Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Identification of Clinical Variants beyond the Exome in Inborn Errors of Metabolism.

Inborn errors of metabolism (IEM) constitute a huge group of rare diseases affecting 1 in every 1000 newborns. Next-generation sequencing has transformed the diagnosis of IEM, leading to its proposed use as a second-tier technology for confirming cases detected by clinical/biochemical studies or newborn screening. The diagnosis rate is, however, still not 100%. This paper reports the use of a personalized multi-omics (metabolomic, genomic and transcriptomic) pipeline plus functional genomics to aid in the genetic diagnosis of six unsolved cases, with a clinical and/or biochemical diagnosis of galactosemia, mucopolysaccharidosis type I (MPS I), maple syrup urine disease (MSUD), hyperphenylalaninemia (HPA), citrullinemia, or urea cycle deficiency. Eight novel variants in six genes were identified: six (four of them deep intronic) located in GALE, IDUA, PTS, ASS1 and OTC, all affecting the splicing process, and two located in the promoters of IDUA and PTS, thus affecting these genes' expression. All the new variants were subjected to functional analysis to verify their pathogenic effects. This work underscores how the combination of different omics technologies and functional analysis can solve elusive cases in clinical practice.

Diagnosis of inborn errors of metabolism within the expanded newborn screening in the Madrid region.

We present the results of our experience in the diagnosis of inborn errors of metabolism (IEM) since the Expanded Newborn Screening was implemented in our Region. Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by mass spectrometry (MS)/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed. In the period 2011 to 2019, 592 822 children were screened: 902 of them were referred for abnormal results. An IEM was confirmed in 222 (1/2670): aminoacidopathies: 89 hyperphenylalaninemia (HPA) (51 benign HPA, 32 phenylketonuria, 4 DNAJC12 defect, and 2 primapterinuria), 6 hypermethioninemia, 3 tyrosinemia type 1 (TYR-1), 1 TYR-3, 4 maple syrup urine disease (MSUD), 2 branched-chain amino acid transferase 2 deficiency, 2 homocystinuria, 1 cystinuria, 2 ornithine transcarbamylase (OTC) deficiency, 2 citrullinemia type I (CTLN1); FAO defects: 43 medium-chain acyl-CoA dehydrogenase deficiency (MCADD), 13 very long-chain acyl-CoA dehydrogenase deficiency, 2 long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), 1 multiple acyl-coA dehydrogenation deficiency, 11 systemic primary carnitine deficiency, 2 carnitine palmitoyltransferase type 2 (CPT-II) deficiency, 1 CPT-I deficiency; organic acidurias: 12 glutaric aciduria type 1 (GA-1), 4 methylmalonic acidemia (MMA), 7 MMA including combined cases with homocystinuria (MMAHC), 6 propionic acidemia (PA), 7 3-methylcrotonyl-CoA carboxylase, 1 3-hydroxy-3-methylglutaryl-CoA lyase deficiency lyase deficiency. Only 19 infants (8.5%) were symptomatic at newborn screening result (1 LCHADD, 5 PA, 1 CPT-II deficiency, 1 MMA, 3 MMAHC, 2 MSUD, 2 OTC deficiency, 1 CTLN1, 1 MCADD, 2 TYR-1). No false negative cases were identified. Genetic diagnosis was conclusive in all biochemically confirmed cases, except for two infants with HPA, identifying pathogenic variants in 32 different genes. The conditions with the highest incidence were HPA (1/6661) and MCAD deficiencies (1/13 787).

Efficient processing of complex XSD using Hive and Spark.

The eXtensible Markup Language (XML) files are widely used by the industry due to their flexibility in representing numerous kinds of data. Multiple applications such as financial records, social networks, and mobile networks use complex XML schemas with nested types, contents, and/or extension bases on existing complex elements or large real-world files. A great number of these files are generated each day and this has influenced the development of Big Data tools for their parsing and reporting, such as Apache Hive and Apache Spark. For these reasons, multiple studies have proposed new techniques and evaluated the processing of XML files with Big Data systems. However, a more usual approach in such works involves the simplest XML schemas, even though, real data sets are composed of complex schemas. Therefore, to shed light on complex XML schema processing for real-life applications with Big Data tools, we present an approach that combines three techniques. This comprises three main methods for parsing XML files: cataloging, deserialization, and positional explode. For cataloging, the elements of the XML schema are mapped into root, arrays, structures, values, and attributes. Based on these elements, the deserialization and positional explode are straightforwardly implemented. To demonstrate the validity of our proposal, we develop a case study by implementing a test environment to illustrate the methods using real data sets provided from performance management of two mobile network vendors. Our main results state the validity of the proposed method for different versions of Apache Hive and Apache Spark, obtain the query execution times for Apache Hive internal and external tables and Apache Spark data frames, and compare the query performance in Apache Hive with that of Apache Spark. Another contribution made is a case study in which a novel solution is proposed for data analysis in the performance management systems of mobile networks.

