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WD repeat domain 83 opposite strand (WDR83OS) encodes the 106-aa (amino acid) protein Asterix, which heterodimerizes with CCDC47 to form the PAT (protein associated with ER translocon) complex. This complex functions as a chaperone for large proteins containing transmembrane domains to ensure proper folding. Until recently, little was known about the role of WDR83OS or CCDC47 in human disease traits. However, biallelic variants in CCDC47 were identified in four unrelated families with trichohepatoneurodevelopmental syndrome, characterized by a neurodevelopmental disorder (NDD) with liver dysfunction. Three affected siblings in an additional family share a homozygous truncating WDR83OS variant and a phenotype of NDD, dysmorphic features, and liver dysfunction. Using family-based rare variant analyses of exome sequencing (ES) data and case matching through GeneMatcher, we describe the clinical phenotypes of 11 additional individuals in eight unrelated families (nine unrelated families, 14 individuals in total) with biallelic putative truncating variants in WDR83OS. Consistent clinical features include NDD (14/14), facial dysmorphism (13/14), intractable itching (9/14), and elevated bile acids (5/6). Whereas bile acids were significantly elevated in 5/6 of individuals tested, bilirubin was normal and liver enzymes were normal to mildly elevated in all 14 individuals. In three of six individuals for whom longitudinal data were available, we observed a progressive reduction in relative head circumference. A zebrafish model lacking Wdr83os function further supports its role in the nervous system, craniofacial development, and lipid absorption. Taken together, our data support a disease-gene association between biallelic loss-of-function of WDR83OS and a neurological disease trait with hypercholanemia., Competing Interests: Declaration of interests The Department of Molecular & Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories. J.R.L. serves on the Scientific Advisory Board of Baylor Genetics. J.R.L. has stock ownership in 23andMe, is a paid consultant for Genome International, and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more., Methods: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics., Results: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father., Conclusion: This is the first report linking UXS1 to short-limbed short stature in humans., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Genetic repeat expansions cause neuronal degeneration in amyotrophic lateral sclerosis as well as other neurodegenerative disorders such as spinocerebellar ataxia, Huntington's disease and Kennedy's disease. Repeat expansions in the same gene can cause multiple clinical phenotypes. We aimed to characterize repeat expansions in a Norwegian amyotrophic lateral sclerosis cohort. Norwegian amyotrophic lateral sclerosis patients ( n = 414) and neurologically healthy controls adjusted for age and gender ( n = 713) were investigated for repeat expansions in AR , ATXN1 , ATXN2 and HTT using short read exome sequencing and the ExpansionHunter software. Five amyotrophic lateral sclerosis patients (1.2%) and two controls (0.3%) carried ≥36 repeats in HTT ( P = 0.032), and seven amyotrophic lateral sclerosis patients (1.7%) and three controls (0.4%) carried ≥29 repeats in ATXN2 ( P = 0.038). One male diagnosed with amyotrophic lateral sclerosis carried a pathogenic repeat expansion in AR , and his diagnosis was revised to Kennedy's disease. In ATXN1 , 50 amyotrophic lateral sclerosis patients (12.1%) and 96 controls (13.5%) carried ≥33 repeats ( P = 0.753). None of the patients with repeat expansions in ATXN2 or HTT had signs of Huntington's disease or spinocerebellar ataxia type 2, based on a re-evaluation of medical records. The diagnosis of amyotrophic lateral sclerosis was confirmed in all patients, with the exception of one patient who had primary lateral sclerosis. Our findings indicate that repeat expansions in HTT and ATXN2 are associated with increased likelihood of developing amyotrophic lateral sclerosis. Further studies are required to investigate the potential relationship between HTT repeat expansions and amyotrophic lateral sclerosis., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Objective: In Norway, 89% of patients with Amyotrophic lateral sclerosis (ALS) lacks a genetic diagnose. ALS genes and genes that cause other neuromuscular or neurodegenerative disorders extensively overlap. This population-based study examined whether patients with ALS have a family history of neurological disorders and explored the occurrence of rare genetic variants associated with other neurodegenerative or neuromuscular disorders., Methods: During a two-year period, blood samples and clinical data from patients with ALS were collected from all 17 neurological departments in Norway. Our genetic analysis involved exome sequencing and bioinformatics filtering of 510 genes associated with neurodegenerative and neuromuscular disorders. The variants were interpreted using genotype-phenotype correlations and bioinformatics tools., Results: A total of 279 patients from a Norwegian population-based ALS cohort participated in this study. Thirty-one percent of the patients had first- or second-degree relatives with other neurodegenerative disorders, most commonly dementia and Parkinson's disease. The genetic analysis identified 20 possible pathogenic variants, in ATL3, AFG3L2, ATP7A, BICD2, HARS1, KIF1A, LRRK2, MSTO1, NEK1, NEFH, and SORL1, in 25 patients . NEK1 risk variants were present in 2.5% of this ALS cohort. Only four of the 25 patients reported relatives with other neurodegenerative or neuromuscular disorders., Conclusion: Gene variants known to cause other neurodegenerative or neuromuscular disorders, most frequently in NEK1 , were identified in 9% of the patients with ALS. Most of these patients had no family history of other neurodegenerative or neuromuscular disorders. Our findings indicated that AFG3L2, ATP7A, BICD2, KIF1A , and MSTO1 should be further explored as potential ALS-causing genes.

