Heterozygous variants in CTR9, which encodes a major component of the PAF1 complex, are associated with a neurodevelopmental disorder.

PURPOSE: CTR9 is a subunit of the PAF1 complex (PAF1C) that plays a crucial role in transcription regulation by binding CTR9 to RNA polymerase II. It is involved in transcription-coupled histone modification through promoting H3K4 and H3K36 methylation. We describe the clinical and molecular studies in 13 probands, harboring likely pathogenic CTR9 missense variants, collected through GeneMatcher. METHODS: Exome sequencing was performed in all individuals. CTR9 variants were assessed through 3-dimensional modeling of the activated human transcription complex Pol II-DSIF-PAF-SPT6 and the PAF1/CTR9 complex. H3K4/H3K36 methylation analysis, mitophagy assessment based on tetramethylrhodamine ethyl ester perchlorate immunofluorescence, and RNA-sequencing in skin fibroblasts from 4 patients was performed. RESULTS: Common clinical findings were variable degrees of intellectual disability, hypotonia, joint hyperlaxity, speech delay, coordination problems, tremor, and autism spectrum disorder. Mild dysmorphism and cardiac anomalies were less frequent. For 11 CTR9 variants, de novo occurrence was shown. Three-dimensional modeling predicted a likely disruptive effect of the variants on local CTR9 structure and protein interaction. Additional studies in fibroblasts did not unveil the downstream functional consequences of the identified variants. CONCLUSION: We describe a neurodevelopmental disorder caused by (mainly) de novo variants in CTR9, likely affecting PAF1C function.

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

Familial motor neuron disease: co-occurrence of PLS and ALS (-FTD).

OBJECTIVE: To report the frequency and characteristics of patients diagnosed with primary lateral sclerosis (PLS) with a positive family history for motor neuron diseases (MND) in the Netherlands and to compare our findings to the literature. METHODS: Patients were identified through our ongoing, prospective population-based study on MND in The Netherlands, which also includes a standardized collection of patient characteristics, genetic testing, and family history. Only patients meeting the latest consensus criteria for definite PLS were included. The family history was considered positive for MND if any family members had been diagnosed with PLS, amyotrophic lateral sclerosis (ALS)(-FTD), or progressive muscular atrophy (PMA). Additionally, the literature was reviewed on PLS cases in which MND co-occurred within the same family. RESULTS: We identified 392 definite PLS cases, resulting in 9 families with a PLS patient and a positive family history for MND (2.3%). In only one of these pedigrees, a pathogenic variant (C9orf72 repeat expansion) was found. Our literature review revealed 23 families with a co-occurrence of PLS and MND, with 12 of them having a potentially pathogenic genetic variant. CONCLUSIONS: The consistent observation of PLS patients with a positive family history for MND, evident in both our study and the literature, implies the presence of shared underlying genetic factors between PLS and ALS. However, these factors are yet to be elucidated.

A new variant in the ZCCHC8 gene: diverse clinical phenotypes and expression in the lung.

Introduction: Pulmonary fibrosis is a severe disease which can be familial. A genetic cause can only be found in ∼40% of families. Searching for shared novel genetic variants may aid the discovery of new genetic causes of disease. Methods: Whole-exome sequencing was performed in 152 unrelated patients with a suspected genetic cause of pulmonary fibrosis from the St Antonius interstitial lung disease biobank. Variants of interest were selected by filtering for novel, potentially deleterious variants that were present in at least three unrelated pulmonary fibrosis patients. Results: The novel c.586G>A p.(E196K) variant in the ZCCHC8 gene was observed in three unrelated patients: two familial patients and one sporadic patient, who was later genealogically linked to one of the families. The variant was identified in nine additional relatives with pulmonary fibrosis and other telomere-related phenotypes, such as pulmonary arterial venous malformations, emphysema, myelodysplastic syndrome, acute myeloid leukaemia and dyskeratosis congenita. One family showed incomplete segregation, with absence of the variant in one pulmonary fibrosis patient who carried a PARN variant. The majority of ZCCHC8 variant carriers showed short telomeres in blood. ZCCHC8 protein was located in different lung cell types, including alveolar type 2 (AT2) pneumocytes, the culprit cells in pulmonary fibrosis. AT2 cells showed telomere shortening and increased DNA damage, which was comparable to patients with sporadic pulmonary fibrosis and those with pulmonary fibrosis carrying a telomere-related gene variant, respectively. Discussion: The ZCCHC8 c.586G>A variant confirms the involvement of ZCCHC8 in pulmonary fibrosis and short-telomere syndromes and underlines the importance of including the ZCCHC8 gene in diagnostic gene panels for these diseases.