Impact of COVID-19 Lockdown in Eating Disorders: A Multicentre Collaborative International Study.

BACKGROUND: The COVID-19 lockdown has had a significant impact on mental health. Patients with eating disorders (ED) have been particularly vulnerable. AIMS: (1) To explore changes in eating-related symptoms and general psychopathology during lockdown in patients with an ED from various European and Asian countries; and (2) to assess differences related to diagnostic ED subtypes, age, and geography. METHODS: The sample comprised 829 participants, diagnosed with an ED according to DSM-5 criteria from specialized ED units in Europe and Asia. Participants were assessed using the COVID-19 Isolation Scale (CIES). RESULTS: Patients with binge eating disorder (BED) experienced the highest impact on weight and ED symptoms in comparison with other ED subtypes during lockdown, whereas individuals with other specified feeding and eating disorders (OFSED) had greater deterioration in general psychological functioning than subjects with other ED subtypes. Finally, Asian and younger individuals appeared to be more resilient. CONCLUSIONS: The psychopathological changes in ED patients during the COVID-19 lockdown varied by cultural context and individual variation in age and ED diagnosis. Clinical services may need to target preventive measures and adapt therapeutic approaches for the most vulnerable patients.

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia.

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298-2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation-specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation-specific treatments for PKU.

Genes and Variants Underlying Human Congenital Lactic Acidosis-From Genetics to Personalized Treatment.

Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group's experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system's workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system's use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.

Generation and characterization of a human iPSC line (UAMi004-A) from a patient with propionic acidemia due to defects in the PCCB gene.

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.

Value of genetic analysis for confirming inborn errors of metabolism detected through the Spanish neonatal screening program.

The present work describes the value of genetic analysis as a confirmatory measure following the detection of suspected inborn errors of metabolism in the Spanish newborn mass spectrometry screening program. One hundred and forty-one consecutive DNA samples were analyzed by next-generation sequencing using a customized exome sequencing panel. When required, the Illumina extended clinical exome panel was used, as was Sanger sequencing or transcriptional profiling. Biochemical tests were used to confirm the results of the genetic analysis. Using the customized panel, the metabolic disease suspected in 83 newborns (59%) was confirmed. In three further cases, two monoallelic variants were detected for two genes involved in the same biochemical pathway. In the remainder, either a single variant or no variant was identified. Given the persistent absence of biochemical alterations, carrier status was assigned in 39 cases. False positives were recorded for 11. In five cases in which the biochemical pattern was persistently altered, further genetic analysis allowed the detection of two variants affecting the function of BCAT2, ACSF3, and DNAJC12, as well as a second, deep intronic variant in ETFDH or PTS. The present results suggest that genetic analysis using extended next-generation sequencing panels can be used as a confirmatory test for suspected inborn errors of metabolism detected in newborn screening programs. Biochemical tests can be very helpful when a diagnosis is unclear. In summary, simultaneous genomic and metabolomic analyses can increase the number of inborn errors of metabolism that can be confirmed following suggestive newborn screening results.

Clinical and molecular diagnosis of non-phosphomannomutase 2 N-linked congenital disorders of glycosylation in Spain.

The congenital disorders of glycosylation (CDG) are defects in glycoprotein and glycolipid glycan synthesis and attachment. They affect multiple organ/systems, but non-specific symptoms render the diagnosis of the different CDG very challenging. Phosphomannomutase 2 (PMM2)-CDG is the most common CDG, but advances in genetic analysis have shown others to occur more commonly than previously thought. The present work reports the clinical and mutational spectrum of 25 non-PMM2 CDG patients. The most common clinical symptoms were hypotonia (80%), motor or psychomotor disability (80%) and craniofacial dysmorphism (76%). Based on their serum transferrin isoform profile, 18 were classified as CDG-I and 7 as CDG-II. Pathogenic variations were found in 16 genes (ALG1, ALG6, ATP6V0A2, B4GALT1, CCDC115, COG7, DOLK, DPAGT1, DPM1, GFPT1, MPI, PGM1, RFT1, SLC35A2, SRD5A3, and SSR4). Overall, 27 variants were identified, 12 of which are novel. The results highlight the importance of combining genetic and biochemical analyses for the early diagnosis of this heterogeneous group of disorders.

Ada Carmen Ovies García, paradigma de la Ginecología y la Obstetricia en Latinoamérica / Ada Carmen Ovies García, paradigm of Gynecology and Obstetrics in Latin America

Se presenta la vida y obra de la Dra. Ada Carmen Ovies García, figura relevante de la Ginecología y la Obstetricia tanto en nuestro país como en el mundo, haciéndose énfasis en su labor docente, asistencial y directiva dentro del Sistema Nacional de Salud (AU)

Identification of 34 novel mutations in propionic acidemia: Functional characterization of missense variants and phenotype associations.