Valosin-containing protein (VCP) is an AAA+ ATPase that plays critical roles in multiple ubiquitin-dependent cellular processes. Dominant pathogenic variants in VCP are associated with adult-onset multisystem proteinopathy (MSP), which manifests as myopathy, bone disease, dementia, and/or motor neuron disease. Through GeneMatcher, we identified 13 unrelated individuals who harbor heterozygous VCP variants (12 de novo and 1 inherited) associated with a childhood-onset disorder characterized by developmental delay, intellectual disability, hypotonia, and macrocephaly. Trio exome sequencing or a multigene panel identified nine missense variants, two in-frame deletions, one frameshift, and one splicing variant. We performed in vitro functional studies and in silico modeling to investigate the impact of these variants on protein function. In contrast to MSP variants, most missense variants had decreased ATPase activity, and one caused hyperactivation. Other variants were predicted to cause haploinsufficiency, suggesting a loss-of-function mechanism. This cohort expands the spectrum of VCP-related disease to include neurodevelopmental disease presenting in childhood., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Hydrops fetalis, a pathological fluid accumulation in two or more body compartments, is aetiologically heterogeneous. We investigated a consanguineous family with recurrent pregnancy loss due to severe early-onset non-immune hydrops fetalis., Methods and Results: Whole exome sequencing in four fetuses with hydrops fetalis revealed that they were homozygous for the angiopoietin-2 ( ANGPT2 ) variant Chr8 (GRCh37/Hg19): 6385085T>C, NM_001147.2:c.557A>G. The substitution introduces a cryptic, exonic splice site predicted to result in loss of 10 nucleotides with subsequent shift in reading frame, leading to a premature stop codon. RNA analysis in the heterozygous parents demonstrated loss of detectable mutant allele, indicative of loss-of-function via nonsense-mediated mRNA decay. Serum ANGPT2 levels were reduced in the parents. In a pregnancy with a healthy, heterozygous child, transiently increased fetal nuchal translucency was noted., Conclusion: Pathogenic heterozygous ANGPT2 missense variants were recently shown to cause autosomal dominant primary lymphoedema. ANGPT2 is a ligand of the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) pathway. It is critical to the formation and remodelling of blood and lymphatic vessels and is involved in vessel maintenance. ANGPT2 knockout mice die from generalised lymphatic dysfunction. We show here that a homozygous pathogenic variant causes loss-of-function and results in severe early-onset hydrops fetalis. This is the first report of an autosomal recessive ANGPT2 -related disorder in humans., Competing Interests: Competing interests: PB has received support from Belgian Fonds de la Recherche Scientifique for attending meetings and/or travel., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Competing Interests: Competing interests: None declared.

Purpose: GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT., Methods: All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records., Results: Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively., Conclusion: Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described., (© 2021. The Author(s).)

