may form the basis for future studies on the regulation of plasma homocysteine in health and disease, and should motivate the evaluation of a homocysteine loading test as a diagnostic tool.

We administered reduced L-homocysteine perorally (67 mumol/kg of body wt) to 12 healthy subjects and injected the same dose into one person, and determined the kinetics of the alterations in reduced, oxidized, and protein-bound concentrations of homocysteine, cysteine, and cysteinylglycine. After oral intake, reduced homocysteine increased rapidly (tmax < or = 15 min), reaching concentrations [3.97 (SD 2.99) mumol/L] 20-fold above fasting values, and then declined towards the normal concentration within 2 h. There was a similar increase in reduced cysteine and a moderate increase in reduced cysteinylglycine. During this response, we observed a positive correlation between the reduced/total ratio for homocysteine and cysteine. When homocysteine was injected, the increase in reduced homocysteine preceded the increase in reduced cysteine by about 3 min. After oral loading, oxidized homocysteine showed a transient increase (tmax = 30 min) that lagged behind the increase of reduced homocysteine. Oxidized cysteine and cysteinylglycine were stable or decreased slightly. Protein-bound homocysteine increased the least rapidly after homocysteine administration (tmax = 1-2 h), and returned to normal values slowly. Changes in protein-bound homocysteine essentially mirrored a concurrent decrease in protein-bound cysteine, suggesting displacement of bound cysteine. These data show that plasma homocysteine has a pronounced, direct effect on the redox status and protein binding of other plasma thiol components. Such effects should be recognized when studying the mechanisms behind the atherogenic effect of increased plasma homocysteine.

The present study describes the effects of 2 folate antagonists, methotrexate (MTX) and the lipophilic antifolate trimetrexate (TMX) on 2 permanent human glioma cell lines (GaMg and D-54Mg) grown as monolayers and as multicellular tumor spheroids. In addition, the effects of drug exposure on tumor cell invasion was studied using a three-dimensional organ co-culture system. In monolayer cultures, TMX was a more potent inhibitor of cell growth than MTX, especially towards the GaMg cell line. The 2 drugs, however, showed similar cytotoxicity as assessed by the plating efficiency assay. Reduced ability of directional migration of cells on a plastic surface was seen by either antifolate usually at concentrations to 10-fold higher than those exerting a cytotoxic effect in the plating efficiency assay. TMX was somewhat more potent than MTX as an inhibitor of spheroid growth. When tumor spheroids were exposed to MTX or TMX at concentrations that caused 65 to 70% inhibition of cell migration, there was a latent period of 4 to 5 days before inhibition of spheroid growth ensued. Invasion was investigated in a co-culture system, where tumor spheroids were confronted with fetal rat brain cell aggregates. Neither drug reduced tumor cell invasion, although histological examination revealed toxic effects both in GaMg and in D-54Mg spheroids. We conclude that spheroids from human glioma cells were less sensitive to the antifolates than monolayers. For both drugs a latency period was observed before inhibition of spheroid growth. The spheroids retained their ability to invade normal brain tissue when exposed to levels of folate antagonists inhibiting spheroid growth.

We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sample output is approximately 70 samples in 24 h. Serum and heparin plasma can also be analyzed. Hemolysis up to approximately 2.0 g/L of hemoglobin did not interfere with the analytical recovery of homocysteine or cysteine. Collection of blood, separation of plasma from whole blood, and acid precipitation must be standardized to obtain reproducible thiol results. Our modifications and the standardization of blood-sampling procedures have substantially improved the method and broadened its applications.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a neurodegenerative disease marked by early-onset cataract and hearing loss, retinitis pigmentosa, and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia. Previously, we mapped this Refsum-like disorder to a 16 Mb region on chromosome 20. Here we report that mutations in the ABHD12 gene cause PHARC disease and we describe the clinical manifestations in a total of 19 patients from four different countries. The ABHD12 enzyme was recently shown to hydrolyze 2-arachidonoyl glycerol (2-AG), the main endocannabinoid lipid transmitter that acts on cannabinoid receptors CB1 and CB2. Our data therefore represent an example of an inherited disorder related to endocannabinoid metabolism. The endocannabinoid system is involved in a wide range of physiological processes including neurotransmission, mood, appetite, pain appreciation, addiction behavior, and inflammation, and several potential drugs targeting these pathways are in development for clinical applications. Our findings show that ABHD12 performs essential functions in both the central and peripheral nervous systems and the eye. Any future drug-mediated interference with this enzyme should consider the potential risk of long-term adverse effects.