Propionic acidemia (PA) is caused by mutations in the PCCA and PCCB genes, encoding α and ß subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Up to date, >200 pathogenic mutations have been identified, mostly missense defects. Genetic analysis in PA patients referred to the laboratory for the past 15 years identified 20 novel variants in the PCCA gene and 14 in the PCCB gene. 21 missense variants were predicted as probably disease-causing by different bioinformatics algorithms. Structural analysis in the available 3D model of the PCC enzyme indicated potential instability for most of them. Functional analysis in a eukaryotic system confirmed the pathogenic effect for the missense variants and for one amino acid deletion, as they all exhibited reduced or null PCC activity and protein levels compared to wild-type constructs. PCCB variants p.E168del, p.Q58P and p.I460T resulted in medium-high protein levels and no activity. Variants p.R230C and p.C712S in PCCA, and p.G188A, p.R272W and p.H534R in PCCB retained both partial PCC activity and medium-high protein levels. Available patients-derived fibroblasts carriers of some of these mutations were grown at 28 °C or 37 °C and a slight increase in PCC activity or protein could be detected in some cases at the folding-permissive conditions. Examination of available clinical data showed correlation of the results of the functional analysis with disease severity for most mutations, with some notable exceptions, confirming the notion that the final phenotypic outcome in PA is not easily predicted.

Improving the diagnosis of cobalamin and related defects by genomic analysis, plus functional and structural assessment of novel variants.

BACKGROUND: Cellular cobalamin defects are a locus and allelic heterogeneous disorder. The gold standard for coming to genetic diagnoses of cobalamin defects has for some time been gene-by-gene Sanger sequencing of individual DNA fragments. Enzymatic and cellular methods are employed before such sequencing to help in the selection of the gene defects to be sought, but this is time-consuming and laborious. Furthermore some cases remain undiagnosed because no biochemical methods have been available to test for cobalamin absorption and transport defects. RESULTS: This paper reports the use of massive parallel sequencing of DNA (exome analysis) for the accurate and rapid genetic diagnosis of cobalamin-related defects in a cohort of affected patients. The method was first validated in an initial cohort with different cobalamin defects. Mendelian segregation, the frequency of mutations, and the comprehensive structural and functional analysis of gene variants, identified disease-causing mutations in 12 genes involved in the absorption and synthesis of active cofactors of vitamin B12 (22 cases), and in the non-cobalamin metabolism-related genes ACSF3 (in four biochemically misdiagnosed patients) and SUCLA2 (in one patient with an unusual presentation). We have identified thirteen new variants all classified as pathogenic according to the ACGM recommendation but four were classified as variant likely pathogenic in MUT and SUCLA2. Functional and structural analysis provided evidences to classify them as pathogenic variants. CONCLUSIONS: The present findings suggest that the technology used is sufficiently sensitive and specific, and the results it provides sufficiently reproducible, to recommend its use as a second-tier test after the biochemical detection of cobalamin disorder markers in the first days of life. However, for accurate diagnoses to be made, biochemical and functional tests that allow comprehensive clinical phenotyping are also needed.

Generation and characterization of a human iPSC line from a patient with propionic acidemia due to defects in the PCCA gene.

Human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia carrying mutations in the PCCA gene: c.1899+4_1899+7delAGTA; p.(Cys616_Val633del) and c.1430--?_1643+?del; p.(Gly477Glufs*9). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.

Efectividad de la Página Web Historia de la Salud en Mayabeque / Effectiveness of the Health History in Mayabeque Web Page

En el 2011 la Facultad de Ciencias Médicas de Mayabeque no contaba con conexión a la red Infomed y a solicitud del departamento de Historia y Filosofía se diseñó una página web estática. Objetivo: Valorar la efectividad de la página web estática Historia de la Salud en Mayabeque a partir del uso que han hecho los usuarios de la red de bibliotecas. Método: Se realizó un estudio descriptivo de corte transversal en la red de bibliotecas de la provincia en el periodo comprendido desde septiembre del 2011 a julio del 2015. El universo estuvo constituido por los usuarios que acudieron a las 26 unidades de la red en la provincia y utilizaron la página, de las que se tomó una muestra intencional de 15 bibliotecas docentes y aleatoria de 281 usuarios. Resultados: Se operó con las variables: uso de la página, categoría de usuario, sexo y edad. Los indicadores para determinar la efectividad de la página fueron: para qué se ha usado la página y evaluación del usuario a partir de una escala de 1 a 5. Discusión: Son los estudiantes del sexo femenino los que más usan la página. Se evidencia el poco uso que hacen los profesores. La página web es efectiva atendiendo al uso realizado por sus usuarios, contribuye a su alfabetización informacional y constituye un medio de apoyo a la enseñanza.