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. In Europe, disease-causing genetic variants have been identified in 40-70% of familial ALS patients and approximately 5% of sporadic ALS patients. In Norway, the contribution of genetic variants to ALS has not yet been studied. In light of the potential development of personalized medicine, knowledge of the genetic causes of ALS in a population is becoming increasingly important. The present study provides clinical and genetic data on familial and sporadic ALS patients in a Norwegian population-based cohort., Methods: Blood samples and clinical information from ALS patients were obtained at all 17 neurological departments throughout Norway during a 2-year period. Genetic analysis of the samples involved expansion analysis of C9orf72 and exome sequencing targeting 30 known ALS-linked genes. The variants were classified using genotype-phenotype correlations and bioinformatics tools., Results: A total of 279 ALS patients were included in the study. Of these, 11.5% had one or several family members affected by ALS, whereas 88.5% had no known family history of ALS. A genetic cause of ALS was identified in 31 individuals (11.1%), among which 18 (58.1%) were familial and 13 (41.9%) were sporadic. The most common genetic cause was the C9orf72 expansion (6.8%), which was identified in 8 familial and 11 sporadic ALS patients. Pathogenic or likely pathogenic variants of SOD1 and TBK1 were identified in 10 familial and 2 sporadic cases. C9orf72 expansions dominated in patients from the Northern and Central regions, whereas SOD1 variants dominated in patients from the South-Eastern region., Conclusion: In the present study, we identified several pathogenic gene variants in both familial and sporadic ALS patients. Restricting genetic analysis to only familial cases would miss more than 40 percent of those with a disease-causing genetic variant, indicating the need for genetic analysis in sporadic cases as well., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Background: Although asthma and obesity are each associated with adverse respiratory outcomes, a possible interaction between them is less studied. This study assessed the extent to which asthma and overweight/obese status were independently associated with respiratory symptoms, lung function, Work Ability Score (WAS) and sick leave; and whether there was an interaction between asthma and body mass index (BMI) ≥25 kg/m 2 regarding these outcomes., Methods: In a cross-sectional study, 626 participants with physician-diagnosed asthma and 691 without asthma were examined. All participants completed a questionnaire and performed spirometry. The association of outcome variables with asthma and BMI category were assessed using regression models adjusted for age, sex, smoking status and education., Results: Asthma was associated with reduced WAS (OR=1.9 (95% CI 1.4 to 2.5)), increased sick leave in the last 12 months (OR=1.4 (95% CI 1.1 to 1.8)) and increased symptom score (OR=7.3 (95% CI 5.5 to 9.7)). Obesity was associated with an increased symptom score (OR=1.7 (95% CI 1.2 to 2.4)). Asthma was associated with reduced prebronchodilator and postbronchodilator forced expiratory volume in 1 s (FEV 1 ) (β=-6.6 (95% CI -8.2 to -5.1) and -5.2 (95% CI -6.7 to -3.4), respectively) and prebronchodilator forced vital capacity (FVC) (β=-2.3 (95% CI -3.6 to -0.96)). Obesity was associated with reduced prebronchodilator and postbronchodilator FEV 1 (β=-2.9 (95% CI -5.1 to -0.7) and -2.8 (95% CI -4.9 to -0.7), respectively) and FVC (-5.2 (95% CI -7.0 to -3.4) and -4.2 (95% CI -6.1 to -2.3), respectively). The only significant interaction was between asthma and overweight status for prebronchodilator FVC (β=-3.6 (95% CI -6.6 to -0.6))., Conclusions: Asthma and obesity had independent associations with increased symptom scores, reduced prebronchodilator and postbronchodilator FEV 1 and reduced prebronchodilator FVC. Reduced WAS and higher odds of sick leave in the last 12 months were associated with asthma, but not with increased BMI. Besides a possible association with reduced FVC, we found no interactions between asthma and increased BMI., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Aims: A prolonged corrected QT interval (QTc) ≥500 ms is associated with high all-cause mortality in hospitalized patients. We aimed to explore any difference in short- and long-term mortality in patients with QTc ≥500 ms compared with patients with QTc <500 ms after adjustment for comorbidity and main diagnosis., Methods and Results: Patients with QTc ≥500 ms who were hospitalized at Telemark Hospital Trust, Norway between January 2007 and April 2014 were identified. Thirty-day and 3-year all-cause mortality in 980 patients with QTc ≥500 ms were compared with 980 patients with QTc <500 ms, matched for age and sex and adjusting for Charlson comorbidity index (CCI), previous admissions, and main diagnoses. QTc ≥500 ms was associated with increased 30-day all-cause mortality [hazard ratio (HR) 1.90, 95% confidence interval (CI) 1.38-2.62; P < 0.001]. There was no significant difference in mortality between patients with QTc ≥500 ms and patients with QTc <500 ms who died between 30 days and 3 years; 32% vs. 29%, P = 0.20. Graded CCI was associated with increased 3-year all-cause mortality (CCI 1-2: HR 1.62, 95% CI 1.34-1.96; P < 0.001; CCI 3-4: HR 2.50, 95% CI 1.95-3.21; P < 0.001; CCI ≥5: HR 3.76, 95% CI 2.85-4.96; P < 0.001) but was not associated with 30-day all-cause mortality., Conclusion: QTc ≥500 ms is a powerful predictor of short-term mortality overruling comorbidities. QTc ≥500 ms also predicted long-term mortality, but this effect was mainly caused by the increased short-term mortality. For long-term mortality, comorbidity was more important., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Asthma is defined by variable respiratory symptoms and lung function, and may influence work ability. Similarly, obesity may contribute to respiratory symptoms, affect lung function, and reduce work ability. Thus, assessment of the influence of obesity on work ability, respiratory symptoms, and lung function in adults with asthma is needed., Objectives: We hypothesized that patients with obesity and asthma have more respiratory symptoms and reduced work ability and lung function compared with normal-weight patients with asthma., Methods: We examined 626 participants with physician-diagnosed asthma, aged 18-52 years, recruited from a cross-sectional general population study using a comprehensive questionnaire including work ability score, the asthma control test (ACT), height and weight, and spirometry with reversibility testing., Results: Participants with a body mass index (BMI) ≥30 kg/m2 (i.e., obese) had a higher symptom score (OR 1.78, 95% CI 1.14-2.80), current use of asthma medication (1.60, 1.05-2.46), and incidence of ACT scores ≤19 (poor asthma control) (1.81, 1.03-3.18) than participants with BMI ≤24.9 kg/m2 (i.e., normal weight). Post-bronchodilator forced vital capacity (FVC) as a percentage of predicted (β coefficient -4.5) and pre-bronchodilator forced expiratory volume in 1 s as a percentage of predicted (FEV1) (β coefficient -4.6) were negatively associated with BMI ≥30 kg/m2. We found no statistically significant association of BMI >30 kg/m2 (compared to BMI <24.9 kg/m2) with sick leave (1.21, 0.75-1.70) or reduced work ability (1.23, 0.74-2.04)., Conclusions: There were indications that patients with obesity had a higher symptom burden, poorer asthma control, higher consumption of asthma medication, and reduced lung function, in particular for FVC, compared with normal-weight patients., (© 2019 S. Karger AG, Basel.)