SMDT1 variants impair EMRE-mediated mitochondrial calcium uptake in patients with muscle involvement.

Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca2+ uptake.

Diagnostic workup of paediatric patients with inflammatory bowel disease in Europe: results of a 5-year audit of the EUROKIDS registry.

OBJECTIVE: In 2005, the Inflammatory Bowel Disease (IBD) Working Group of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition published consensus guidelines on the diagnostic workup of paediatric IBD, the Porto criteria. According to these guidelines, children suspected of having IBD should undergo an oesophagogastroduodenoscopy (OGD), ileocolonoscopy, and (except in cases of definitive ulcerative colitis) adequate imaging of the small bowel. To audit and evaluate the diagnostic workup of paediatric patients with IBD in Europe, the Working Group created EUROKIDS, a prospective, Web-based registry of newly diagnosed paediatric patients with IBD. METHODS: Patients with IBD (ages 0-18 years) were registered in 44 centres in 18 countries. Data on diagnostic workup were analysed according to the year of diagnosis, type of IBD, and centre size. Diagnostic yield of OGD and ileal intubation were evaluated. RESULTS: Between 2004 and 2009, 2087 newly diagnosed patients were correctly registered. Both OGD and ileocolonoscopy had been performed in 64% of all of the patients and increased significantly from year 1 (52 %) to 5 (71%, P  <  0.001). Small-bowel follow-through use decreased during the years (year 1 n = 213, year 5 n = 108; P < 0.001), whereas magnetic resonance imaging use increased (year 1 n  = 25, year 5 n  = 171; P < 0.001). Patients diagnosed as having Crohn disease (CD, 59%) and ulcerative colitis (58%) were more likely to have had a complete diagnostic workup than patients diagnosed as having IBD unclassified (45%). In CD, the diagnostic yield of OGD was 7.5% and the yield of ileal intubation was 13%. CONCLUSIONS: The quality of diagnostic workup in paediatric patients with IBD increased steadily between 2004 and 2009. Small-bowel imaging by magnetic resonance imaging superseded the use of small-bowel follow-through. OGD and ileal intubation contributed to a definitive diagnosis of CD.

Distal spinal muscular atrophy featured by predominant calf muscle involvement in VRK1 associated disease - Case series and review.

We describe the shared clinical, biochemical, radiological and myopathological characteristics of four patients with distal spinal muscular atrophy (dSMA) caused by vaccinia-related kinase 1 (VRK1) variants and provide a review of the literature on phenotype-genotype correlations in VRK1-related disease. The clinical phenotype was characterized by adult-onset dSMA with predominant calf muscle involvement and mildly elevated serum creatinine kinase (CK) levels. Muscle imaging showed predominant atrophy and fatty replacement of calf muscles. We identified the novel compound heterozygous variants c.607C>T (p.Arg203Trp) and c.858G>T (p.Met286Ile) in two siblings with adult-onset dSMA. Additionally, two unrelated patients both carried the known c.583T>G (p.Leu195Val) VRK1 variant, with either c.197C>G (p.Ala66Gly) or c.701A>G (p.Asn234Ser) as a second variant. We conclude that compound heterozygous VRK1 variants cause distal spinal muscular atrophy with predominant posterior leg muscle involvement.

B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.

Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding-deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3ß, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance.

Disease phenotype at diagnosis in pediatric Crohn's disease: 5-year analyses of the EUROKIDS Registry.

BACKGROUND: It has been speculated that pediatric Crohn's disease (CD) is a distinct disease entity, with probably different disease subtypes. We therefore aimed to accurately phenotype newly diagnosed pediatric CD by using the pediatric modification of the Montreal classification, the Paris classification. METHODS: Information was collected from the EUROKIDS registry, a prospective, web-based registry of new-onset pediatric IBD patients in 17 European countries and Israel. When a complete diagnostic workup was performed (ileocolonoscopy, upper gastrointestinal [GI] endoscopy, small bowel imaging), CD patients were evaluated for ileocolonic disease extent, esophagogastroduodenal involvement, and jejunal/proximal ileal involvement. Disease behavior and the occurrence of granulomas were also analyzed. RESULTS: In all, 582 pediatric CD patients could be classified according to the Paris classification. Isolated terminal ileal disease (± limited cecal disease) was seen at presentation in 16%, isolated colonic disease in 27%, ileocolonic disease in 53%, and isolated upper GI disease in 4% of patients. In total, 30% had esophagogastroduodenal involvement and 24% jejunal/proximal ileal disease. Patients with L2 disease were less likely to have esophagogastroduodenal involvement or stricturing disease than patients with L1 or L3 disease. Terminal ileal disease and stricturing disease behavior were more common in children diagnosed after 10 years of age than in younger patients. Granulomas were identified in 43% of patients. CONCLUSIONS: Accurate phenotyping is essential in pediatric CD, as this affects the management of individual patients. Disease phenotypes differ according to age at disease onset. The Paris classification is a useful tool to capture the variety of phenotypic characteristics of pediatric CD.