The Faculty of Medical Sciences in Mayabeque province didn't have an Infomed network connection back in 2011, therefore, as a request of the History and Philosophy Department, a static web page was designed. Objective: assessing the effectiveness of the Health History in Mayabeque static web page starting from how libraries network users have made use of it. Method: A cross section descriptive study of the libraries network was made in the province from September 2011 to July 2015. The population consisted of users who went to the 26 libraries in the province to use the page. An intentional sample of 15 teaching libraries and a random sample of 281 users was taken. Results: The following variables were taken into account: web page use, user category, sex and age. In order to determine the effectiveness of the page, the subsequent indicators were used: "what was the page used for" and assessment of the user from a 1 to 5 scale. Discussion: Female students use the page the most. The study showed professors make little use of it. The web page is effective according to the use users make of it; it contributes to their information literacy and it is a teaching aid.

Efectividad de la Página Web Historia de la Salud en Mayabeque / Effectiveness of the Health History in Mayabeque Web Page

En el 2011 la Facultad de Ciencias Médicas de Mayabeque no contaba con conexión a la red Infomed y a solicitud del departamento de Historia y Filosofía se diseñó una página web estática.Objetivo: Valorar la efectividad de la página web estática Historia de la Salud en Mayabeque a partir del uso que han hecho los usuarios de la red de bibliotecas.Método: Se realizó un estudio descriptivo de corte transversal en la red de bibliotecas de la provincia en el periodo comprendido desde septiembre del 2011 a julio del 2015. El universo estuvo constituido por los usuarios que acudieron a las 26 unidades de la red en la provincia y utilizaron la página, de las que se tomó una muestra intencional de 15 bibliotecas docentes y aleatoria de 281 usuarios.Resultados: Se operó con las variables: uso de la página, categoría de usuario, sexo y edad. Los indicadores para determinar la efectividad de la página fueron: para qué se ha usado la página y evaluación del usuario a partir de una escala de 1 a 5.Discusión: Son los estudiantes del sexo femenino los que más usan la página. Se evidencia el poco uso que hacen los profesores. La página web es efectiva atendiendo al uso realizado por sus usuarios, contribuye a su alfabetización informacional y constituye un medio de apoyo a la enseñanza(AU)

The Faculty of Medical Sciences in Mayabeque province didn't have an Infomed network connection back in 2011, therefore, as a request of the History and Philosophy Department, a static web page was designed.Objective: assessing the effectiveness of the Health History in Mayabeque static web page starting from how libraries network users have made use of it.Method: A cross section descriptive study of the libraries network was made in the province from September 2011 to July 2015. The population consisted of users who went to the 26 libraries in the province to use the page. An intentional sample of 15 teaching libraries and a random sample of 281 users was taken.Results: The following variables were taken into account: web page use, user category, sex and age. In order to determine the effectiveness of the page, the subsequent indicators were used: "what was the page used for" and assessment of the user from a 1 to 5 scale.Discussion: Female students use the page the most. The study showed professors make little use of it. The web page is effective according to the use users make of it; it contributes to their information literacy and it is a teaching aid(AU)

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

Genotype and phenotype characterization in a Spanish cohort with isovaleric acidemia.

Isovaleric acidemia (IVA) is a rare disorder of leucine metabolism. We carried out a multicenter study of IVA patients diagnosed by newborn screening (NBS) or symptoms clinics over a period of 28 years in Spain. Evaluated at diagnosis, data included age, detection method, levels of C5 and IVG, enzymatic studies, clinical presentation parameters and genotype in 16 patients. Follow-up data included C5 levels, intellectual quotient and correlation genotype-phenotype. IVA was detected by NBS in 8 patients (prevalence of 1/326 629). Except 1, all the 8 patients identified by NBS were asymptomatic at diagnosis and had isovalerylcarnitine (C5) levels of 1.6-6.4 µM and isovalerylglycine (IVG) levels <1100 mmol per mol creatinine; they remained asymptomatic with a natural protein intake ⩾1.5 g kg-1 per day. Symptomatic patients with chronic intermittent or acute neonatal IVA had C5 levels of 3.9-16.3 µM and IVG levels >3400 mmol per mol creatinine. The percentage of isovalerate incorporation in fibroblasts was 64-80% in patients detected by NBS and 4.9-13% in symptomatic patients. Cognitive function was within normal ranges in all patients but was negatively correlated with IVG at detection (-0.592; P<0.05). The genetic analysis revealed nine novel mutations. The clinical/biochemical phenotype correlated fairly well with the phenotype predicted by the mutations found. In conclusion, although blood C5 levels have traditionally been considered the prognostic marker of choice, urine IVG levels would appear to be a better predictor, as they correlated well with severity of mutations and were associated with a lower incorporation rate of IVA in fibroblasts and a less favorable clinical course.