Background Congenital long- QT syndrome ( LQTS ) is a genetic disorder characterized by prolongation of the corrected QT interval ( QT c) on an ECG . The aim of the present study was to estimate the prevalence of pathogenic and likely pathogenic sequence variants in patients who had at least 1 ECG with a QT c ≥500 ms. Methods and Results Telemark Hospital Trust is a community hospital within the Norwegian national health system, serving ≈173 000 inhabitants. We searched the ECG database at Telemark Hospital Trust, Norway, from January 2004 to December 2014, and identified 1531 patients with at least 1 ECG with a QT c ≥500 ms. At the time of inclusion in this study (2015), 766 patients were alive. A total of 733 patients were invited to participate, and 475 accepted. The 17 genes that have been reported to cause monogenic LQTS were sequenced among the patients. Pro- QT c score was calculated for each patient. A molecular genetic cause of LQTS was detected in 31 (6.5%) of 475 patients. These patients had a lower pro- QT c score than those without pathogenic or likely pathogenic variants (1.7±1.0 versus 2.8±1.6; P<0.001). Conclusions Compared with the general population, hospitalized patients with a QT c ≥500 ms in at least 1 ECG recording had an increased likelihood for pathogenic and likely pathogenic variants in LQTS genes. We recommend increased awareness of the possibility of LQTS in patients with at least 1 ECG with a QT c ≥500 ms.

Aims: To determine predictors of mortality in patients with corrected QT interval (QTc) ≥ 500 ms in a community hospital., Methods and Results: In this retrospective observational study, we searched the electrocardiogram (ECG) database at Telemark Hospital Trust, Norway, from January 2004 to December 2014. Medication, electrolyte abnormalities, and medical conditions known to prolong the QT interval were recorded. From the medical records, we assessed whether the prolonged QTc was noted by the health care providers. We identified 1531 patients (age = 70 ± 15 years, 59% female) with an ECG with QTc ≥ 500 ms. All-cause mortality during 952 (range 0-4161) days of follow-up was 50% (n =  765/1531). Main predictors of mortality were aborted cardiac arrest [hazard ratio (HR) 2.40, 95% confidence interval (CI) 1.44-4.01; P = 0.001], cerebral stroke/head trauma (HR 2.28, 95% CI 1.70-3.05; P < 0.001), and heart failure (HR 1.74, 95% CI 1.43-2.12; P< 0.001). Females with prolonged QTc had better survival compared with males (P = 0.006). We constructed a risk-weighted QTc mortality score. QT prolongation was acknowledged in the medical records in 12% of the cases., Conclusions: QTc ≥ 500 ms was associated with high all-cause mortality with increased mortality in males compared with females. A new QTc mortality score was constructed to predict mortality. Only a minority of cases with prolonged QTc ≥ 500 ms were acknowledged in the medical records.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Rare sequence variants in at least five genes are known to cause monogenic obesity. In this study we aimed to investigate the prevalence of, and characterize, rare coding and splice site variants in LEP, LEPR, MC4R, PCSK1 and POMC in patients with morbid obesity and normal weight controls., Method: Targeted next-generation sequencing of all exons in LEP, LEPR, MC4R, PCSK1 and POMC was performed in 485 patients with morbid obesity and 327 normal weight population-based controls from Norway., Results: In total 151 variants were detected. Twenty-eight (18.5%) of these were rare, coding or splice variants and five (3.3%) were novel. All individuals, except one control, were heterozygous for the 28 variants, and the distribution of the rare variants showed a significantly higher carrier frequency among cases than controls (9.9% vs. 4.9%, p=0.011). Four variants in MC4R were classified as pathogenic or likely pathogenic., Conclusion: Four cases (0.8%) of monogenic obesity were detected, all due to MC4R variants previously linked to monogenic obesity. Significant differences in carrier frequencies among patients with morbid obesity and normal weight controls suggest an association between heterozygous rare coding variants in these five genes and morbid obesity. However, additional studies in larger cohorts and functional testing of the novel variants identified are required to confirm the findings., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Next-generation sequencing (NGS) is a genetic technique used to determine the order of nucleotides in DNA. The technique has proved to be more efficient than the traditional method, Sanger sequencing, for sequencing multiple genes. NGS is now being used to diagnose disorders in which multiple genes are involved. This study has examined whether next-generation sequencing produces a greater number of positive diagnoses than its traditional counterpart in patients with suspected hereditary peripheral neuropathy., Material and Method: This study is a retrospective review of samples from 103 patients investigated for hereditary peripheral neuropathy, received by Telemark Hospital in the period 2012-14. After exclusion of duplication/deletion of PMP22, 96 samples were analysed by NGS with physical enrichment of 52 hereditary peripheral neuropathy genes., Results: A genetic cause was identified in 35 patients (34%) with peripheral neuropathy, of which 28 (27%) were point mutations identified by NGS., Interpretation: Of the pathogenic point mutations identified in this study, 12 were in genes that would previously have been analysed by Sanger sequencing in our department, whereas 16 were in genes that would not previously have been tested.