Atypical disease phenotypes in pediatric ulcerative colitis: 5-year analyses of the EUROKIDS Registry.

BACKGROUND: Definitive diagnosis of pediatric ulcerative colitis (UC) may be particularly challenging since isolated colitis with overlapping features is common in pediatric Crohn's disease (CD), while atypical phenotypes of UC are not uncommon. The Paris classification allows more accurate phenotyping of atypical inflammatory bowel disease (IBD) patients. Our aim was to identify the prevalence of atypical disease patterns in new-onset pediatric UC using the Paris classification. METHODS: Information was collected from the EUROKIDS Registry, an inception cohort of untreated pediatric IBD patients undergoing evaluation at diagnosis. Patients with IBD-unclassified were excluded. Patients with isolated Crohn's colitis served as a control group. RESULTS: Data from 898 pediatric patients (643 UC, 255 CD colitis) were included. Extensive or pancolitis was present in 77% of UC patients and macroscopic rectal sparing in 5%. Rectal sparing was inversely associated with age (mean age with rectal sparing 9.9 years vs. 11.8 without; P = 0.02). Upper gastrointestinal (UGI) involvement occurred in 4% of patients. Erosions in the stomach were present in 3.1% of children, but frank ulcerations in 0.4%; 0.8% of children had erosions or ulcerations limited to the esophagus or duodenum. The corresponding UGI involvement in Crohn's colitis was 22%. A cecal patch occurred in 2% of patients. CONCLUSIONS: Extensive disease and rectal sparing are age-dependent phenotypes in pediatric UC. Rectal sparing, cecal patch, backwash ileitis, and gastric erosions are not uncommon at diagnosis, while gastric ulcerations and erosions in the duodenum or esophagus are. Recognition of atypical phenotypes in pediatric-onset UC is crucial to prevent misclassification of IBD.

Gene expression analysis of peripheral cells for subclassification of pediatric inflammatory bowel disease in remission.

OBJECTIVE: In current clinical practice, optimal treatment of inflammatory bowel disease (IBD) aims at the induction and maintenance of clinical remission. Clinical remission is apparent when laboratory markers of inflammation are normal and clinical symptoms are absent. However, sub-clinical inflammation can still be present. A detailed analysis of the immune status during this inactive state of disease may provide a useful tool to categorize patients with clinical remission into subsets with variable states of immune activation. DESIGN: By using Affymetrix GeneChips, we analysed RNA gene expression profiles of peripheral blood leukocytes from pediatric IBD patients in clinical remission and controls. We performed (un)supervised clustering analysis of IBD-associated genes and applied Ingenuity® pathway software to identify specific molecular profiles between patients. RESULTS: Pediatric IBD patients with disease in clinical remission display heterogeneously distributed gene expression profiles that are significantly distinct from controls. We identified three clusters of IBD patients, each displaying specific expression profiles of IBD-associated genes. CONCLUSION: The expression of immune- and IBD-associated genes in peripheral blood leukocytes from pediatric IBD patients in clinical remission was different from healthy controls, indicating that sub-clinical immune mechanisms are still active during remission. As such, RNA profiling of peripheral blood may allow for non-invasive patient subclassification and new perspectives in treatment regimes of IBD patients in the future.

Antitumor necrosis factor treatment for pediatric inflammatory bowel disease.

Infliximab, adalimumab, and certolizumab are monoclonal antibodies against tumor necrosis factor-α (TNFα), a proinflammatory cytokine with an increased expression in the inflamed tissues of inflammatory bowel disease (IBD) patients. Currently, infliximab is the only anti-TNF drug that has been approved for use in refractory pediatric Crohn's disease (CD). Nevertheless, adalimumab and certolizumab have been used off-label to treat refractory pediatric IBD. Over the past 10 years, anti-TNF treatment has been of great benefit to many pediatric IBD patients, but their use is not without risks (infections, autoimmune diseases, malignancies). Despite the growing experience with these drugs in children with IBD, optimal treatment strategies still need to be determined. The purpose of this review is to summarize the current knowledge on the use of anti-TNF drugs in pediatric IBD and to discuss the yet-unsolved issues.