We have investigated a family where two siblings had a developmental disorder associated with polycystic dysplastic kidney disease that was incompatible with postnatal survival. Additional features observed were ductal plate malformation in the liver, dysplasia of the pancreas, and (in one individual) complete situs inversus and polymicrogyria of the cingulate gyri. The autopsy findings were compatible with renal-hepatic-pancreatic dysplasia, a condition with unknown genetic cause at the time of autopsy but with similarities to the Meckel-Gruber/Joubert group of recessive ciliopathies. Consanguinity between the parents made it likely that the mutated gene (with known or potential function in cilia) was located within a rather large region of homozygosity in the affected individuals (identical by descent). Using genetic markers (50K single nucleotide polymorphism microarrays), we found a single large homozygous region of 21.16 Mb containing approximately 200 genes on the long arm of chromosome 3. This region contained two known ciliopathy genes: NPHP3 (adolescent nephronophthisis) and IQCB1 (NPHP5), which is associated with Senior-Löken syndrome. In NPHP3, homozygosity for a deletion of the conserved splice acceptor dinucleotide (AG) preceding exon 20 was found. Our finding confirms the recent report that NPHP3-null mutations cause renal-hepatic-pancreatic dysplasia. Also, our case illustrates that genes for rare and genetically heterogeneous recessive conditions may be identified by homozygosity mapping using single nucleotide polymorphism arrays in the routine clinical setting.

Cobalamin metabolism and function were investigated at the levels from transcobalamin II (TCII) receptor to the cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, in a methionine-dependent (P60) and a methionine-independent (P60H) glioma cell line. Using P60H as reference, the P60 cells cultured in a methionine medium had slightly lower TCII receptor activity and normal total cobalamin content, a moderately reduced microsomal and mitochondrial cobalamin(III) reductase activity but only trace amounts of the methylcobalamin and adenosylcobalamin cofactors. When transferred to a homocysteine medium without methionine, P60H cells showed a slightly enhanced TCII receptor activity, but the other cobalamin-related functions were essentially unchanged. In contrast, the methionine-dependent P60 cells responded to homocysteine medium with a nearly 6-fold enhancement of TCII receptor expression and a doubling of both the hydroxycobalamin content and the microsomal reductase activity. The mitochondrial reductase and the cobalamin-related processes further down the pathway did not change markedly. In both cell lines, TCII receptor activity was further increased when growth in homocysteine medium was combined with N2O exposure. These data suggest that low methionine and/or high homocysteine exert a positive feedback control on TCII receptor activity. The concurrent increase in hydroxycobalamin content and in microsomal reductase activity are either subjected to similar regulation or secondary to increased cobalamin transport. This regulatory network is most prominent in the methionine-dependent P60 cells harboring a disruption of the network in the proximity of cobalamin(III) reductase.

The plasma level of homocysteine (Hcy) and its oxidized products, i.e., plasma total Hcy (tHcy), is a function of the influx rate of Hcy to plasma and the plasma tHcy clearance. In vitro experiments show that proliferating cells usually export more Hcy than stationary cells and that the Hcy export increases in response to high methionine, low folate or low cobalamin level, and to agents interfering with Hcy remethylation. Comparison between various cell types suggests that hepatocytes have a unique ability to increase the Hcy export in response to extracellular methionine, probably due to its capacity to form adenosylmethionine. Some but not all cell types have an ability to use extracellular Hcy as a methionine source. Clearance studies in healthy subjects indicate that about 1.2 mmol Hcy is supplied from the cells to plasma per 24 h, which is only about 5-10% of total Hcy formed. Comparison of area under the curves after administration of Hcy and methionine shows that about 10% of the methionine administered is released to plasma as Hcy. Notably, only a few percent of Hcy from plasma is excreted unchanged in the urine, and this shows that most tHcy in plasma is metabolized. Folate or cobalamin deficient patients have normal plasma tHcy clearance, which suggests that their elevated tHcy level is due to increased Hcy export from tissues into the plasma compartment. In contrast, the hyperhomocysteinemia in renal failure is accounted for by a marked reduction in tHcy clearance, suggesting an important role of kidney in elimination of Hcy from plasma.

The total concentration of homocysteine (tHcy)* in plasma is a useful marker of impaired function of cobalamin and folate. Moreover, it is an independent risk factor for atherosclerotic disease [1]. These findings have encouraged the search for determinants of plasma tHcy level [1, 2].