Background: New DNA-sequencing technology is revolutionising medical diagnostics. Through the use of exome sequencing, it is now possible to sequence all human genes in parallel. This technology has been widely used in research over the last few years and is now also being applied to diagnostics. The aim of this study was to systematically examine initial experiences with diagnostic exome sequencing in Norway., Material and Method: This is a retrospective observational study of the results of all exome sequencing performed by the Section of Medical Genetics at Telemark Hospital between December 2012 and October 2014, and includes 125 persons in 46 families. The majority of these families were being investigated for a syndrome (n = 35, 76%) or neurological disease (n = 9, 20%)., Results: Exome sequencing detected pathogenic sequence variants in 15 of 46 probands, and variants of unknown significance in 12 probands. Of the 100 patients who stated their wishes regarding feedback of any incidental findings, six indicated that they did not wish to receive such information. There were no incidental findings in this study, but neither were such sequence variants actively looked for., Interpretation: Exome sequencing can enable more patients with syndromes or neurological diseases to receive a causal diagnosis, and to receive this diagnosis at an earlier stage. However, the patients in this study were quite highly selected, and the results must therefore be interpreted with caution.

Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited neuropathy. Today more than 40 CMT genes have been identified. Diagnosing heterogeneous diseases by conventional Sanger sequencing is time consuming and expensive. Thus, more efficient and less costly methods are needed in clinical diagnostics. We included a population based sample of 81 CMT families. Gene mutations had previously been identified in 22 families; the remaining 59 families were analysed by next-generation sequencing. Thirty-two CMT genes and 19 genes causing other inherited neuropathies were included in a custom panel. Variants were classified into five pathogenicity classes by genotype-phenotype correlations and bioinformatics tools. Gene mutations, classified certainly or likely pathogenic, were identified in 37 (46%) of the 81 families. Point mutations in known CMT genes were identified in 21 families (26%), whereas four families (5%) had point mutations in other neuropathy genes, ARHGEF10, POLG, SETX, and SOD1. Eleven families (14%) carried the PMP22 duplication and one family carried a MPZ duplication (1%). Most mutations were identified not only in known CMT genes but also in other neuropathy genes, emphasising that genetic analysis should not be restricted to CMT genes only. Next-generation sequencing is a cost-effective tool in diagnosis of CMT improving diagnostic precision and time efficiency.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor homology domain repeat A of the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts recycling of the internalized LDLR. As a consequence, the LDLR is rerouted to the lysosomes for degradation. Although PCSK9 may bind to an LDLR lacking the ligand-binding domain, at least three ligand-binding repeats of the ligand-binding domain are required for PCSK9 to reroute the LDLR to the lysosomes. In this study, we have studied the binding of PCSK9 to an LDLR with or without the ligand-binding domain at increasingly acidic conditions in order to mimic the milieu of the LDLR:PCSK9 complex as it translocates from the cell membrane to the sorting endosomes. These studies have shown that PCSK9 is rapidly released from an LDLR lacking the ligand-binding domain at pH in the range of 6.9-6.1. A similar pattern of release at acidic pH was also observed for the binding to the normal LDLR of mutant PCSK9 lacking the C-terminal domain. Together these data indicate that an interaction between the negatively charged ligand-binding domain of the LDLR and the positively charged C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDLR during the early phase of endosomal acidification as the LDLR translocates from the cell membrane to the sorting endosome.

In this study we have characterized a naturally occurring truncated form of the low density lipoprotein receptor (LDLR). Western blot analysis of transfected cells indicated that the truncated form (∆N-LDLR) is a degradation product of the full-length LDLR generated by cleavage in the linker region between ligand-binding repeats 4 and 5 of the ligand-binding domain. The cleavage of the linker was not caused by components of the culture media, as heat inactivation of the media did not prevent cleavage. Rather, it is assumed that cleavage was caused by an enzyme secreted from the cells. Biotinylation experiments showed that ∆N-LDLR is located on the cell surface and is detectable approximately 5 h after synthesis of the full-length LDLR. Flow cytometric analysis showed that ∆N-LDLR was not able to bind and internalize low density lipoprotein (LDL). ∆N-LDLR appeared to be equally stable as the full-length LDLR. Thus, generation of ∆N-LDLR does not appear to be the first signal for degradation of the LDLR. The existence of two functionally different populations of LDLRs on the cell surface, of which ∆N-LDLR constitutes 28%, must be taken into account when interpreting results of experiments to study LDLRs on the cell surface. Furthermore, if the cleavage of the linker between ligand-binding repeats 4 and 5 could be prevented by an enzyme inhibitor, this could represent a novel therapeutic strategy to increase the number of functioning LDLRs and thereby decrease the levels of plasma LDL cholesterol., (Copyright © 2011. Published by Elsevier Inc.)

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE(814) and/or ExD(837) had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.

The low density lipoprotein receptor (LDLR) binds and internalizes low density lipoprotein (LDL). At the mildly acidic pH of the sorting endosomes, LDL is released from the receptor and the receptor recycles back to the cell membrane. Mutations in the LDLR gene may disrupt the normal function of the LDLR in different ways. Class 5 mutations result in receptors that are able to bind and internalize LDL, but they fail to release LDL in the sorting endosomes and fail to recycle. Instead they are rerouted to the lysosomes for degradation. However, the underlying mechanism remains to be determined. To study the role of the cytoplasmic domain of the LDLR for rerouting Class 5 mutants to the lysosomes, we have performed studies to determine whether Class 5 mutants caused by mutations E387K or V408M are degraded when the cytoplasmic domain has been altered or deleted. As determined by confocal laser-scanning microscopy, these mutant LDLR were inserted into the cell membrane and were able to internalize LDL. As determined by Western blot analysis, Class 5 mutants without a cytoplasmic domain still were degraded after binding LDL. Thus, the cytoplasmic domain does not play a role in rerouting Class 5 mutant LDLR to the lysosomes. Rather, one may speculate that sterical hindrance may prevent Class 5 mutants with bound LDL from entering the narrow recycling tubules of the sorting endosome., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31-53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32-40 and 48-50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs. Deletion of the acidic residues of the longest negatively charged segment increased PCSK9's ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9's activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9's activity by 36%. These findings indicate that the inhibitory effect of residues 31-53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9's activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-translationally regulates the number of cell-surface low density lipoprotein receptors (LDLR). This is accomplished by the ability of PCSK9 to mediate degradation of the LDLR. The underlying mechanism involves binding of secreted PCSK9 to the epidermal growth factor-like repeat A of the extracellular domain of the LDLR at the cell surface, followed by lysosomal degradation of the internalized LDLR:PCSK9 complex. However, the mechanism by which the normal recycling of the LDLR is disrupted by PCSK9, remains to be determined. In this study we have investigated the role of the cytoplasmic domain of the LDLR for this process. This has been done by studying the ability of a mutant LDLR (K811X-LDLR) which lacks the cytoplasmic domain, to be degraded by PCSK9. We show that this mutant receptor is degraded by PCSK9. Thus, the machinery which directs the LDLR:PCSK9 complex to the lysosomes for degradation, does not interact with the cytoplasmic domain of the LDLR.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the extracellular domain of the low density lipoprotein receptor (LDLR) at the cell surface, and disrupts the normal recycling of the LDLR. However, the exact mechanism by which the LDLR is re-routed for lysosomal degradation remains to be determined. To clarify the role of the cytoplasmic domain of the LDLR for re-routing to the lysosomes, we have studied the ability of PCSK9 to degrade a chimeric receptor which contains the extracellular and transmembrane domains of the LDLR and the cytoplasmic domain of the transferrin receptor. These studies were performed in CHO T-REx cells stably transfected with a plasmid encoding the chimeric receptor and a novel assay was developed to study the effect of PCSK9 on the LDLR in these cells. Localization, function and stability of the chimeric receptor were similar to that of the wild-type LDLR. The addition of purified gain-of-function mutant D374Y-PCSK9 to the culture medium of stably transfected CHO T-REx cells showed that the chimeric receptor was degraded, albeit to a lower extent than the wild-type LDLR. In addition, a mutant LDLR, which has the three lysines in the intracellular domain substituted with arginines, was also degraded by D374Y-PCSK9. Thus, the mechanism for the PCSK9-mediated degradation of the LDLR does not appear to involve an interaction between the endosomal sorting machinery and LDLR-specific motifs in the cytoplasmic domain. Moreover, ubiquitination of lysines in the cytoplasmic domain does not appear to play a critical role in the PCSK9-mediated degradation of the LDLR., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Objective: Published data may suggest that the cholesterol-lowering effect of mutation R46L in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene in familial hypercholesterolemia (FH) heterozygotes, is less pronounced than in normocholesterolemic subjects., Methods: 1130 unrelated subjects with molecularly defined FH were screened for mutation R46L in the PCSK9 gene and cell culture experiments were performed to study the effect of high concentrations of low density lipoprotein (LDL) on the binding of PCSK9 to the LDL receptor (LDLR)., Results: 2.7% of the subjects were carriers of the R46L mutation and they had a non-significant 6% lower value for total serum cholesterol than non-carriers. This reduction is lower than the 8-9% reduction in total serum cholesterol levels previously observed in normocholesterolemic subjects. Cell culture experiments showed that increasing concentrations of low density lipoprotein (LDL) in the media, decreased the amount of PCSK9 internalized and decreased the PCSK9-mediated degradation of the LDLR., Conclusion: High levels of LDL, as seen in untreated FH heterozygotes, compete against wild-type PCSK9 for binding to the LDLR. Thus, in the presence of high LDL levels, wild-type-PCSK9, which has twice the binding affinity of R46L-PCSK9 to bind to the LDLR, may not be significantly more potent in degrading the LDLR than R46L-PCSK9. These data may suggest that targeting PCSK9 as monotherapy in FH heterozygotes, may not prove to be very effective., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: Familial hypercholesterolemia (FH) patients with the splice site mutation c.313+1, G>A in intron 3 of the low density lipoprotein receptor (LDLR) gene, present with a phenotype similar to that of FH patients in general. However, a mild phenotype would have been expected from the published data showing that the mutation only causes skipping of exon 3., Methods: Epstein Barr virus-transformed lymphocytes from eight c.313+1, G>A heterozygotes and two c.313+1, G>A homozygotes were subjected to studies of the LDLR at the mRNA and protein levels., Results: Mutation c.313+1, G>A not only causes skipping of exon 3, but also causes inclusion of intron 3. No functional LDLR was produced from the transcript with inclusion of intron 3. The transcript with skipping of exon 3 produced a receptor which had markedly reduced ability to internalize low density lipoprotein., Conclusion: The findings that the mutation c.313+1, G>A in the LDLR gene also generates a mutant transcript with inclusion of intron 3, explains why the mutation c.313+1, G>A may result in a severe phenotype.

Screening for mutations in the low density lipoprotein receptor (LDLR) gene has identified more than 1000 mutations as the cause of familial hypercholesterolemia (FH). In addition, numerous intronic mutations with uncertain effects on pre-mRNA splicing have also been identified. In this study, we have selected 18 intronic mutations in the LDLR gene for comprehensive studies of their effects on pre-mRNA splicing. Epstein-Barr virus (EBV) transformed lymphocytes from subjects heterozygous for these mutations were established and mRNA was studied by Northern blot analyses and reverse transcription polymerase chain reactions. Furthermore, functional studies of the LDLRs were performed by flow cytometry. The results of the wet-lab analyses were compared to the predictions obtained from bioinformatics analyses using the programs MaxEntScan, NetGene2 and NNSplice 0.9, which are commonly used software packages for prediction of abnormal splice sites. Thirteen of the 18 intronic mutations were found to affect pre-mRNA splicing in a biologically relevant way as determined by wet-lab analyses. Skipping of one or two exons was observed for eight of the mutations, intron inclusion was observed for four of the mutations and activation of a cryptic splice site was observed for two of the mutations. Transcripts from eight of the mutant alleles were subjected to degradation. The computational analyses of the normal and mutant splice sites, predicted abnormal splicing with a sensitivity of 100% and a specificity of 60%. Thus, bioinformatics analyses are valuable tools as a first screening of the effects of intronic mutations in the LDLR gene on pre-mRNA splicing.

Objective: The normal function of proprotein convertase subtilisin/kexin type 9 (PCSK9) is to mediate degradation of the low density lipoprotein (LDL) receptors. However, the exact mechanism for this function remains to be determined. Characterization of how the naturally occurring mutations in the PCSK9 gene affect the function of PCSK9, may provide important insight into the mechanism by which PCSK9 degrades the LDL receptors., Methods: DNA sequencing of the 12 exons with flanking intron sequences of the PCSK9 gene was performed in 1336 unrelated hypercholesterolemic subjects. In vitro assays and bioinformatics analysis were employed to characterize the functional consequences of a novel mutation. EXPERIMENTAL RESULTS: One subject was heterozygous for the novel mutation S462P in exon 9 of the PCSK9 gene. Based upon Western blot analysis of transiently transfected HepG2 cells, S462P-PCSK9 was almost completely retained in the endoplasmic reticulum (ER) even though it did undergo autocatalytic cleavage. Thus, only trace amounts of S462P-PCSK9 were detected in the culture media of transfected cells. Flow cytometric experiments revealed that the S462P-PCSK9 mutant was unable to degrade the LDL receptors., Discussion: The markedly reduced secretion of S462P-PCSK9 makes S462P a loss-of-function mutation. Ser462 is one of the few residues in the C-terminal domain which is conserved in all known PCSK9 homologs. A hydrogen bond between the side-chain of Ser462 and the backbone of beta-strand 6 of the C-terminal domain, appears to be essential for the proper folding of the C-terminal domain. The S462P mutation is believed to disrupt the normal folding of the C-terminal domain leading to retention of the mutant protein in the ER.

Objective: The objective of this project was to determine whether nonsense mutation in the low density lipoprotein receptor (LDLR) induces nonsense-mediated mRNA decay (NMD)., Material and Methods: Four known nonsense mutations (W23X, S78X, E207X and W541X) in the LDLR gene, which are found in Norwegian familial hypercholesterolaemia (FH) patients, were investigated. Epstein-Barr virus (EBV) transformed lymphocytes from patients heterozygous for these mutations in the LDLR gene were analysed. Flow cytometric analysis was used to determine the amount and function of the cell surface LDLRs. The expression of LDLR mRNA in lymphocytes was quantified by real-time polymerase chain reaction (PCR). The presence of NMD was tested using the inhibitors gentamicin, emetine or cycloheximide., Results: Cells from heterozygous FH patients with nonsense mutations in the LDLR gene contained significantly less LDLR protein (p<0.05). In addition, flow cytometric analysis revealed that these patients had a reduced LDL-uptake compared to controls (p<0.005). Cells from heterozygous FH patients with nonsense mutations W23X, S78X or W541X in the LDLR gene showed significantly decreased levels of LDLR mRNA (p<0.005). LDLR mRNA was reduced in the mutant lymphocyte S78X prior to treatment with pharmacological inhibitors, and after treatment the level of LDLR mRNA increased to the same level as that of normal cells., Conclusion: In the present study, NMD was confirmed in the LDLR gene. Translation inhibitors showed reduced NMD caused by nonsense mutated LDLR transcripts. Knowledge of NMD might have an important impact in clinical medicine as genetic intervention develops.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low-density lipoprotein receptor. To date, more than 900 different mutations have been described. Transport-defective mutations (class 2) causing partial or complete retention of the receptor in the endoplasmic reticulum are the predominant class of mutations. In a cell culture system (Chinese hamster ovary cells), we show that chemical chaperones are able to mediate rescue of a transport-defective mutant (G544V), and that the ability to obtain rescue is mutation dependent. In particular, the low molecular mass fatty acid derivative 4-phenylbutyrate mediated a marked increase in the transport of G544V-mutant low-density lipoprotein receptor to the plasma membrane. Thirty per cent of the mutant receptor was able to escape from the endoplasmic reticulum and reach the cell surface. The rescued receptor had reduced stability, but was found to be as efficient as the wild-type low-density lipoprotein receptor in binding and internalizing low-density lipoprotein. In addition to 4-phenylbutyrate, we also studied 3-phenylpropionate and 5-phenylvalerate, and compared their effect on rescue of the G544V-mutant low-density lipoprotein receptor with their ability to increase overall gene expression caused by their histone deacetylase inhibitor activity. No correlation was found. Our results indicate that the effect of these agents was not solely mediated by their ability to induce gene expression of proteins involved in intracellular transport, but rather could be due to a direct chemical chaperone activity. These data suggest that rescue of mutant low-density lipoprotein receptor is possible and that it might be feasible to develop pharmacologic chaperones to treat familial hypercholesterolemia patients with class 2 mutations.

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). However, it is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR., Results: We have transiently transfected HepG2 cells with wild-type and mutant D374Y-PCSK9 plasmids to study the effect of the conditioned medium on the LDLR of untransfected HepG2 cells. The ability of the conditioned medium to reduce the internalization of LDL was abolished by removal of recombinant PCSK9 from the conditioned medium by affinity chromatography. Thus, PCSK9 is the only factor in the conditioned medium able to mediate degradation of the LDLR. Moreover, fractionation of the conditioned medium by gel filtration showed that the ability of the fractions to reduce the internalization of LDL, closely paralleled the amount of D374Y-PCSK9 in the fractions. Incubation of a secreted, truncated LDLR without cytoplasmic and transmembrane domains, as well as membrane fractions from HepG2 cells, with conditioned medium containing PCSK9, did not reduce the amount of LDLR as determined by western blot analysis. Thus, the LDLR is not degraded by PCSK9 on the cell surface. The LDLR of HepG2 cells incubated with conditioned medium was protected from PCSK9-mediated degradation by the addition of nocodazole or ammonium chloride, but was not protected when the conditioned medium was made hypertonic. These findings indicate that the intracellular degradation of the LDLR involves intracellular transport along microtubules, an acidic intracellular compartment and that it occurs even when endocytosis through clathrin-coated pits has been blocked., Conclusion: Degradation of the LDLR by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly. Also the PCSK9-mediated degradation of the LDLR does not take place on the cell surface. Rather, the PCSK9-mediated degradation of the LDLR appears to take place intracellularly and occurs even when endocytosis through clathrin-coated pits is blocked by hypertonic medium.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through 'loss-of-function' and 'gain-of-function' mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.

Objective: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia., Material and Methods: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells., Results: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls., Conclusions: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.